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Characterization and comparison of plant and crustacean polyphenol oxidases

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Title:
Characterization and comparison of plant and crustacean polyphenol oxidases kinetics and inhibition by chemical methods
Creator:
Chen, Jon-shang, 1958-
Publication Date:
Language:
English
Physical Description:
xii, 216 leaves : ill., photos ; 29 cm.

Subjects

Subjects / Keywords:
Apples ( jstor )
Enzymes ( jstor )
Gels ( jstor )
Lobsters ( jstor )
Mushrooms ( jstor )
Oxidases ( jstor )
Phosphates ( jstor )
Polyphenols ( jstor )
Shrimp ( jstor )
Sodium ( jstor )
Crustacea -- Analysis ( lcsh )
Dissertations, Academic -- Food Science and Human Nutrition -- UF
Food Science and Human Nutrition thesis Ph. D
Oxidases -- Analysis ( lcsh )
Plants -- Analysis ( lcsh )
City of Gainesville ( local )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1991.
Bibliography:
Includes bibliographical references (leaves 199-215).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Jon-Shang Chen.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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26272856 ( OCLC )
AJF8221 ( NOTIS )
AA00004748_00001 ( sobekcm )

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CHARACTERIZATION AND COMPARISON OF PLANT AND CRUSTACEAN
POLYPHENOL OXIDASES: KINETICS AND INHIBITION BY CHEMICAL METHODS

















By

JON-SHANG CHEN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1991


UNIVERSITY OF FLORIDA !! .PI~ES














ACKNOWLEDGEMENTS


I wish to express my sincere appreciation to Drs. Marty R. Marshall

(the chairman of the graduate supervisory committee) and Cheng-I Wei (the

cochairman) for their scientific guidance and financial support during my

graduate study. I would also like to extend my gratitude to the other

members of my graduate committee, Dr. James F. Preston, Dr. Murat 0.

Balaban, and Dr. Steven Otwell for their advice and assistance in

improving the depth of this research work. My special thanks are extended

to my wife, Shaw-Lien Hsia, for her invaluable support and encouragement.















TABLE OF CONTENTS


ACKNOWLEDGEMENTS. . ... ....... .ii

LIST OF TABLES. . ... ....... vii

LIST OF FIGURES . ... ... .. .viii

ABSTRACT. . ... xii

INTRODUCTION. . ... 1

LITERATURE REVIEW . 4

Polyphenol Oxidase . 4
Enzymatic Browning (Melanosis) . 6
Inhibition/Prevention of Melanosis ... 9
Kojic Acid. . 12
Supercritical Fluid (SCF) Treatment 12

CHARACTERIZATION AND COMPARISON OF CRUSTACEAN AND PLANT POLYPHENOL
OXIDASES: KINETICS AND SOME PROPERTIES. ... 16

Introduction . .. 16
Materials and Methods . 17
Extraction and Purification of Lobster Polyphenol Oxidase
(PPO) . 18
Extraction and Purification of Potato PPO .. 19
Extraction and Purification of Apple PPO .. 20
Extraction and Purification of White Shrimp and Grass Prawn
PPO . 21
Protein Quantitation and Enzyme Purity Determination. .. .. 23
PPO Activity Determination .. 23
pH Optima and Stability of Lobster PPO ... 24
Activation Energy and Thermostability of Lobster PPO. .. .. 24
Molecular Weight Determination of Lobster PPO ... 25
Enzyme Kinetics Study .... 26
Results and Discussion . .. 26
Effect of pH on Lobster PPO Activity ... 26
Effect of pH on Lobster PPO Stability .. 31
Effect of Temperature on Lobster PPO Activity and Stability 31
Activation Energy (Ea) of Lobster PPO .. 39
Molecular Weight and Isoform Determination of Lobster PPO 40
Enzyme Kinetics of Lobster PPO. ... 43









Conclusion. .. .. .51

STRUCTURAL COMPARISON OF CRUSTACEAN, PLANT, AND MUSHROOM POLYPHENOL
OXIDASES . ... 52

Introduction. . ... 52
Materials and Methods . 53
Extraction of Mushroom, Potato, Lobster, and Shrimp PPO 53
Purification of Mushroom, Potato, Lobster, and Shrimp PPO 54
Protein Quantitation and Enzyme Purity Determination. .. .. 54
Enzyme Activity Assay . .. 55
Anti-lobster PPO Antibody Production and Purification 55
Molecular Weight Determination of Anti-lobster PPO Antibody 56
Antibody Titer Determination by Enzyme-linked Immunosorbent
Assay (ELISA) ................ .. 57
Analysis of Antigenic Properties of PPO .. ... 58
Immunoblotting . .. 58
Spectropolarimetric Analysis of PPO 59
Results and Discussion. . ... 60
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE) Profile of PPO . .. 60
Antibody Production and Molecular Weight Determination. 63
Antibody Titer Determination .... .63
Immunological Characteristics of PPO. ... 70
Spectropolarimetric Analysis of PPO .. 75
Conclusion. . .. 89

INHIBITION MECHANISM OF KOJIC ACID ON SOME PLANT AND CRUSTACEAN
POLYPHENOL OXIDASES . 90

Introduction . 90
Materials and Methods . 91
Extraction and Purification of Potato PPO .. 92
DEAE-cellulose Chromatography .. 93
Gel Filtration . .. 93
Extraction and Purification of Apple PPO .. 94
Hydroxylapatite Chromatography ... 94
Extraction and Purification of Grass Prawn PPO ... 95
Extraction and Purification of Lobster PPO ... 96
Extraction and Purification of white shrimp PPO .. 97
Protein Quantitation and Enzyme Purity Determination. ... 98
Enzyme Activity Assay . .. 98
Effect of Kojic Acid on Enzyme Activity 99
Enzyme Kinetics Study . 100
Effect of Pre-incubation Temperature on PPO Inhibition by
Kojic Acid . 102
Effect of Kojic Acid on the Hydroxylation Capability of PPO 103
Effect of Kojic Acid on Oxygen Uptake by PPO Reaction 103
Effect of Kojic Acid on Reduction of Cupric Copper .... 104
Effect of Kojic Acid on Quinone Products. 104
Enzymatic Activities of Kojic Acid-treated PPO ... 105
Statistical Analysis . ... 106









Results and Discussion. . ... 107
Effect of Kojic Acid on Mushroom PPO Activity ... 107
Effect of Kojic Acid on Potato PPO Activity ... 107
Effect of Kojic Acid on Apple PPO Activity. .. 114
Effect of Kojic acid on Crustacean PPO Activity ...... 117
Enzyme Kinetics . .. .. 117
Effect of Pre-incubation Temperature on PPO Inhibition by
Kojic Acid. . .. 129
Effect of Kojic Acid on the Hydroxylation Capability of PPO 129
Effect of Kojic Acid on Oxygen Uptake by PPO Reaction 131
Effect of Kojic Acid on Reduction of Cupric Copper. .131
Effect of Kojic Acid on Quinone Products. ... 135
Enzymatic Activities of Kojic Acid-treated PPO. .. 140
Conclusion. . ... ....... 140

EFFECT OF CARBON DIOXIDE ON THE INACTIVATION OF PLANT AND
CRUSTACEAN POLYPHENOL OXIDASES. ... 143

Introduction. . ... ....... 143
Materials and Methods . 144
Extraction and Purification of Lobster, Shrimp, and Potato
PPO .. ... .144
Protein Quantitation and Enzyme Purity Determination. ... 145
Enzyme Activity Assay .......... .. 146
Effect of Carbon Dioxide (1 atm) on PPO Activity. ...... 146
pH Control Study. ... 147
Effect of High Pressure (58 atm) Carbon Dioxide on PPO
Activity. . 147
Kinetics of PPO Inactivation. ... 150
Nondenaturing Polyacrylamide Gel Electrophoresis of Carbon
Dioxide (1 atm)-treated PPO .. ..... 151
Mass Balance of High Pressure Carbon Dioxide-treated and
Nontreated PPO. . .. 151
Polyacrylamide Gel Isoelectric Focusing of Carbon Dioxide-
treated PPO .. ... .152
Spectropolarimetric Analysis of PPO .. 153
Study of Restoration of Carbon Dioxide-treated PPO Activity 153
Results and Discussion .................. .. 154
Effect of Carbon Dioxide (1 atm) on PPO Activity. ... 154
Effect of Nitrogen on PPO Activity. ... 157
Effect of High Pressure Carbon Dioxide on PPO Activity. 159
Kinetics of PPO Inactivation .. 166
Polyacrylamide Gel Electrophoresis of Carbon Dioxide (1 atm)-
treated PPO . 168
Mass Balance of High Pressure Carbon Dioxide-treated and
Nontreated PPO . 168
Polyacrylamide Gel Isoelectric Focusing of Carbon Dioxide-
treated PPO . 172
Spectropolarimetric Analysis of High Pressure Carbon Dioxide-
treated PPO . 172
Restorative Ability of Carbon Dioxide-treated PPO Activity. 186
Conclusion . 189









CONCLUSIONS . 197

REFERENCE LIST..................... ..... .199

BIOGRAPHICAL SKETCH .. .. .. 216














LIST OF TABLES


TABLE PAGE

1 Comparison of Kinetic Properties of Polyphenol Oxidase (PPO)
from Various Sources . .. 30

2 Comparison of Polyphenol Oxidase (PPO) Activity between
Florida Spiny Lobster and Western Australian Lobster 49
3 Secondary Structure Estimates of Various Polyphenol Oxidases
(PPOs) from Far UV Circular Dichroic Spectra ... 88
4 Inhibitory Mechanism of Kojic Acid on Polyphenol Oxidase
Obtained from Various Sources. ... 124

5 Effect of Different Pre-incubation Temperatures on the
Inhibition of Various Polyphenol Oxidases (PPOs) by Kojic
Acid . .. ... .130

6 Inhibitory Effect of Kojic Acid on the Consumption of Oxygen
by Polyphenol Oxidase. . ... 134

7 Enzymatic Activity of Kojic Acid-treated Polyphenol Oxidase
Following Kojic Acid Removal .... 141

8 Effect of Nitrogen on Florida Spiny Lobster Polyphenol Oxidase
Activity . ... .158

9 Kinetic Parameters of Florida Spiny Lobster Polyphenol Oxidase
Inactivation by Carbon Dioxide (1 atm) at Various
Temperatures . ... ...... 167

10 Mass Balance of Protein Contents from High Pressure CO2-
treated and Nontreated PPO . 171

11 Secondary Structure Estimates of Nontreated Control and High
Pressure CO -treated Florida Spiny Lobster, Brown Shrimp,
and Potato Polyphenol Oxidases (PPOs) from Far UV Circular
Dichroic Spectra .. .. 185















LIST OF FIGURES


FIGURE PAGE

1 Mechanism of Polyphenol Oxidase-catalyzed Reaction .. 8

2 Structure of Kojic Acid (5-hydroxy-2-hydroxymethyl-y-
pyrone). . ... ...... .14
3 Effect of pH on the Activity of Polyphenol Oxidase (PPO)
Isolated from Florida Spiny Lobster and Western
Australian Lobster .... 28

4 Effect of pH on the Stability of Polyphenol Oxidase (PPO)
Isolated from Florida Spiny Lobster and Western
Australian Lobster . 33

5 Effect of Temperature on the Activity of Polyphenol Oxidase
(PPO) Isolated from Florida Spiny Lobster and Western
Australian Lobster . .. 35

6 Effect of Temperature on the Stability of Polyphenol
Oxidase (PPO) Isolated from Florida Spiny Lobster and
Western Australian Lobster .. 38

7 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) Profile of Polyphenol Oxidase from
Florida Spiny Lobster (FSL) and Western Australian
Lobster (WAL). . 42

8 Double Reciprocal Plots for the Oxidation of DL-DOPA and
Catechol by Florida Spiny Lobster Polyphenol Oxidase
(PPO). . 45

9 Double Reciprocal Plots for the Oxidation of DL-DOPA and
Catechol by Western Australian Lobster Polyphenol
Oxidase (PPO). . ... .. 47

10 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) Profile of Polyphenol Oxidase from
Mushroom, Potato, Florida Spiny Lobster, White Shrimp,
and Brown Shrimp .... 62

11 Profile of Anti-lobster PPO Antibody Production in
Immunized Hen during the Immunization Period .. 65


viii









12 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) Profile of Anti-lobster PPO
Antibody . .. 67

13 The Titer Determination of Antibody against Florida Spiny
Lobster PPO versus Mushroom, Potato, Florida Spiny
Lobster, White Shrimp, and Brown Shrimp PPO. .. 69

14 Analysis of Antigenic Properties of Purified Mushroom,
Potato, Florida Spiny Lobster, White Shrimp, and Brown
Shrimp PPO by Competitive ELISA. ... 72

15 Profile of Protein Standard (SDS-6H), Purified Mushroom,
Potato, Florida Spiny Lobster, White Shrimp, and Brown
Shrimp PPO, and Crude Lobster PPO Preparation on a
Nitrocellulose Membrane. ... 74

16 Determination of the Specific Reactivity of Anti-lobster
PPO Antibody with Crude Lobster PPO Preparation,
Purified Mushroom, Potato, Florida Spiny Lobster, White
Shrimp, and Brown Shrimp PPO as well as SDS-6H Protein
Standard as Analyzed by Immunoblotting Technique 77

17 The far UV Circular Dichroic Spectra of Mushroom PPO 79

18 The far UV Circular Dichroic Spectra of Potato PPO 81

19 The far UV Circular Dichroic Spectra of Florida Spiny
Lobster PPO. .. .. 83

20 The far UV Circular Dichroic Spectra of White Shrimp PPO 85

21 The far UV Circular Dichroic Spectra of Brown Shrimp PPO 87

22 Effect of Concentration-related Inhibitory Effect of Kojic
Acid on Mushroom Tyrosinase (PPO) Activity on DL-P-3,4-
dihydroxyphenylalanine (DL-DOPA) .... .... 109

23 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of DL-DOPA and L-Tyrosine by Mushroom
PPO. . .111

24 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of Chlorogenic Acid and Catechol by
Potato PPO . .. 113

25 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of 4-Methylcatechol and Chlorogenic
Acid by Apple PPO. . ... 116









26 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of L-DOPA and Catechol by White Shrimp
PPO. . ... 119
27 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of DL-DOPA and Catechol by Grass Prawn
PPO. . ... ....... 121

28 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of DL-DOPA and Catechol by Lobster PPO. 123

29 Effect of Kojic Acid on the Hydroxylation of Monohydroxy-
phenol by Mushroom Tyrosinase (PPO). ... 133
30 Effect of Kojic Acid on Reduction of Cu2+ to Cu+ in a
Model System . 137

31 Change in Absorption Spectra of Dopaquinone due to the
Addition of Kojic Acid. (a) Dopaquinone Produced from
the Action of Mushroom PPO on DL-DOPA; and
(b) Dopaquinone Plus Kojic Acid. ... 139

32 Apparatus Used for Studying Polyphenol Oxidase (PPO)
Inactivation by High Pressure CO .. 149
33 Effect of Carbon Dioxide (1 atm) on the Change in pH and
Enzyme Activity of Florida Spiny Lobster PPO Heated at
330 (a), 380 (b), or 430C (c). ... 156
34 Effect of High Pressure (58 atm) Carbon Dioxide on the
Change in pH and Enzyme Activity of Florida Spiny
Lobster PPO heated at 43C .. 161
35 Effect of High Pressure (58 atm) Carbon Dioxide on the
Change in pH and Enzyme Activity of Brown Shrimp PPO
heated at 43C . 163
36 Effect of High Pressure (58 atm) Carbon Dioxide on the
Change in pH and Enzyme Activity of Potato PPO heated at
430C . 165

37 Nondenaturing Polyacrylamide Gel Electrophoresis (PAGE,
7.5% Gel) Profile of Carbon Dioxide (1 atm)-treated
Florida Spiny Lobster PPO 170
38 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) Profile of High Pressure (58 atm)
Carbon Dioxide-treated Florida Spiny Lobster PPO 174









39 Polyacrylamide Gel Isoelectric Focusing (IEF, 5% Gel)
Profile of Carbon Dioxide (1 atm)-treated Florida Spiny
Lobster PPO ..... 176

40 Polyacrylamide Gel Isoelectric Focusing (IEF, 5% Gel)
Profile of High Pressure (58 atm) Carbon Dioxide-treated
Florida Spiny Lobster PPO. 178

41 Comparison of Far UV Circular Dichroic Spectra of
Nontreated Control and High Pressure (58 atm) Carbon
Dioxide-treated Florida Spiny Lobster PPO. .. 180

42 Comparison of Far UV Circular Dichroic Spectra of
Nontreated Control and High Pressure (58 atm) Carbon
Dioxide-treated Brown Shrimp PPO .... 182

43 Comparison of Far UV Circular Dichroic Spectra of
Nontreated Control and High Pressure (58 atm) Carbon
Dioxide-treated Potato PPO .... 184

44 The Restorative Ability of Carbon Dioxide (1 atm)-treated
Florida Spiny Lobster PPO Activity and the Pertinent
Environmental pH Changes during Frozen-storage .. 188

45 The Restorative Ability of High Pressure (58 atm) Carbon
Dioxide-treated Florida Spiny Lobster PPO Activity and
the Pertinent Environmental pH Changes during Frozen-
storage. ... ..... 191

46 The Restorative Ability of High Pressure (58 atm) Carbon
Dioxide-treated Brown Shrimp PPO Activity and the
Pertinent Environmental pH Changes during Frozen-storage 193

47 The Restorative Ability of High Pressure (58 atm) Carbon
Dioxide-treated Potato PPO Activity and the Pertinent
Environmental pH Changes during Frozen-storage .195














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

CHARACTERIZATION AND COMPARISON OF PLANT AND CRUSTACEAN
POLYPHENOL OXIDASES: KINETICS AND INHIBITION BY CHEMICAL METHODS

By

JON-SHANG CHEN

December, 1991

Chairman: M.R. Marshall
Cochair: C.I. Wei
Major Department: Food Science and Human Nutrition

The characterization and comparison of polyphenol oxidase (PPO, E.C.

1.14.18.1) from crustaceans (lobster and shrimp) and plants were made.

PPO from various plant and crustacean sources varied with respect to

molecular weight, pH and temperature effects on activity and stability,

substrate specificity, and other kinetic parameters. Differences in

conformational structure among PPOs from mushroom, potato, lobster, white

shrimp, and brown shrimp were investigated using spectropolarimetric and

immunological methods. Varied secondary structures were observed among

these PPOs, although they were shown to possess similar antigenic

determinants.

Kojic acid [5-hydroxy-2-(hydroxymethyl)---pyrone], a fungal

metabolite, exhibited competitive and/or mixed types of inhibition on the

oxidation of various phenolic substrates (L-tyrosine, DL-P-3,4-dihydroxy

phenylalanine, 4-methylcatechol, catechol, and chlorogenic acid) by

mushroom, potato, apple, and crustacean (lobster, grass prawn, and white
xii









shrimp) PPOs. In addition, kojic acid was capable of reducing o-quinones

back to diphenols and prevented melanin formation.

The inactivation of lobster, brown shrimp, and potato PPO by CO2 was

studied. When exposed to CO2 (1 atm) at 330, 380, or 430C, lobster PPO

showed a decline in enzyme activity with treatment time. Studies on

inactivation kinetics revealed that PPO was more labile to CO2 and heat

than to heat alone. The use of polyacrylamide gel electrophoresis showed

that there were no differences in protein patterns and isoelectric

profiles between the CO2 (1 atm)-treated and untreated PPO. When lobster,

brown shrimp, and potato PPOs were subjected to high pressure (58 atm) CO2

at 430C, the inactivation of these PPOs followed trends similar to the

atmospheric CO, experiments. Crustacean PPOs, however, were more

susceptible to inactivation by high pressure CO2 than by atmospheric CO,.

Differences in the secondary structures between the high pressure CO2-

treated and the nontreated PPO were evident by spectropolarimetric

analysis.














INTRODUCTION


Undesirable enzymatic browning causing the discoloration or
formation of black spots melanosiss) by polyphenol oxidase (E.C.

1.14.18.1; PPO) on the surface of many vegetable, fruit, and seafood

(crustacean) products has been of great concern to food scientists. For

food processors, the formation of melanin pigments not only imparts the

problems in sensory attributes and, hence, the marketability of the

product, but often lower its nutritive value as well (Synge, 1975).

Kinetic properties have been studied for PPO from various sources of
vegetables and fruits (Schwimmer, 1981) and more recently from crustaceans

(Ferrer et al., 1989a; Rolle et al., 1991; Simpson et al., 1987, 1988a).

However, little comparative biochemical information exists between PPO

from plants and crustaceans. A preliminary study conducted in this

laboratory revealed that Western Australian lobster (Panulirus cygnus) was

far less susceptible to melanosis during storage at refrigeration

temperature than Florida spiny lobster (Panulirus argus) maintained under

the same conditions. It is speculated that the susceptibility to

melanosis could be attributed to the difference in PPO activity between

these two lobster species. Thus, the first objective of this study was to

characterize and determine the kinetic properties of PPO enzymes from

these two lobsters as well as from other crustacean and plant sources.

Many chemical and physical methods have been studied for their

effectiveness on the inhibition of enzymatic browning. Browning may be

1









2

prevented not only by inactivating the enzyme, but also by eliminating

substrate (02 or polyphenol) necessary for the reaction, or by reacting

with the products of the enzyme reaction thus preventing the formation of

the quinones necessary for the preceding non-enzymatic steps. Although

some chemicals have been shown to inhibit PPO activities, their use in

food processing is restricted by many concerns such as toxicity,

wholesomeness, effect on taste, flavor, texture, etc. (Vamos-Vigyazo,

1981). Kojic acid (5-hydroxy-2-hydroxymethyl---pyrone), a fungal

metabolite produced by many species of Aspergillus and Penicillium

(Kinosita and Shikata, 1964), has been reported for its inhibitory effect

on mushroom PPO (Saruno et al., 1979). Kojic acid mixed with ascorbic

acid and citric acid constitutes a Japanese commercial product which is

used as a tyrosinase inhibitor in foods. Since only limited information

was available on the inhibitory activity and mechanism of kojic acid on

PPO, the second objective of this study was to investigate the inhibitory

activity of this compound on crustacean (Florida spiny lobster, white

shrimp, and grass prawn) mushroom, and plant (potato and apple) PPO and to

elaborate the mechanisms of inhibition.

In past years, treatment in an atmosphere modified with carbon

dioxide has been used as an application for retarding enzyme activity, to

preserve food quality and to extend the shelf-life of food products.

Recently, inactivation of peroxidase, PPO, pectinesterase, a-amylase,

glucose oxidase, lipase, or catalase by supercritical fluid using CO2 as

the solvent has been reported by many workers (Arreola, 1990; Christianson

et al., 1984; Taniguchi et al., 1987; Zemel, 1989). However, information

concerning the inhibitory effect and the inhibition mechanism of CO2 on








3

plant and crustacean PPO was limited. Thus, the third objective of this

study was to investigate the effect of CO2 (atmospheric and high pressure)

on the inhibition of lobster, brown shrimp, and potato PPO.














LITERATURE REVIEW


Polyphenol Oxidase


Polyphenol oxidase (PPO) (E.C. 1.14.18.1.), also known as

tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, and

catecholase, is widely distributed in nature (Schwimmer, 1981). In

addition to its general occurrence in plants, it can also be found in

microorganisms, especially fungi, and in some animal tissue (Brown,

1967). PPO plays an important role in the resistance of plants to

microbial or viral infections and probably to adverse climate (Vamos-

Vigyazo, 1981). The vast literature dealing with the role of the PPO-

polyphenol system in plant pathology has been extensively reviewed

(Bonner, 1957; Farkas and Kiraly, 1962). A simplified explanation of this

role in the resistance of plants to infections is that the quinones formed

upon the action of the enzyme undergo secondary polymerization reactions

yielding dark, insoluble polymers; the tissues impregnated with these

polymers act as barriers to prevent further spreading of the infection.

This is considered by some authors to be the main function of the enzyme

(Macrae and Duggleby, 1968). However, plants resistant to adverse

climatic conditions have, in general, higher PPO activities than

susceptible varieties (Khrushcheva and Krehin, 1965).

In addition to the involvement in phenolic compounds biosynthesis,

the PPO enzymes also indirectly participate in auxin biosynthesis. The











primary products of its action on o-diphenols, the o-quinones, react with

tryptophan to form indole acetic acid via indolepyruvic acid (Gordon and

Paleg, 1961). Thus, PPO together with auxin degrading enzyme

(peroxidase), might play a role in plant growth regulation (Vamos-Vigyazo,

1981). The quinones formed upon PPO action may also participate in

reactions similar to those leading to nonenzymatic browning and

humification and thus contribute to producing organic matter of soil

(Synge, 1975).

PPO from insects and crustaceans plays an important role in the
sclerotization during molting (Andersen, 1971; Brunet, 1980; Cobb, 1977;

Ferrer et al., 1989a; Summers, 1967; Vinayakam and Nellaiappan, 1987). In

this process, PPO oxidizes diphenols to quinones, which interact with

certain side groups on adjacent proteins, thus linking them together

(Stevenson, 1985). N-Acetyldopamine (Andersen, 1979) and 3,4-

dihydroxybenzoic acid (Pryor et al., 1962) have been identified as the

cross-linking agents in crustacean and cockroach, respectively. Another

function closely related to plant systems was PPO's involvement in the

process of wound repair and calcification of the cuticle (Stevenson,

1985).

Most PPO's are copper-containing enzymes which catalyze two entirely
different reactions: (a) the hydroxylation of monophenols to the

corresponding o-dihydroxy compounds, which is called cresolase activity,

and (b) the oxidation of o-dihydroxy phenols to o-quinones, which is

termed catechol activity. The activity of cresolase involves three steps

which can be represented by the following equation (Mason, 1956):

Protein-Cu2+-02 + monophenol Protein-Cu2+ + o-quinone + H20









6
The protein-copper-oxygen complex is formed by combining one molecule of

oxygen with the protein to which two adjacent cuprous atoms are attached.

Catecholase activity involves the oxidation of 2 molecules of o-diphenols

to 2 molecules of o-quinones, resulting in the reduction of one molecule

of oxygen to two molecules of water. The sequence for the PPO-catalyzed

reaction proposed by Mason (1957) is shown in Figure 1. The enzyme-oxygen

complex serves as the hydroxylating or dehydroxylating intermediate, and

(Cu)n represents the actual charge designation of the copper at the active
site. The overall reaction involves the use of one molecule of oxygen;

one atom of which goes into the formation of diphenol, and the other is

reduced to water. Substrate specificity varies considerably for PPO from

various sources (Aurand and Woods, 1973).


Enzymatic Browning (Melanosis)

Food scientists' primary interest in PPO is in the enzymatic

browning phenomenon and its effect on food quality. o-Quinones, the

primary products of PPO oxidative reaction can (a) interact with each

other to form high molecular weight polymers, (b) form macromolecular

complexes with amino acids or proteins, and (c) oxidize compounds of lower

oxidation-reduction potentials (Mathew and Parpia, 1971). Enzymatic

browning is a desirable process for the manufacture of black tea, sultana

grape, and ground coffee bean (De Amorim and Silva, 1968; Grncarevic and
Hawker, 1971; Takeo, 1966).

Unfavorable darkening on the surface of many fruits, vegetables, and
seafood products is primarily due to the indirect consequence of PPO

oxidizing phenols to orthoquinones, which in turn rapidly polymerize to


















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Ol- c
t<- 0
0 0 *- V
u-, +- r
e "as



Cr +J X








8


cr

00
C



I +
I 0


xc
+ 0
S+:3
0" CL )0
< X, o



00 cI 6 0

00





t-
*L_
0 w





+ I
C =: .1

0+ ZI )


co









9

form brown pigments or melanins (Joslyn and Ponting, 1951). Enzymatic

browning of fruits, vegetables, and seafood products (mainly shrimp and

lobster) due to PPO activity has been extensively studied (Ferrer et al.,

1989a; Flurkey and Jen, 1978; Harel et al., 1966; Koburger et al., 1985;

Macrae and Duggleby, 1968; Madero and Finne, 1982; Mapson et al.,1963;

Mosel and Hermann, 1974; Palmer, 1963; Patil and Zucker, 1965; Savagaon

and Sreenivasan, 1978; Sciancalepore and Longone, 1984; Simpson et al.,

1987, 1988a; Walker, 1962, 1964; Weurman and Swain, 1955). Although not

harmful to consumers, it is the formation of melanins on the surface of

these products by PPO that decreases their market value, making them

unappealing and the perception of spoilage (Alford and Fieger, 1952;

Bailey and Fieger, 1954; Bailey et al., 1960a, 1960b; Faulkner et al.,

1954; Ogawa et al., 1984).

Inhibition/Prevention of Melanosis


Undesirable enzymatic browning occurring in agricultural products

causes an enormous economical loss for food industries. Thus, in order to

reduce this loss, the occurrence of melanosis on food products must be

prevented or inhibited. Inhibition of PPO and prevention of enzymatic

browning are often treated as one and the same subject. Browning can be

prevented by inactivating PPO, by eliminating one of the substrates

necessary for the reaction (02 and the polyphenols), or by reacting the

products of PPO metabolism which in turn inhibit the formation of the

colored compounds produced in the secondary, non-enzymatic reaction steps.

As PPO is a metalloprotein with copper as the prosthetic group (Bailey et

al., 1960a; Bendall and Gregory, 1966; Mayer, 1962; Smith and Kruger,









10

1962), the activity of this enzyme can be inhibited by metal-chelating

agents. The effects of cyanide, carbon monoxide, sodium-diethyl-dithio-

carbamate (DIECA), mercaptobenzothiazole, dimercaptopropanol, potassium

methyl xanthate, fluoride, azide, borate, benzoic acid, small peptides,

polyvinylpyrrolidone (PVP), 2,3-naphthalenediol, ascorbic acid, and

dichlorodifluoromethane on plant PPO activity have been extensively

studied; these chemical inhibitors act primarily on the enzyme (Bedrosian

et al., 1959; Harel et al., 1967; Jones et al., 1965; Mayer et al., 1964;

Palmer and Roberts, 1967; Pierpoint, 1966; Pifferi et al., 1974; Robb et

al., 1966; Taeufel and Voigt, 1964; Walker, 1964, 1975, 1976; Walker and

Wilson, 1975; Yasunobu and Norris, 1957).

Other compounds acting as reducing agents which reduce o-quinones to

diphenols or react with plant PPO substrates to lessen the extent of

enzymatic browning are ascorbic acid, SO2, sodium sulfite, sodium

bisulfite, sodium metabisulfite, potassium metabisulfite, cysteine,

2-mercaptoethanol, glutathione, benzenesulphinic acid, DIECA, Na-ethyl

xanthate, and PVP (Cash et al., 1976; Embs and Markakis, 1965; Feinberg et

al., 1976; Haisman, 1974; Hope, 1961; Markakis and Embs, 1966; Mapson,

1965; Mapson and Wager, 1961; Muneta, 1966; Muneta and Walradt, 1968;

Muneta and Wang, 1977; Ponting, 1960; Ponting and Jackson, 1972;

Sayavedra-Soto and Montgomery, 1986; Singh and Ahlawati, 1974; Taeufel and

Voigt, 1964; Vamos-Vigyazo, 1981; Walker, 1964). For the prevention of

melanosis in crustaceans, numerous agents including ascorbic acid and

sodium ascorbate, ascorbate and citrate, L-tyrosine, L-methionine, citric

acid, L-cysteine, sodium diethyl dithiocarbamate, sodium tripolyphosphate

(STP), sodium sulfite, and sodium bisulfite have been broadly studied









11

(Antony and Nair, 1975; Bailey and Fieger, 1954; Faulkner et al., 1954;

Ferrer et al., 1989b; Fink, 1988; Madero, 1982; Madero and Finne, 1982).

Many methods are available for inhibiting/inactivating PPO. The

application of these methods or chemicals for inhibition in the food

industry, however, is limited. Problems related to off-flavor, off-odor,

toxicity, and economic feasibility all affect the application of chemicals

(Eskin et al., 1971). Sulfiting agents listed as Generally Recognized As

Safe (GRAS) by the Food and Drug Administration (FDA) since 1959 (FDA,

1984) have been widely used to prevent melanosis of agricultural and

seafood products. However, due to the recent health concern and questions

regarding the safety of sulfiting agents, the FDA currently proposed

revoking the GRAS status of these additives for use on fruits and

vegetables intended to be served or sold fresh or raw to consumers. The

FDA also requires label declaration of these agents added to food or as an

ingredient whenever they are present in the finished product at a level

equal to or higher than 10 ppm as SO2 (FDA, 1985). Furthermore, the FDA

requires that sulfite-treated shrimp products having residue levels

greater than 10 ppm must bear a label stating the presence of these

additives (FDA, 1985).

The maximum allowable residual level of sulfite on shrimp in the raw

edible portion is 100 ppm as SO2 (Marshall et al., 1984), which is not

hazardous to the majority of the population. After exposure to these
additives even at this level, however, people still suffer adverse

reactions ranging from mild to severe symptoms (Lecos, 1986). Therefore,

the search for an alternative which can inhibit melanosis but does not

cause adverse reactions becomes a necessity.











Kojic Acid

The inhibitory effect of some tyrosine-3-hydroxylase inhibitors of

microbial origins such as aquayamycin, chrothiomycin, and oudenone on PPO

activity has been studied (Ayukawa et al., 1968, 1969; Nagatsu et al.,

1968; Sezaki et al., 1968; Umezawa et al., 1970). Kojic acid (5-hydroxy-

2-hydroxymethyl-7-pyrone) (Figure 2) produced in the culture filtrate of

Aspergillus albus was shown in vitro to inhibit the activity of mushroom

tyrosinase using DOPA and tyrosine as substrates (Saruno et al., 1979).

At low concentration, kojic acid was found to inhibit the in vitro

oxidation of a number of D-amino acids, L-phenylalanine and a few related

compounds (Bajpai et al., 1981). Production of kojic acid, generally

considered as a secondary metabolite from carbohydrate metabolism under

aerobic conditions, has been extensively studied (Bajpai et al., 1981,

1982a, 1982b; Morton et al., 1945; Saruno et al., 1979). Although kojic

acid can be considered a potential candidate to inhibit melanosis, the use

of this chemical, depends not only on its production but also its

potential toxicity to humans.


Supercritical fluid (SCF) Treatment

Intensive study of supercritical fluids for extraction of food

components has mostly concentrated on decaffeination, deodorization, aroma

recovery, oil recovery, oil refining, and fractionation (Bulley et al.,

1984; Christianson et al., 1984; Friedrich and Pryde, 1984; Lee et al.,

1986; Maddocks and Gibson, 1977; Peter and Brunner, 1978; Snyder et al.,

1984; Schultz et al., 1967; Stahl et al., 1978, 1980, 1984; Taniguchi et

al., 1985; Vollbrecht, 1982; Zosel, 1979, 1981). Modified atmospheres






























C
0
O
S.-



I






I


0-
X
O








.0




Lf



0


'-a
u

u





*4-

U





l..







0)














17
0
I
0


0


0

Q.
a)
E






0
0t






C'
I
-o

0
"0
--
I
LC)


O)


O
10


0
I:









15

(carbon dioxide or C02-saturated brines) have been used successfully to

preserve food quality and extend shelf-life (Barnett et al., 1978; Brown

et al., 1980; Bullard and Collins, 1978; Gee and Brown, 1987a, 1987b;

Lannelongue et al., 1982; Longard and Reiger, 1974; Shewan, 1950; Vernath

and Robe, 1979; Villemure et al., 1986; Woyewoda et al., 1984; Yokoseki et

al., 1956). Studies of seafood preservation using a modified atmosphere

of CO2 showed that the lower internal pH of the tissue was due to exposure

of the external CO2 present and resulted in a rapid acidification of the

internal cellular environment (Aickin and Thomas, 1975; Boron and DeWeer,

1976; Thomas, 1974; Thomas and Ellis, 1976; Turin and Warner, 1977).

Intracellular and extracellular acidification of tissue could produce an

antimicrobial effect and also influence many different enzymatic

activities (Parkin et al., 1981). SCF possesses physicochemical

properties intermediate between those of liquids and gases which enhance

its efficacy as solvents; the higher gas density gives good solvent power,

while the lower viscosity and higher diffusivity provide the SCF with

higher gas permeability (Rizvi et al., 1986). Carbon dioxide is used as

a solvent for SCF because it is nontoxic, nonflammable, inexpensive and

readily available (Hardardottir and Kinsella, 1988) and it has a

relatively low critical temperature (33.1C) and pressure (72 atm) (Rizvi

et al., 1986). Eldridge et al. (1986) reported that SCF produced minimal

detrimental effects on the functional properties of proteins. The

application of SCF using CO2 as a solvent in seafood processing could be

a benefit because it possesses characteristics of antimicrobial action,

lipid (cholesterol) reduction, and enzymatic inhibitory activities.














CHARACTERIZATION AND COMPARISON OF CRUSTACEAN AND PLANT POLYPHENOL
OXIDASES: KINETICS AND SOME PROPERTIES

Introduction


Polyphenol oxidase (PPO) (E.C. 1.14.18.1.), also known as

tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, and

catecholase, is widely distributed in nature (Schwimmer, 1981). In

addition to its general occurrence in plants, it can also be found in

microorganisms, especially in fungi, and in some animal organs (Brown,

1967). Unfavorable darkening of many fruits and vegetables after cutting

is primarily due to the action of this enzyme. Enzymatic browning of

fruits and vegetables due to PPO activity has been widely reported

(Flurkey and Jen, 1978; Harel et al., 1966; Macrae and Duggleby, 1968;

Mapson et al., 1963; Mosel and Herrman, 1974; Palmer, 1963; Patil and

Zucker, 1965; Sciancalepore and Longone, 1984; Walker, 1962, 1964; Weurman

and Swain, 1955). However, the role of PPO in crustaceans is not well

documented considering the variation in susceptibility to melanosis.

PPO plays an important role in the sclerotization of insects and

crustaceans during the molting cycle (Andersen, 1971; Brunet, 1980;

Summers, 1967; Vinayakam and Nellaiappan, 1987). However, it is the

formation of melanins causing darkening on the surface of seafood products

due to PPO action which is of concern to the seafood industry. PPO

enzymes from various crustaceans have been partially characterized (Madero

and Finne, 1982; Nakagawa and Nagayama, 1981; Simpson et al., 1987,









17

1988a). Although kinetic properties have been studied for PPO from

various sources of vegetables and fruits (Schwimmer, 1981) and more

recently from crustaceans (Chen et al., 1991a; Ferrer et al., 1989a; Rolle

et al., 1991), little comparative biochemical information exists between

PPO from plants and crustaceans. A preliminary study conducted in this

laboratory revealed that Western Australian lobsters were far less

susceptible than the Florida spiny lobsters to melanosis during storage at

refrigeration temperature. A similar phenomenon was observed on pink

shrimp (Penaeus duorarum) in comparison to white shrimp (Penaeus

setiferus) (Simpson et al., 1988a). These workers characterized the PPO

properties and they attributed the difference in susceptibility to

melanosis to the varied physiological efficiency of the enzyme and the

distribution of phenolic substrates in the shrimp (Simpson et al., 1988a).

Koburger et al. (1985) also showed that South African lobster tails were

less susceptible to melanosis than Florida spiny lobster tails. Since the

water habitat of Western Australian lobster is colder than that of Florida

spiny lobster, it is speculated that the PPO activity for these two

species could be different. Thus, the objectives of this study were to

characterize the PPO from these two lobster species and to compare their

kinetic properties to PPOs from other plant and crustacean sources.


Materials and Methods

Fresh Florida spiny lobster (Panulirus argus) tails obtained from

the Whitney Marine Laboratory at Marineland, FL were transported in ice to

the laboratory and stored at -200C. Frozen Western Australian lobster

(Panulirus cygnus) tails were purchased from the Beaver Street Foods









18
(Jacksonville, FL); these lobsters were found to contain less than 5 ppm

sulfite background residue when randomly selected samples from each batch

were checked using the method of Simpson et al. (1988b). Mushroom

(Agaricus bispora) tyrosinase with an activity of 2,200 units/mg solid was

purchased from Sigma Chemical Co. (St. Louis, MO). Russet potatoes and Red

Delicious apples were purchased from a local supermarket. White shrimp

(Penaeus setiferus) and brown shrimp (Penaeus aztecus) were obtained from

a local seafood store. Grass prawn or Taiwanese black tiger shrimp

(Penaeus monodon) frozen in dry ice was provided by Dr. J. S. Yang, Food

Industry Research and Development Institute, Hsinchu, Taiwan, Republic of

China. Lobster cuticle, shrimp cephalothorax (head), and potato and apple

peels were frozen in liquid nitrogen and ground into a fine powder using

a Waring blender. The ground powder was stored at -200C until needed.


Extraction and Purification of Lobster PPO

Frozen lobster tails were thawed at room temperature. After the

cuticle was separated from the flesh, it was frozen in liquid nitrogen and

ground into a fine powder in a Waring blender. The cuticle powder was

stored at -200C and used as required.

PPO was extracted according to the procedure of Simpson et al.

(1988a). One part lobster cuticle powder was added to three parts (w/v)

0.05 M sodium phosphate buffer (pH 7.2) containing 1 M NaCl and 0.2% Brij

35 (Fisher Scientific Co., Orlando, FL). The extract was stirred for 3 hr

at 40C and the suspension was centrifuged at 8,000g (40C) for 30 min. The

supernatant was then dialyzed at 40C overnight against 3 changes (4L) of

0.05 M sodium phosphate buffer (pH 6.5).


I








19

Each enzyme was purified further using a nondenaturing preparative

polyacrylamide gel electrophoresis (PAGE) system. Equipment utilized

included a gel tube chamber (Bio-Rad Model 175, Richmond, CA) and a Bio-

Rad power supply (Model EPS 500/400). A one-mL aliquot of crude enzyme
extract was applied to each of eight gel tubes (1.4 cm I.D. x 12 cm

length) containing 5% acrylamide/ 0.13% bisacrylamide gel prepared

according to the method of Sigma Bulletin No. MKR-137 (Sigma Chemical Co.,
1984), and ran at a constant current of 10 mA/tube in a buffer (pH 8.3)

containing 5 mM Tris-HC1 and 38 mM glycine. PPO was visualized using a

specific enzyme-substrate staining method (Constantinides and Bedford,
1967); 10 mM DL-P-3,4-dihydroxyphenylalanine (DL-DOPA) in 0.05 M sodium

phosphate buffer (pH 6.5) was used as a substrate. One tube was used to

determine the migration of the enzyme relative to the dye front (Rf). The

remaining gels were then sectioned at the determined Rf and the enzyme was

eluted from the gel by homogenization in 0.05 M sodium phosphate buffer

(pH 6.5) utilizing a Dounce manual tissue grinder (Wheaton, Millville,

NJ). The homogenates were filtered through Whatman No. 4 filter papers,

pooled, and concentrated using an Amicon stirred cell (Model 8050, Amicon

Co., Danvers, MA) fitted with a 10 K filter (Pharmacia LBK Biotechnology

Inc., Piscataway, NJ).

Extraction and Purification of Potato PPO

The method of Patil and Zucker (1965) with some modifications was

used. After ammonium sulfate precipitation and dialysis, crude PPO
preparation was subjected to chromatography with a DEAE-cellulose (0.95

meq/g, Sigma) column (40 cm length x 26 mm i.d., K 26/40 Pharmacia Fine









20
Chemicals) which had been equilibrated with 1.0 mM potassium phosphate

buffer (pH 7.0). Unbound phenolic compounds and proteins were washed off

using 250 mL 1 mM phosphate buffer (pH 7.0) at 24 mL/hr for 3 hr. Elution

of PPO was performed using a linear gradient (0 1.0 M) of NaC1 in 1.0 mM

potassium phosphate buffer at 24 mL/hr for 18 hr. Four-mL fractions were

collected, and protein was estimated by spectrophotometry at 280 nm.
Fractions showing PPO activity were pooled and concentrated using an

Amicon stirred cell fitted with an Amicon YM 10 filter.

The partially purified enzyme preparation was loaded onto a Sephadex
G-100 (Pharmacia) gel filtration column (Pharmacia K 26/40)

pre-equilibrated with 1.0 mM potassium phosphate buffer (pH 7.0). The

column was eluted at 40C with 400 mL 1.0 mM potassium phosphate buffer (pH
7.0) at 24 mL/hr for 15 hr. Four-mL fractions were collected and protein

estimated as above; fractions showing PPO activity were pooled and

concentrated using an Amicon stirred cell. Concentrated samples were

dialyzed at 40C overnight against 3 changes of 2L elution buffer.

Extraction and Purification of Apple PPO

The method of Stelzig et al. (1972) with some modification was
followed. Crude apple PPO after partial purification and dialysis against

H20 was loaded onto an HT hydroxylapatite (Bio-Rad) column (K 26/40). The
enzyme was desorbed from the gel using 250 mL 0.005 0.3 M (linear

gradient) sodium phosphate buffer (pH 7.6) containing 5% ammonium sulfate
at 24 mL/hr for 18 hr. Four-mL fractions were collected and protein
estimated by absorbance at 280 nm; fractions showing PPO activity were

pooled and dialyzed overnight (4C) against 2 changes of 4L H20. The









21
dialysate was further concentrated using an Amicon stirred cell fitted
with a YM 10 filter.

Extraction and Purification of White Shrimp and Grass Prawn PPO

The methods of Chen et al. (1991a) and Rolle et al. (1991) with
slight modification were followed to purify white shrimp and grass prawn

PPO, respectively. Shrimp cephalothorax powder was suspended in 3 volumes

(w/v) 0.05 M sodium phosphate buffer (pH 7.2) containing 1 M NaCl
(extraction buffer) and 0.2% (v/v) Brij 35, and stirred at 40C for 3 hr.
Following centrifugation at 23,000g (40C) for 30 min, the supernatant was
fractionated with ammonium sulfate between 0 40% saturation; protein

precipitate was collected by centrifugation at 23,500g at 40C for 30 min.

For white shrimp, the precipitate was dissolved in 0.05 M phosphate
buffer (pH 7.2) and dialyzed at 40C overnight against 3 changes of 4L of
0.05 M phosphate buffer (pH 7.2). The dialyzed PPO was loaded onto a

DEAE-cellulose (0.95 meq/g) column (K 26/40) pre-equilibrated with 0.05 M
phosphate buffer (pH 7.2). Sixty-mL of 0.05 M sodium phosphate buffer (pH

7.2) was used to desorb unbound phenolic compounds and proteins at 0.2

mL/min for 1.5 hr. Elution of PPO was performed using a 300 mL 0.05 M
sodium phosphate buffer (pH 7.2) containing a linear gradient (0 1.0 M)
of NaC1. Three-mL fractions were collected and the protein estimated by

absorbance at 280 nm. Fractions possessing PPO activity were pooled and
concentrated using an Amicon stirred cell fitted with a YM 10 filter.









22
PPO was loaded onto a Sephadex G-100 gel column (K 26/40) pre-

equilibrated with 0.05 M sodium phosphate buffer (pH 7.2) and then eluted

with 300 mL 0.05 M sodium phosphate buffer (pH 7.2) at 0.15 mL/min.

Three-mL fractions were collected and protein was estimated by absorbance

at 280 nm; fractions showing PPO activity were pooled and concentrated

using an Amicon stirred cell fitted with YM 10 filter. Concentrated PPO

was then dialyzed at 40C overnight against 3 changes of 2L H20.

For grass prawn, the precipitate was resuspended in extraction

buffer containing 40% ammonium sulfate. After homogenization using a

Dounce manual tissue grinder, the sample was centrifuged at 23,500g (40C)

for 20 min. The precipitate was homogenized in extraction buffer and

centrifuged as previously described. The resulting precipitate was

homogenized in extraction buffer, then subjected to high performance

hydrophobic interaction chromatography at 40C using a preparative Phenyl

Sepharose CL-4B (Sigma) column (K 16/40) attached to a Dionex gradient

pump (Dionex Corp., Sunnyvale, CA). The column was pre-equilibrated with

extraction buffer.

PPO was eluted with a stepwise gradient of elution buffer [100%

extraction buffer (9 mL), 50% extraction buffer in water (24 mL), and 10%

extraction buffer in water (24 mL)], 50% ethylene glycol (12 mL), and then

distilled water (150 mL) at a flow of 0.2 mL/min. Four-mL fractions were

collected and fractions exhibiting PPO activity were pooled and

concentrated via ultrafiltration utilizing an Amicon stirred cell fitted

with an XM 50 filter (Amicon).


I











Protein Ouantitation and Enzyme Purity Determination


The protein contents of the various PPO preparations were

quantitated using the Bio-Rad Protein Assay kit with bovine serum albumin

(Sigma) as standard. Enzyme purity was examined using a mini gel system

(Mini-Protean II Dual Slab Cell) (Bio-Rad, 1985b). Plant and crustacean

PPO's (20 pg protein/well) were loaded and electrophoresis was carried out

at constant voltage (200 V) in a buffer (pH 8.3) containing 25 mM Tris-HC1

and 0.19 M glycine for 35 min. The purity of enzyme preparations was

determined by comparing gels stained with 10 mM DL-DOPA in 0.05 M sodium

phosphate buffer (pH 6.5) and then with a Commassie blue R-250 (Eastman

Kodak Co., Rochester, NY) solution.


PPO Activity Determination


Lobster PPO activity was determined spectrophotometrically by

monitoring at 475 nm the rate of dopachrome formation from DL-DOPA

(Savagaon and Sreenivasan, 1978). The assay was run at 250C for 10 min by

mixing 40 pL of enzyme extract with 560 pL of 10 mM DL-DOPA in 0.05 M

sodium phosphate buffer (pH 6.5). Two molecules of DOPA produce one

molecule of dopaquinone which has a molar absorption coefficient (a,,) of

3,600 M'cmI' (Fling et al., 1963). PPO activity was defined as pmoles

dopachrome formed per min at 250C.

PPO activities of shrimp, potato, and apple were measured by adding

60 pL enzyme preparations to 840 pL 10 mM DL-DOPA in 0.05 M sodium

phosphate buffer (pH 6.5) and monitored at 250C for 5 min. Maximal initial

velocity was determined as AA4 rn/min and one unit of PPO activity was

defined as an increase in absorbance of 0.001/min at 25C. Unless


- I









24
otherwise specified, experiments were carried out three times in

duplicate. For this study, the enzyme activities of potato, apple, spiny

lobster, Australian lobster, white shrimp, and grass prawn PPOs were

determined to be 10,900, 97,400, 7,000, 3,000, 5,400, and 900 units/mg

protein, respectively.

DH Optima and Stability of Lobster PPO

The modified method of Gormori (1955) was followed for preparation

of various buffer solutions including 0.1 M sodium citrate-0.1 M HC1, pH

2.0, 3.0, and 4.0; 0.05 M sodium phosphate, pH 5.0, 6.0, 6.5, 7.0, 7.5,

and 8.0; 0.1 M glycine-0.1 M NaOH, pH 9.0, and 10.0; and 0.1 M sodium

phosphate-0.1 M NaOH, pH 11.0 and 12.0. The assay was performed at 250C

by adding 40 AL enzyme solution to a mixture containing 280 pL of buffer

solution and an equal volume of 10 mM DL-DOPA in distilled water. The

mean velocity for dopachrome formation was determined at 475 nm using a

DU-7 spectrophotometer (Beckman Instruments, Inc., Irvine, CA).

After enzyme mixtures containing 40 AL enzyme preparation and 120 pL

of each of the previously described buffer systems were incubated at 250C

for 30 min, a 40 AL aliquot was removed and added to 560 pL of 10 mM DL-

DOPA solution in distilled water. Dopachrome formation was monitored

spectrophotometrically.

Activation Enerqy and Thermostabilitv of Lobster PPO


Reaction mixtures containing 40 pL enzyme extract and 560 AL 10 mM
DL-DOPA solution were incubated at various temperatures ranging from 200

to 600C. Activation energy (Ea) was determined according to the Arrhenius









25

equation by measuring the initial rate of reaction at different

temperatures and plotting the logarithmic value of Vm versus 1/T (Segel,

1976).

To determine the thermostability, a 40 pL aliquot of lobster PPO was

sealed in a quartz cell and incubated in a DU-7 spectrophotometer for 30

min at different temperatures ranging from 20-600C. Following

equilibration to room temperature, the enzyme extract was mixed with 560

AL of 10 mM DL-DOPA and then monitored spectrophotometrically as described
above.

Molecular Weight Determination of Lobster PPO

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

was used for molecular weight determination of the enzyme isoforms. Slab

gels (16 cm x 20 cm) at 1.50 mm thickness, consisting of stacking gel (4%

acrylamide/ 0.1% bisacrylamide) and separating gel (7.5% acrylamide/ 0.2%

bisacrylamide), were prepared according to the ProteanTM II Slab Cell

Instruction Manual (Bio-Rad Labs., 1985a). Electrophoresis was carried

out in a Protean II Slab Cell system equipped with a Bio-Rad Model

3000/300 power supply. A constant current of 13 mA/gel and 18 mA/gel was

applied to the stacking and separating gel, respectively in a buffer (pH
8.3) containing 5 mM Tris-HC1 and 38 mM glycine. Enzyme samples were

diluted with 4 volumes of SDS reducing buffer and then heated at 950C for

4 min. Fifty-&g aliquots were applied to each sample well. An SDS-6H

Molecular Weight Marker Kit (Sigma) containing carbonic anhydrase

(29,000), egg albumin (45,000), bovine albumin (66,000), phosphorylase B

(97,000), f-galactosidase (116,000), and myosin (205,000) was used. The









26

molecular weights of the proteins were determined following the methods of

Weber and Osborn (1969) and Weber et al. (1972).

Enzyme Kinetics Study

Kinetic parameters (K, and Vma) of the two lobster PPO's were

determined using the Lineweaver-Burk equation (Lineweaver and Burk, 1934).

DL-DOPA and catechol solutions at concentrations varying from 1.67 to 9.92

mM in 0.05 M phosphate buffer (pH 6.5) were used as substrates. PPO

activity on catechol was defined as Amoles of benzoquinone formed per min

at 250C. One molecule of catechol produced one molecule of benzoquinone,

which has a molar absorption coefficient (am,) of 1,350 M"1 cm"1 (Whitaker,

1972).

In addition, Michaelis constants (K,) for mushroom, potato, apple,

white shrimp, and grass prawn PPOs were determined. Sixty-pL PPO was

added to 10 mM DL-DOPA in 0.05 M sodium phosphate buffer (pH 6.5). The

total volume in the cuvette was 1.0 mL and the final concentration of DL-

DOPA varied from 1.4 to 8.9 mM. The reaction was monitored at 475 nm and

250C for 10 min.

Results and Discussion


Effect of pH on Lobster PPO Activity

PPO enzymes isolated from Western Australian lobster and Florida

spiny lobster exhibited similar patterns of sensitivity to pH changes.

Florida spiny lobster PPO had a pH optimum of 6.5, which was a half unit

less than that for Western Australian lobster PPO (Figure 3). These

values for the two lobster PPO's were similar to that of gulf (brown)














s.0 s-

Q) q0) -







0 0
4> --J
C L






.- VI V)

I-g




C~J
-41



=U











S.- =
X- X
..o CO









0 00






o 4)m









4 .4 *L
+> 0








S- -
L- )0-
o W





















4. CO







0. uI -




U. 0 =
1->
C C S
0 i 3
0) *I-1
r+ _
Q > *- -I
^^^-O
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u-O~













L.

0 0
o c





Go
9 C-




LL.


Ic


















co CDt cv
> 0 000

m$ S O









29
shrimp (Madero and Finne, 1982). PPO from white shrimp (Penaeus

setiferus) had a pH optimum at 7.5, while that from pink shrimp (Penaeus

duorarum) was 8.0 (Simpson et al., 1987, 1988a). Ohshima and Nagayama

(1980) showed that catecholase isolated from antarctic krill (Euphausia

superba) had a pH optimum at 6.5. PPO isolated from grass prawn was shown

to have a pH optimum at 6.0 (Rolle et al., 1991). For plant PPO, the pH

optimum of 7.0 was observed for mushroom (Dawson and Mager, 1962), apple

(Stelzig et al., 1972), and banana (Galeazzi and Sgarbieri, 1978),

respectively, while pear PPO was reported to have a pH optimum at 4.0

(Rivas and Whitaker, 1973).

With respect to the pH-related relative activity, the Western

Australian lobster PPO had a broader pH range (from 5 to 9) than that

observed for the Florida spiny lobster PPO. The behavior of Florida

lobster PPO to various pH environments observed in this study was similar

to that of IP01 (inert phenoloxidase) and TAP01 (trypsin activated

phenoloxidase) forms from a study by Ferrer et al. (1989a). For white and

pink shrimp PPO, the optimal pH environments ranged from 6-7.5 and 6.5-

9.0, respectively (Simpson et al., 1988a).

Data in Table 1 indicated that crustacean (Florida spiny lobster,

Western Australian lobster, pink shrimp, white shrimp, brown shrimp, and

grass prawn) PPOs had a narrow range of optimum pH between 6 to 8.

However, a broad range of pH optimum between 4 and 7 was observed for

mushroom and other plant (potato, apple, peach, pear, and banana) PPOs.

Aylward and Haisman (1969) proposed that the optimum pH of PPO activity,

which usually ranged between pH 4 and 7, varied with enzyme sources and

substrates used.



































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Effect of DH on Lobster PPO Stability

Enzyme stability over a broad pH range (2 12) revealed that PPO

obtained from both lobster species exhibited optimum stability at pH 7

(Figure 4). When PPO was pre-incubated between pH of 5 and 9, both

Florida spiny lobster and Western Australian lobster PPOs still retained

at least 60% of their relative activity when compared at pH 6.5.

Conformational changes at the active site due to dramatic pH changes may

have caused the significant decline in enzyme activity between pH 2 and 5,

and between 9 and 12. Similar changes were reported to occur with TAP02

(trypsin activated phenoloxidase) form of the Florida spiny lobster PPO

(Ferrer et al., 1989a). PPO from pink shrimp (Simpson et al., 1988a),

white shrimp (Simpson et al., 1987), and grass prawn (Rolle et al., 1991)

all exhibited optimal activity within the neutral to alkaline pH range (pH

6-8), and showed maximal stability at pH 8.

Effect of Temperature on Lobster PPO Activity and Stability

Both lobster PPOs showed temperature-related changes in enzyme

activity (Figure 5). Results obtained for this study showed that Florida

spiny lobster and Western Australian lobster PPO had the temperature

optimum at 350 and 300C, respectively, (Figure 5). These values were lower

than those observed for the PPO of pink shrimp (40C, Simpson et al.,

1988a), white shrimp (450C, Simpson et al., 1987), and grass prawn (450C,

Rolle et al., 1991). Compared to crustacean PPO, the activity of peach

PPO was found to increase from 30 to 370C and then declined up to 450C
(Vamos-Vigyazo, 1981). In apple, the enzyme reached its maximum activity

at 300C when chlorogenic acid was used as substrate (Vamos-Vigyazo, 1981);














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36

; while 37C was reported for banana PPO using dopamine as substrate

(Palmer, 1963). Regarding potato and grape PPO activity, the optimum

temperature of 25C was reported for the former (Lavollay et al., 1963),

while 10-15C for the latter (Cash et al., 1976). The data available

indicate that the temperature optimum of the enzyme also depends

essentially on the same factors as the pH optimum (Vamos-Vigyazo, 1981).

It is noted that most crustacean PPOs had higher temperature optima than

those observed for plant PPOs. With the exception of banana PPO, which

showed an unusually high temperature optimum at 370C (Palmer, 1963).

Both lobster PPOs exhibited similar thermostability characteristics,

although the Australian lobster PPO showed decreased activity when pre-

incubated at temperatures greater than 300C (Figure 6). However, Florida

lobster PPO showed greater stability at a preincubation temperature of

350C, which was within the range of EAPO (endogenously activated

phenoloxidase) and TAPO1 forms but slightly different from the IP01 form

of the Florida spiny lobster PPO (Ferrer et al., 1989a). The

thermostability characteristics of both lobster PPO's behave similarly to

that of grass prawn (Rolle et al., 1991) and pink shrimp PPO (20-300C,

Simpson et al., 1988a) but varied from that of white shrimp PPO (25-500C,

Simpson et al., 1987). Florida spiny lobsters grew in warm water while

Western Australian lobsters are found in cold water. These differences in

their habitats may account for the difference in the optimal

thermostability between these two enzymes. PPO enzymes from shrimp are

usually stable at temperatures ranging between 300 and 500C (Madero and

Finne, 1982; Simpson et al., 1987, 1988a). Most PPO enzymes are heat














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39

labile; a short exposure of the enzyme to temperatures at 70-900C is

sufficient to cause partial or total irreversible denaturation. Crude PPO

isolated from deep sea crab was shown to be inactivated at 70C (Marshall

et al., 1984). Similarly, apple PPO (Walker, 1964) and grape PPO (Vamos-

Vigyazo, 1981) were found to be markedly inactivated at temperatures above

700C. For banana PPO, an exposure to 800C for 15 min was required to

inactivate enzymes completely (Galeazzi and Sgarbieri, 1978). Out of 22

cultivars of different stone fruits, peach PPO was found the least

thermostable and the greater heat stable enzyme from plum and cherry was

accompanied by higher activity when compared to peach PPO (Dang and

Yankov, 1970).

Vamos-Vigyazo (1981) pointed out that the thermotolerance of PPO is

dependent on the source of enzyme. In addition, different molecular forms

of PPO from the same source may behave differently in thermostabilities.

Activation Energy (Ea) of Lobster PPO

The activation energies, Ea, for Florida and Australian lobster PPO

were 6.9 and 7.5 Kcal/mole, respectively. These Ea values were similar to

TAP01 (7.8 Kcal/mole) from Florida spiny lobster PPO (Ferrer et al.,

1989a), and comparable to shrimp PPO (Ea = 5.2 Kcal/mole) (Bailey et al.,

1954), but somewhat different from those of the PPO prepared from white

shrimp (Ea = 13.9 Kcal/mole; Simpson et al., 1987), pink shrimp (Ea = 11.5

Kcal/mole; Simpson et al., 1988a), and grass prawn (E, = 13.3 Kcal/mole;
Rolle et al., 1991). The Ea of banana PPO was found to be 4.4 Kcal/mole

when catechol was used as substrate (Palmer, 1963).











Molecular Weight and Isoform Determination of Lobster PPO

Figure 7 shows the SDS-PAGE (reduction condition) patterns for both

lobster PPO enzymes; the Florida spiny lobster PPO had three isoforms and

the Western Australian lobster PPO had two. The molecular masses of the

Florida lobster PPO subunits were determined to be 82, 88, and 97 kD,

while those of the Australian lobster were 87 and 92 kD. The molecular

masses of these subunits were higher than those of white shrimp (30 kD),

pink shrimp (40 kD), grass prawn (63 and 80 kD) and antarctic krill (75

and 83 kD) (Simpson et al., 1987, 1988a; Ohshima and Nagayama, 1980; Rolle

et al., 1991), but lower than that of brown shrimp (210 kD) (Madero and

Finne, 1982). Multiple molecular forms with different molecular masses

were observed for PPO from various plant sources (Vamos-Vigyazo, 1981).

Mushroom PPO was reported to have 4 isoforms (isozymes), each had

molecular masses of 34.5 kD (Bouchilloux et al., 1963). Apple PPO was

shown to have 3 isoforms with molecular masses of 24, 67, and 134 kD,

respectively (Demenyuk et al., 1974). PPO from banana was reported to

possess two isoforms with molecular masses of 12 and 60 kD, respectively

(Palmer, 1963). Regarding potato, multiple isoforms of PPO bearing varied

molecular masses have been reported by many workers (Anisimov et al.,1978;

Constantinides and Bedford, 1967; Patil and Evans, 1963; Thomas et al.,

1978). Although Florida spiny lobster and Western Australian lobster were

grown in different habitats, a recent study using immunological techniques

with rabbit antisera against the Florida spiny lobster PPO revealed that

these two lobsters shared cross-reactivity (Rolle et al., 1991).





























Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-
PAGE, 7.5% Gel) Profile of Polyphenol Oxidase from Florida
Spiny Lobster (FSL) and Western Australian Lobster (WAL); PPO
Samples Were Diluted with 4 Volumes of SDS Reducing Buffer
Containing #-Mercaptoethanol and Heated at 950C for 4 Min.
Fifty-pg PPO Was Loaded onto Each Sample Well.


Figure 7.

















200 kDa


116 kDa
97.4 kDa
i '
66 kDa

43 kDa


Protein WAL
Standard PPO


FSL
PPO


.- fMH e ^








43

PPO from most sources, as mentioned previously, have been reported

to be present in different molecular forms. The number of these forms

depends on the enzyme source and on methods used to prepare them. It has

been revealed that part of molecular forms are due to association-
dissociation phenomena which is attributed to (1) association of various
degrees of polymerization of similar units; (2) various combinations of

different subunits; (3) conformational changes of a single protein; or
combinations of these three possibilities (Vamos-Vigyazo, 1981). Much

attention has been paid to differences in the properties of multiple forms

of the enzyme and to the possible physiological significance of such
differences. These include differences in affinity and specificity to

phenolic substrates and to oxygen (Harel et al., 1964; Kahn, 1976; Taneja

and Sachar, 1974), sensitivity to inhibitors (Constantinides and Bedford,

1967; Harel et al., 1964), pH optima (Takeo and Uritani, 1965; Wong et

al., 1971), and inactivation by heat (Ben-Shalom et al., 1977; Fling et

al., 1963; Sussman, 1961). In addition, differences in isozyme patterns
were also reported in connection with subcellular organelles (Harel et

al., 1965), the stage of tissue development (Takeo and Baker, 1973; Taneja

et al., 1974), as result of attack by pathogens (Hyodo and Uritani, 1964),

or of treatment with plant hormones (Taneja and Sachar, 1977a, b).

Enzyme Kinetics of Lobster PPO

Double-reciprocal plots for the oxidation of DL-DOPA and catechol by

both lobster PPOs are shown in Figures 8 and 9, respectively. Both

lobster PPOs were capable of catalyzing DL-DOPA and catechol. Australian

lobster PPO displayed a relatively greater Michaelis constant (Km = 3.57














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48
mM) and a lower maximum velocity (Vmx = 0.008 mmol/liter/min) with DL-DOPA

than with catechol (Km = 3.09 mM and V, = 0.041 mmol/liter/min) (Table

2). Similar results were also noted for the Florida lobster PPO (Table

2). These two PPOs had a higher affinity for catechol than for DL-DOPA.

Using DL-DOPA as a substrate, Simpson et al. (1987, 1988a) showed that

pink shrimp PPO had a lower Michaelis constant (1.6 mM) than white shrimp

(2.8 mM). Rolle et al. (1991) characterized grass prawn PPO and they

determined the Km of enzyme was 4.45 mM when DL-DOPA was used as substrate.

Summers (1967) showed that blood PPO from fiddler crab had a Km value of

0.50 mM.

Australian lobster PPO exhibited a higher affinity for DL-DOPA and

catechol than Florida lobster PPO. However, the latter showed a greater

rate for oxidizing DL-DOPA and catechol than the former. It has been

reported by Lavollay et al. (1963) that no relationship could be found

between Km and V., values obtained for a substrate with a given PPO

preparation. Instead, the term of Vx/Km has been recommended by some

authors (Lavollay et al., 1963; Pollock, 1965) to express the efficiency

of a given substrate for a given enzyme. Table 2 indicates that Florida

lobster PPO not only showed a higher specific activity but also had a

higher turnover number than the Australian lobster PPO. The Florida

lobster PPO also showed a higher physiological efficiency for both

substrates than the Australian lobster PPO (Table 2), which could account

for why Florida spiny lobsters were more susceptible to melanosis than

Western Australian lobsters. The kinetic and molecular properties of PPO

from other sources are also summarized in Table 1. With DL-DOPA as

substrate, different Michaelis constants (Km) were observed for mushroom,


I
















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50

potato, apple, grass prawn, pink shrimp, and white shrimp PPOs. Varied Km

values were also reported for peach (K, = 4.2 mM, Wong et al., 1971), pear

(K, = 20.9 mM, Rivas and Whitaker, 1973), and banana (K, = 0.63 mM, Palmer,
1963) PPOs when different phenolic compounds were used as substrates

(Table 2). Data obtained from this study showed that mushroom, potato,

and apple PPOs had comparatively lower K, than those of crustacean PPOs,

indicating that the former PPOs had a higher affinity for DL-DOPA than the

latter ones. It has been reported that the affinity of PPO towards a

given substrate may vary widely, even if isozymes of the same origin are

used. The differences might be due, at least partly, to steric factors

connected to differences in protein structure (Vamos-Vigyazo, 1981).

Substrate specificity varies considerably for PPO from various

sources (Aurand and Wood, 1977; deMan, 1985). Simpson et al. (1988a)

identified higher levels of free tyrosine and phenylalanine as natural

substrates of melanosis in the carapace of pink shrimp. Further studies

by Ali (1991) showed PPO isolated from Florida spiny lobster was capable

of hydroxylating free tyrosine to DOPA. Tyramine, produced by bacterial

activity on free L-tyrosine (Veciana-Nogues et al., 1989; Santos-Buelga et

al., 1986) was also identified as the substrate of crab PPO (Summers,

1967). For mushroom (Agaricus bisporum) PPO, DOPA and catechol were,

respectively, reported by Bouchilloux et al. (1962) and Nakamura et al.

(1966) as specific substrates. For potato and apple PPOs, chlorogenic

acid (Patil and Zucker, 1965) was considered a specific substrate for the

former, while 4-methylcatechol for the latter (Stelzig, 1972). Compared

to pear PPO with pyrocatechol (Rivas and Whitaker, 1973) and banana PPO

with dopamine (Palmer, 1963), D-catechin was demonstrated as the specific









51

substrate of peach (Luh and Phithakpol, 1972). Results from this study

show that DOPA was the unanimous substrate of crustacean PPOs. For

mushroom and other plant PPOs, the specific substrate varied with the

sources of enzyme. Mason (1955) reported that tyrosine and DOPA were

specific substrates of animal tissue PPO, while Mayer and Harel (1979)

listed a wide variety of mono- and o-diphenols as the substrates of fungal

and plant PPOs. Besides the previously discussed characteristics

(substrate specificity, K,, pH and temperature effects, enzyme isoforms,

and molecular weights), other biochemical properties concerning the

catalytical function (mechanism), response to the activator/inhibitor, and

isoelectric profile (pI) of PPO also varied with the enzyme sources.

Conclusion

PPO isolated from Florida spiny lobster and Western Australian

lobster showed very similar patterns in response to effects of pH and

temperature on enzyme activity and in SDS-PAGE profile. Using DL-DOPA and

catechol as substrates, the Western Australian lobster PPO was shown to

have higher affinity but lower physiological efficiency than the Florida

spiny lobster PPO. These two PPOs showed distinctly different properties

for catalyzing the oxidation of phenolic substrates; this may explain

differences in susceptibility of spiny lobster to melanosis compared to

Western Australian lobster. Results indicate that PPO from various plant

and crustacean sources vary in substrate specificity, kinetic properties,

molecular weights, isoforms, activity and stability to pH and temperature

effects, activation energy, and isoelectric profiles.














STRUCTURAL COMPARISON OF CRUSTACEAN, PLANT, AND MUSHROOM
POLYPHENOL OXIDASES

Introduction


Polyphenol oxidase (PPO) (E.C. 1.14.18.1.), also known as

tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, and

catecholase, is widely distributed in nature (Schwimmer, 1981). The

unfavorable enzymatic browning caused by PPO on the surface of many plants

and seafood products has been of a great concern to food processors and

scientists. Although the formation of melanin (blackening spot) does not

affect the nutrient content of food products, it does however connote

spoilage by consumers (Eskin et al., 1971). Economic loss resulting from

this action has caused great concern among food processors. Enzymatic

browning of fruits, vegetables, and crustaceans due to PPO activity has

been extensively studied (Chen et al., 1991a; Ferrer et al., 1989a;

Flurkey and Jen, 1978; Macrae and Duggleby, 1968; Sciancalepore and

Longone, 1984; Simpson et al., 1989a; Walker, 1964).

Differences in the secondary structure between endogenously

activated (EAPO) and trypsin activated (TAPO) forms of Florida spiny

lobster PPO was recently demonstrated by Rolle et al. (1991) using

circular dichroism (CD) spectropolarimetry. A study conducted by Chen et

al. (1991b) showed that PPOs from plant and crustacean sources not only

varied with respect to catalytic activity in the oxidation of DL-0-3,4-

dihydroxyphenylalanine (DL-DOPA) but also had different sensitivities to
52









53
the inhibitor, kojic acid (5-hydroxy-2-hydroxymethyl--y-pyrone). Since the

conformational structures of these fungal, plant and crustacean PPOs has

not been well documented, this study was undertaken to elucidate whether

conformational differences exists among these PPOs using immunological

techniques and circular dichroism spectropolarimetry.


Materials and Methods

Fresh Florida spiny lobster (Panulirus argus) tails obtained from

the Whitney Marine Laboratory at Marineland, FL, were transported in ice

to the laboratory and stored at -200C. Mushroom (Agaricus bispora)

tyrosinase with an activity of 2,200 units/mg solid was purchased from

Sigma Chemical Co. Russet potato was purchased from a local supermarket.

White shrimp (Penaeus setiferus) and brown shrimp (Penaeus aztecus) were

obtained from a local seafood store. Lobster cuticle, shrimp

cephalothorax (head), and potato peel were each frozen in liquid nitrogen

and ground into a fine powder using a Waring blender. The individual

ground powder was stored at -200C until needed.

Extraction of Mushroom, Potato, Lobster, and Shrimp PPO


PPO was extracted according to the procedure of Simpson et al.

(1988a). One part ground powder was added to three parts (w/v) 0.05 M

sodium phosphate buffer (pH 7.2) containing 1 M NaCl and 0.2% Brij 35.

The extract was stirred for 5 min at 40C and the suspension was centrifuged

at 8,000g (40C) for 30 min. The supernatant was then dialyzed at 40C

overnight against 3 changes of 4L 0.05 M sodium phosphate buffer (pH 6.5).


I










Purification of Mushroom, Potato, Lobster, and Shrimp PPO

Crude PPO preparation was purified further using a nondenaturing

preparative polyacrylamide gel electrophoresis (PAGE) system. Equipment

utilized included a gel tube chamber (Model 175, Bio-Rad Labs.) and a

power supply (Model EPS 500/400, Pharmacia LKB Biotechnology Inc.). A

one-mL aliquot of crude enzyme extract (potato, lobster, or shrimp) was

applied to each of eight gel tubes (1.4 cm I.D. x 12 cm length) containing

5% acrylamide/ 0.13% bisacrylamide gel and subjected to a constant current

of 10 mA/tube in a buffer (pH 8.3) containing 5 mM Tris-HC1 and 38 mM

glycine (Sigma Chemical CO., 1984). PPO was visualized using a specific

enzyme-substrate staining method (Constantinides and Bedford, 1967); 10 mM

DL-DOPA in 0.05 M sodium phosphate buffer (pH 6.5) was used as substrate.

After the migration of the enzyme relative to the dye front (Rf) was

determined using one of the eight gels, the remaining gels were sectioned

at the determined R,. PPO was eluted from the gel by homogenization in

0.05 M sodium phosphate buffer (pH 6.5) utilizing a Dounce manual tissue

grinder. The homogenates were filtered through Whatman No. 4 filter

paper, pooled, and concentrated using an Amicon stirred cell (Model 8050).

Mushroom PPO (0.25 mg/mL) in 0.05 M sodium phosphate buffer (pH 6.5) was

further purified according to the procedures previously described.

Protein Quantitation and Enzyme Purity Determination

Protein content of the various PPO preparations was quantitated

using the Bio-Rad Protein Assay kit with bovine serum albumin as standard.

Enzyme purity was examined using a mini gel system (Mini-Protean II Dual

Slab Cell) (Bio-Rad, 1985b). Mushroom, potato, and crustacean PPOs (20 Ag









55

protein/well) were loaded and electrophoresis was carried out at constant

voltage (200 V) in a buffer (pH 8.3) containing 25 mM Tris-HCl and 0.19 M

glycine for 35 min. Purity of the preparations was determined by
comparing gels stained with 10 mM DL-DOPA in 0.05 M sodium phosphate
buffer (pH 6.5) and then with a Coomassie blue R-250 solution.

Enzyme Activity Assay

PPO activities were measured by adding 60 AL PPO to 840 iL 10 mM DL-

DOPA in 0.05 M sodium phosphate buffer (pH 6.5) and monitoring at 250C for

5 min in a Beckman DU7 spectrophotometer at 475 nm. Maximal initial

velocity was determined as AA475 n/min and one unit of PPO activity was

defined as an increase in absorbance of 0.001/min at 250C. Unless

otherwise specified, experiments were carried out twice in triplicates.

For this study, the enzyme activities of mushroom, potato, lobster, white

shrimp, and brown shrimp PPO were determined to be 96,000, 120,000, 4,500,
1,000, and 1,200 units/mg protein, respectively.

Anti-lobster PPO Antibody Production and Purification

One-mL purified lobster PPO containing 100 ag protein was used as an

antigen to inject into a hen biweekly. Eggs laid by the immunized hen

were collected and anti-lobster PPO antibody was isolated and purified
from the egg yolk using the method of Polson et al. (1985). One part egg
yolk was added to 4 parts (v/v) 0.1 M sodium phosphate buffer (pH 7.6).

The mixture was made up 3.5% (w/v) with polyethyleneglycol (PEG) and

stirred for 5 min. Following centrifugation at 5,000g (10C) for 20 min,

the supernatant collected was made up with 8.5% (w/v) PEG. The suspension









56

was allowed to stand for 10 min followed by centrifugation at 5,000g (100C)

for 25 min. The pellet was dissolved in 2.5 volumes (v/v) phosphate

buffer (pH 7.6) and the suspension was made up with PEG to 12% (v/v).

Again, the suspension was allowed to stand for 10 min and then centrifuged

at 5,000g (100C) for 25 min. The pellet was resuspended in 1/4 volume
phosphate buffer and cooled to 00C before adding an equivalent volume of
50% ethanol (-200C). Following centrifugation at 10,000g (40C) for 25 min,

the precipitate was dissolved in 1/4 volume phosphate buffer and the

suspension was dialyzed overnight (40C) against 4L 0.1 M sodium phosphate

buffer (pH 7.6). After dialysis, the antibody preparation was made up

with NaN3 to 0.1% and stored in the refrigerator until needed.

Molecular Weight Determination of Anti-lobster PPO Antibody

Protein content of the antibody preparation was also quantitated

using the Bio-Rad Protein Assay kit. The molecular weight of the antibody

preparation was determined using SDS-PAGE (reduction condition) according

to the method of Laemmli (1970). Mini slab gels (7 cm x 8 cm) at 1.0 mm
thickness, consisting of stacking gel (4% acrylamide/ 0.1% bisacrylamide)

and separating gel (7.5% acrylamide/ 0.2% bisacrylamide) were prepared

according to the Mini-Protein II Dual Slab Cell Instruction Manual (Bio-

Rad, 1985b). Antibody preparations were diluted with 4 volumes of SDS

sample buffer containing P-mercaptoethanol, and heated for 4 min at 950C.
After 30 pg aliquots were applied to each sample well and electrophoresis
was carried out for 35 min at a constant voltages of 200 V. An SDS-6H

high Molecular Weight Protein Standards kit (Sigma) containing carbonic

anhydrase (29,000), egg albumin (45,000), bovine albumin (66,000),








57

phosphorylase (97,400), #-galactosidase (116,000), and myosin (205,000)
was used. Molecular weights of the antibody proteins were determined

following the methods of Weber and Osborn (1969) and Weber et al. (1972).

Antibody Titer Determination by Enzyme-linked Immunosorbent Assay (ELISA)

One hundred-pL lobster PPO containing 2.5 100 ng protein in 0.1 M

NaHCO3, pH 8.6, (coating buffer) was applied to the sample well of a
microplate (Immulon 2, Dynatech). Following overnight incubation at 40C,

the well was aspirated and washed 4 times with PBS-Tween [0.01 M sodium

phosphate, pH 7.4, containing 0.15 M NaC1 and 0.2% (v/v) Tween 20] using

the Nunc-Immuno Wash (A/S NUNC, Denmark). After 100 pL primary antibody

(anti-lobster PPO antibody) at amounts of 0.01 10 Ag in PBS-Tween was

added to the well, incubation was allowed to proceed at ambient

temperature for 1 hr. The aspirations and washings were repeated as

previously described before 0.1 mL secondary antibody (antichicken IgG-

alkaline phosphatase conjugate, Sigma) was added. Following another one-

hour incubation, the microplate was aspirated and washed again with PBS-

Tween. Following the addition of 0.1 mL p-nitrophenyl phosphate disodium

(1.0 mg/mL) in assay buffer (0.05 M Na CO3 and 0.05 M NaHCO3 containing
0.0005 M MgCl) to the well, the plate was incubated at ambient temperature

till a yellow color developed. The absorbance of the plate at 405 nm was

monitored every hour using an ELISA reader (Model 2550, Bio-Rad). In this

study, coating buffer without antigen was used as the negative control.










Analysis of Antigenic Properties of PPO

The competitive ELISA adopted from Seymour et al. (1991) was

employed to study whether PPO from mushroom, potato, white shrimp, and

brown shrimp possessed similar antigenic determinants as the Florida spiny

lobster. After the microplate was coated with lobster PPO at 10 ng/well
for one hour at room temperature, 100 AL aliquot of antibody-competitive

PPO mixture was added. The antibody-PPO mixture was prepared by mixing

the competitive PPO (either mushroom, potato, or lobster, white shrimp,

and brown shrimp) (0.2 2.0 Ag/mL) with an equal volume of primary

antibody solution (10 or 20 Ag/mL of IgY) for 1 hr at ambient temperature.

The assay procedures were conducted as previously described.

Immunoblotting

Purified mushroom, potato, lobster, white shrimp, and brown shrimp

PPO and a crude lobster PPO preparation were subjected to SDS-PAGE under

reduction condition (Laemmli, 1970) and then electro-transferred to

nitrocellulose membrane (Bio-Rad). Slab gels (16 cm x 20 cm) at 1.0 mm

thickness, consisting of stacking gel (4% acrylamide/ 0.1% bisacrylamide)

and separating gel (7.5% acrylamide/ 0.2% bisacrylamide), were prepared

according to the ProteanM II Slab Cell Instruction Manual (Bio-Rad,

1985a). A constant current of 13 mA/gel was applied during stacking and

18 mA/gel during separation. Fifty-pg aliquots of test samples were

applied to each well and run with the protein standards (SDS-6H Molecular

Weight Marker Kit).

Following electrophoresis, the gel was equilibrated in 500 mL of

Towbin transfer buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, and 20%









59

methanol; pH 8.3) for 15 min. Electro-transfer was performed according to

the Trans-Blot Electrophoretic Transfer Cell Instruction Manual (Bio-Rad,

1989) at a constant voltage of 50 V for 1.5 hr using Towbin buffer as the

electrolytic buffer. Complete transfer of proteins was verified by

staining the gel and the nitrocellulose membrane with a Coomassie blue

solution.

The nitrocellulose membrane following electro-transferring was

rinsed with phosphate-buffered saline (PBS) 3 times, and then incubated

with Blotto/Tween blocking solution (5% w/v nonfat dry milk, 0.2% v/v

Tween 20, and 0.02% w/v NaN3 in PBS; Harlow and Lane, 1988) at ambient

temperature with agitation for 2 hr. After washing twice for 5 min each

in PBS, the membrane was incubated overnight in the primary antibody (IgY)

solution (10 pg/mL). Following washing with 4 changes of PBS for 5 min

each, the membrane was treated for 1 hr with the secondary antibody

(antichicken IgG-alkaline phosphatase conjugate, Sigma) at a dilution of

1/2000. The membrane was then washed again with PBS as previously

described, and incubated with 100 mL alkaline phosphatase buffer (100 mM

NaCl, 5 mM MgCl2, and 100 mM Tris; pH 9.5) containing 0.016%

bromochloroindolyl phosphate (BCIP) and 0.032% nitro blue tetrazolium

(NBT) (Harlow and Lane, 1988) for 45 min. The reaction was stopped by

rinsing the membrane with PBS containing 20 mM EDTA.

Spectropolarimetric Analysis of PPO

The circular dichromic spectra of PPO was scanned at the far UV (250

- 200 nm) range using a Jasco J-20 automatic recording spectropolarimeter

(Japan Spectroscopic Co., Tokyo, Japan). A 1.0-cm Suprasil (Helma Cells)









60

cuvette with a 1.0-cm light path was used. Four-mL of PPO (10 20 pg/mL)

in 0.5 mM sodium phosphate buffer (pH 6.5) was analyzed at ambient

temperature. Calculations of the secondary structures were carried out by

computer analysis of the spectra using the SSE program (Japan

Spectroscopic Co., 1985) with myoglobin, cytochrome c, ribonuclease A,

lysozyme, and papain as references.

Results and Discussion

SDS-PAGE Profile of PPO

Various enzyme subunits with different molecular masses were

observed for crude mushroom (7 isozymes), potato (5 isozymes), lobster (3

isozymes), white shrimp (2 isozymes), and brown shrimp (2 isozymes) PPO

preparations after the nondenaturing preparative polyacrylamide gel was

stained with DL-DOPA. After subjecting to SDS-PAGE (without treatment of

f-mercaptoethanol), mushroom and potato PPO chosen for this study were

shown to have a subunit of lower molecular mass than crustacean (lobster,

white shrimp, and brown shrimp) PPO, estimated as 66 and 148 kD,

respectively (Figure 10). These are in agreement with the previously

reported data of Anisimov et al. (1978) and Bouchilloux et al. (1963). As

to lobster, white shrimp, and brown shrimp PPO, the molecular masses were

determined as 200, 190, and 190 kD, respectively. Brown shrimp PPO of 190

kD was close to that of the Gulf brown shrimp PPO (210 kD) (Madero and

Finne, 1982). However, the molecular mass of white shrimp PPO determined

in this study varied with that reported previously by Simpson et al.

(1988). The use of different preparation and analytical methods in these

studies could have contributed to such discrepancy.
















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Antibody Production and Molecular Weight Determination


The production of lobster PPO specific antibody did not occur until

after the second boosting; peak activity occurred on day 18 (Figure 11).

Antibody activity decreased gradually with time following the third and

fourth boosting. The serum eventually lost antibody activity after day

35. The animals apparently had become tolerant to the antigen after the

third boosting.

Antibody produced on day 17 and 18 having the highest activities was

pooled for molecular weight determination. Three distinct bands were

observed on the SDS-PAGE gel (Figure 12). The molecular weight for the

lower band was estimated as 59,000 which was close to the value reported

for the heavy chain of IgY (Jensenius et al., 1981).

Antibody Titer Determination

Dose-related interactions of various PPO (mushroom, potato, lobster,

white shrimp, and brown shrimp) at 2.5 10 ng with lobster PPO-specific

antibody at 2.5 jig are shown in Figure 13. All test samples showed

affinity for the antibody. Therefore, these antigens were partially

cross-reactive and shared similar antigenic determinants. The appropriate

dose range of the interaction of the various PPOs with the antibody was

determined to be 2.5 10 ng/well. When PPO was added at more than 10

ng/well, the antigen-antibody reaction was saturated and the sensitivity

of the ELISA became dull.















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Standard (SDS-6H); Anti-lobster PPO Antibody Was Diluted with
4 Volumes of SDS Sample Buffer Containing P-Mercaptoethanol
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Immunological Characteristics of PPO

The absorbance at 405 nm of a lobster PPO-antibody reaction mixture

in the presence of various competitors (lobster, white shrimp, brown

shrimp, mushroom, and potato PPO) was determined by ELISA (Figure 14).

The color intensity was reduced by 18 79% of the control (Abs. = 0.967)

when the various competitor-antibody mixtures containing 12.5 ng

competitor and 5 pg antibody per sample well were added to the microplate.

The increase of the competitor up to 100 ng/well slightly reduced the

color intensity. For mushroom PPO, the competition with lobster PPO for

antibody however was not enhanced even when mushroom PPO was added at 100

ng/well. The result again indicated that these PPOs were partially cross-

reactive and therefore shared similar structural components. From the

extent of competition for lobster PPO-specific antibody, white shrimp and

brown shrimp PPO were shown to share more antigenic similarity to lobster

PPO than potato and mushroom PPO (in descending order).

Complete transfer of PPO bands along with protein standards from an

acrylamide gel onto a nitrocellulose membrane was achieved following

electro-transferring; the staining of the treated gels with Coomassie blue

revealed no protein bands. The purified lobster, white shrimp, brown

shrimp, mushroom, and potato PPO had only one protein band, while crude

lobster PPO had 3 isozymes (Figure 15). The nitrocellulose membrane,

following staining with lobster PPO-specific antibody, was found to

contain dark bands corresponding to potato, lobster, white shrimp, and

brown shrimp PPO and the crude lobster PPO preparation. Three dark bands

were observed for both the potato PPO and the crude lobster PPO

preparation, while only one dark band was found for purified lobster,














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SDS-PAGE Profile of Protein Standard (SDS-6H), Purified
Mushroom (M), Potato (P), Florida Spiny Lobster (L), White
Shrimp (W), and Brown Shrimp (B) PPO, and a Crude Lobster PPO
Preparation (CL) under Reduction Condition on a Nitrocellulose
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75

white shrimp, and brown shrimp PPO (Figure 16). Incomplete protein

transfer or torsional changes in protein structure following electro-

transfer process was possibly responsible for the failure of mushroom PPO

that not to form a dark band with lobster PPO-antibody. Result from this

study did not support the competitive ELISA experiment obtained for

mushroom PPO. However, our previous findings that PPO from potato and

crustacean sources shared similar structural components was further

demonstrated.


Spectropolarimetric Analysis of PPO

Lobster, white shrimp, and brown shrimp PPO had similar circular

dichroic spectra (Figures 19, 20, and 21), which were different from those

of mushroom and potato (Figures 17 and 18). They all varied in their

secondary structures (a-helix, P-sheet, f-turn, and random coil) (Table

3). For example, white shrimp PPO had a higher percentage of

a-helix than brown shrimp PPO; they both showed the same broad negative

ellipticity between 207 and 220 nm. The percentage of a-helix of

mushroom, potato, and crustacean PPOs estimated using the SSE program were

close to the values that calculated according to the formula of Greenfield

and Fasman (1969). The crustacean PPO, in general, had a higher

percentage of a-helix and lower percentage of f-turn than mushroom and

potato PPO (Table 3). The percentages of the secondary structures of

these PPO estimated from the SSE program may not represent the absolute

values. However, the results from this study showed that PPO from various

sources possessed varied secondary structures, with the crustacean sources

producing PPO which showed very similar secondary structure.






























Figure 16.


Determination of the Specific Reactivity of Anti-lobster PPO
Antibody with a Crude Lobster PPO Preparation (CL), Purified
Mushroom (M), Potato (P), Florida Spiny Lobster (L), White
Shrimp (W), and Brown Shrimp (B) PPO as well as SDS-6H Protein
Standards as Analyzed by Immunoblotting Following SDS-PAGE


















































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Full Text
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FILES


CHARACTERIZATION AND COMPARISON OF PLANT AND CRUSTACEAN
POLYPHENOL OXIDASES: KINETICS AND INHIBITION BY CHEMICAL METHODS
By
JON-SHANG.CHEN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1991
UNIVERSITY OF FLORIDA LIBRARIES

ACKNOWLEDGEMENTS
I wish to express my sincere appreciation to Drs. Marty R. Marshall
(the chairman of the graduate supervisory committee) and Cheng-I Wei (the
cochairman) for their scientific guidance and financial support during my
graduate study. I would also like to extend my gratitude to the other
members of my graduate committee, Dr. James F. Preston, Dr. Murat 0.
Balaban, and Dr. Steven Otwell for their advice and assistance in
improving the depth of this research work. My special thanks are extended
to my wife, Shaw-Lien Hsia, for her invaluable support and encouragement.

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF TABLES vii
LIST OF FIGURES viii
ABSTRACT xii
INTRODUCTION 1
LITERATURE REVIEW 4
Polyphenol Oxidase 4
Enzymatic Browning (Melanosis) 6
Inhibition/Prevention of Melanosis 9
Kojic Acid 12
Supercritical Fluid (SCF) Treatment 12
CHARACTERIZATION AND COMPARISON OF CRUSTACEAN AND PLANT POLYPHENOL
OXIDASES: KINETICS AND SOME PROPERTIES 16
Introduction 16
Materials and Methods 17
Extraction and Purification of Lobster Polyphenol Oxidase
(PPO) 18
Extraction and Purification of Potato PPO 19
Extraction and Purification of Apple PPO 20
Extraction and Purification of White Shrimp and Grass Prawn
PPO 21
Protein Quantitation and Enzyme Purity Determination 23
PPO Activity Determination 23
pH Optima and Stability of Lobster PPO 24
Activation Energy and Thermostability of Lobster PPO 24
Molecular Weight Determination of Lobster PPO 25
Enzyme Kinetics Study 26
Results and Discussion 26
Effect of pH on Lobster PPO Activity 26
Effect of pH on Lobster PPO Stability 31
Effect of Temperature on Lobster PPO Activity and Stability . 31
Activation Energy (EJ of Lobster PPO 39
Molecular Weight and Isoform Determination of Lobster PPO . . 40
Enzyme Kinetics of Lobster PPO 43
i i i

Conclusion 51
STRUCTURAL COMPARISON OF CRUSTACEAN, PLANT, AND MUSHROOM POLYPHENOL
OXIDASES 52
Introduction 52
Materials and Methods 53
Extraction of Mushroom, Potato, Lobster, and Shrimp PPO ... 53
Purification of Mushroom, Potato, Lobster, and Shrimp PPO . . 54
Protein Quantitation and Enzyme Purity Determination 54
Enzyme Activity Assay 55
Anti-lobster PPO Antibody Production and Purification .... 55
Molecular Weight Determination of Anti-lobster PPO Antibody . 56
Antibody Titer Determination by Enzyme-linked Immunosorbent
Assay (ELISA) 57
Analysis of Antigenic Properties of PPO 58
Immunoblotting 58
Spectropolarimetric Analysis of PPO 59
Results and Discussion 60
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE) Profile of PPO 60
Antibody Production and Molecular Weight Determination. ... 63
Antibody Titer Determination 63
Immunological Characteristics of PPO 70
Spectropolarimetric Analysis of PPO 75
Conclusion 89
INHIBITION MECHANISM OF KOJIC ACID ON SOME PLANT AND CRUSTACEAN
POLYPHENOL OXIDASES 90
Introduction 90
Materials and Methods 91
Extraction and Purification of Potato PPO 92
DEAE-cellulose Chromatography 93
Gel Filtration 93
Extraction and Purification of Apple PPO 94
Hydroxyl apatite Chromatography 94
Extraction and Purification of Grass Prawn PPO 95
Extraction and Purification of Lobster PPO 96
Extraction and Purification of white shrimp PPO 97
Protein Quantitation and Enzyme Purity Determination 98
Enzyme Activity Assay 98
Effect of Kojic Acid on Enzyme Activity 99
Enzyme Kinetics Study 100
Effect of Pre-incubation Temperature on PPO Inhibition by
Kojic Acid 102
Effect of Kojic Acid on the Hydroxylation Capability of PPO . 103
Effect of Kojic Acid on Oxygen Uptake by PPO Reaction .... 103
Effect of Kojic Acid on Reduction of Cupric Copper 104
Effect of Kojic Acid on Quinone Products 104
Enzymatic Activities of Kojic Acid-treated PPO 105
Statistical Analysis 106
IV

Results and Discussion 107
Effect of Kojic Acid on Mushroom PPO Activity 107
Effect of Kojic Acid on Potato PPO Activity 107
Effect of Kojic Acid on Apple PPO Activity 114
Effect of Kojic acid on Crustacean PPO Activity 117
Enzyme Kinetics 117
Effect of Pre-incubation Temperature on PPO Inhibition by
Kojic Acid 129
Effect of Kojic Acid on the Hydroxylation Capability of PPO . 129
Effect of Kojic Acid on Oxygen Uptake by PPO Reaction .... 131
Effect of Kojic Acid on Reduction of Cupric Copper 131
Effect of Kojic Acid on Quinone Products 135
Enzymatic Activities of Kojic Acid-treated PPO 140
Conclusion 140
EFFECT OF CARBON DIOXIDE ON THE INACTIVATION OF PLANT AND
CRUSTACEAN POLYPHENOL OXIDASES 143
Introduction 143
Materials and Methods 144
Extraction and Purification of Lobster, Shrimp, and Potato
PPO 144
Protein Quantitation and Enzyme Purity Determination 145
Enzyme Activity Assay 146
Effect of Carbon Dioxide (1 atm) on PPO Activity 146
pH Control Study 147
Effect of High Pressure (58 atm) Carbon Dioxide on PPO
Activity 147
Kinetics of PPO Inactivation 150
Nondenaturing Polyacrylamide Gel Electrophoresis of Carbon
Dioxide (1 atm)-treated PPO 151
Mass Balance of High Pressure Carbon Dioxide-treated and
Nontreated PPO 151
Polyacrylamide Gel Isoelectric Focusing of Carbon Dioxide-
treated PPO 152
Spectropolarimetric Analysis of PPO 153
Study of Restoration of Carbon Dioxide-treated PPO Activity . 153
Results and Discussion 154
Effect of Carbon Dioxide (1 atm) on PPO Activity 154
Effect of Nitrogen on PPO Activity 157
Effect of High Pressure Carbon Dioxide on PPO Activity. ... 159
Kinetics of PPO Inactivation 166
Polyacrylamide Gel Electrophoresis of Carbon Dioxide (1 atm)-
treated PPO 168
Mass Balance of High Pressure Carbon Dioxide-treated and
Nontreated PPO 168
Polyacrylamide Gel Isoelectric Focusing of Carbon Dioxide-
treated PPO 172
Spectropolarimetric Analysis of High Pressure Carbon Dioxide-
treated PPO 172
Restorative Ability of Carbon Dioxide-treated PPO Activity. . 186
Conclusion 189
v

CONCLUSIONS 197
REFERENCE LIST 199
BIOGRAPHICAL SKETCH 216
vi

LIST OF TABLES
TABLE PAGE
1 Comparison of Kinetic Properties of Polyphenol Oxidase (PPO)
from Various Sources 30
2 Comparison of Polyphenol Oxidase (PPO) Activity between
Florida Spiny Lobster and Western Australian Lobster .... 49
3 Secondary Structure Estimates of Various Polyphenol Oxidases
(PPOs) from Far UV Circular Dichroic Spectra 88
4 Inhibitory Mechanism of Kojic Acid on Polyphenol Oxidase
Obtained from Various Sources 124
5 Effect of Different Pre-incubation Temperatures on the
Inhibition of Various Polyphenol Oxidases (PPOs) by Kojic
Acid 130
6 Inhibitory Effect of Kojic Acid on the Consumption of Oxygen
by Polyphenol Oxidase 134
7 Enzymatic Activity of Kojic Acid-treated Polyphenol Oxidase
Following Kojic Acid Removal 141
8 Effect of Nitrogen on Florida Spiny Lobster Polyphenol Oxidase
Activity 158
9 Kinetic Parameters of Florida Spiny Lobster Polyphenol Oxidase
Inactivation by Carbon Dioxide (1 atm) at Various
Temperatures 167
10 Mass Balance of Protein Contents from High Pressure C02-
treated and Nontreated PPO 171
11 Secondary Structure Estimates of Nontreated Control and High
Pressure CO~-treated Florida Spiny Lobster, Brown Shrimp,
and Potato Polyphenol Oxidases (PPOs) from Far UV Circular
Dichroic Spectra 185
vi i

LIST OF FIGURES
FIGURE PAGE
1 Mechanism of Polyphenol Oxidase-catalyzed Reaction .... 8
2 Structure of Kojic Acid (5-hydroxy-2-hydroxymethyl-y-
pyrone) 14
3 Effect of pH on the Activity of Polyphenol Oxidase (PPO)
Isolated from Florida Spiny Lobster and Western
Australian Lobster 28
4 Effect of pH on the Stability of Polyphenol Oxidase (PPO)
Isolated from Florida Spiny Lobster and Western
Australian Lobster 33
5 Effect of Temperature on the Activity of Polyphenol Oxidase
(PPO) Isolated from Florida Spiny Lobster and Western
Australian Lobster 35
6 Effect of Temperature on the Stability of Polyphenol
Oxidase (PPO) Isolated from Florida Spiny Lobster and
Western Australian Lobster 38
7 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) Profile of Polyphenol Oxidase from
Florida Spiny Lobster (FSL) and Western Australian
Lobster (WAL) 42
8 Double Reciprocal Plots for the Oxidation of DL-DOPA and
Catechol by Florida Spiny Lobster Polyphenol Oxidase
(PPO) 45
9 Double Reciprocal Plots for the Oxidation of DL-DOPA and
Catechol by Western Australian Lobster Polyphenol
Oxidase (PPO) 47
10 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) Profile of Polyphenol Oxidase from
Mushroom, Potato, Florida Spiny Lobster, White Shrimp,
and Brown Shrimp 62
11 Profile of Anti-lobster PPO Antibody Production in
Immunized Hen during the Immunization Period 65
vi i i

12 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) Profile of Anti-lobster PPO
Antibody 67
13 The Titer Determination of Antibody against Florida Spiny
Lobster PPO versus Mushroom, Potato, Florida Spiny
Lobster, White Shrimp, and Brown Shrimp PPO 69
14 Analysis of Antigenic Properties of Purified Mushroom,
Potato, Florida Spiny Lobster, White Shrimp, and Brown
Shrimp PPO by Competitive ELISA 72
15 Profile of Protein Standard (SDS-6H), Purified Mushroom,
Potato, Florida Spiny Lobster, White Shrimp, and Brown
Shrimp PPO, and Crude Lobster PPO Preparation on a
Nitrocellulose Membrane 74
16 Determination of the Specific Reactivity of Anti-lobster
PPO Antibody with Crude Lobster PPO Preparation,
Purified Mushroom, Potato, Florida Spiny Lobster, White
Shrimp, and Brown Shrimp PPO as well as SDS-6H Protein
Standard as Analyzed by Immunoblotting Technique .... 77
17 The far UV Circular Dichroic Spectra of Mushroom PPO ... 79
18 The far UV Circular Dichroic Spectra of Potato PPO .... 81
19 The far UV Circular Dichroic Spectra of Florida Spiny
Lobster PPO 83
20 The far UV Circular Dichroic Spectra of White Shrimp PPO . 85
21 The far UV Circular Dichroic Spectra of Brown Shrimp PPO . 87
22 Effect of Concentration-related Inhibitory Effect of Kojic
Acid on Mushroom Tyrosinase (PPO) Activity on DL-/J-3,4-
dihydroxyphenylalanine (DL-DOPA) 109
23 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of DL-DOPA and L-Tyrosine by Mushroom
PPO Ill
24 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of Chlorogenic Acid and Catechol by
Potato PPO 113
25 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of 4-Methylcatechol and Chlorogenic
Acid by Apple PPO 116
IX

26
27
28
29
30
31
32
33
34
35
36
37
38
119
121
123
133
137
139
149
156
161
163
165
170
174
The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of L-DOPA and Catechol by White Shrimp
PPO
The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of DL-DOPA and Catechol by Grass Prawn
PPO
The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of DL-DOPA and Catechol by Lobster PPO.
Effect of Kojic Acid on the Hydroxylation of Monohydroxy-
phenol by Mushroom Tyrosinase (PPO)
Effect of Kojic Acid on Reduction of Cu2+ to Cu+ in a
Model System
Change in Absorption Spectra of Dopaquinone due to the
Addition of Kojic Acid, (a) Dopaquinone Produced from
the Action of Mushroom PPO on DL-DOPA; and
(b) Dopaquinone Plus Kojic Acid
Apparatus Used for Studying Polyphenol Oxidase (PPO)
Inactivation by High Pressure C02
Effect of Carbon Dioxide (1 atm) on the Change in pH and
Enzyme Activity of Florida Spiny Lobster PPO Heated at
33° (a), 38° (b), or 43°C (c)
Effect of High Pressure (58 atm) Carbon Dioxide on the
Change in pH and Enzyme Activity of Florida Spiny
Lobster PPO heated at 43°C
Effect of High Pressure (58 atm) Carbon Dioxide on the
Change in pH and Enzyme Activity of Brown Shrimp PPO
heated at 43°C
Effect of High Pressure (58 atm) Carbon Dioxide on the
Change in pH and Enzyme Activity of Potato PPO heated at
43°C
Nondenaturing Polyacrylamide Gel Electrophoresis (PAGE,
7.5% Gel) Profile of Carbon Dioxide (1 atm)-treated
Florida Spiny Lobster PPO
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) Profile of High Pressure (58 atm)
Carbon Dioxide-treated Florida Spiny Lobster PPO . . . .
x

39
Polyacrylamide Gel Isoelectric Focusing (IEF, 5% Gel)
Profile of Carbon Dioxide (1 atm)-treated Florida Spiny
Lobster PPO 176
40 Polyacrylamide Gel Isoelectric Focusing (IEF, 5% Gel)
Profile of High Pressure (58 atm) Carbon Dioxide-treated
Florida Spiny Lobster PPO 178
41 Comparison of Far UV Circular Dichroic Spectra of
Nontreated Control and High Pressure (58 atm) Carbon
Dioxide-treated Florida Spiny Lobster PPO 180
42 Comparison of Far UV Circular Dichroic Spectra of
Nontreated Control and High Pressure (58 atm) Carbon
Dioxide-treated Brown Shrimp PPO 182
43 Comparison of Far UV Circular Dichroic Spectra of
Nontreated Control and High Pressure (58 atm) Carbon
Dioxide-treated Potato PPO 184
44 The Restorative Ability of Carbon Dioxide (1 atm)-treated
Florida Spiny Lobster PPO Activity and the Pertinent
Environmental pH Changes during Frozen-storage 188
45 The Restorative Ability of High Pressure (58 atm) Carbon
Dioxide-treated Florida Spiny Lobster PPO Activity and
the Pertinent Environmental pH Changes during Frozen-
storage 191
46 The Restorative Ability of High Pressure (58 atm) Carbon
Dioxide-treated Brown Shrimp PPO Activity and the
Pertinent Environmental pH Changes during Frozen-storage 193
47 The Restorative Ability of High Pressure (58 atm) Carbon
Dioxide-treated Potato PPO Activity and the Pertinent
Environmental pH Changes during Frozen-storage 195
xi

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
CHARACTERIZATION AND COMPARISON OF PLANT AND CRUSTACEAN
POLYPHENOL OXIDASES: KINETICS AND INHIBITION BY CHEMICAL METHODS
By
JON-SHANG CHEN
December, 1991
Chairman: M.R. Marshall
Cochair: C.I. Wei
Major Department: Food Science and Human Nutrition
The characterization and comparison of polyphenol oxidase (PPO, E.C.
1.14.18.1) from crustaceans (lobster and shrimp) and plants were made.
PPO from various plant and crustacean sources varied with respect to
molecular weight, pH and temperature effects on activity and stability,
substrate specificity, and other kinetic parameters. Differences in
conformational structure among PPOs from mushroom, potato, lobster, white
shrimp, and brown shrimp were investigated using spectropolarimetric and
immunological methods. Varied secondary structures were observed among
these PPOs, although they were shown to possess similar antigenic
determinants.
Kojic acid [5-hydroxy-2-(hydroxymethyl)-7-pyrone], a fungal
metabolite, exhibited competitive and/or mixed types of inhibition on the
oxidation of various phenolic substrates (L-tyrosine, DL-0-3,4-dihydroxy
phenylalanine, 4-methylcatechol, catechol, and chlorogenic acid) by
mushroom, potato, apple, and crustacean (lobster, grass prawn, and white

shrimp) PPOs. In addition, kojic acid was capable of reducing o-quinones
back to diphenols and prevented melanin formation.
The inactivation of lobster, brown shrimp, and potato PPO by C02 was
studied. When exposed to C02 (1 atm) at 33°, 38°, or 43°C, lobster PPO
showed a decline in enzyme activity with treatment time. Studies on
inactivation kinetics revealed that PPO was more labile to C02 and heat
than to heat alone. The use of polyacrylamide gel electrophoresis showed
that there were no differences in protein patterns and isoelectric
profiles between the C02 (1 atm)-treated and untreated PPO. When lobster,
brown shrimp, and potato PPOs were subjected to high pressure (58 atm) C02
at 43°C, the inactivation of these PPOs followed trends similar to the
atmospheric C02 experiments. Crustacean PPOs, however, were more
susceptible to inactivation by high pressure C02 than by atmospheric C02.
Differences in the secondary structures between the high pressure C02-
treated and the nontreated PPO were evident by spectropolarimetric
analysis.

INTRODUCTION
Undesirable enzymatic browning causing the discoloration or
formation of black spots (melanosis) by polyphenol oxidase (E.C.
1.14.18.1; PPO) on the surface of many vegetable, fruit, and seafood
(crustacean) products has been of great concern to food scientists. For
food processors, the formation of melanin pigments not only imparts the
problems in sensory attributes and, hence, the marketability of the
product, but often lower its nutritive value as well (Synge, 1975).
Kinetic properties have been studied for PPO from various sources of
vegetables and fruits (Schwimmer, 1981) and more recently from crustaceans
(Ferrer et al., 1989a; Rolle et al., 1991; Simpson et al., 1987, 1988a).
However, little comparative biochemical information exists between PPO
from plants and crustaceans. A preliminary study conducted in this
laboratory revealed that Western Australian lobster (Panul i rus cygnus) was
far less susceptible to melanosis during storage at refrigeration
temperature than Florida spiny lobster (Panulirus argus) maintained under
the same conditions. It is speculated that the susceptibility to
melanosis could be attributed to the difference in PPO activity between
these two lobster species. Thus, the first objective of this study was to
characterize and determine the kinetic properties of PPO enzymes from
these two lobsters as well as from other crustacean and plant sources.
Many chemical and physical methods have been studied for their
effectiveness on the inhibition of enzymatic browning. Browning may be
1

2
prevented not only by inactivating the enzyme, but also by eliminating
substrate (02 or polyphenol) necessary for the reaction, or by reacting
with the products of the enzyme reaction thus preventing the formation of
the quiñones necessary for the preceding non-enzymatic steps. Although
some chemicals have been shown to inhibit PPO activities, their use in
food processing is restricted by many concerns such as toxicity,
wholesomeness, effect on taste, flavor, texture, etc. (Vamos-Vigyazo,
1981). Kojic acid (5-hydroxy-2-hydroxymethyl-y-pyrone), a fungal
metabolite produced by many species of Aspergillus and Penicillium
(Kinosita and Shikata, 1964), has been reported for its inhibitory effect
on mushroom PPO (Saruno et al., 1979). Kojic acid mixed with ascorbic
acid and citric acid constitutes a Japanese commercial product which is
used as a tyrosinase inhibitor in foods. Since only limited information
was available on the inhibitory activity and mechanism of kojic acid on
PPO, the second objective of this study was to investigate the inhibitory
activity of this compound on crustacean (Florida spiny lobster, white
shrimp, and grass prawn) mushroom, and plant (potato and apple) PPO and to
elaborate the mechanisms of inhibition.
In past years, treatment in an atmosphere modified with carbon
dioxide has been used as an application for retarding enzyme activity, to
preserve food quality and to extend the shelf-life of food products.
Recently, inactivation of peroxidase, PPO, pectinesterase, a-amylase,
glucose oxidase, lipase, or catalase by supercritical fluid using C02 as
the solvent has been reported by many workers (Arreola, 1990; Christianson
et al., 1984; Taniguchi et al., 1987; Zemel, 1989). However, information
concerning the inhibitory effect and the inhibition mechanism of C02 on

3
plant and crustacean PPO was limited. Thus, the third objective of this
study was to investigate the effect of C02 (atmospheric and high pressure)
on the inhibition of lobster, brown shrimp, and potato PPO.

LITERATURE REVIEW
Polyphenol Oxidase
Polyphenol oxidase (PPO) (E.C. 1.14.18.1.), also known as
tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, and
catecholase, is widely distributed in nature (Schwimmer, 1981). In
addition to its general occurrence in plants, it can also be found in
microorganisms, especially fungi, and in some animal tissue (Brown,
1967). PPO plays an important role in the resistance of plants to
microbial or viral infections and probably to adverse climate (Vamos-
Vigyazo, 1981). The vast literature dealing with the role of the PPO-
polyphenol system in plant pathology has been extensively reviewed
(Bonner, 1957; Farkas and Kiraly, 1962). A simplified explanation of this
role in the resistance of plants to infections is that the quiñones formed
upon the action of the enzyme undergo secondary polymerization reactions
yielding dark, insoluble polymers; the tissues impregnated with these
polymers act as barriers to prevent further spreading of the infection.
This is considered by some authors to be the main function of the enzyme
(Macrae and Duggleby, 1968). However, plants resistant to adverse
climatic conditions have, in general, higher PPO activities than
susceptible varieties (Khrushcheva and Krehin, 1965).
In addition to the involvement in phenolic compounds biosynthesis,
the PPO enzymes also indirectly participate in auxin biosynthesis. The
4

5
primary products of its action on o-diphenols, the o-quinones, react with
tryptophan to form indole acetic acid via indolepyruvic acid (Gordon and
Paleg, 1961). Thus, PPO together with auxin degrading enzyme
(peroxidase), might play a role in plant growth regulation (Vamos-Vigyazo,
1981). The quiñones formed upon PPO action may also participate in
reactions similar to those leading to nonenzymatic browning and
humification and thus contribute to producing organic matter of soil
(Synge, 1975).
PPO from insects and crustaceans plays an important role in the
sclerotization during molting (Andersen, 1971; Brunet, 1980; Cobb, 1977;
Ferrer et al., 1989a; Summers, 1967; Vinayakam and Nellaiappan, 1987). In
this process, PPO oxidizes diphenols to quiñones, which interact with
certain side groups on adjacent proteins, thus linking them together
(Stevenson, 1985). N-Acetyldopamine (Andersen, 1979) and 3,4-
dihydroxybenzoic acid (Pryor et al., 1962) have been identified as the
cross-linking agents in crustacean and cockroach, respectively. Another
function closely related to plant systems was PPO's involvement in the
process of wound repair and calcification of the cuticle (Stevenson,
1985).
Most PPO's are copper-containing enzymes which catalyze two entirely
different reactions: (a) the hydroxylation of monophenols to the
corresponding o-dihydroxy compounds, which is called cresolase activity,
and (b) the oxidation of o-dihydroxy phenols to o-quinones, which is
termed catechol activity. The activity of cresolase involves three steps
which can be represented by the following equation (Mason, 1956):
Protein-Cu2+-02 + monophenol -*â–  Protein-Cu2+ + o-quinone + H20

6
The protein-copper-oxygen complex is formed by combining one molecule of
oxygen with the protein to which two adjacent cuprous atoms are attached.
Catecholase activity involves the oxidation of 2 molecules of o-diphenols
to 2 molecules of o-quinones, resulting in the reduction of one molecule
of oxygen to two molecules of water. The sequence for the PPO-catalyzed
reaction proposed by Mason (1957) is shown in Figure 1. The enzyme-oxygen
complex serves as the hydroxylating or dehydroxylating intermediate, and
(Cu)n represents the actual charge designation of the copper at the active
site. The overall reaction involves the use of one molecule of oxygen;
one atom of which goes into the formation of diphenol, and the other is
reduced to water. Substrate specificity varies considerably for PPO from
various sources (Aurand and Woods, 1973).
Enzymatic Browning (Melanosis)
Food scientists' primary interest in PPO is in the enzymatic
browning phenomenon and its effect on food quality. o-Quinones, the
primary products of PPO oxidative reaction can (a) interact with each
other to form high molecular weight polymers, (b) form macromolecular
complexes with amino acids or proteins, and (c) oxidize compounds of lower
oxidation-reduction potentials (Mathew and Parpia, 1971). Enzymatic
browning is a desirable process for the manufacture of black tea, sultana
grape, and ground coffee bean (De Amorim and Silva, 1968; Grncarevic and
Hawker, 1971; Takeo, 1966).
Unfavorable darkening on the surface of many fruits, vegetables, and
seafood products is primarily due to the indirect consequence of PPO
oxidizing phenols to orthoquinones, which in turn rapidly polymerize to

Figure 1. Mechanism of Polyphenol Oxidase-catalyzed Reaction: Activation of Polyphenol Oxidase (A),
Two-step Four-Electron Reduction of Oxycupropolyphenoloxidase (B-C-D), and the Associated
Hydroxylation of Monophenols (B-C-E)
Source: Mason, 1957

Protein-(Cü
(A)
+ ne'
(B)
)n — » Protein-(Cu+)n „ Prote¡n-(Cu+)n02
Activation
o-D¡phenol
Monophenol
(E)
O
o-Diphenol
(D)
Protein-(Cu+)nO + OH
o-Diphenol
o-Quinone
oo

9
form brown pigments or melanins (Joslyn and Ponting, 1951). Enzymatic
browning of fruits, vegetables, and seafood products (mainly shrimp and
lobster) due to PPO activity has been extensively studied (Ferrer et al.,
1989a; Flurkey and Jen, 1978; Harel et al., 1966; Koburger et al., 1985;
Macrae and Duggleby, 1968; Madero and Finne, 1982; Mapson et al.,1963;
Mosel and Hermann, 1974; Palmer, 1963; Patil and Zucker, 1965; Savagaon
and Sreenivasan, 1978; Sciancalepore and Longone, 1984; Simpson et al.,
1987, 1988a; Walker, 1962, 1964; Weurman and Swain, 1955). Although not
harmful to consumers, it is the formation of melanins on the surface of
these products by PPO that decreases their market value, making them
unappealing and the perception of spoilage (Alford and Fieger, 1952;
Bailey and Fieger, 1954; Bailey et al., 1960a, 1960b; Faulkner et al.,
1954; Ogawa et al., 1984).
Inhibition/Prevention of Melanosis
Undesirable enzymatic browning occurring in agricultural products
causes an enormous economical loss for food industries. Thus, in order to
reduce this loss, the occurrence of melanosis on food products must be
prevented or inhibited. Inhibition of PPO and prevention of enzymatic
browning are often treated as one and the same subject. Browning can be
prevented by inactivating PPO, by eliminating one of the substrates
necessary for the reaction (02 and the polyphenols), or by reacting the
products of PPO metabolism which in turn inhibit the formation of the
colored compounds produced in the secondary, non-enzymatic reaction steps.
As PPO is a metalloprotein with copper as the prosthetic group (Bailey et
al., 1960a; Benda!1 and Gregory, 1966; Mayer, 1962; Smith and Kruger,

10
1962), the activity of this enzyme can be inhibited by metal-chelating
agents. The effects of cyanide, carbon monoxide, sodium-diethyl-dithio-
carbamate (DIECA), mercaptobenzothiazole, dimercaptopropanol, potassium
methyl xanthate, fluoride, azide, borate, benzoic acid, small peptides,
polyvinylpyrrolidone (PVP), 2,3-naphthalenediol, ascorbic acid, and
dichlorodifluoromethane on plant PPO activity have been extensively
studied; these chemical inhibitors act primarily on the enzyme (Bedrosian
et al., 1959; Harel et al., 1967; Jones et al., 1965; Mayer et al., 1964;
Palmer and Roberts, 1967; Pierpoint, 1966; Pifferi et al., 1974; Robb et
al., 1966; Taeufel and Voigt, 1964; Walker, 1964, 1975, 1976; Walker and
Wilson, 1975; Yasunobu and Norris, 1957).
Other compounds acting as reducing agents which reduce o-quinones to
diphenols or react with plant PPO substrates to lessen the extent of
enzymatic browning are ascorbic acid, S02, sodium sulfite, sodium
bisulfite, sodium metabisulfite, potassium metabisulfite, cysteine,
2-mercaptoethanol, glutathione, benzenesulphinic acid, DIECA, Na-ethyl
xanthate, and PVP (Cash et al., 1976; Embs and Markakis, 1965; Feinberg et
al., 1976; Haisman, 1974; Hope, 1961; Markakis and Embs, 1966; Mapson,
1965; Mapson and Wager, 1961; Muneta, 1966; Muneta and Walradt, 1968;
Muneta and Wang, 1977; Ponting, 1960; Ponting and Jackson, 1972;
Sayavedra-Soto and Montgomery, 1986; Singh and Ahlawati, 1974; Taeufel and
Voigt, 1964; Vamos-Vigyazo, 1981; Walker, 1964). For the prevention of
melanosis in crustaceans, numerous agents including ascorbic acid and
sodium ascorbate, ascorbate and citrate, L-tyrosine, L-methionine, citric
acid, L-cysteine, sodium diethyl dithiocarbamate, sodium tripolyphosphate
(STP), sodium sulfite, and sodium bisulfite have been broadly studied

11
(Antony and Nair, 1975; Bailey and Fieger, 1954; Faulkner et al., 1954;
Ferrer et al., 1989b; Fink, 1988; Madero, 1982; Madero and Finne, 1982).
Many methods are available for inhibiting/inactivating PPO. The
application of these methods or chemicals for inhibition in the food
industry, however, is limited. Problems related to off-flavor, off-odor,
toxicity, and economic feasibility all affect the application of chemicals
(Eskin et al., 1971). Sulfiting agents listed as Generally Recognized As
Safe (GRAS) by the Food and Drug Administration (FDA) since 1959 (FDA,
1984) have been widely used to prevent melanosis of agricultural and
seafood products. However, due to the recent health concern and questions
regarding the safety of sulfiting agents, the FDA currently proposed
revoking the GRAS status of these additives for use on fruits and
vegetables intended to be served or sold fresh or raw to consumers. The
FDA also requires label declaration of these agents added to food or as an
ingredient whenever they are present in the finished product at a level
equal to or higher than 10 ppm as S02 (FDA, 1985). Furthermore, the FDA
requires that sulfite-treated shrimp products having residue levels
greater than 10 ppm must bear a label stating the presence of these
additives (FDA, 1985).
The maximum allowable residual level of sulfite on shrimp in the raw
edible portion is 100 ppm as S02 (Marshall et al., 1984), which is not
hazardous to the majority of the population. After exposure to these
additives even at this level, however, people still suffer adverse
reactions ranging from mild to severe symptoms (Leeos, 1986). Therefore,
the search for an alternative which can inhibit melanosis but does not
cause adverse reactions becomes a necessity.

12
Ko.iic Acid
The inhibitory effect of some tyrosine-3-hydroxylase inhibitors of
microbial origins such as aquayamycin, chrothiomycin, and oudenone on PPO
activity has been studied (Ayukawa et al., 1968, 1969; Nagatsu et al.,
1968; Sezaki et al., 1968; Umezawa et al., 1970). Kojic acid (5-hydroxy-
2-hydroxymethyl-7-pyrone) (Figure 2) produced in the culture filtrate of
Aspergillus albus was shown in vitro to inhibit the activity of mushroom
tyrosinase using DOPA and tyrosine as substrates (Saruno et al., 1979).
At low concentration, kojic acid was found to inhibit the in vitro
oxidation of a number of D-amino acids, L-phenylalanine and a few related
compounds (Bajpai et al., 1981). Production of kojic acid, generally
considered as a secondary metabolite from carbohydrate metabolism under
aerobic conditions, has been extensively studied (Bajpai et al., 1981,
1982a, 1982b; Morton et al., 1945; Saruno et al., 1979). Although kojic
acid can be considered a potential candidate to inhibit melanosis, the use
of this chemical, depends not only on its production but also its
potential toxicity to humans.
Supercritical fluid (SCF) Treatment
Intensive study of supercritical fluids for extraction of food
components has mostly concentrated on decaffeination, deodorization, aroma
recovery, oil recovery, oil refining, and fractionation (Bui ley et al.,
1984; Christianson et al., 1984; Friedrich and Pryde, 1984; Lee et al.,
1986; Maddocks and Gibson, 1977; Peter and Brunner, 1978; Snyder et al.,
1984; Schultz et al., 1967; Stahl et al., 1978, 1980, 1984; Taniguchi et
al., 1985; Vollbrecht, 1982; Zosel, 1979, 1981). Modified atmospheres

Figure 2. Structure of Kojic Acid (5-hydroxy-2-hydroxymethyl-y-pyrone)

Kojic acid
(5-hydroxy-2-hydroxymethyl-gamma-pyrone)

15
(carbon dioxide or C02-saturated brines) have been used successfully to
preserve food quality and extend shelf-life (Barnett et al., 1978; Brown
et al., 1980; Bullard and Collins, 1978; Gee and Brown, 1987a, 1987b;
Lannelongue et al., 1982; Longard and Reiger, 1974; Shewan, 1950; Vernath
and Robe, 1979; Villemure et al., 1986; Woyewoda et al., 1984; Yokoseki et
al., 1956). Studies of seafood preservation using a modified atmosphere
of C02 showed that the lower internal pH of the tissue was due to exposure
of the external C02 present and resulted in a rapid acidification of the
internal cellular environment (Aickin and Thomas, 1975; Boron and DeWeer,
1976; Thomas, 1974; Thomas and Ellis, 1976; Turin and Warner, 1977).
Intracellular and extracellular acidification of tissue could produce an
antimicrobial effect and also influence many different enzymatic
activities (Parkin et al., 1981). SCF possesses physicochemical
properties intermediate between those of liquids and gases which enhance
its efficacy as solvents; the higher gas density gives good solvent power,
while the lower viscosity and higher diffusivity provide the SCF with
higher gas permeability (Rizvi et al., 1986). Carbon dioxide is used as
a solvent for SCF because it is nontoxic, nonflammable, inexpensive and
readily available (Hardardottir and Kinsella, 1988) and it has a
relatively low critical temperature (33.1°C) and pressure (72 atm) (Rizvi
et al., 1986). Eldridge et al. (1986) reported that SCF produced minimal
detrimental effects on the functional properties of proteins. The
application of SCF using C02 as a solvent in seafood processing could be
a benefit because it possesses characteristics of antimicrobial action,
lipid (cholesterol) reduction, and enzymatic inhibitory activities.

CHARACTERIZATION AND COMPARISON OF CRUSTACEAN AND PLANT POLYPHENOL
OXIDASES: KINETICS AND SOME PROPERTIES
Introduction
Polyphenol oxidase (PPO) (E.C. 1.14.18.1.), also known as
tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, and
catecholase, is widely distributed in nature (Schwimmer, 1981). In
addition to its general occurrence in plants, it can also be found in
microorganisms, especially in fungi, and in some animal organs (Brown,
1967). Unfavorable darkening of many fruits and vegetables after cutting
is primarily due to the action of this enzyme. Enzymatic browning of
fruits and vegetables due to PPO activity has been widely reported
(Flurkey and Jen, 1978; Harel et al., 1966; Macrae and Duggleby, 1968;
Mapson et al., 1963; Mosel and Herrman, 1974; Palmer, 1963; Patil and
Zucker, 1965; Sciancalepore and Longone, 1984; Walker, 1962, 1964; Weurman
and Swain, 1955). However, the role of PPO in crustaceans is not well
documented considering the variation in susceptibility to melanosis.
PPO plays an important role in the sclerotization of insects and
crustaceans during the molting cycle (Andersen, 1971; Brunet, 1980;
Summers, 1967; Vinayakam and Nellaiappan, 1987). However, it is the
formation of melanins causing darkening on the surface of seafood products
due to PPO action which is of concern to the seafood industry. PPO
enzymes from various crustaceans have been partially characterized (Madero
and Finne, 1982; Nakagawa and Nagayama, 1981; Simpson et al., 1987,
16

17
1988a). Although kinetic properties have been studied for PPO from
various sources of vegetables and fruits (Schwimmer, 1981) and more
recently from crustaceans (Chen et al., 1991a; Ferrer et al., 1989a; Rolle
et al., 1991), little comparative biochemical information exists between
PPO from plants and crustaceans. A preliminary study conducted in this
laboratory revealed that Western Australian lobsters were far less
susceptible than the Florida spiny lobsters to melanosis during storage at
refrigeration temperature. A similar phenomenon was observed on pink
shrimp (Penaeus duorarum) in comparison to white shrimp (Penaeus
setiferus) (Simpson et al., 1988a). These workers characterized the PPO
properties and they attributed the difference in susceptibility to
melanosis to the varied physiological efficiency of the enzyme and the
distribution of phenolic substrates in the shrimp (Simpson et al., 1988a).
Koburger et al. (1985) also showed that South African lobster tails were
less susceptible to melanosis than Florida spiny lobster tails. Since the
water habitat of Western Australian lobster is colder than that of Florida
spiny lobster, it is speculated that the PPO activity for these two
species could be different. Thus, the objectives of this study were to
characterize the PPO from these two lobster species and to compare their
kinetic properties to PPOs from other plant and crustacean sources.
Materials and Methods
Fresh Florida spiny lobster [Panulirus argus) tails obtained from
the Whitney Marine Laboratory at Marineland, FL were transported in ice to
the laboratory and stored at -20°C. Frozen Western Australian lobster
[Panulirus cygnus) tails were purchased from the Beaver Street Foods

18
(Jacksonville, FL); these lobsters were found to contain less than 5 ppm
sulfite background residue when randomly selected samples from each batch
were checked using the method of Simpson et al. (1988b). Mushroom
(Agaricus bispora) tyrosinase with an activity of 2,200 units/mg solid was
purchased from Sigma Chemical Co. (St. Louis, MO). Russet potatos and Red
Delicious apples were purchased from a local supermarket. White shrimp
(Penaeus setiferus) and brown shrimp (Penaeus aztecus) were obtained from
a local seafood store. Grass prawn or Taiwanese black tiger shrimp
(Penaeus monodon) frozen in dry ice was provided by Dr. J. S. Yang, Food
Industry Research and Development Institute, Hsinchu, Taiwan, Republic of
China. Lobster cuticle, shrimp cephalothorax (head), and potato and apple
peels were frozen in liquid nitrogen and ground into a fine powder using
a Waring blender. The ground powder was stored at -20°C until needed.
Extraction and Purification of Lobster PPO
Frozen lobster tails were thawed at room temperature. After the
cuticle was separated from the flesh, it was frozen in liquid nitrogen and
ground into a fine powder in a Waring blender. The cuticle powder was
stored at -20°C and used as required.
PPO was extracted according to the procedure of Simpson et al.
(1988a). One part lobster cuticle powder was added to three parts (w/v)
0.05 M sodium phosphate buffer (pH 7.2) containing 1 M NaCl and 0.2% Brij
35 (Fisher Scientific Co., Orlando, FL). The extract was stirred for 3 hr
at 4°C and the suspension was centrifuged at 8,000g (4°C) for 30 min. The
supernatant was then dialyzed at 4°C overnight against 3 changes (4L) of
0.05 M sodium phosphate buffer (pH 6.5).

19
Each enzyme was purified further using a nondenaturing preparative
polyacrylamide gel electrophoresis (PAGE) system. Equipment utilized
included a gel tube chamber (Bio-Rad Model 175, Richmond, CA) and a Bio-
Rad power supply (Model EPS 500/400). A one-mL aliquot of crude enzyme
extract was applied to each of eight gel tubes (1.4 cm I.D. x 12 cm
length) containing 5% acrylamide/ 0.13% bisacrylamide gel prepared
according to the method of Sigma Bulletin No. MKR-137 (Sigma Chemical Co.,
1984), and ran at a constant current of 10 mA/tube in a buffer (pH 8.3)
containing 5 mM Tris-HCl and 38 mM glycine. PP0 was visualized using a
specific enzyme-substrate staining method (Constantinides and Bedford,
1967); 10 mM DL-/J-3,4-dihydroxyphenylalanine (DL-DOPA) in 0.05 M sodium
phosphate buffer (pH 6.5) was used as a substrate. One tube was used to
determine the migration of the enzyme relative to the dye front (Rf). The
remaining gels were then sectioned at the determined Rf and the enzyme was
eluted from the gel by homogenization in 0.05 M sodium phosphate buffer
(pH 6.5) utilizing a Dounce manual tissue grinder (Wheaton, Millville,
NJ). The homogenates were filtered through Whatman No. 4 filter papers,
pooled, and concentrated using an Amicon stirred cell (Model 8050, Amicon
Co., Danvers, MA) fitted with a 10 K filter (Pharmacia LBK Biotechnology
Inc., Piscataway, NJ).
Extraction and Purification of Potato PPO
The method of Pati1 and Zucker (1965) with some modifications was
used. After ammonium sulfate precipitation and dialysis, crude PPO
preparation was subjected to chromatography with a DEAE-cel1ulose (0.95
meq/g, Sigma) column (40 cm length x 26 mm i.d., K 26/40 Pharmacia Fine

20
Chemicals) which had been equilibrated with 1.0 mM potassium phosphate
buffer (pH 7.0). Unbound phenolic compounds and proteins were washed off
using 250 mL 1 mM phosphate buffer (pH 7.0) at 24 mL/hr for 3 hr. Elution
of PPO was performed using a linear gradient (0-1.0 M) of NaCl in 1.0 mM
potassium phosphate buffer at 24 mL/hr for 18 hr. Four-mL fractions were
collected, and protein was estimated by spectrophotometry at 280 nm.
Fractions showing PPO activity were pooled and concentrated using an
Amicon stirred cell fitted with an Amicon YM 10 filter.
The partially purified enzyme preparation was loaded onto a Sephadex
G-100 (Pharmacia) gel filtration column (Pharmacia K 26/40)
pre-equilibrated with 1.0 mM potassium phosphate buffer (pH 7.0). The
column was eluted at 4°C with 400 mL 1.0 mM potassium phosphate buffer (pH
7.0) at 24 mL/hr for 15 hr. Four-mL fractions were collected and protein
estimated as above; fractions showing PPO activity were pooled and
concentrated using an Amicon stirred cell. Concentrated samples were
dialyzed at 4°C overnight against 3 changes of 2L elution buffer.
Extraction and Purification of Apple PPO
The method of Stelzig et al. (1972) with some modification was
followed. Crude apple PPO after partial purification and dialysis against
H20 was loaded onto an HT hydroxyl apatite (Bio-Rad) column (K 26/40). The
enzyme was desorbed from the gel using 250 mL 0.005 - 0.3 M (linear
gradient) sodium phosphate buffer (pH 7.6) containing 5% ammonium sulfate
at 24 mL/hr for 18 hr. Four-mL fractions were collected and protein
estimated by absorbance at 280 nm; fractions showing PPO activity were
pooled and dialyzed overnight (4°C) against 2 changes of 4L H20. The

21
dialysate was further concentrated using an Amicon stirred cell fitted
with a YM 10 filter.
Extraction and Purification of White Shrimp and Grass Prawn PPO
The methods of Chen et al. (1991a) and Rolle et al. (1991) with
slight modification were followed to purify white shrimp and grass prawn
PPO, respectively. Shrimp cephalothorax powder was suspended in 3 volumes
(w/v) 0.05 M sodium phosphate buffer (pH 7.2) containing 1 M NaCl
(extraction buffer) and 0.2% (v/v) Brij 35, and stirred at 4°C for 3 hr.
Following centrifugation at 23,000g (4°C) for 30 min, the supernatant was
fractionated with ammonium sulfate between 0 - 40% saturation; protein
precipitate was collected by centrifugation at 23,500g at 4°C for 30 min.
For white shrimp, the precipitate was dissolved in 0.05 M phosphate
buffer (pH 7.2) and dialyzed at 4°C overnight against 3 changes of 4L of
0.05 M phosphate buffer (pH 7.2). The dialyzed PPO was loaded onto a
DEAE-cellulose (0.95 meq/g) column (K 26/40) pre-equilibrated with 0.05 M
phosphate buffer (pH 7.2). Sixty-mL of 0.05 M sodium phosphate buffer (pH
7.2) was used to desorb unbound phenolic compounds and proteins at 0.2
mL/min for 1.5 hr. Elution of PPO was performed using a 300 mL 0.05 M
sodium phosphate buffer (pH 7.2) containing a linear gradient (0 - 1.0 M)
of NaCl. Three-mL fractions were collected and the protein estimated by
absorbance at 280 nm. Fractions possessing PPO activity were pooled and
concentrated using an Amicon stirred cell fitted with a YM 10 filter.

22
PPO was loaded onto a Sephadex G-100 gel column (K 26/40) pre¬
equilibrated with 0.05 M sodium phosphate buffer (pH 7.2) and then eluted
with 300 mL 0.05 M sodium phosphate buffer (pH 7.2) at 0.15 mL/min.
Three-mL fractions were collected and protein was estimated by absorbance
at 280 nm; fractions showing PPO activity were pooled and concentrated
using an Amicon stirred cell fitted with YM 10 filter. Concentrated PPO
was then dialyzed at 4°C overnight against 3 changes of 2L H20.
For grass prawn, the precipitate was resuspended in extraction
buffer containing 40% ammonium sulfate. After homogenization using a
Dounce manual tissue grinder, the sample was centrifuged at 23,500g (4°C)
for 20 min. The precipitate was homogenized in extraction buffer and
centrifuged as previously described. The resulting precipitate was
homogenized in extraction buffer, then subjected to high performance
hydrophobic interaction chromatography at 4°C using a preparative Phenyl
Sepharose CL-4B (Sigma) column (K 16/40) attached to a Dionex gradient
pump (Dionex Corp., Sunnyvale, CA). The column was pre-equilibrated with
extraction buffer.
PPO was eluted with a stepwise gradient of elution buffer [100%
extraction buffer (9 mL), 50% extraction buffer in water (24 mL), and 10%
extraction buffer in water (24 mL)], 50% ethylene glycol (12 mL), and then
distilled water (150 mL) at a flow of 0.2 mL/min. Four-mL fractions were
collected and fractions exhibiting PPO activity were pooled and
concentrated via ultrafiltration utilizing an Amicon stirred cell fitted
with an XM 50 filter (Amicon).

23
Protein Quantitation and Enzyme Purity Determination
The protein contents of the various PPO preparations were
quantitated using the Bio-Rad Protein Assay kit with bovine serum albumin
(Sigma) as standard. Enzyme purity was examined using a mini gel system
(Mini-Protean II Dual Slab Cell) (Bio-Rad, 1985b). Plant and crustacean
PPO's (20 ng protein/well) were loaded and electrophoresis was carried out
at constant voltage (200 V) in a buffer (pH 8.3) containing 25 mM Tris-HCl
and 0.19 M glycine for 35 min. The purity of enzyme preparations was
determined by comparing gels stained with 10 mM DL-DOPA in 0.05 M sodium
phosphate buffer (pH 6.5) and then with a Commassie blue R-250 (Eastman
Kodak Co., Rochester, NY) solution.
PPO Activity Determination
Lobster PPO activity was determined spectrophotometrically by
monitoring at 475 nm the rate of dopachrome formation from DL-DOPA
(Savagaon and Sreenivasan, 1978). The assay was run at 25°C for 10 min by
mixing 40 /¿L of enzyme extract with 560 /zL of 10 mM DL-DOPA in 0.05 M
sodium phosphate buffer (pH 6.5). Two molecules of DOPA produce one
molecule of dopaquinone which has a molar absorption coefficient (a^^) of
3,600 M'1cm‘1 (Fling et al., 1963). PPO activity was defined as /¿moles
dopachrome formed per min at 25°C.
PPO activities of shrimp, potato, and apple were measured by adding
60 nl enzyme preparations to 840 /¿L 10 mM DL-DOPA in 0.05 M sodium
phosphate buffer (pH 6.5) and monitored at 25°C for 5 min. Maximal initial
velocity was determined as AA475 ^min and one unit of PPO activity was
defined as an increase in absorbance of 0.001/min at 25°C. Unless

24
otherwise specified, experiments were carried out three times in
duplicate. For this study, the enzyme activities of potato, apple, spiny
lobster, Australian lobster, white shrimp, and grass prawn PPOs were
determined to be 10,900, 97,400, 7,000, 3,000, 5,400, and 900 units/mg
protein, respectively.
pH Optima and Stability of Lobster PPO
The modified method of Gormori (1955) was followed for preparation
of various buffer solutions including 0.1 M sodium citrate-0.1 M HC1, pH
2.0, 3.0, and 4.0; 0.05 M sodium phosphate, pH 5.0, 6.0, 6.5, 7.0, 7.5,
and 8.0; 0.1 M glycine-0.1 M NaOH, pH 9.0, and 10.0; and 0.1 M sodium
phosphate-0.1 M NaOH, pH 11.0 and 12.0. The assay was performed at 25°C
by adding 40 /xL enzyme solution to a mixture containing 280 /xL of buffer
solution and an equal volume of 10 mM DL-D0PA in distilled water. The
mean velocity for dopachrome formation was determined at 475 nm using a
DU-7 spectrophotometer (Beckman Instruments, Inc., Irvine, CA).
After enzyme mixtures containing 40 /xL enzyme preparation and 120 /xL
of each of the previously described buffer systems were incubated at 25°C
for 30 min, a 40 /xL aliquot was removed and added to 560 /xL of 10 mM DL-
DOPA solution in distilled water. Dopachrome formation was monitored
spectrophotometrically.
Activation Energy and Thermostability of Lobster PPO
Reaction mixtures containing 40 /xL enzyme extract and 560 /xL 10 mM
DL-DOPA solution were incubated at various temperatures ranging from 20°
to 60°C. Activation energy (Ea) was determined according to the Arrhenius

25
equation by measuring the initial rate of reaction at different
temperatures and plotting the logarithmic value of Vinax versus 1/T (Segel,
1976).
To determine the thermostability, a 40 aliquot of lobster PPO was
sealed in a quartz cell and incubated in a DU-7 spectrophotometer for 30
min at different temperatures ranging from 20-60°C. Following
equilibration to room temperature, the enzyme extract was mixed with 560
/xL of 10 mM DL-DOPA and then monitored spectrophotometrically as described
above.
Molecular Weight Determination of Lobster PPO
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
was used for molecular weight determination of the enzyme isoforms. Slab
gels (16 cm x 20 cm) at 1.50 mm thickness, consisting of stacking gel (4%
acrylamide/ 0.1% bisacrylamide) and separating gel (7.5% acrylamide/ 0.2%
bisacrylamide), were prepared according to the Proteanâ„¢ II Slab Cell
Instruction Manual (Bio-Rad Labs., 1985a). Electrophoresis was carried
out in a Protean II Slab Cell system equipped with a Bio-Rad Model
3000/300 power supply. A constant current of 13 mA/gel and 18 mA/gel was
applied to the stacking and separating gel, respectively in a buffer (pH
8.3) containing 5 mM Tris-HCl and 38 mM glycine. Enzyme samples were
diluted with 4 volumes of SDS reducing buffer and then heated at 95°C for
4 min. Fifty-/xg aliquots were applied to each sample well. An SDS-6H
Molecular Weight Marker Kit (Sigma) containing carbonic anhydrase
(29,000), egg albumin (45,000), bovine albumin (66,000), phosphorylase B
(97,000), /3-galactosidase (116,000), and myosin (205,000) was used. The

26
molecular weights of the proteins were determined following the methods of
Weber and Osborn (1969) and Weber et al. (1972).
Enzyme Kinetics Study
Kinetic parameters (Km and Vmax) of the two lobster PPO's were
determined using the Lineweaver-Burk equation (Lineweaver and Burk, 1934).
DL-DOPA and catechol solutions at concentrations varying from 1.67 to 9.92
mM in 0.05 M phosphate buffer (pH 6.5) were used as substrates. PPO
activity on catechol was defined as ¿¿moles of benzoquinone formed per min
at 25°C. One molecule of catechol produced one molecule of benzoquinone,
which has a molar absorption coefficient (a^^) of 1,350 M'1 cm'1 (Whitaker,
1972).
In addition, Michael is constants (KJ for mushroom, potato, apple,
white shrimp, and grass prawn PPOs were determined. Sixty-/iL PPO was
added to 10 mM DL-DOPA in 0.05 M sodium phosphate buffer (pH 6.5). The
total volume in the cuvette was 1.0 mL and the final concentration of DL-
DOPA varied from 1.4 to 8.9 mM. The reaction was monitored at 475 nm and
25°C for 10 min.
Results and Discussion
Effect of pH on Lobster PPO Activity
PPO enzymes isolated from Western Australian lobster and Florida
spiny lobster exhibited similar patterns of sensitivity to pH changes.
Florida spiny lobster PPO had a pH optimum of 6.5, which was a half unit
less than that for Western Australian lobster PPO (Figure 3). These
values for the two lobster PPO's were similar to that of gulf (brown)

Figure 3. Effect of pH on the Activity of Polyphenol Oxidase (PPO) Isolated from Florida Spiny Lobster
(a) and Western Australian Lobster (o); the Assay Was Performed at 25°C by Adding 40 ni Lobster
PPO to a Mixture Containing 280 nl of Buffer Solution and an Equal Volume of 10 mM DL-DOPA in
Distilled H20.

100
80
60
40
20
0 &
2
4
6
Florida Spiny Lobster
—
Western Australian Lobster
e—

29
shrimp (Madero and Finne, 1982). PPO from white shrimp (Penaeus
setiferus) had a pH optimum at 7.5, while that from pink shrimp (Penaeus
duorarum) was 8.0 (Simpson et al., 1987, 1988a). Ohshima and Nagayama
(1980) showed that catecholase isolated from antarctic krill (Euphausia
superba) had a pH optimum at 6.5. PPO isolated from grass prawn was shown
to have a pH optimum at 6.0 (Rolle et al., 1991). For plant PPO, the pH
optimum of 7.0 was observed for mushroom (Dawson and Mager, 1962), apple
(Stelzig et al., 1972), and banana (Galeazzi and Sgarbieri, 1978),
respectively, while pear PPO was reported to have a pH optimum at 4.0
(Rivas and Whitaker, 1973).
With respect to the pH-related relative activity, the Western
Australian lobster PPO had a broader pH range (from 5 to 9) than that
observed for the Florida spiny lobster PPO. The behavior of Florida
lobster PPO to various pH environments observed in this study was similar
to that of IP01 (inert phenoloxidase) and TAPOl (trypsin activated
phenoloxidase) forms from a study by Ferrer et al. (1989a). For white and
pink shrimp PPO, the optimal pH environments ranged from 6-7.5 and 6.5-
9.0, respectively (Simpson et al., 1988a).
Data in Table 1 indicated that crustacean (Florida spiny lobster,
Western Australian lobster, pink shrimp, white shrimp, brown shrimp, and
grass prawn) PPOs had a narrow range of optimum pH between 6 to 8.
However, a broad range of pH optimum between 4 and 7 was observed for
mushroom and other plant (potato, apple, peach, pear, and banana) PPOs.
Aylward and Haisman (1969) proposed that the optimum pH of PPO activity,
which usually ranged between pH 4 and 7, varied with enzyme sources and
substrates used.

Table 1. Comparison of Kinetic Properties of Polyphenol Oxidase (PPO) from Various Sources
Enzyme
source
Substrate
specificity
Km (mM)
pH optima
Temperature
optima (°C)
Molecular
Masses (kD)
Isoforms
Mushroom
DOPA1
Catechol4,5
0.201
5.5 - 72
--
34.53
43
Potato
Chlorogenic acid6
0.061
6.07
228
36 - 5409
59
Apple
4-methylcatechol10
0.041
4.5/7.010
301°
24 - 13411
311
Peach
D-catechin12
4.2012
6.213
--
70 - 9012
412
Pear
Pyrocatechol14
20.914
4.014
--
--
214
Banana
Dopamine15
0.6315
7.015
3715
12/6015
2
S. lobster1
DL-DOPA
9.85
6.5
35
82 - 97
3
A. lobster1
DL-DOPA
3.57
7.0
30
87 - 92
2
Grass prawn16
DL-DOPA
4.45
6.0
45
63 - 80
2
Pink shrimp17
DL-DOPA
1.60
8.0
40
40
1
White shrimp18
DL-DOPA
2.80
7.5
45
30
1
Brown shrimp19
DL-DOPA
--
6.5
--
213
2
Obtained from this study; Dawson and Mager (1962);
Nakamura et al. (1966); 6Lavollay et al. (1963); 'Macrae
and Belitz (1975); 1°Stelzig et al. (1972); 11Demenyuk et
Matheis
13Luh
16Rol le et
3Bouchilloux et al.
and Dubbleby
(1963); 'Harrison et al. (1967);
(1968); 8Schaller (1972);
. , al. (1974); 12Wong et al. (1971);
and Phithakpol (1972); 4Rivas and Whitaker (1973); 5Palmer (1963);
. 17.isc._„ ,i moot iqoo,i. 19Madero and Finne (1982) £
al. (1991); 17,18Simpson et al. (1987, 1988a);

31
Effect of pH on Lobster PPO Stability
Enzyme stability over a broad pH range (2 - 12) revealed that PPO
obtained from both lobster species exhibited optimum stability at pH 7
(Figure 4). When PPO was pre-incubated between pH of 5 and 9, both
Florida spiny lobster and Western Australian lobster PPOs still retained
at least 60% of their relative activity when compared at pH 6.5.
Conformational changes at the active site due to dramatic pH changes may
have caused the significant decline in enzyme activity between pH 2 and 5,
and between 9 and 12. Similar changes were reported to occur with TAP02
(trypsin activated phenoloxidase) form of the Florida spiny lobster PPO
(Ferrer et al., 1989a). PPO from pink shrimp (Simpson et al., 1988a),
white shrimp (Simpson et al., 1987), and grass prawn (Rolle et al., 1991)
all exhibited optimal activity within the neutral to alkaline pH range (pH
6-8), and showed maximal stability at pH 8.
Effect of Temperature on Lobster PPO Activity and Stability
Both lobster PPOs showed temperature-related changes in enzyme
activity (Figure 5). Results obtained for this study showed that Florida
spiny lobster and Western Australian lobster PPO had the temperature
optimum at 35° and 30°C, respectively, (Figure 5). These values were lower
than those observed for the PPO of pink shrimp (40°C, Simpson et al.,
1988a), white shrimp (45°C, Simpson et al., 1987), and grass prawn (45°C,
Rolle et al., 1991). Compared to crustacean PPO, the activity of peach
PPO was found to increase from 3° to 37°C and then declined up to 45°C
(Vamos-Vigyazo, 1981). In apple, the enzyme reached its maximum activity
at 30°C when chlorogenic acid was used as substrate (Vamos-Vigyazo, 1981);

Figure 4. Effect of pH on the Stability of Polyphenol Oxidase (PPO) Isolated from Florida Spiny Lobster
(a) and Western Australian Lobster (o); Forty-/iL PPO Aliquot Removed from the Various Enzyme-
buffer Mixture Was Added to 560 ¿tL of 10 mM DL-DOPA in Distilled H20 and the Assay Was
Performed at 25°C.

100
Florida Spiny Lobster
A—
Western Australian Lobster

Figure 5. Effect of Temperature on the Activity of Polyphenol Oxidase (PPO) Isolated from Florida Spiny
Lobster (a) and Western Australian Lobster (o); the Assay Was Performed by Incubating 560 /xL
10 mM DL-DOPA with 40 /xL PPO at Various Temperatures.

100
80
60
40
20
0
Florida Spiny Lobster
A—
Western Australian Lobster
30 40 50
Temperature (°C)
I
60
u>
U1

36
; while 37°C was reported for banana PPO using dopamine as substrate
(Palmer, 1963). Regarding potato and grape PPO activity, the optimum
temperature of 25°C was reported for the former (Lavollay et al., 1963),
while 10-15°C for the latter (Cash et al., 1976). The data available
indicate that the temperature optimum of the enzyme also depends
essentially on the same factors as the pH optimum (Vamos-Vigyazo, 1981).
It is noted that most crustacean PPOs had higher temperature optima than
those observed for plant PPOs. With the exception of banana PPO, which
showed an unusually high temperature optimum at 37°C (Palmer, 1963).
Both lobster PPOs exhibited similar thermostability characteristics,
although the Australian lobster PPO showed decreased activity when pre¬
incubated at temperatures greater than 30°C (Figure 6). However, Florida
lobster PPO showed greater stability at a preincubation temperature of
35°C, which was within the range of EAPO (endogenously activated
phenoloxidase) and TAP01 forms but slightly different from the IP01 form
of the Florida spiny lobster PPO (Ferrer et al., 1989a). The
thermostability characteristics of both lobster PPO's behave similarly to
that of grass prawn (Rolle et al., 1991) and pink shrimp PPO (20-30°C,
Simpson et al., 1988a) but varied from that of white shrimp PPO (25-50°C,
Simpson et al., 1987). Florida spiny lobsters grew in warm water while
Western Australian lobsters are found in cold water. These differences in
their habitats may account for the difference in the optimal
thermostability between these two enzymes. PPO enzymes from shrimp are
usually stable at temperatures ranging between 30° and 50°C (Madero and
Finne, 1982; Simpson et al., 1987, 1988a). Most PPO enzymes are heat

Figure 6. Effect of Temperature on the Stability of Polyphenol Oxidase (PPO) Isolated from Florida Spiny
Lobster (a) and Western Australian Lobster (o); Following Equilibration to Room Temperature,
40 ill Lobster PPO Incubated at Various Temperatures (20° - 60°C) Was Added to 560 ill of 10 mM
DL-DOPA and the Assay Was Performed at 25°C.

% Relative Activity
w
00

39
labile; a short exposure of the enzyme to temperatures at 70-90°C is
sufficient to cause partial or total irreversible denaturation. Crude PPO
isolated from deep sea crab was shown to be inactivated at 70°C (Marshall
et al., 1984). Similarly, apple PPO (Walker, 1964) and grape PPO (Vamos-
Vigyazo, 1981) were found to be markedly inactivated at temperatures above
70°C. For banana PPO, an exposure to 80°C for 15 min was required to
inactivate enzymes completely (Galeazzi and Sgarbieri, 1978). Out of 22
cultivars of different stone fruits, peach PPO was found the least
thermostable and the greater heat stable enzyme from plum and cherry was
accompanied by higher activity when compared to peach PPO (Dang and
Yankov, 1970).
Vamos-Vigyazo (1981) pointed out that the thermotolerance of PPO is
dependent on the source of enzyme. In addition, different molecular forms
of PPO from the same source may behave differently in thermostabilities.
Activation Energy (E3) of Lobster PPO
The activation energies, Ea, for Florida and Australian lobster PPO
were 6.9 and 7.5 Kcal/mole, respectively. These Ea values were similar to
TAP01 (7.8 Kcal/mole) from Florida spiny lobster PPO (Ferrer et al.,
1989a), and comparable to shrimp PPO (Ea = 5.2 Kcal/mole) (Bailey et al.,
1954), but somewhat different from those of the PPO prepared from white
shrimp (Ea = 13.9 Kcal/mole; Simpson et al., 1987), pink shrimp (Ea = 11.5
Kcal/mole; Simpson et al., 1988a), and grass prawn (Ea = 13.3 Kcal/mole;
Rolle et al., 1991). The Ea of banana PPO was found to be 4.4 Kcal/mole
when catechol was used as substrate (Palmer, 1963).

40
Molecular Weight and Isoform Determination of Lobster PPO
Figure 7 shows the SDS-PAGE (reduction condition) patterns for both
lobster PPO enzymes; the Florida spiny lobster PPO had three isoforms and
the Western Australian lobster PPO had two. The molecular masses of the
Florida lobster PPO subunits were determined to be 82, 88, and 97 kD,
while those of the Australian lobster were 87 and 92 kD. The molecular
masses of these subunits were higher than those of white shrimp (30 kD),
pink shrimp (40 kD), grass prawn (63 and 80 kD) and antarctic krill (75
and 83 kD) (Simpson et al., 1987, 1988a; Ohshima and Nagayama, 1980; Rolle
et al., 1991), but lower than that of brown shrimp (210 kD) (Madero and
Finne, 1982). Multiple molecular forms with different molecular masses
were observed for PPO from various plant sources (Vamos-Vigyazo, 1981).
Mushroom PPO was reported to have 4 isoforms (isozymes), each had
molecular masses of 34.5 kD (Bouchilloux et al., 1963). Apple PPO was
shown to have 3 isoforms with molecular masses of 24, 67, and 134 kD,
respectively (Demenyuk et al., 1974). PPO from banana was reported to
possess two isoforms with molecular masses of 12 and 60 kD, respectively
(Palmer, 1963). Regarding potato, multiple isoforms of PPO bearing varied
molecular masses have been reported by many workers (Anisimov et al.,1978;
Constantinides and Bedford, 1967; Patil and Evans, 1963; Thomas et al.,
1978). Although Florida spiny lobster and Western Australian lobster were
grown in different habitats, a recent study using immunological techniques
with rabbit antisera against the Florida spiny lobster PPO revealed that
these two lobsters shared cross-reactivity (Rolle et al., 1991).

Figure 7. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-
PAGE, 7.5% Gel) Profile of Polyphenol Oxidase from Florida
Spiny Lobster (FSL) and Western Australian Lobster (WAL); PPO
Samples Were Diluted with 4 Volumes of SDS Reducing Buffer
Containing /J-Mercaptoethanol and Heated at 95°C for 4 Min.
Fifty-/xg PPO Was Loaded onto Each Sample Well.

42
200 kDa
116 kDa
97.4 kDa
66 kDa
43 kDa
Protein WAL FSL
Standard PPO PPO

43
PPO from most sources, as mentioned previously, have been reported
to be present in different molecular forms. The number of these forms
depends on the enzyme source and on methods used to prepare them. It has
been revealed that part of molecular forms are due to association-
dissociation phenomena which is attributed to (1) association of various
degrees of polymerization of similar units; (2) various combinations of
different subunits; (3) conformational changes of a single protein; or
combinations of these three possibilities (Vamos-Vigyazo, 1981). Much
attention has been paid to differences in the properties of multiple forms
of the enzyme and to the possible physiological significance of such
differences. These include differences in affinity and specificity to
phenolic substrates and to oxygen (Harel et al., 1964; Kahn, 1976; Taneja
and Sachar, 1974), sensitivity to inhibitors (Constantinides and Bedford,
1967; Harel et al., 1964), pH optima (Takeo and Uritani, 1965; Wong et
al., 1971), and inactivation by heat (Ben-Shalom et al., 1977; Fling et
al., 1963; Sussman, 1961). In addition, differences in isozyme patterns
were also reported in connection with subcellular organelles (Harel et
al., 1965), the stage of tissue development (Takeo and Baker, 1973; Taneja
et al., 1974), as result of attack by pathogens (Hyodo and Uritani, 1964),
or of treatment with plant hormones (Taneja and Sachar, 1977a, b).
Enzyme Kinetics of Lobster PPO
Double-reciprocal plots for the oxidation of DL-DOPA and catechol by
both lobster PPOs are shown in Figures 8 and 9, respectively. Both
lobster PPOs were capable of catalyzing DL-DOPA and catechol. Australian
lobster PPO displayed a relatively greater Michael is constant (Km = 3.57

Figure 8. Double Reciprocal Plots for the Oxidation of DL-DOPA (•) and Catechol (0) by Florida Spiny
Lobster Polyphenol Oxidase (PPO); DL-DOPA or Catechol at Concentrations of 1.67 - 9.92 mM in
0.05 M Sodium Phosphate Buffer (pH 6.5) Was Used as Substrate and the Assay Was Performed at
25°C.

1/[S] (mM)1
4*
U1

Figure 9. Double Reciprocal Plots for the Oxidation of DL-DOPA (•) and Catechol (0) by Western
Australian Lobster Polyphenol Oxidase (PPO); DL-DOPA or Catechol at Concentrations of 1.67 -
9.92 mM in 0.05 M Sodium Phosphate Buffer (pH 6.5) Was Used as Substrate and the Assay Was
Performed at 25°C.

'J

48
mM) and a lower maximum velocity (Vmax = 0.008 mmol/1 iter/min) with DL-DOPA
than with catechol (Km = 3.09 mM and Vmax = 0.041 mmol/1iter/min) (Table
2). Similar results were also noted for the Florida lobster PP0 (Table
2). These two PPOs had a higher affinity for catechol than for DL-DOPA.
Using DL-DOPA as a substrate, Simpson et al. (1987, 1988a) showed that
pink shrimp PPO had a lower Michael is constant (1.6 mM) than white shrimp
(2.8 mM). Rolle et al. (1991) characterized grass prawn PPO and they
determined the K,,, of enzyme was 4.45 mM when DL-DOPA was used as substrate.
Summers (1967) showed that blood PPO from fiddler crab had a Km value of
0.50 mM.
Australian lobster PPO exhibited a higher affinity for DL-DOPA and
catechol than Florida lobster PPO. However, the latter showed a greater
rate for oxidizing DL-DOPA and catechol than the former. It has been
reported by Lavollay et al. (1963) that no relationship could be found
between Km and Vmax values obtained for a substrate with a given PPO
preparation. Instead, the term of V^/K,,, has been recommended by some
authors (Lavollay et al., 1963; Pollock, 1965) to express the efficiency
of a given substrate for a given enzyme. Table 2 indicates that Florida
lobster PPO not only showed a higher specific activity but also had a
higher turnover number than the Australian lobster PPO. The Florida
lobster PPO also showed a higher physiological efficiency for both
substrates than the Australian lobster PPO (Table 2), which could account
for why Florida spiny lobsters were more susceptible to melanosis than
Western Australian lobsters. The kinetic and molecular properties of PPO
from other sources are also summarized in Table 1. With DL-DOPA as
substrate, different Michael is constants (KJ were observed for mushroom,

Table 2. Comparison of Polyphenol Oxidase (PPO) Activity between Florida Spiny Lobster and Western
Australian Lobster
Specific activity8
PPO (AA/min/mg protein)
Turnover3
No.
Vmax (mmole/1 iter/min)
«m (mM)
Vmax/Km (min'1)6
D0PAa
Catechol
D0PA3 Catechol
DOPA3 Catechol
Florida
Spiny
Lobster 0.36
5.3 x 107
0.48
0.72
9.85 4.58
0.05 0.16
Western
Australian
Lobster 0.03
4.5 x 106
0.01
0.04
3.57 3.09
0.01 0.01
aSpiny lobster and Australian lobster PPO with activity of 7,000 and 3,000 units/mg protein, respectively,
were used for this study; DL-D0PA was used as substrate and the unit of turnover number was expressed
as mmoles DL-D0PA/min/mole of lobster PPO.
Physiological efficiency
VD

50
potato, apple, grass prawn, pink shrimp, and white shrimp PPOs. Varied Km
values were also reported for peach (Km = 4.2 mM, Wong et al., 1971), pear
(Km = 20.9 mM, Rivas and Whitaker, 1973), and banana (Km = 0.63 mM, Palmer,
1963) PPOs when different phenolic compounds were used as substrates
(Table 2). Data obtained from this study showed that mushroom, potato,
and apple PPOs had comparatively lower Km than those of crustacean PPOs,
indicating that the former PPOs had a higher affinity for DL-DOPA than the
latter ones. It has been reported that the affinity of PPO towards a
given substrate may vary widely, even if isozymes of the same origin are
used. The differences might be due, at least partly, to steric factors
connected to differences in protein structure (Vamos-Vigyazo, 1981).
Substrate specificity varies considerably for PPO from various
sources (Aurand and Wood, 1977; deMan, 1985). Simpson et al. (1988a)
identified higher levels of free tyrosine and phenylalanine as natural
substrates of melanosis in the carapace of pink shrimp. Further studies
by Ali (1991) showed PPO isolated from Florida spiny lobster was capable
of hydroxylating free tyrosine to DOPA. Tyramine, produced by bacterial
activity on free L-tyrosine (Veciana-Nogues et al., 1989; Santos-Buelga et
al., 1986) was also identified as the substrate of crab PPO (Summers,
1967). For mushroom (Agaricus bisporum) PPO, DOPA and catechol were,
respectively, reported by Bouchilloux et al. (1962) and Nakamura et al.
(1966) as specific substrates. For potato and apple PPOs, chlorogenic
acid (Patil and Zucker, 1965) was considered a specific substrate for the
former, while 4-methyl catechol for the latter (Stelzig, 1972). Compared
to pear PPO with pyrocatechol (Rivas and Whitaker, 1973) and banana PPO
with dopamine (Palmer, 1963), D-catechin was demonstrated as the specific

51
substrate of peach (Luh and Phithakpol, 1972). Results from this study
show that DOPA was the unanimous substrate of crustacean PPOs. For
mushroom and other plant PPOs, the specific substrate varied with the
sources of enzyme. Mason (1955) reported that tyrosine and DOPA were
specific substrates of animal tissue PPO, while Mayer and Harel (1979)
listed a wide variety of mono- and o-diphenols as the substrates of fungal
and plant PPOs. Besides the previously discussed characteristics
(substrate specificity, Km, pH and temperature effects, enzyme isoforms,
and molecular weights), other biochemical properties concerning the
catalytical function (mechanism), response to the activator/inhibitor, and
isoelectric profile (pi) of PPO also varied with the enzyme sources.
Conclusion
PPO isolated from Florida spiny lobster and Western Australian
lobster showed very similar patterns in response to effects of pH and
temperature on enzyme activity and in SDS-PAGE profile. Using DL-DOPA and
catechol as substrates, the Western Australian lobster PPO was shown to
have higher affinity but lower physiological efficiency than the Florida
spiny lobster PPO. These two PPOs showed distinctly different properties
for catalyzing the oxidation of phenolic substrates; this may explain
differences in susceptibility of spiny lobster to melanosis compared to
Western Australian lobster. Results indicate that PPO from various plant
and crustacean sources vary in substrate specificity, kinetic properties,
molecular weights, isoforms, activity and stability to pH and temperature
effects, activation energy, and isoelectric profiles.

STRUCTURAL COMPARISON OF CRUSTACEAN, PLANT, AND MUSHROOM
POLYPHENOL OXIDASES
Introduction
Polyphenol oxidase (PPO) (E.C. 1.14.18.1.), also known as
tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, and
catecholase, is widely distributed in nature (Schwimmer, 1981). The
unfavorable enzymatic browning caused by PPO on the surface of many plants
and seafood products has been of a great concern to food processors and
scientists. Although the formation of melanin (blackening spot) does not
affect the nutrient content of food products, it does however connote
spoilage by consumers (Eskin et al., 1971). Economic loss resulting from
this action has caused great concern among food processors. Enzymatic
browning of fruits, vegetables, and crustaceans due to PPO activity has
been extensively studied (Chen et al., 1991a; Ferrer et al., 1989a;
Flurkey and Jen, 1978; Macrae and Duggleby, 1968; Sciancalepore and
Longone, 1984; Simpson et al., 1989a; Walker, 1964).
Differences in the secondary structure between endogenously
activated (EAPO) and trypsin activated (TAPO) forms of Florida spiny
lobster PPO was recently demonstrated by Rolle et al. (1991) using
circular dichroism (CD) spectropolarimetry. A study conducted by Chen et
al. (1991b) showed that PPOs from plant and crustacean sources not only
varied with respect to catalytic activity in the oxidation of DL->3-3,4-
dihydroxyphenylalanine (DL-DOPA) but also had different sensitivities to
52

53
the inhibitor, kojic acid (5-hydroxy-2-hydroxymethyl-y-pyrone). Since the
conformational structures of these fungal, plant and crustacean PPOs has
not been well documented, this study was undertaken to elucidate whether
conformational differences exists among these PPOs using immunological
techniques and circular dichroism spectropolarimetry.
Materials and Methods
Fresh Florida spiny lobster (Panulirus argus) tails obtained from
the Whitney Marine Laboratory at Marineland, FL, were transported in ice
to the laboratory and stored at -20°C. Mushroom (Agaricus bispora)
tyrosinase with an activity of 2,200 units/mg solid was purchased from
Sigma Chemical Co. Russet potato was purchased from a local supermarket.
White shrimp (Penaeus setiferus) and brown shrimp (Penaeus aztecus) were
obtained from a local seafood store. Lobster cuticle, shrimp
cephalothorax (head), and potato peel were each frozen in liquid nitrogen
and ground into a fine powder using a Waring blender. The individual
ground powder was stored at -20°C until needed.
Extraction of Mushroom. Potato. Lobster, and Shrimp PPO
PPO was extracted according to the procedure of Simpson et al.
(1988a). One part ground powder was added to three parts (w/v) 0.05 M
sodium phosphate buffer (pH 7.2) containing 1 M NaCl and 0.2% Brij 35.
The extract was stirred for 5 min at 4°C and the suspension was centrifuged
at 8,000g (4°C) for 30 min. The supernatant was then dialyzed at 4°C
overnight against 3 changes of 4L 0.05 M sodium phosphate buffer (pH 6.5).

54
Purification of Mushroom, Potato, Lobster, and Shrimp PPO
Crude PPO preparation was purified further using a nondenaturing
preparative polyacrylamide gel electrophoresis (PAGE) system. Equipment
utilized included a gel tube chamber (Model 175, Bio-Rad Labs.) and a
power supply (Model EPS 500/400, Pharmacia LKB Biotechnology Inc.). A
one-mL aliquot of crude enzyme extract (potato, lobster, or shrimp) was
applied to each of eight gel tubes (1.4 cm I.D. x 12 cm length) containing
5% acrylamide/ 0.13% bisacrylamide gel and subjected to a constant current
of 10 mA/tube in a buffer (pH 8.3) containing 5 mM Tris-HCl and 38 mM
glycine (Sigma Chemical CO., 1984). PPO was visualized using a specific
enzyme-substrate staining method (Constantinides and Bedford, 1967); 10 mM
DL-DOPA in 0.05 M sodium phosphate buffer (pH 6.5) was used as substrate.
After the migration of the enzyme relative to the dye front (Rf) was
determined using one of the eight gels, the remaining gels were sectioned
at the determined Rf. PPO was eluted from the gel by homogenization in
0.05 M sodium phosphate buffer (pH 6.5) utilizing a Dounce manual tissue
grinder. The homogenates were filtered through Whatman No. 4 filter
paper, pooled, and concentrated using an Amicon stirred cell (Model 8050).
Mushroom PPO (0.25 mg/mL) in 0.05 M sodium phosphate buffer (pH 6.5) was
further purified according to the procedures previously described.
Protein Quantitation and Enzyme Purity Determination
Protein content of the various PPO preparations was quantitated
using the Bio-Rad Protein Assay kit with bovine serum albumin as standard.
Enzyme purity was examined using a mini gel system (Mini-Protean II Dual
Slab Cell) (Bio-Rad, 1985b). Mushroom, potato, and crustacean PPOs (20 /xg

55
protein/well) were loaded and electrophoresis was carried out at constant
voltage (200 V) in a buffer (pH 8.3) containing 25 mM Tris-HCl and 0.19 M
glycine for 35 min. Purity of the preparations was determined by
comparing gels stained with 10 mM DL-DOPA in 0.05 M sodium phosphate
buffer (pH 6.5) and then with a Coomassie blue R-250 solution.
Enzyme Activity Assay
PP0 activities were measured by adding 60 ¿íL PP0 to 840 ni 10 mM DL-
DOPA in 0.05 M sodium phosphate buffer (pH 6.5) and monitoring at 25°C for
5 min in a Beckman DU7 spectrophotometer at 475 nm. Maximal initial
velocity was determined as AA475 ,^/min and one unit of PP0 activity was
defined as an increase in absorbance of 0.001/min at 25°C. Unless
otherwise specified, experiments were carried out twice in triplicates.
For this study, the enzyme activities of mushroom, potato, lobster, white
shrimp, and brown shrimp PP0 were determined to be 96,000, 120,000, 4,500,
1,000, and 1,200 units/mg protein, respectively.
Anti-lobster PPO Antibody Production and Purification
One-mL purified lobster PPO containing 100 /zg protein was used as an
antigen to inject into a hen biweekly. Eggs laid by the immunized hen
were collected and anti-lobster PPO antibody was isolated and purified
from the egg yolk using the method of Poison et al. (1985). One part egg
yolk was added to 4 parts (v/v) 0.1 M sodium phosphate buffer (pH 7.6).
The mixture was made up 3.5% (w/v) with polyethyleneglycol (PEG) and
stirred for 5 min. Following centrifugation at 5,000g (10°C) for 20 min,
the supernatant collected was made up with 8.5% (w/v) PEG. The suspension

56
was allowed to stand for 10 min followed by centrifugation at 5,000g (10°C)
for 25 min. The pellet was dissolved in 2.5 volumes (v/v) phosphate
buffer (pH 7.6) and the suspension was made up with PEG to 12% (v/v).
Again, the suspension was allowed to stand for 10 min and then centrifuged
at 5,000g (10°C) for 25 min. The pellet was resuspended in 1/4 volume
phosphate buffer and cooled to 0°C before adding an equivalent volume of
50% ethanol (-20°C). Following centrifugation at 10,000g (4°C) for 25 min,
the precipitate was dissolved in 1/4 volume phosphate buffer and the
suspension was dialyzed overnight (4°C) against 4L 0.1 M sodium phosphate
buffer (pH 7.6). After dialysis, the antibody preparation was made up
with NaN3 to 0.1% and stored in the refrigerator until needed.
Molecular Weight Determination of Anti-lobster PPO Antibody
Protein content of the antibody preparation was also quantitated
using the Bio-Rad Protein Assay kit. The molecular weight of the antibody
preparation was determined using SDS-PAGE (reduction condition) according
to the method of Laemmli (1970). Mini slab gels (7 cm x 8 cm) at 1.0 mm
thickness, consisting of stacking gel (4% acrylamide/ 0.1% bisacrylamide)
and separating gel (7.5% acrylamide/ 0.2% bisacrylamide) were prepared
according to the Mini-Protein II Dual Slab Cell Instruction Manual (Bio-
Rad, 1985b). Antibody preparations were diluted with 4 volumes of SDS
sample buffer containing /J-mercaptoethanol, and heated for 4 min at 95°C.
After 30 /xg aliquots were applied to each sample well and electrophoresis
was carried out for 35 min at a constant voltages of 200 V. An SDS-6H
high Molecular Weight Protein Standards kit (Sigma) containing carbonic
anhydrase (29,000), egg albumin (45,000), bovine albumin (66,000),

57
phosphorylase (97,400), 0-galactosidase (116,000), and myosin (205,000)
was used. Molecular weights of the antibody proteins were determined
following the methods of Weber and Osborn (1969) and Weber et al. (1972).
Antibody Titer Determination bv Enzvme-1inked Immunosorbent Assay (ELISA)
One hundred-pL lobster PP0 containing 2.5 - 100 ng protein in 0.1 M
NaHC03, pH 8.6, (coating buffer) was applied to the sample well of a
microplate (Immulon 2, Dynatech). Following overnight incubation at 4°C,
the well was aspirated and washed 4 times with PBS-Tween [0.01 M sodium
phosphate, pH 7.4, containing 0.15 M NaCl and 0.2% (v/v) Tween 20] using
the Nunc-Immuno Wash (A/S NUNC, Denmark). After 100 pL primary antibody
(anti-lobster PP0 antibody) at amounts of 0.01 - 10 /xg in PBS-Tween was
added to the well, incubation was allowed to proceed at ambient
temperature for 1 hr. The aspirations and washings were repeated as
previously described before 0.1 mL secondary antibody (antichicken IgG-
alkaline phosphatase conjugate, Sigma) was added. Following another one-
hour incubation, the microplate was aspirated and washed again with PBS-
Tween. Following the addition of 0.1 mL p-nitrophenyl phosphate disodium
(1.0 mg/mL) in assay buffer (0.05 M Na2C03 and 0.05 M NaHC03 containing
0.0005 M MgCl2) to the well, the plate was incubated at ambient temperature
till a yellow color developed. The absorbance of the plate at 405 nm was
monitored every hour using an ELISA reader (Model 2550, Bio-Rad). In this
study, coating buffer without antigen was used as the negative control.

58
Analysis of Antigenic Properties of PPO
The competitive ELISA adopted from Seymour et al. (1991) was
employed to study whether PPO from mushroom, potato, white shrimp, and
brown shrimp possessed similar antigenic determinants as the Florida spiny
lobster. After the microplate was coated with lobster PPO at 10 ng/well
for one hour at room temperature, 100 /xL aliquot of antibody-competitive
PPO mixture was added. The antibody-PPO mixture was prepared by mixing
the competitive PPO (either mushroom, potato, or lobster, white shrimp,
and brown shrimp) (0.2 - 2.0 /xg/mL) with an equal volume of primary
antibody solution (10 or 20 jig/mL of IgY) for 1 hr at ambient temperature.
The assay procedures were conducted as previously described.
Immunoblotting
Purified mushroom, potato, lobster, white shrimp, and brown shrimp
PPO and a crude lobster PPO preparation were subjected to SDS-PAGE under
reduction condition (Laemmli, 1970) and then electro-transferred to
nitrocellulose membrane (Bio-Rad). Slab gels (16 cm x 20 cm) at 1.0 mm
thickness, consisting of stacking gel (4% acrylamide/ 0.1% bisacrylamide)
and separating gel (7.5% acrylamide/ 0.2% bisacrylamide), were prepared
according to the Proteanâ„¢ II Slab Cell Instruction Manual (Bio-Rad,
1985a). A constant current of 13 mA/gel was applied during stacking and
18 mA/gel during separation. Fifty-jig aliquots of test samples were
applied to each well and run with the protein standards (SDS-6H Molecular
Weight Marker Kit).
Following electrophoresis, the gel was equilibrated in 500 mL of
Towbin transfer buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, and 20%

59
methanol; pH 8.3) for 15 min. Electro-transfer was performed according to
the Trans-Blot Electrophoretic Transfer Cell Instruction Manual (Bio-Rad,
1989) at a constant voltage of 50 V for 1.5 hr using Towbin buffer as the
electrolytic buffer. Complete transfer of proteins was verified by
staining the gel and the nitrocellulose membrane with a Coomassie blue
solution.
The nitrocellulose membrane following electro-transferring was
rinsed with phosphate-buffered saline (PBS) 3 times, and then incubated
with Blotto/Tween blocking solution (5% w/v nonfat dry milk, 0.2% v/v
Tween 20, and 0.02% w/v NaN3 in PBS; Harlow and Lane, 1988) at ambient
temperature with agitation for 2 hr. After washing twice for 5 min each
in PBS, the membrane was incubated overnight in the primary antibody (IgY)
solution (10 /xg/mL). Following washing with 4 changes of PBS for 5 min
each, the membrane was treated for 1 hr with the secondary antibody
(antichicken IgG-alkaline phosphatase conjugate, Sigma) at a dilution of
1/2000. The membrane was then washed again with PBS as previously
described, and incubated with 100 mL alkaline phosphatase buffer (100 mM
NaCl, 5 mM MgCl2, and 100 mM Tris; pH 9.5) containing 0.016%
bromochloroindolyl phosphate (BCIP) and 0.032% nitro blue tetrazolium
(NBT) (Harlow and Lane, 1988) for 45 min. The reaction was stopped by
rinsing the membrane with PBS containing 20 mM EDTA.
Spectropolarimetric Analysis of PPO
The circular dichromic spectra of PPO was scanned at the far UV (250
- 200 nm) range using a Jasco J-20 automatic recording spectropolarimeter
(Japan Spectroscopic Co., Tokyo, Japan). A 1.0-cm Suprasil (Helma Cells)

60
cuvette with a 1.0-cm light path was used. Four-mL of PPO (10 - 20 /xg/mL)
in 0.5 mM sodium phosphate buffer (pH 6.5) was analyzed at ambient
temperature. Calculations of the secondary structures were carried out by
computer analysis of the spectra using the SSE program (Japan
Spectroscopic Co., 1985) with myoglobin, cytochrome c, ribonuclease A,
lysozyme, and papain as references.
Results and Discussion
SDS-PAGE Profile of PPO
Various enzyme subunits with different molecular masses were
observed for crude mushroom (7 isozymes), potato (5 isozymes), lobster (3
isozymes), white shrimp (2 isozymes), and brown shrimp (2 isozymes) PPO
preparations after the nondenaturing preparative polyacrylamide gel was
stained with DL-DOPA. After subjecting to SDS-PAGE (without treatment of
/1-mercaptoethanol), mushroom and potato PPO chosen for this study were
shown to have a subunit of lower molecular mass than crustacean (lobster,
white shrimp, and brown shrimp) PPO, estimated as 66 and 148 kD,
respectively (Figure 10). These are in agreement with the previously
reported data of Anisimov et al. (1978) and Bouchilloux et al. (1963). As
to lobster, white shrimp, and brown shrimp PPO, the molecular masses were
determined as 200, 190, and 190 kD, respectively. Brown shrimp PPO of 190
kD was close to that of the Gulf brown shrimp PPO (210 kD) (Madero and
Finne, 1982). However, the molecular mass of white shrimp PPO determined
in this study varied with that reported previously by Simpson et al.
(1988). The use of different preparation and analytical methods in these
studies could have contributed to such discrepancy.

Figure 10. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE, 7.5% Gel) Profile of
Polyphenol Oxidase from Mushroom (M), Potato (P), Florida Spiny Lobster (L), White Shrimp
(W), and Brown Shrimp (B) and High Molecular Weight Protein Standards (SDS-6H); PPO
Preparations Diluted with 4 Volumes of SDS Sample Buffer without /3-Mercaptoethanol Was Heated
at 95°C for 4 Min. Twenty-/xg PPO Aliquots Was Loaded onto Sample Well.

B W L P
205 kU
116 kD
97 kD
66 kD
45 kD
29 kD
M SDS-6H
o\
to

63
Antibody Production and Molecular Weight Determination
The production of lobster PPO specific antibody did not occur until
after the second boosting; peak activity occurred on day 18 (Figure 11).
Antibody activity decreased gradually with time following the third and
fourth boostings. The serum eventually lost antibody activity after day
35. The animals apparently had become tolerant to the antigen after the
third boosting.
Antibody produced on day 17 and 18 having the highest activities was
pooled for molecular weight determination. Three distinct bands were
observed on the SDS-PAGE gel (Figure 12). The molecular weight for the
lower band was estimated as 59,000 which was close to the value reported
for the heavy chain of IgY (Jensenius et al., 1981).
Antibody Titer Determination
Dose-related interactions of various PPO (mushroom, potato, lobster,
white shrimp, and brown shrimp) at 2.5 - 10 ng with lobster PPO-specific
antibody at 2.5 H9 are shown in Figure 13. All test samples showed
affinity for the antibody. Therefore, these antigens were partially
cross-reactive and shared similar antigenic determinants. The appropriate
dose range of the interaction of the various PPOs with the antibody was
determined to be 2.5 - 10 ng/well. When PPO was added at more than 10
ng/well, the antigen-antibody reaction was saturated and the sensitivity
of the ELISA became dull.

Figure 11. Profile of Anti-lobster PPO Antibody Produced from the Immunized Hen during the Immunization
Period; Purified Lobster PPO (100 /¿g Protein) Used as Antigen Was Biweekly Injected into the
Animal.

Absorbance (405 nm)
o\

Figure 12. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) of Anti-lobster PPO Antibody and Protein
Standard (SDS-6H); Anti-lobster PPO Antibody Was Diluted with
4 Volumes of SDS Sample Buffer Containing /3-Mercaptoethanol
and Heated at 95°C for 4 Min. Thirty-jug Antibody Preparation
Was Loaded onto Each Sample Well.

r -4
205 kD -
116 kD -
97 kD -
SDS-6H
Anti-lobster
PPO antibody

Figure 13. The Titer Determination of Antibody against Florida Spiny Lobster PPO versus Mushroom,
Potato, Florida Spiny Lobster, White Shrimp, and Brown Shrimp PPO; Mushroom, Potato,
Lobster, White Shrimp, and Brown Shrimp PPO at 2.5 - 100 ng/well Were Used to React with
Anti-lobster PPO Antibody at 2.5 /^g/well.

SOP) e
2
CTl
VO

70
Immunological Characteristics of PPO
The absorbance at 405 nm of a lobster PPO-antibody reaction mixture
in the presence of various competitors (lobster, white shrimp, brown
shrimp, mushroom, and potato PPO) was determined by ELISA (Figure 14).
The color intensity was reduced by 18 - 79% of the control (Abs. = 0.967)
when the various competitor-antibody mixtures containing 12.5 ng
competitor and 5 /xg antibody per sample well were added to the microplate.
The increase of the competitor up to 100 ng/well slightly reduced the
color intensity. For mushroom PPO, the competition with lobster PPO for
antibody however was not enhanced even when mushroom PPO was added at 100
ng/well. The result again indicated that these PPOs were partially cross¬
reactive and therefore shared similar structural components. From the
extent of competition for lobster PPO-specific antibody, white shrimp and
brown shrimp PPO were shown to share more antigenic similarity to lobster
PPO than potato and mushroom PPO (in descending order).
Complete transfer of PPO bands along with protein standards from an
acrylamide gel onto a nitrocellulose membrane was achieved following
electro-transferring; the staining of the treated gels with Coomassie blue
revealed no protein bands. The purified lobster, white shrimp, brown
shrimp, mushroom, and potato PPO had only one protein band, while crude
lobster PPO had 3 isozymes (Figure 15). The nitrocellulose membrane,
following staining with lobster PPO-specific antibody, was found to
contain dark bands corresponding to potato, lobster, white shrimp, and
brown shrimp PPO and the crude lobster PPO preparation. Three dark bands
were observed for both the potato PPO and the crude lobster PPO
preparation, while only one dark band was found for purified lobster,

Figure 14. Analysis of Antigenic Properties of Purified Mushroom, Potato, Florida Spiny Lobster, White
Shrimp, and Brown Shrimp Polyphenol Oxidase (PPO) by Competitive ELISA. Lobster PPO (10
ng/well) as the Antigen Was Plated and Reacted with Anti-lobster PPO Antibody (5 /xg/well) in
the Presence of 12.5 - 100 ng/well of Mushroom, Potato, Florida Spiny Lobster, White Shrimp,
and Brown Shrimp PPO as Competitors.

Absorbance (405 nm)
to

Figure 15. SDS-PAGE Profile of Protein Standard (SDS-6H), Purified
Mushroom (M), Potato (P), Florida Spiny Lobster (L), White
Shrimp (W), and Brown Shrimp (B) PPO, and a Crude Lobster PPO
Preparation (CL) under Reduction Condition on a Nitrocellulose
Membrane

74
P M B W L CL SDS-6H

75
white shrimp, and brown shrimp PPO (Figure 16). Incomplete protein
transfer or torsional changes in protein structure following electro¬
transfer process was possibly responsible for the failure of mushroom PPO
that not to form a dark band with lobster PPO-antibody. Result from this
study did not support the competitive ELISA experiment obtained for
mushroom PPO. However, our previous findings that PPO from potato and
crustacean sources shared similar structural components was further
demonstrated.
Spectropolarimetric Analysis of PPO
Lobster, white shrimp, and brown shrimp PPO had similar circular
dichroic spectra (Figures 19, 20, and 21), which were different from those
of mushroom and potato (Figures 17 and 18). They all varied in their
secondary structures (a-helix, ¿S-sheet, /J-turn, and random coil) (Table
3). For example, white shrimp PPO had a higher percentage of
a-helix than brown shrimp PPO; they both showed the same broad negative
ellipticity between 207 and 220 nm. The percentage of a-helix of
mushroom, potato, and crustacean PPOs estimated using the SSE program were
close to the values that calculated according to the formula of Greenfield
and Fasman (1969). The crustacean PPO, in general, had a higher
percentage of a-helix and lower percentage of /J-turn than mushroom and
potato PPO (Table 3). The percentages of the secondary strucutures of
these PPO estimated from the SSE program may not represent the absolute
values. However, the results from this study showed that PPO from various
sources possessed varied secondary structures, with the crustacean sources
producing PPO which showed very similar secondary structure.

Figure 16. Determination of the Specific Reactivity of Anti-lobster PPO
Antibody with a Crude Lobster PPO Preparation (CL), Purified
Mushroom (M), Potato (P), Florida Spiny Lobster (L), White
Shrimp (W), and Brown Shrimp (B) PPO as well as SDS-6H Protein
Standards as Analyzed by Immunoblotting Following SDS-PAGE

77
P
M
B
W
L CL SDS-6H

Figure 17.
The Far UV (250 - 200 nm) Circular Dichroic
20 ng/ml) in 0.5 mM Sodium Phosphate Buffer
Spectra of Mushroom PP0; Four-mL
(pH 6.5) Was Analyzed at Ambient
of PP0 (10
Temperature

Wavelength (nm)

igure 18. The Far UV (250 - 200 nm) Circular Dichroic Spectra of Potato PPO; Four-mL of PPO (10 -
20 /ig/mL) in 0.5 mM Sodium Phosphate Buffer (pH 6.5) Was Analyzed at Ambient Temperature.

o
-10 L-
200
i i i
220 230
Wavelength (nm)
210
240
250

Figure 19. The Far UV (250 - 200 nm) Circular Dichroic Spectra of Florida Spiny Lobster PPO; Four-mL of
PPO (10 - 20 ng/ml) in 0.5 mM Sodium Phosphate Buffer (pH 6.5) Was Analyzed at Ambient
Temperature.

o
n -2
0)
O
E
â– O
E -4
O
CD
2 -6
CD
CD
~o
O
-8
X
O -10
-12
200
210
220 230 240 250
Wavelength (nm)
03
U>

Figure 20.
The Far UV (250 - 200 nm) Circular Dichroic Spectra of White Shrimp PPO; Four-mL of
PPO (10 - 20 ng/ml) in 0.5 mM Sodium Phosphate Buffer (pH 6.5) Was Analyzed at Ambient
Temperature.

o
220 230 240 250
Wavelength (nm)
00

Figure 21
PPn nn ^n250/. ?°° n!)cC1üCcl5r Dichroic sPectra of Brown Shrimp PPO; Four-mL of
Temperature0 M9/mL in °‘5 mM Sodlum Ph°sphate Buffer (pH 6.5) Was Analyzed at Ambient

Wavelength (nm)
m
'J

88
Table 3. Secondary Structure Estimates of Various Polyphenol Oxidases
(PPOs)
from
Far UV
Circular Dichroic Spectra
% of Secondary
structure
PPO
a-l
^1 ix
/3-sheet
/}-turn
random coil
Mushroom
18.3
18.1*
17.3
24.8
39.5
Potato
14.8
★
13.1
34.6
28.4
22.2
Lobster
24.4
25.1*
26.2
21.4
29.9
White shrimp
29.0
29.7*
30.0
11.3
29.7
Brown shrimp
20.1
20.0*
22.3
15.2
42.4
The circular dichroic spectra of PPO was scanned at the far UV (250 -
200 nm) range. Four-mL PPO (10 - 20 /xg/mL) in 0.5 mM sodium phosphate
buffer (pH 6.5) was analyzed at ambient temperature
Calculated according to the formula proposed by Greenfield and Fasman
(1969)

89
Conclusion
Differences in the percentage of secondary structure (o-helix,
/J-sheet, /1-turn, and random coil) among polyphenol oxidases from mushroom,
potato, and crustacean were revealed by spectropolarimetric analysis.
Using competitive ELISA technique, white shrimp and brown shrimp PPOs were
shown to share more antigenic determinants similar to that of lobster PPO
than potato and mushroom PPOs when these enzymes were tested against anti-
lobster PPO antibody. This result was correlated to the previous finding
that crustaceans PPO had higher percentage of a-helix than that of potato
and mushroom PPOs.

INHIBITION MECHANISM OF KOJIC ACID ON SOME PLANT AND
CRUSTACEAN POLYPHENOL OXIDASES
Introduction
Unfavorable darkening of many food products resulting from enzymatic
oxidation of phenols to orthoquinones by polyphenol oxidase (PPO, E.C.
1.14.18.1) has been of great concern to food technologists and processors
(Joslyn and Ponting, 1951). The darkening of food products, although
innocuous to consumers, causes a decrease in market value and economic
loss because it connotes spoilage.
Enzymatic browning of plant products and crustaceans due to PPO
activity has been widely studied (Bailey et al., 1960a, 1960b; Ferrer et
al., 1989a; Flurkey and Jen, 1978; Hare! et al., 1966; Ogawa et al., 1984;
Walker, 1962, 1964). Compounds capable of inhibiting melanosis in these
products through the interference of PPO mediated reactions or through the
reduction of orthoquinones to diphenols have been identified (Bailey and
Fieger, 1954; Ferrer et al., 1989b; Golan-Goldhhirsh and Whitaker, 1984;
Harel et al., 1967; Madero and Finne, 1982; Palmer and Roberts, 1967; Robb
et al., 1966; Sayavedra-Soto and Montgomery, 1986; Wagner and Finne, 1984;
Walker, 1975, 1976). However, the number of chemicals that can actually
be used in food systems to inhibit melanosis is limited due to off-
flavors, off-odors, toxicity, and economic feasibility (Eskin et al.,
1971).
90

91
Sulfiting agents have been widely used to prevent melanosis in
agricultural and seafood products. Due to health concerns, the use of
sulfiting agents as food additives has been re-evaluated by FDA and in
some products, banned for use (Leeos, 1986). It has become necessary to
search for alternatives that show effective inhibition on melanosis but
are devoid of health concerns to consumers.
Kojic acid (5-hydroxy-2-hydroxymethyl-y-pyrone) is a fungal
metabolite produced by many species of Aspergillus and PenicilHum
(Kinosita and Shikata, 1964; Parrish et al., 1966). It possesses
antibacterial and antifungal activity (Morton et al., 1945). Saruno et
al. (1979) demonstrated that kojic acid from Aspergillus albus inhibited
mushroom tyrosinase activity. This compound was also shown to inhibit
melanosis in pink shrimp (Applewhite et al., 1990). Kojic acid mixed with
ascorbic acid and citric acid constitutes a Japanese product which is used
as a tyrosinase inhibitor in foods. Since only limited information was
available on the inhibitory effect of kojic acid on PPO, this study was
undertaken to investigate the inhibitory activity of this compound on
mushroom, plant (potato and apple), and crustacean (white shrimp, Florida
spiny lobster, and grass prawn) PPOs and to elaborate on mechanisms
involved.
Materials and Methods
Mushroom (Agaricus bispora) tyrosinase with an activity of 2,200
units/mg solid was purchased from Sigma Chemical Co. Russet potatoes and
Red Delicious apples were purchased from a local supermarket. Fresh
Florida spiny lobster (Panulirus argus) tails were obtained from Dr. R. A.

92
Gleeson, Whitney Marine Laboratory, Marineland, FL, and transported in ice
to the laboratory. Fresh, nonsulfited white shrimp (Penaeus setiferus)
was obtained from a local seafood store. Grass prawn (Penaeus monodon)
frozen in dry ice was provided by Dr. J. S. Yang, Food Industry Research
and Development Institute, Hsinchu, Taiwan, Republic of China.
Extraction and Purification of Potato PPO
Potato PPO was extracted following the methods of Patil and Zucker
(1965) with slight modification. Potato peel (130 g) was suspended
overnight in 3 volumes of cold acetone (w/v, -15°C). Frozen tissue was
macerated in a Waring blender with 2 volumes of cold acetone (w/v), and
the residue obtained following filtration was homogenized again with cold
acetone. After centrifugation at 2,500g (4°C) for 20 min, the precipitate
was collected and dried at this temperature. Dried powder was suspended
and stirred for 10 min in 50 mL of 0.02 M acetate buffer (pH 5.7) and then
poured through cheesecloth. The filtrate was centrifuged at 2,500g (4°C)
for 20 min and the supernatant was treated with 1.5 volumes (v/v) of cold
acetone (-15°C). The precipitate was dissolved in 40 mL of 0.02 M acetate
buffer (pH 5.7), and then was treated again with an equal volume of cold
acetone. The acetone-precipitate was dissolved in 0.01 M acetate buffer
(pH 5.7) to make a 0.5% (w/v) solution. After removing brown precipitate
by centrifugation, one volume of cold acetone was added to the
supernatant. The mixture was centrifuged again at 2,500g (4°C) for 20 min
and the precipitate was redissolved in 0.01 M acetate buffer (pH 5.7).
Ammonium sulfate precipitation between 0 and 60% saturation was obtained
by centrifugation at 2,500g (4°C) for 20 min. The ammonium sulfate-

93
precipitate was redissolved in 0.05 M potassium phosphate buffer (pH 7.0)
and then dialyzed overnight at 4°C against 2 changes of 4L 0.001 M
potassium phosphate buffer (pH 7.0).
DEAE-cellulose Chromatography
Crude potato PP0 preparation was loaded onto a DEAE-cellulose (0.95
meq/g, Sigma) column (K 26/40, 40 cm length x 26 mm i.d., Pharmacia) which
had been equilibrated with 0.001 M potassium phosphate buffer (pH 7.0).
Unbound phenolic compounds and proteins were washed off using 250 mL of
0.001 M phosphate buffer (pH 7.0) at a flow rate of 24 mL/hr for 2 hr.
Elution of PPO was performed using a linear concentration (0-1.0 M) of
NaCl. Four-mL fractions were collected using a fraction collector (Model
2110, Bio-Rad), and protein profile of fractions was determined at 280 nm.
Fractions showing PPO activity were pooled and concentrated using an
Amicon stirred cell (Model 8050) fitted with a YM 10 membrane filter.
Gel Filtration
Partially purified enzyme preparation after DEAE-cellulose
chromatography was loaded onto a Sephadex G-100 gel column (K 26/40,
Pharmacia) pre-equilibrated with 0.001 M potassium phosphate buffer (pH
7.0). Elution with 400 mL 0.001 M phosphate buffer (pH 7.0) was then
performed at a flow rate of 2.4 mL/hr for 15 hr. Four-mL fractions were
collected and protein profile of fractions was determined at 280 nm;
fractions showing PPO activity were pooled and concentrated using an
Amicon stirred cell. Concentrated samples were dialyzed at 4°C overnight

94
against 3 changes of 2L of elution buffer. Column chromatography was
carried out at 4°C.
Extraction and Purification of Apple PPO
The modified method of Stelzig et al. (1972) was followed to purify
apple PPO. Apple peel (150 g) was mixed with liquid nitrogen and ground
into fine powder using a Waring blender. Powder (4 g) was homogenized at
4°C in 200 mL of 0.1 M sodium phosphate buffer (pH 6.0) containing 0.3 M
sucrose, 0.2% cysteine hydrochloride and 1 mL of 2 x 10‘2 M 2-mercapto-
benzothiazole (MBT) in 95% ethanol, followed by centrifugation at 20,000g
(4°C) for 20 min. The pellet was washed twice with 50 mL 0.1 M phosphate
buffer (pH 6.0) containing 0.2% cysteine-HCl and resuspended in this
buffer. This suspension was made to 2% with Triton X-100 (Sigma), and
then incubated at 25°C for 15 min. Following centrifugation at 40,000g
(4°C) for 30 min, supernatant was collected and dialyzed at 4°C overnight
against 3 changes of 4L distilled H20. The dialysate was extracted with
cold (-20°C) n-butanol, the aqueous phase was collected and further
dialyzed overnight at 4°C against H20.
Hvdroxvlapatite Chromatography
A hydroxyl apatite gel (Bio-Gel HT hydroxyl apatite, Bio-Rad)
suspension (200 mL) was poured into a K 40/26 column. It was then washed
with 0.001 M sodium phosphate buffer (pH 7.6). Crude apple PPO was loaded
onto the column, and 250 mL of 0.005 - 0.3 M (linear gradient) sodium
phosphate buffer (pH 7.6) containing 5% ammonium sulfate was used to
desorb the enzyme at a flow rate of 24 mL/hr. Four-mL fractions were

95
collected and protein profile of fractions were recorded at 280 nm;
fractions showing PPO activity were pooled and dialyzed overnight (4°C)
against 2L H20. Dialysate was further concentrated using an Amicon stirred
cell fitted with a YM 10 filter.
Extraction and Purification of Grass Prawn PPO
The method of Rolle et al. (1991) was followed. Heads of grass
prawn were frozen in liquid N2 and then ground to a fine powder in liquid
nitrogen using a Waring blender. Powder was suspended in 3 volumes (w/v)
of 0.05 M sodium phosphate buffer (pH 7.2) containing 1 M sodium chloride
(extraction buffer) and 0.2% (v/v) Brij 35, and stirred at 4°C for 3 hr.
Following centrifugation at 23,000g at 4°C for 30 min, supernatant was
fractionated with ammonium sulfate. Ammonium sulfate precipitating
between 0 - 40% saturation was collected by centrifugation at 23,500g at
4°C for 30 min. Precipitate was resuspended in 50 mL extraction buffer
containing 40% ammonium sulfate. After homogenization using a Dounce
manual tissue grinder, the sample was centrifuged at 23,500g (4°C) for 20
min. The precipitate was homogenized in extraction buffer and centrifuged
as previously described. The resulting precipitate was homogenized in
extraction buffer, then subjected to high performance hydrophobic
interaction chromatography at 4°C using a preparative Phenyl Sepharose
CL-4B (Sigma) column (K 16/40) attached to a Dionex gradient pump (Dionex
Corp., Sunnyvale, CA). The column was pre-equilibrated with extraction
buffer.
PPO in the column was eluted with a stepwise gradient of elution
buffer [100% extraction buffer (9 mL), 50% extraction buffer in water (24

96
mL), and 10% extraction buffer in water (24 mL)], 50% ethylene glycol (12
mL), and then distilled water (150 mL) at a flow rate of 0.2 mL/min.
Four-mL fractions were collected and fractions exhibiting PPO activity
were pooled and concentrated via ultrafiltration utilizing a YM 10 filter.
Extraction and Purification of Lobster PPO
The modified procedures of Simpson et al. (1987) were followed.
Lobster cuticle was emerged in liquid nitrogen and ground into a fine
powder using a Waring blender. It was then extracted with 3 parts (w/v)
0.05 M sodium phosphate buffer (pH 7.2) containing 1 M NaCl and 0.2% Brij
35 at 4°C for 3 hr. Following centrifugation at 8,000g at 4°C for 30 min,
the supernatant was dialyzed at 4°C against 3 changes of 4L of 0.05 M
sodium phosphate buffer (pH 6.5).
PPO was purified using a nondenaturing preparative polyacrylamide
gel electrophoresis (PAGE). A one-mL aliquot of crude enzyme extract was
applied to each of eight gel tubes (12 cm length x 14 mm i.d.) containing
5% acrylamide/ 0.13% bisacrylamide gel, and ran at a constant current of
10 mA/tube. PPO was visualized using a specific enzyme-substrate staining
method (Constantidines and Bedford, 1967) using 10 mM DL-/J-3,4-
dihydroxyphenylalanine (DL-D0PA) in 0.05 M sodium phosphate buffer (pH
6.5). Gels containing PPO were sectioned and homogenized in 0.05 M
phosphate buffer (pH 6.5) utilizing a Dounce tissue grinder. Following
filtration through a Whatman No. 4 filter paper, the filtrate was
concentrated using an Amicon stirred cell fitted with 10 K filter. The
lobster PPO was further purified by subjecting the concentrated filtrate
to PAGE at 7.5% acrylamide/ 0.2% bisacrylamide gel.

97
Extraction and Purification of White Shrimp PPO
The combined methods of Simpson et al. (1987) and Rolle et al.
(1991) with slight modification were followed to purify white shrimp PPO.
Shrimp cephalothorax was frozen in liquid nitrogen and ground using a
Waring blender. Shrimp powder was suspended in 3 volumes (w/v) 0.05 M
sodium phosphate buffer (pH 7.2) containing 1 M NaCl (extraction buffer)
and 0.2% (v/v) Brij 35, and stirred at 4°C for 3 hr. Following
centrifugation at 23,000g (4°C) for 30 min, the supernatant was
fractionated with ammonium sulfate between 0 - 40% saturation; protein
precipitate was collected by centrifugation at 23,500g at 4°C for 30 min.
Precipitate collected was dissolved in 0.05 M phosphate buffer (pH
7.2) and dialyzed at 4°C overnight against 3 changes of 4L of 0.05 M
phosphate buffer (pH 7.2). The dialyzed PPO was loaded onto a DEAE-
cellulose (0.95 meq/g) column (K 26/40) pre-equilibrated with 0.05 M
phosphate buffer (pH 7.2). Sixty-mL of 0.05 M sodium phosphate buffer (pH
7.2) was used to desorb unbound phenolic compounds and proteins at 0.2
mL/min. Elution of PPO was performed by a linear gradient (0 - 1.0 M) of
NaCl with 300 mL 0.05 M sodium phosphate buffer (pH 7.2). Three-mL
fractions were collected and the protein estimated by absorbance at 280
nm. Fractions possessing PPO activity were pooled and concentrated using
an Amicon stirred cell fitted with YM 10 filter.
PPO was loaded onto a Sephadex G-100 gel column (K 26/40) pre¬
equilibrated with 0.05 M sodium phosphate buffer (pH 7.2) and then eluted
with 300 mL 0.05 M sodium phosphate buffer (pH 7.2) at 0.15 mL/min.
Three-mL fractions were collected and protein was estimated by absorbance
at 280 nm; fractions showing PPO activity were pooled and concentrated

98
using an Amicon stirred cell fitted with YM 10 filter. Concentrated PPO
was then dialyzed at 4°C overnight against 3 changes of 2L H20.
Protein Quantitation and Enzyme Purity Determination
Protein content of all preparations was quantitated using the Bio-
Rad Protein Assay kit with bovine serum albumin as standard. Enzyme
purity was examined using a mini gel system (Mini-Protean II Dual Slab
Cell) (Bio-Rad, 1985b). Plant and crustacean PPO (20 /ig protein/well) was
loaded and electrophoresis was carried out at constant voltage (200 V) for
35 min. The purity of enzyme preparations were determined by comparing
gels stained with 10 mM DL-D0PA in 0.05 M sodium phosphate buffer (pH 6.5)
(Constantidines and Bedford, 1967) and then with a Commassie brilliant
blue R-250 solution.
Enzvme Activity Assay
Potato PPO activity was determined at 25°C for 5 min by mixing 2.9
mL 0.97 mM chlorogenic acid in 1 mM potassium phosphate buffer (pH 7.0)
with 0.1 mL enzyme. Maximal initial velocity for quinone formation was
monitored at 395 nm using a DU-7 spectrophotometer. One unit of PPO
activity was defined as an increase in absorbance of 0.001/min at 395 nm
and 25°C. Apple PPO activity was measured at 30°C for 5 min by mixing 0.2
mL PPO preparation with 1.8 mL of 0.05 M 4-methylcatechol in 0.1 M sodium
phosphate buffer (pH 6.0). Maximal initial velocity for quinone formation
was determined at 395 nm and one unit of PPO activity was defined as an
increase in absorbance of 0.001/min.

99
White shrimp PPO activity was carried out at 40°C for 5 min by adding
80 fil of PPO to 1.12 ml. 10 mM L-DOPA in 0.05 M sodium phosphate buffer (pH
6.5). Maximal initial velocity for dopachrome formation was determined at
475 nm. One unit of PPO activity was defined as an increase in absorbance
of 0.001/min at 40°C. Grass prawn and spiny lobster PPO activities were
measured by adding 0.1 mL of enzyme to 1.4 mL 10 mM DL-DOPA in 0.05 M
sodium phosphate buffer (pH 6.5). The reaction was monitored at 25°C for
5 min. Maximal initial velocity was determined as (AA475nm/rnin) and one
unit of PPO activity was defined as an increase in absorbance of 0.001/min
at 25°C.
Effect of Ko.iic Acid on Enzyme Activity
The method of Saruno et al. (1979) was adopted to study the
inhibitory effect of kojic acid on PPO activities. PPO preparation was
pre-incubated with sodium acetate, potassium or sodium phosphate buffer,
and kojic acid (Sigma) solutions in respective buffer at 37°C for 15 min.
Following equilibration to ambient temperature, specific substrate for
each system in buffer solution was added to the mixture and the change in
absorbance of the reaction product, benzoquinone, was
spectrophotometrically monitored for 5 min. For control sample, an
equivalent volume of buffer was used to replace kojic acid solution.
Percentage inhibition (I) was expressed as [(T -T*)/T] x 100, where T* and
T were enzyme activities in the presence and absence of kojic acid,
respectively (Saruno et al., 1979).
The mushroom PPO system was composed of 0.9 mL of kojic acid (20 -
200 ng/ml), 0.1 mL enzyme (1 mg/mL) in distilled H20, and 2.0 mL 0.83 mM

100
L-tyrosine or 5 mM DL-DOPA in 0.05 M sodium acetate buffer (pH 6.8). The
reaction was monitored at 475 nm and 25°C. The potato PPO system contained
0.9 mL of kojic acid (20 - 800 /xg/mL), 0.1 mL of enzyme, and 2.0 mL 1.4 mM
chlorogenic acid or 5 mM catechol in 1.0 mM sodium phosphate buffer (pH
7.0). The reaction was monitored at 395 nm and 25°C. The apple PPO system
contained 1.15 mL of kojic acid (0.02 - 2.0 mg/mL), 0.1 mL enzyme, and 2.0
mL of 1.4 mM chlorogenic acid or 5 mM 4-methylcatechol in 0.1 M sodium
phosphate buffer (pH 6.0). The reaction was monitored at 395 nm and 30°C.
White shrimp PPO system contained 0.45 mL of kojic acid (20 - 200
/xg/mL), 50 /xL of enzyme, and 1.0 mL of 5 mM L-D0PA or catechol in 0.05 M
sodium phosphate buffer (pH 6.5). The reaction was monitored at 40°C and
at 395 and 475 nm, respectively, for catechol and L-D0PA oxidation. Grass
prawn and lobster PPO systems contained 0.9 mL of kojic acid (20 - 150
/xg/mL), 0.1 mL of enzyme, and 2.0 mL of 5 mM DL-DOPA or catechol in
0.05 M sodium phosphate buffer (pH 6.5). The reaction was monitored at
25°C and 395 and 475 nm, respectively.
Enzyme Kinetics Study
Michael is constants, Km, for the various mushroom, plant, and
crustacean PPOs were determined using the Lineweaver-Burk equation
(Lineweaver and Burk, 1934). The substrates used for mushroom PPO, potato
PPO (10,900 units/mg of protein), and apple PPO (97,400 units/mg of
protein) were DL-DOPA (0.30 - 3.33 mM) or L-tyrosine (13.8 - 153 /xM) in
0.05 M sodium acetate buffer (pH 6.8), chlorogenic acid (0.60 - 6.67 mM)
or catechol (0.90 - 10.0 mM) in 1 mM sodium phosphate buffer (pH 7.0), and
4-Methyl catechol (1.0 - 9.5 mM) or chlorogenic acid (1.0 - 7.0 mM) in

101
0.1 M sodium phosphate buffer (pH 6.0), respectively. Enzyme activity for
each plant PPO was monitored as previously described.
L-DOPA and catechol at 1.5 - 7.0 mM and DL-DOPA and catechol at 1.67
- 9.92 mM in 0.05 M sodium phosphate buffer (pH 6.5) were respectively
used as substrate for white shrimp PPO (5,400 units/mg of protein) and
grass prawn (900 units/mg of protein) and lobster PPO (7,000 units/mg of
protein). The assay for white shrimp PPO was conducted for 5 min at 40°C
and at 25°C for grass prawn and lobster PPO. Enzyme activity on L-DOPA or
DL-DOPA and catechol was monitored at 475 and 395 nm, respectively.
The inhibitory mechanism of kojic acid on enzyme activities was
also investigated. Except for the substitution of 0.5 mL of kojic acid
solutions for buffer, substrate concentrations used in the previous study
were employed. Kojic acid solutions at 0.28, 0.56, and 1.06 mM were used
in the mushroom, white shrimp, grass prawn, and lobster PPO assay systems,
while those at 0.56, 1.06, and 1.41 mM were used in the potato and apple
PPO systems. Prior to the addition of the substrate, an enzyme-inhibitor
mixture was pre-incubated at 37°C for 15 min. The assays for mushroom,
potato, grass prawn, and lobster PPO were carried out as previously
described. Kinetic parameters of apparent Km (Kmapp) and K. for enzyme
activities were also determined according to the equations of Lineweaver-
Burk (1934) and Dixon (1953).
The kinetic constants (Km, Kmapp, and K^ of potato, apple, and white
shrimp PPO with DL-DOPA as the substrate were also determined. Sixty
microliters of PPO was added to 940 /jL of DL-DOPA in 0.05 M sodium
phosphate buffer (pH 6.5). The final concentration of DL-DOPA varied from
1.4 to 8.9 mM. The reaction was monitored at 475 nm and 25°C for 10 min.

102
The inhibitory effect of kojic acid on the enzyme activity in oxidizing
DL-DOPA in these systems was determined by adding 50 /iL of kojic acid at
0.56 or 1.12 mM to the cuvette containing PPO and sodium phosphate buffer
(pH 6.5). The mixture was pre-incubated at 37°C for 15 min. Following the
equilibration to ambient temperature, DL-DOPA was added and the reaction
was monitored at 475 nm (25°C) for 10 min. All the assays to determine
enzyme activity, the inhibitory effect of kojic acid on the various
enzymes, and the enzyme kinetics were conducted at least three times with
three different sample preparations.
Effect of Pre-incubation Temperature on PPO Inhibition by Ko.iic Acid
A reaction mixture containing 950 /jL of 0.13 mM kojic acid in 0.05
M sodium phosphate buffer (pH 6.5) and 50 nl mushroom tyrosinase (0.5
mg/mL) or spiny lobster PPO (0.5 mg/mL) was pre-incubated for 15 min in a
cuvette at 0°, 25°, or 37°C. After the mixture was equilibrated to ambient
temperature, 500 nl 10 mM DL-DOPA in 0.05 M sodium phosphate buffer (pH
6.5) was added and the reaction monitored at 475 nm (25°C) for 5 min. For
controls, kojic acid was replaced by phosphate buffer. Percent inhibition
(I) was expressed as [(A - A*)/A] x 100, where A and A* were enzyme
activities in the absence and presence of the inhibitor (kojic acid),
respectively (Saruno et al., 1979).
Similarly, 50 nl potato PPO was added to a cuvette containing 500 /iL
1 mM sodium phosphate buffer (pH 7.0) and 450 nl 0.56 mM kojic acid.
After incubation at 0°, 25°, or 37°C for 15 min, and equilibration back to
ambient temperature, 500 /xL 20 mM catechol in 1 mM phosphate buffer (pH
7.0) was added. The reaction was monitored at 395 nm (25°C) for 5 min.

103
Percent inhibition was determined as described above. For apple PPO, the
reaction was carried out at 30°C in 0.1 M sodium phosphate buffer (pH 6.0).
Effect of Ko.iic Acid on the Hydroxylation Capability of PPO
A 100 nl kojic acid solution (0.35 - 5.63 mM) in 0.05 M sodium
phosphate buffer (pH 6.5) with 60 /iL mushroom PPO (0.5 mg/mL) in water at
ambient temperature for 15 min, 840 ni of 1 mM L-tyrosine in the same
buffer was added. The reaction was monitored at 475 nm (25°C) for 90 min.
For control sample, kojic acid was replaced with buffer.
Effect of Ko.iic Acid on 0. Uptake by PPO Reaction
The effect of kojic acid on the inhibition of PPO was also conducted
using a polarographic method. A 0.1 mL aliquot of apple PPO was added to
0.1 mL kojic acid solution (0.28, 0.56, or 1.06 mM) in 0.1 M sodium
phosphate buffer (pH 6.0) into the sample chamber of a biological oxygen
monitor (YSI model 53, Yellow Springs Instrument Co., Yellow Springs, OH).
Following incubation at ambient temperature for 30 min, 2.9 mL of 0.1 M 4-
methylcatechol or chlorogenic acid in 0.1 M sodium phosphate buffer (pH
6.0) was added. The reaction was allowed to proceed at ambient
temperature for 10 min and the consumption rate of 02 was monitored using
a Brinkmann Servogor 210 recorder at a chart speed of 1 cm/min. The rate
of 02 consumption was determined as the initial slope of the curve; the
percent change in 02 was measured against time. For the control sample,
phosphate buffer was used in place of kojic acid. Background 02

104
consumption for kojic acid, substrate, and a mixture of the two was also
carried out.
A similar study was conducted using lobster PPO; in which 10 mM DL-
DOPA or catechol in 0.05 M sodium phosphate buffer (pH 6.5) was used as
the substrate. Percent inhibition (I) on the rate of 02 consumption was
defined as [(U -U*)/U] x 100, where U and U* were the rate of 02
consumption in the absence or presence of kojic acid.
Effect of Ko.iic Acid on Reduction of Cu2+
The method of Andrawis and Kahn (1990) with slight modification was
followed. Ten minutes after incubating at ambient temperature, a mixture
containing 1 mL kojic acid in 0.05 M sodium phosphate buffer (pH 7.0) and
0.5 mL 0.4 mM cupric sulfate (Fisher) in the same buffer was added to a
0.5 mL aliquot of 4 mM aqueous bathocuproine disulfonic acid. The final
concentration of kojic acid in the mixture was between 0.02 to 1.40 mM.
After the mixture was incubated at ambient temperature for another 20 min,
the absorbance at 483 nm was determined using a DU-40 spectrophotometer
(Beckman). For the control sample, 1 mL phosphate buffer was substituted
for kojic acid.
Effect of Kojic Acid on Quinone Products
A reaction mixture containing 1.2 mL 10 mM DL-DOPA and 0.8 mL
mushroom PPO (0.125 mg/mL) in 0.05 M sodium phosphate buffer (pH 6.5) was
incubated at ambient temperature for 30 min. Following red color
development due to dopaquinone formation, the mixture was scanned from 220
to 700 nm using a spectrophotometer. The effect of kojic acid on

105
dopaquinone was also studied by adding 0.6 mL 5.63 mM kojic acid in 0.05
M sodium phosphate buffer (pH 6.5) to a mixture containing 0.2 mL mushroom
PPO (0.5 mg/mL) and 1.2 mL 10 mM DL-D0PA. Following incubation at ambient
temperature for 30 min, the solution was scanned as previously described.
An similar study was also conducted on spiny lobster PPO.
The effect of kojic acid on dopaquinone formation was further
investigated using thin-layer chromatography. One-mL mushroom PPO (0.5
mg/mL) was mixed with 1.0 mL 5 mM DL-D0PA in 0.05 M sodium phosphate
buffer (pH 6.5) and the reaction was allowed to proceed at ambient
temperature for 2 hr. Following the reaction, 0.5 mL kojic acid (1 mg/mL)
in 0.05 M sodium phosphate buffer (pH 6.5) was added to 0.5 mL aliquot of
the reaction mixture and incubated at ambient temperature. After 2 hr,
80-/iL aliquots were spotted on TLC plates (20 x 20 cm Redi/plt Sil, Gel G,
Fisher) and the plates developed using a butanol-acetic acid-water (4: 1:
5, v/v) solvent system. An equivalent volume of 10 mM DL-DOPA in 0.05 M
sodium phosphate buffer (pH 6.5) was used as standard. The
chromatographic pattern was examined and the Rf value for each compound was
determined after spraying the plate with a ninhydrin reagent (Sigma). For
control sample, an equivalent volume of phosphate buffer was in place of
kojic acid.
Enzymatic Activities of Ko.iic Acid-treated PPO
Two-mL of mushroom, white shrimp, and lobster PPO was individually
incubated at ambient temperature for 30 min with 0.5 mL kojic acid (0.56
or 1.12 mM) in 0.05 M sodium phosphate buffer (pH 6.5) in a Spectra/Por
Membrane (Spectrum Medical Industries Inc., Los Angels, CA) with molecular

106
weight cutoff of 12,000 - 14,000. The chamber containing the enzyme-kojic
acid mixture was then dialyzed overnight (4°C) against 3 changes of 2 L
phosphate buffer. For control sample, kojic acid was replaced with an
equivalent volume of buffer. Studies on potato and apple PP0 were
similarly conducted except kojic acid at 1.12 or 5.60 mM was used. The
enzyme activity of mushroom, white shrimp, and lobster PPO was determined
by adding 60 /xL PPO to the cuvette containing 840 /xL of 10 mM DL-D0PA in
0.05 M sodium phosphate buffer (pH 6.5). The reaction was monitored for
5 min at 475 nm and 25°C for mushroom and lobster, and 40°C for white
shrimp PPO. For potato and apple PPO, the activity was determined by
adding 60 /xL enzyme to the cuvette containing 840 /xL of 10 mM catechol in
1 mM sodium phosphate buffer (pH 7.0) or 0.1 M sodium phosphate buffer (pH
6.0), respectively. The reaction was monitored for 5 min at 395 nm and
25°C for potato PPO, and 30°C for apple PPO.
Kojic acid residual in enzyme preparations was quantitated using the
method of Bentley (1957) with slight modification. Five-hundred /xL enzyme
preparation was incubated with 1.5 mL of 1% (w/v) FeCl3 solution at ambient
temperature for 40 min and absorbance measured at 505 nm. Kojic acid at
various concentrations (10-250 /zg/mL) was used as standard.
Statistical Analysis
Statistical analysis was carried out using a PC SAS package (SAS,
1985). Duncan's multiple range test was performed at a level of a = 0.05
to determine any significant difference among treatments. Unless
otherwise specified, all experiments were carried out twice in
triplicates.

107
Results and Discussion
Effect of Ko.iic Acid on Mushroom PPO Activity
Kojic acid showed a concentration-dependent inhibitory effect on
mushroom tyrosinase oxidation of DL-DOPA (Figure 22). Addition of kojic
acid (20 ng/ml) to the assay system containing DL-DOPA and
L-tyrosine inhibited tyrosinase by 72 and 64%, respectively (Figure 23).
This inhibitory effect was elevated to 90% when kojic acid at 80 ng/mi was
added. Saruno et al. (1979) reported only 20 to 30% mushroom PPO
inhibition when kojic acid at 20 /xg/mL was used. The use of different
enzyme preparations in these two studies could have affected the
inhibitory effectiveness of kojic acid.
Effect of Kojic Acid on Potato PPO Activity
Potato PPO was not as effectively inhibited by kojic acid as
mushroom tyrosinase especially when catechol was used as substrate (Figure
24). Approximately 95% of mushroom tyrosinase activity was suppressed
when kojic acid at 200 /xg/mL was used (Figure 23). However, only 55 and
25% inhibition of potato PPO was achieved for chlorogenic acid and
catechol, respectively, under the same conditions. As kojic acid
concentration was increased to 800 /xg/mL, the inhibition of potato PPO on
the oxidation of chlorogenic acid approached 90%. However, the inhibition
was only 53% for the oxidation of catechol. Abukharma and Woolhouse
(1966) demonstrated that 0.1 mM potassium cyanide or sodium azide and 1 mM
3,4-dichlorophenyl serine gave about 60% inhibition on potato PPO when
chlorogenic acid was used as substrate.

Figure 22. Effect of Concentration-related Inhibitory Effect of Kojic
Acid on Mushroom Tyrosinase (PPO) Activity on DL-/J-3,4-
dihydroxyphenylalanine (DL-DOPA); A 0.9 ml. Aliquot of Kojic
Acid Solution at 20 - 150 /xg/mL Was Added to the Assay System.

Absorbance (475 nm)
109
Control
—*—
20 pg/mL Kojlc acid
—©—
80 pg/mL Kojic acid
40 pg/mL kojic acid
B
150 pg/mL Kojic acid
—A

Figure 23. The Concentration-related Inhibitory Effect of Kojic Acid on the Oxidation of DL-DOPA (â–¡) and
L-Tyrosine (•) by Mushroom PPO; A 0.9-mL Aliquot of Kojic Acid at 20 - 200 /zg/mL Was Added
to the Assay Mixture.


Figure 24. The Concentration-related Inhibitory Effect of Kojic Acid on the Oxidation of Chlorogenic
Acid (□) and Catechol (•) by Potato PPO; A 0.9-mL Aliquot of Kojic Acid at 20 - 800 [ig/mL Was
Added to the Assay Mixture.

% Inhibition
100 h-
80
60 -
40
20
Chlorogenic acid
B
Catechol
-—□
J3
ET
0'
0
0
0 *—
0
,0'
100
200 300
400
500
600 700
800
Kojic acid (ng/mL)
113

114
Cysteine and other sulfhydryl-related compounds have been studied
for their inhibitory effect on enzymatic browning (Lerner, 1953). Roston
(1960) proposed that cysteine inhibits enzymatic blackening caused by the
oxidation of L-tyrosine and DL-DOPA by forming addition products with
quiñones. Muneta and Walradt (1968) studied potato PPO and found that the
concentration of cysteine required for comparable inhibition was 5-fold
higher with cysteine than for sulfite. These authors also noted that
cysteine did not inhibit chlorogenic acid oxidation; and the oxidation of
tyrosine to DOPA is more sensitive to cysteine inhibition than the
oxidation of DOPA to dopaquinone. Both Muneta and Walradt (1968) and
Henze (1956) proposed that cysteine inhibited enzymatic browning by
combining with the quinone. The sulfiting agents have been postulated
toreact with the enzyme or the quiñones to exert the inhibitory effect
(Joslyn and Ponting, 1951; Joslyn and Braverman, 1954). Although sulfite
agents can effectively inhibit enzymatic browning, they affect the
nutritive value either by preserving ascorbic acid or destroying thiamine
(Mapson and Wager, 1961; Markakis and Embs, 1966).
Effect of Ko.iic Acid on Apple PPO Activity
Oxidation rates of 4-methyl catechol and chlorogenic acid by apple
PPO decreased as kojic acid concentration increased. Only 50% inhibition
occurred for kojic acid at 200 /xg/mL (Figure 25). Kojic acid inhibition
of apple PPO was similar to potato PPO (Figure 24), but was far less
pronounced than for mushroom PPO (Figure 23). Harel et al. (1965)
demonstrated that 2,3-naphthalenediol at 5 mM and N-vinyl-2-pyrrolidone at
2.5% respectively inhibited 64 and 60% PPO prepared from apple

Figure 25. The Concentration-related Inhibitory Effect of Kojic Acid on the Oxidation of
4-Methyl catechol (â–¡) and Chlorogenic Acid (!) by Apple PPO; A 1.15-mL Aliquot of Kojic Acid
at 0.02 - 2.0 mg/mL Was Added to the Assay Mixture.

116

117
chloroplasts. Walker (1964) demonstrated that diethylthiocarbamate or
dimercaptopropanol at 1 mM was able to inhibit 90% of the oxidation of
chlorogenic acid.
Effect of Ko.iic Acid on Crustacean PPO Activity
Kojic acid inhibition on white shrimp (Figure 26) and grass prawn
PPO (Figure 27) was less effective than on spiny lobster (Figure 28). The
former two enzymes were only inhibited by about 20%, while the oxidation
of DL-DOPA and catechol by lobster PPO was inhibited to 80 and 70%,
respectively, when kojic acid at 20 /zg/mL was used. Kojic acid appeared
to be more effective in inhibiting lobster PPO than white shrimp and grass
prawn PPO and plant PPO. L-Tyrosine, L-cysteine, and sodium diethyl
dithiocarbamate effectively inhibited the oxidation of DOPA (Antony and
Nair, 1975). p-Aminobenzoic acid (PABA) and NaN3 was more effective than
EDTA and cysteine in inhibiting pink and white shrimp PPO (Simpson et al.,
1988a). On the basis of the molar ratio between crustacean PPO and
inhibitor used, kojic acid was found to be less effective in inhibiting
white shrimp, grass prawn, and lobster PPO than PABA and NaN3 on pink and
white shrimp PPO.
Enzyme Kinetics
Kinetic parameters (Km, Kmapp, and K.) for the various PPOs utilizing
different substrates in the absence or presence of kojic acid,and the type
of inhibition are listed in Table 4. Kojic acid was a competitive

Figure 26.
The Concentration-related Inhibitory Effect of Kojic Acid on the Oxidation of L-DOPA (â–¡) and
Catechol (•) by White Shrimp PPO; A 0.25-mL Aliquot of Kojic Acid (20 - 200 ng/ml) Was Added
to the Assay Mixture.

80
60
40
20
0
L-DOPA
—e—
Catechol
//
/
/ /
/
/
/
40
80
120
160
200
Kojic acid (/L/g/mL)
119

Figure 27. The Concentration-related Inhibitory Effect of Kojic Acid on the Oxidation of DL-DOPA (â–¡) and
Catechol (•) by Grass Prawn PPO; A 0.9-mL Aliquot Kojic Acid (20 - 150 /xg/mL) Was Added to
the Assay Mixture.

sz
c
100 h
80
Ü 60
40
20
DL-DOPA
EÍ
Catechol
0
/
/
om
0
20
/ /
0 /
40
0
.0
0-
60
80
100
120
140
Kojic acid OL/g/mL)
160
121

Figure 28. The Concentration-related Inhibitory Effect of Kojic Acid on the Oxidation of DL-DOPA (â–¡) and
Catechol (•) by Lobster PPO; A 0.9-mL Aliquot Kojic Acid (20 - 150 /xg/mL) Was Added to
the Assay Mixture.

100
30
60
40
20
0
Eh"
—-Eh
~B-
/
W
L-
i
/
DL-DOPA
B-—
Catechol
20 40 60 80 100 120
Kojic acid (|jg/mL)
140
160
123

Table 4. Inhibitory Mechanism of Kojic Acid on Polyphenol Oxidase Obtained from Various Sources
Enzyme
source
Substrate
Michael is constant
(KJ, mM
Type of
inhibition
Apparent
K , mM
m
Inhibitor constant
(K.), mM
Mushroom
L-tyrosine
0.69
Competitive
2.02
0.03
DL-DOPA
0.24
Mixed
0.66
0.02
Potato
Chlorogenic acid
5.20
Competitive
7.23
0.60
Catechol
7.89
Competitive
10.5
0.71
DL-DOPA
0.06
Competitive
0.08
0.06
Apple
4-methyl catechol
3.85
Competitive
4.28
0.13
Chlorogenic acid
8.20
Competitive
10.3
0.06
DL-DOPA
0.04
Competitive
0.07
0.03
White
L-DOPA
3.48
Mixed
4.29
0.15
Shrimp
Catechol
4.27
Mixed
5.18
0.18
DL-DOPA
3.20
Mixed
4.20
0.09
Spiny
DL-DOPA
3.27
Mixed
4.37
0.07
lobster
Catechol
4.98
Mixed
7.31
0.10
Grass
DL-DOPA
3.64
Mixed
7.77
0.05
prawn
Catechol
5.29
Mixed
7.78
0.07

125
inhibitor for both potato and apple PPO, but a mixed-type inhibitor for
white shrimp, grass prawn and Florida spiny lobster PPO. For mushroom
PPO, it was a competitive inhibitor for L-tyrosine, while a mixed-type
inhibitor for DL-DOPA.
The Michael is constant for the oxidation of DL-DOPA and L-tyrosine
by mushroom tyrosinase was 0.29 mM and 0.69 mM, respectively. The Km value
for DL-DOPA was close to that for the oxidation of catechol (0.22 mM) and
chlorogenic acid (0.22 mM) reported by Sisler and Evans (1958) but was
lower than the values reported by Smith and Kruger (1962) on catechol (2.5
- 4.0 mM). Similarly, the Michael is constant for L-tyrosine was lower
than that for the oxidation of p-cresol (1.5 - 10 mM) (Sisler and Evans,
1958). Mayer et al. (1966) have attributed this discrepancy to different
enzyme preparations and assay methods used. Based on the Km values for
these two substrates, it was noted that mushroom tyrosinase had a higher
affinity for DL-DOPA than for L-tyrosine. Boughilloux et al. (1963)
isolated 4 different forms of tyrosinase from mushroom, all of which were
capable of oxidizing D0PA more actively than tyrosine. A similar
observation was also reported by Harrison et al. (1967) using a
fluorescence spectrophotometric technique.
Apparent Km values for the oxidation of L-tyrosine and DL-DOPA by
mushroom tyrosinase in the presence of kojic acid were determined to be
2.02 and 0.66 mM, respectively. The inhibitor constant (K.) was determined
to be 0.03 mM for the former (competitive inhibition) and 0.02 mM for the
latter (mixed-type inhibition). Since kojic acid was a competitive
inhibitor, it would compete with L-tyrosine for the active site
(Segel,1976). The results also showed that the inhibitory properties of

126
kojic acid on mushroom tyrosinase depends upon whether o-diphenol
(DL-DOPA) or monophenol (L-tyrosine) is used as a substrate. In this
study, a mixed-type inhibition implies that kojic acid affected the
affinity of the enzyme for DL-DOPA but did not bind at the active site
(Webb, 1963).
The Km value for the oxidation of chlorogenic acid by potato PPO was
5.20 mM, while it was 7.89 mM for the oxidation of catechol. These values
were lower than those reported by Abukharma and Wool house (1966),
Alberghina (1964), and Macrae and Duggleby (1968). Variations in enzyme
preparations and assay methods could have contributed to these differences
in Km values (Macrae and Duggleby, 1968).
In the presence of kojic acid, the apparent Michael is constant for
the oxidation of chlorogenic acid became 7.23 mM while it was 10.5 mM for
catechol. The inhibitor constant for the oxidation of chlorogenic acid
was 0.60 mM and 0.71 mM for catechol oxidation. Thus, kojic acid was more
competitive with chlorogenic acid than with catechol for the active site.
The K; values for kojic acid using chlorogenic acid and catechol as
substrate were lower than those reported for p-nitrophenol, ferulic acid,
p-coumaric acid, 2,3-dihydroxy-naphthalene, and cinnamic acid (Macrae and
Duggleby, 1968).
The Michael is constants for the oxidation of 4-methyl catechol and
chlorogenic acid by apple PPO were determined to be 3.85 and 8.20 mM,
respectively. The Km value for 4-methyl catechol was close to that reported
by Harel et al. (1965) and Mayer et al. (1964), whereas the Km for
chlorogenic acid was higher (Walker, 1964).

127
When kojic acid was added, the apparent Michael is constants for
4-methylcatechol and chlorogenic acid were changed to 4.28 and 10.3 mM,
respectively. The inhibitor constant of kojic acid for 4-methylcatechol
and chlorogenic acid oxidation was 0.06 and 0.13 mM, respectively. The
former was similar to the K1 of 2,3-naphthalenediol (Mayer et al., 1964).
However, both Ki values were lower than that of cinnamic acid (1.4 and 0.14
mM, respectively) when it was used as an inhibitor in a solubilized PP0
system (Walker and Wilson, 1975). Using 4-methylcatechol, chlorogenic
acid, and catechin as substrates, Walker and Wilson (1975) observed that
cinnamic acid, ferulic acid, and coumaric acid derivatives behaved as
competitive inhibitors of apple PP0. The Michael is constants for the
oxidation of L-D0PA and catechol by white shrimp PP0 were determined to be
3.48 and 4.27 mM, respectively. The former (L-D0PA) Km value was slightly
higher than that reported for DL-D0PA (2.8 mM) by Simpson et al. (1988a).
Regarding lobster PP0, the Km values for oxidation of DL-DOPA and catechol
were 3.27 and 4.98 mM, respectively. These values were lower than those
reported by Chen et al. (1991a). Lobster PP0 used in this study was
further purified by 7.5% acrylamide gel and thus possessed a higher
specific activity. For grass prawn PP0, the Km values for DL-DOPA and
catechol were 3.64 and 5.29 mM, respectively. The former value was close
to that reported by Rolle et al. (1991). In comparison to pink shrimp
(Simpson et al., 1988a), white shrimp, grass prawn and lobster PPO showed
comparatively higher Km values when either L-D0PA or DL-DOPA was used as
substrate.
When kojic acid was added as an inhibitor, a mixed-type inhibition
was observed for the oxidation of both substrates by these three

128
crustacean PPO. The inhibitor constant for the oxidation of DL-DOPA by
grass prawn PPO was 0.05 mM and 0.07 mM for catechol. Similarly, the Ki
values were determined to be 0.07 mM for DL-DOPA and 0.10 mM for catechol
when lobster PPO was used. For white shrimp PPO, the Ki of L-D0PA and
catechol was determined to be 0.15 and 0.18 mM, respectively. Antony and
Nair (1975) studied the inhibitory effect of several chemicals on prawn
phenolase and found L-tyrosine (Ki = 0.38 mM) was a competitive inhibitor
to the oxidation of DOPA. In contrast, L-cysteine and sodium diethyl
dithiocarbamate were found to behave as mixed-type inhibitors. Madero
(1982) reported that bisulfite was a competitive inhibitor to brown shrimp
PPO. The observation of lower Ki values for grass prawn and lobster PPO
than white shrimp PPO further demonstrate that kojic acid exhibited a
greater inhibitory effect on those two enzymes than the latter one
(Figures 26, 27, and 28). Also, when the same amount of kojic acid was
applied to the assay mixtures, greater inhibition was observed for DL-DOPA
than for catechol (Figures 27 and 28).
It was noted that the Km value of white shrimp PPO for DL-DOPA (3.20
mM) was close to that for L-D0PA (3.48 mM). For potato and apple PPO,
both enzymes showed an extremely high affinity for DL-DOPA than for
chlorogenic acid, 4-methylcatechol, and catechol (Table 4). Also, these
two enzymes showed a higher affinity for DL-DOPA than other PPOs. Kojic
acid also exhibited the same inhibitory mechanism on the oxidation of DL-
DOPA as well as on other diphenolic substrates by white shrimp, potato,
and apple PPO (Table 4). Kojic acid exerts more effective inhibition
(Table 4; K^ on DL-DOPA oxidation by potato and apple PPO than on other
diphenolic substrates.

129
The results thus indicated that kojic acid could inhibit the
oxidation of phenolic substrates by mushroom, potato, apple, white shrimp,
grass prawn, and lobster PPO. Significant inhibitory effects with
different types of inhibition mechanisms were observed with these PPO
activities when DOPA was used as substrate. DOPA is a dominant phenolic
substrate responsible for the formation of melanin (blackening spot) in
crustaceans. It seems likely that kojic acid could be potentially used as
an inhibitor in the prevention of melanosis in seafood products.
Effect of Pre-incubation Temperature on PPO Inhibition by Ko.iic Acid
The pre-incubation temperatures did not affect the inhibitory
activity of kojic acid on various PPOs (Table 5). More than 75%
inhibition was observed with mushroom tyrosinase and lobster PPO when 0.28
mM kojic acid was introduced into the assay mixture containing DL-DOPA and
enzyme. However, only the respective 26% and 41-46% of the potato and
apple PPO activity were inhibited when 0.56 mM kojic acid was added.
These observations were in accordance with earlier findings that kojic
acid was less inhibitory on potato and apple PPO than on mushroom and
lobster PPO. Although the pre-incubation at 0°C usually gave a higher
percentage of inhibition than at higher temperatures, the difference was
insignificant (P > 0.05). Thus, kojic acid can be used to inhibit PPO at
either refrigeration or ambient temperature.
Effect of Kojic Acid on the Hydroxylation Capability of PPO
The lag period associated with the hydroxylation of
monohydroxyphenol by PPO increased with increasing concentrations (35.2 -

130
Table 5. Effect of Different Pre-incubation Temperatures on the
Inhibition of Various Polyphenol Oxidases (PPO) by Kojic Acid
Enzyme source
% Inhibition
0°C
25°C
37°C
Mushroom
78.5
77.3
75.1
Potato
26.2
25.5
25.4
Apple
45.6
41.8
41.1
Lobster
78.1
77.0
76.8

131
563 /íM) of kojic acid (Figure 29). Kojic acid at higher concentrations
was shown to exert a profound inhibitory effect on the oxidation of L-DOPA
by PPO. Thus, kojic acid did not behave as ascorbate, hydroquinone, H202,
and NH20H that were reported to be capable of reducing the lag period of
hydroxylation of monohydroxyphenol when added at small concentrations
(Kahn, 1983; Kahn and Andrawis, 1986; Sato, 1969; Vaughan and Butt, 1970).
Effect of Ko.iic Acid on 02 Uptake by PPO Reaction
Consumption of oxygen did not take place with kojic acid, the
substrates (4-methylcatechol and chlorogenic acid for apple PPO, and DL-
DOPA and catechol for lobster PPO), or the kojic acid-substrate mixtures.
When PPO was added to the mixture containing substrate and buffer, 02
consumption occurred immediately. Although 02 uptake by the PPO-substrate
mixture still took place when kojic acid was added, the percentage of 02
consumption in these mixtures decreased with increasing concentrations of
kojic acid (Table 6). For example, 02 consumption for the oxidation of DL-
DOPA by lobster PPO in the presence of 0.56 and 1.06 mM kojic acid was
inhibited by 60.3 and 80.3%, respectively.
Effect of Ko.iic Acid on Reduction of Cu2+
Bathocuproine disulfonic acid reacts with Cu+ to form a red color
complex having an optimal absorption at 483 nm (Blair and Diehl, 1961).
Thus the reducing ability of kojic acid can be determined from the
measurement of the absorbance at 483 nm in a model reaction mixture. The
absorbance of the reaction mixture increases with increasing
concentrations of kojic acid and then reaches a plateau when kojic acid

Figure 29. Effect of Kojic Acid (t, 0.35; a, 1.41; a, 2.81; and â–¡, 5.63 mM) on the Hydroxylation of
Monohydroxyphenol by Mushroom Tyrosinase (PPO); Control (0) Was Run Similarly Except That
Kojic Was Replaced by an Equivalent Volume of Phosphate Buffer.

Absorbance (475 nm)
133

134
Table 6. Inhibitory Effect of Kojic Acid on the Consumption of Oxygen by
Polyphenol Oxidase
%
Inhibition
Lobster PPO
Apple
PPO
Kojic acid (mM)
DL-DOPA
Catechol
4-Methylcatechol
Chlorogenic acid
0
0
0
0
0
0.28
50.2
47.4
ND
ND
0.56
60.3
65.0
36.7
24.8
1.06
80.3
77.5
50.9
41.9
1.41
ND
ND
55.4
52.1
ND: Not determined

135
exceeds 0.28 mM (Figure 30). On the basis of molar extinction coefficient
for Cu+-bathocuprione disulfonate complex (Blair and Diehl, 1961), all the
Cu2+ present in the reaction mixture was reduced to Cu+ when kojic acid was
added at a concentration greater than 0.28 mM.
Effect of Ko.iic Acid on Quinone Products
Spectrophotometric scanning of the product generated from the
reaction of DL-DOPA and mushroom PPO revealed two distinct absorption
peaks at 316 and 480 nm (Figure 31a). The addition of 5.63 mM kojic acid
to this solution caused the color to change from red-brown to violet, and
the subsequent disappearance of the 480 nm peak which represents
dopaquinone (Figure 31b) (Fling et al., 1963). Similar phenomena occurred
when kojic acid (5.63 mM) was added to the reaction mixture containing
lobster PPO and DL-DOPA. Thus, the formation of dopaquinone from DL-DOPA
through the action of PPO was affected by the presence of kojic acid.
This finding was further verified by the TLC analysis of the DL-DOPA
standard and the reaction mixture (dopaquinone) containing 3.5 mM of kojic
acid. A reddish-purple spot with a Rf value of 0.76 was detected for the
DL-DOPA of these two samples on TLC plate following spraying with
ninhydrin reagent. However, no such reddish-purple spot was detected for
control sample that contained only dopaquinone.
The kojic acid effect on the quinone products formed by the action
of PPO on DL-DOPA is attributed to the reduction of dopaquinones back to
diphenols (DL-DOPA). Many reagents including cysteine, bisulfite, and
ascorbic acid are known to retard enzymatic browning through this

Figure 30. Effect of Kojic Acid on Reduction of Cu2+ to Cu+ in a Model System; One-mL Kojic Acid (0.04 -
2.81 mM) Was Incubated with 0.5 mL of 0.4 mM CuS04 at Ambient Temperature Followed by the
Addition of 0.5 mL of 4 mM Bathocuproinedisulfonic Acid. The Absorbance of the Mixture Was
Measured at 483 nm.

Kojic acid (mM)
Absorbance (483 nm)
¿ex

Figure 31. Change in Absorption Spectra of Dopaquinone due to the Addition of 5.63 mM Kojic Acid,
(a) Dopaquinone Produced from the Action of Mushroom PPO on DL-DOPA; and (b) Dopaquinone
Plus Kojic Acid

Absorbance
139

140
Enzymatic Activities of Ko.iic Acid-treated PPO
The dialysate of the control sample and kojic acid-treated PPO
preparations showed an equivalent volume. Data listed in Table 7 show
that there was no significant difference (P > 0.05) in the enzyme activity
between the control and the kojic acid-treated PPO following dialysis
against phosphate buffer. The restoration of enzymatic activity was
attributed to the removal of kojic acid from the enzyme mixture; the
failure to detect any kojic acid residue following dialysis verified this
assumption. These results thus indirectly suggested that kojic acid did
not bind irreversibly toenzyme and the inhibition on PPO was reversible.
Such inhibitory characteristics are in contrast to that of sulfite and its
derivatives (Sayavedra-soto and Montgomery, 1986; Ferrer et al., 1989b).
Conclusion
Kojic acid exhibited a competitive inhibition for the oxidation of
chlorogenic acid and catechol by potato PPO and of 4-methylcatechol and
chlorogenic acid by apple PPO. This compound showed a mixed-type
inhibition for white shrimp, grass prawn, and lobster PPO when DL-DOPA and
catechol were used as substrates but a mixed-type and a competitive
inhibition for mushroom PPO when DL-DOPA and L-tyrosine were used,
respectively. This compound also showed a competitive inhibition on the
oxidation of DL-DOPA by apple and potato PPOs. The competitive inhibition
suggests that kojic acid might be a nonmetabolizable analog of the
substrates (DL-DOPA, chlorogenic acid, or 4-methylcatechol). The result
also implies that apple, potato, and/or mushroom PPO bear the structural

141
Table 7. Enzymatic Activity of Kojic Acid-treated Polyphenol Oxidase
Following Kojic Acid Removal3
Enzyme
Control
0.56
Kojic acid (mM)
1.12
5.60
Mushroomb
0.80d
0.80
0.79
NDe
Lobsterb
0.124
0.118
0.120
ND
White shrimpb
0.032
0.031
0.032
ND
Potato0
0.059
ND
0.060
0.061
Apple0
0.183
ND
0.188
0.187
aEnzyme was incubated with kojic acid for 30 min followed by
dialyzing against phosphate buffer.
bDL-D0PA was used as substrate and the activity was determined
as AA475 nm/min.
cCatechol was used as substrate and the activity was determined
as AA395 nm/min.
dResult was an average of three observations; values within the
same row are not significantly different (P > 0.05) from each
other.
eND, not determined

142
similarities on their active site. The inhibitory mode of kojic acid on
PPO was shown following actions: (1) by competing with substrate for the
active site of the enzyme; (2) by interfering with the uptake of 02
required for the enzymatic reactions; (3) by reducing o-quinones to
o-diphenols to prevent melanin formation via polymerization; and/or (4) by
combination of the above three methods.

EFFECT OF CARBON DIOXIDE ON THE INACTIVATION OF
PLANT AND CRUSTACEAN POLYPHENOL OXIDASES
Introduction
Undesirable enzymatic browning caused by polyphenol oxidase (E.C.
1.14.18.1; PPO) on the surface of food products has been of great concern
to food scientists and food processors since the melanin formed reduces
the consumers' acceptability of these products. Many chemicals have been
studied extensively for their effectiveness in inhibiting PPO activity
(Jones et al., 1965; Mayer et al., 1964; Palmer and Roberts, 1967; Walker,
1975; Madero and Finne, 1982; Golan-Goldhhirsh and Whitaker, 1984;
Sayavedra-Soto and Montgomery, 1986; Ferrer et al., 1989b; Chen et al.,
1991b) and shown to effectively inhibit melanosis of fruits, vegetables,
and crustaceans. However, problems related to off-flavor, off-odor,
toxicity, and economic feasibility affect the application of these
compounds (Eskin et al., 1971).
A modified atmosphere with carbon dioxide has been used as a
physical application to affect many enzyme activities (Parkin and Brown,
1982; Gee and Brown, 1978a, b), including the inhibition of PPO and thus
the prevention of discoloration in fruits and vegetables (Kader et al.,
1973). Supercritical (SC) fluids exhibiting physicochemical properties
intermediate between those of liquids and gases has been reported by
several workers for its inactivation of peroxidase (Christianson et al.,
1984), PPO (Zemel, 1989), and pectinesterase (Arreola, 1990) when carbon
143

144
dioxide (C02) was used as the fluid. C02 is used in SC fluid because it
is nontoxic, nonflammable, inexpensive, and readily available
(Hardardottir and Kinsella, 1988). C02 has a relatively low critical
temperature and pressure (Rizvi et al., 1986). Taniguchi et al. (1987)
studied the retention of alpha-amylase, glucose oxidase, lipase, and
catalase activity by SC-C02. Although SC-C02 has been shown to inactivate
PPO, information regarding the inhibitory effect and the inactivation
kinetics of SC-C02 on purified PPO has not been elucidated. This study was
undertaken to investigate the effect of C02 (1 or 58 atm) on the
inactivation of Florida spiny lobster, brown shrimp, and potato PPO.
Materials and Methods
Fresh Florida spiny lobster (Panulirus argus) tails were obtained
from the Whitney Marine Laboratory and transported in ice to the
laboratory and stored at -20°C. Russet potato tuber was purchased from a
local supermarket. Non-sulfited fresh brown shrimp (Penaeus aztecus) were
obtained from a local seafood store. Lobster cuticle, shrimp
cephalothorax (head), and potato peel were frozen in liquid nitrogen and
ground into a fine powder using a Waring blender. The ground powder was
stored at -20°C until needed.
Extraction and Purification of Lobster. Shrimp, and Potato PPO
PPO was extracted and purified following the procedures of Chen et
al. (1991a). Ground powder was added to 0.05 M sodium phosphate buffer
(pH 7.2) (1:3, w/v) containing 1 M NaCl and 0.2% Brij 35, stirred for 0.5
hr at 4°C, and then centrifuged at 8,000g (4°C) for 30 min. The

145
supernatant was dialyzed at 4°C overnight against 3 changes of 4L distilled
water.
Crude PPO preparation was purified further using a nondenaturing
preparative polyacrylamide gel electrophoresis (PAGE) system. Equipment
utilized included a gel tube chamber (Model 175, Bio-Rad) and a power
supply (Model EPS 500/400, Pharmacia). A one-mL aliquot of crude enzyme
extract (lobster, shrimp, or potato) was applied to each of eight gel
tubes (1.4 cm I.D. x 12 cm length) containing 5% acrylamide/ 0.13%
bisacrylamide gel and run at a constant current of 10 mA/tube (Sigma
Chemical CO., 1984). PPO was visualized using a specific enzyme-substrate
staining method; 10 mM DL-/3-3,4-dihydroxyphenylalanine (DL-D0PA) in 0.05
M sodium phosphate buffer (pH 6.5) was used as substrate. After the
migration of the enzyme relative to the dye front (Rf) was determined using
one of the eight gels, the remaining gels were sectioned at the determined
Rf. PPO was eluted from the gel by homogenization in distilled water
utilizing a Dounce manual tissue grinder. The homogenates were filtered
through Whatman No. 4 filter paper, pooled, and concentrated using an
Amicon stirred cell (Model 8050) fitted with a YM 10 filter. Concentrated
PPO was dialyzed overnight (4°C) against 2 changes of 4L distilled water.
Protein Quantitation and Enzyme Purity Determination
Protein contents of various PPO preparations were quantitated using
the Bio-Rad Protein Assay kit with bovine serum albumin as standard.
Enzyme purity was examined using a mini gel system (Mini-Protean II Dual
Slab Cell) (Bio-Rad, 1985b). PPO (20 /xg protein/well) was loaded and
electrophoresis was carried out at constant voltage (200 V) for 35 min.

146
Purity of the preparations was determined by comparing gels stained with
10 mM DL-DOPA in 0.05 M sodium phosphate buffer (pH 6.5) and then with a
Coomassie blue R-250 solution.
Enzyme Activity assay
PPO activities were measured by adding 60 /xL of the preparation to
840 /xL 10 mM DL-DOPA in 0.05 M sodium phosphate buffer (pH 6.5) and
monitored at 25°C for 5 min. Maximal initial velocity was determined as
AA475 nm/min and one unit of PPO activity was defined as an increase in
absorbance of 0.001/min at 25°C. Unless otherwise specified, experiments
were replicated three times. The enzyme activities of lobster, brown
shrimp, and potato PPO was determined as 3,750, 740, and 5,400 units/mg
protein, respectively.
Effect of C0: (1 atm) on PPO Activity
Three batches of 20-mL lobster PPO (1,470 units/mg protein) were
loaded in 50-mL polymethylpentene tubes (Nalgene Co.) and were heated
respectively at 33°, 38°, and 43°C in a water-bath. Following the
equilibration of the PPO solution to the desired temperatures, liquid
carbon dioxide [Coleman grade (99.99% C02), Liquid Air Co., Walnut, CA)]
at a flow rate of 110 mL/min was bubbled through the PPO solution for 30
min. PPO (500 /xL) was removed every 5 min from the stock solution heated
at 33° or 38°C. PPO (500 /xL) heated at 43°C was sampled every min during
the first 5 min and then 5 min thereafter. PPO samples were put in 1.5 mL
microcentrifuge tubes (Fisher) and immediately cooled by emerging the tube
in an ice bath. Following equilibration to ambient temperature, PPO

147
activity was determined by adding 60 /¿L PPO into a microcuvette containing
840 /iL 10 mM DL-DOPA in 0.05 M sodium phosphate buffer (pH 6.5). The
reaction was monitored at 475 nm (25°C) for 5 min. The activity of N2-
treated and non-gas-treated controls heated at 33°, 38°, or 43°C were also
determined as previously described. Percentage of relative activity was
determined as (Et/Eo) x 100, where Et and Eo were the PPO activities at
time t and original activity, respectively. Changes in pH resulting from
C02 treatment was monitored using a digital pH meter (E632, Metrohm Ltd.,
Switzerland) equipped with a microelectrode.
pH Control Study
Mixtures containing 1 mL PPO preparation and 3 mL NaHC03 (pH 5.3)
were heated for 30 min at 33°, 38°, and 43°C, respectively, in a water
bath. The mixture was then instantly removed and emerged into a 0°C ice
chest. Following equilibration to ambient temperature, PPO activity was
assayed as previously described.
Effect of high pressure C02 on PPO activity
The apparatus used for PPO inactivation by high pressure C02 is shown
in Figure 32. C02 was connected to a high-pressure resistant stainless
steel vessel (volume = 100 mL) equipped with valves through a metal hose.
After the vessel was immersed into a water bath maintained at 43°C, a
constant pressure of 850 psi (58 atm) inside the vessel chamber was
achieved by adjusting the pressure-regulating valve. For each study, 80
mL lobster, brown shrimp, or potato PPO was placed in the vessel. After

Figure 32. Apparatus Used for Studying Polyphenol Oxidase (PPO) Inactivation by High Pressure C02

Flexible
C02 cyclinder
Pressure
gauge
Enzyme Pressure
solution vessel
149

150
treatment parameters were equilibrated to the desired conditions, PPO (8
mL) was sampled after the vessel was removed from the water bath. Each
time after sampling, the vessel was replaced into the bath. Sampling was
done every minute during the first 5 min and then every 5 min thereafter.
Following equilibration to ambient temperature, PPO activity was
determined as previously described. The activity for temperature control
treatment was also determined. Percentage of relative activity was
determined as (Et/E0) x 100, where Et and Eo were the PPO activities at
time t and the original activity without heat and C02, respectively. The
pH change resulting from C02 treatment during the course of this study was
monitored using a pH meter.
Kinetics of PPO Inactivation
The inactivation reaction constant (k) and the activation energy (EJ
of PPO in the presence or absence of C02 (1 atm) were determined according
to the Arrhenius equation by measuring the initial rate at different
temperatures and plotting the logarithmic value of Vmax versus 1/T (Segel,
1976). The D value (decimal reduction value) defined as the time required
to inactivate 90% of the original enzyme activity at a constant
temperature was determined from the negative reciprocal of the slope from
a plot of logarithmic value of enzyme activity versus time (Richardson and
Hyslop, 1985). The z value which is the number degrees required for the
thermal inactivation curve to traverse one logarithmic cycle was
determined by plotting the logarithmic value of (D^Dj) versus (T^Tj),
where D2 and Dj were the D values at temperature T2 and Tj (fahrenheit, °F),
respectively (Richardson and Hyslop, 1985).

151
Nondenaturino Polyacrylamide Gel Electrophoresis of CCL (1 atm)-treated PPO
Mini polyacrylamide gel with a dimension of 7 x 8 cm (1 mm
thickness) and containing 7.5% acrylamide/ 0.2% bisacrylamide was prepared
according to the Mini-Protean II Dual Slab Cell Instruction Manual (Bio-
Rad, 1985b). After 50 /zl_ PPO was loaded onto the sample well, the
electrophoresis was carried out at a constant voltage of 200 V for 35 min.
Following electrophoresis, the gel was stained with 0.1% Coomassie blue
R-250 in a fixative solution (40% MeOH and 10% HOAc, v/v) for 0.5 hr and
then destained. The molecular weights of PPO were determined by comparing
the Rf values of protein with those of nondenaturing protein molecular
weight standards (Sigma) containing a-lactalbumin (14 kD), bovine
erythrocytes carbonic anhydrase (29 kD), chicken egg albumin (45 kD),
bovine serum albumin (monomer, 66 kD; dimer, 132 kD), and jack bean urease
(dimer, 240 kD; tetramer, 480 kD).
Mass Balance of High Pressure C02-treated and Nontreated PPO
The high pressure C02-treated PPO solution (1.5 mL) was centrifuged
in an Eppendorf 5415 microcentrifuge (Brinkmann Instruments Inc., Hamburg,
Germany) at 13,000 rpm for 30 min. After the supernatant was collected,
the pellet was redissolved in 0.5 mL 0.05 M sodium phosphate buffer (pH
6.5), and the protein contents of both portions quantitated using the Bio-
Rad Protein Assay kit. The combined protein contents of both portions
were then compared to that of an equal volume (1.5 mL) of nontreated PPO.
The protein patterns of high pressure C02-treated and untreated PPO
were also analyzed using mini sodium dodecyl sulfate (SDS) polyacrylamide
gel. Fifty-/zL supernatant and pellet portions of high pressure C02-treated

152
and nontreated PPO were loaded individually onto the sample wells.
Electrophoresis was carried out at a constant voltage of 200 V for 35 min.
Following electrophoresis, the gel was stained with the silver stain kit
(Bio-Rad). The SDS-6H standard (Sigma) containing carbonic anhydrase (29
kD), egg albumin (45 kD), bovine albumin (66 kD), phosphorylase B (97.4
kD), /J-galactosidase (116 kD), and myosin (205 kD) was run together with
the samples for protein molecular weight determination.
Polyacrylamide Gel Isoelectric Focusing of C0:-treated PPO
A gel mixture containing 4% acrylamide, 2.5% Triton X-100, 8 M urea,
and 5.5% ampholyte (Pharmalyte 3-10, Pharmacia) was degassed for 5 min.
After the addition of 5% (v/v) fresh ammonium persulfate and 0.1% (v/v)
N,N,N',N'-tetramethylethylenediamine (TEMED), the gel mixture was poured
into a 16 x 20 cm slab gel plates assembled with a 0.75 mm comb and
allowed to polymerize for 1.5 hr according to the Protean II Slab Cell
Instruction Manual (Bio-Rad Labs., 1985a). Following the removal of the
comb, buffer containing 0.2% (v/v) Pharmalyte 3-10 and 5% (v/v) Triton X-
100 was overlayed onto the polymerized gels and allowed to sit for 1 hr.
Prefocusings at constant voltages of 200 V (15 min), 300 V (30 min), and
400 V (30 min) were alternately carried out after the overlaying buffer
was changed (An et al., 1989). A 50 ¡il of C02 (1 or 58 atm)-treated PPO
was then loaded into the sample well and electrofocusing was performed at
a constant voltage of 400 V for 17 hr (An et al., 1989). The gel was
fixed with the fixative solution (sulfosalicylic acid/ trichloroacetic
acid/ methanol, 4:12.5:30, v/v) and then stained with Coomassie blue R-
250. The isoelectric point (pi) of PPO was determined by comparing the Rf

153
value of the sample with those of the protein standards (Broad pi Kit, pH
3-10, Pharmacia) containing amyloglucosidase, pi 3.50; soybean trypsin
inhibitor, pi 4.55; 0-lactoglobul in, pi 5.20; bovine carbonic anhydrase,
pi 5.85; human carbonic anhydrase B, pi 6.55; horse myoglobin-acidic band,
pi 6.85; -basic band, pi 7.35; lentil lectin-acidic band, pi 8.15; -middle
band, pi 8.45; -basic band, pi 8.65; and trypsinogen, pi 9.30.
Spectropolarimetric Analysis of PPO
Circular dichroic (CD) spectra of high pressure C02-treated and non-
C02-treated PPO were determined at the far UV range (250 - 200 nm) using
a Jasco J-20 automatic recording spectropolarimeter (Japan Spectroscopic
Co., Tokyo, Japan), using a 1.0-cm Suprasil (Helma Cells) cuvette with
1.0-cm light path. Four-mL PPO (10 ¡ig/mL) in 0.05 M sodium phosphate
buffer (pH 6.5) was used as sample and the measurement of CD spectra was
carried out at ambient temperature. Secondary structure calculations were
performed by computer analysis of the CD spectra using the SSE program
(Japan Spectroscopic Co., 1985) with myoglobin, cytochrome c, ribonuclease
A, lysozyme, and papain as CD references.
Study of Restoration of C02-treated PPO Activity
To examine the reactivation ability of PPO following C02 (1 or 58
atm) treatment, a portion of C02-treated sample was stored at -20°C in a
microcentrifuge tube (1.5 mL) for 6 weeks. After thawing at ambient
temperature, the pH was then measured using a digital pH meter. Enzyme
activity was determined as previously described and the assays were
performed weekly. Percentage relative activity was determined as (ERt/ERo)

154
x 100, where ERt and ERo were the activities of C02 (1 or 58 atm)-treated
PPO stored at time t and the original activity of non-C02-treated PPO,
respectively.
Results and Discussion
Effect of C02 (1 atm) on PPO Activity
Enzyme activity of untreated PPO incubated at 33°, 38°, and 43°C
decreased slightly with increased heating times (Figures 33a, 33b, and
33c). If the protein molecule absorbs too much thermal energy, the
secondary and/or tertiary structure will become disrupted and enzyme will
be denatured and lose its catalytic activity (Segel, 1976). PPO (43 ng
protein/mL) solutions exposed to C02 under similar heating conditions
dramatically lost their catalytic activities (Figures 33a, 33b, and 33c).
Only 1.5% of the original activity remained after PPO was treated with C02
at 33°C for 30 min (AA475nm/rnin = 0.0009 vs. 0.059) (Figure 33a). For those
samples subjected to C02 at 38° and 43°C, no enzyme activity was detected
after 25 and 20 min, respectively (Figures 33b and 33c). The spiny
lobster PPO was more vulnerable to C02 than corn germ peroxidase
(Christianson et al., 1984) and nine commercial enzyme preparations
including a-amylase, glucoamylase, /l-galactosidase, glucose oxidase,
glucose isomerase, lipase, thermolysin, alcohol dehydrogenase, and
catalase (Taniguchi et al., 1987).

Figure 33. Effect of Carbon Dioxide (1 atm) on the Change in pH (*) and
Enzyme Activity (a) of Florida Spiny Lobster PPO Incubated at
33° (a), 38° (b), or 43°C (c); the Thermal Effect on the PPO
Activity in the Absence of C02 (â–¡) Was Also Conducted.

80
60
40
20
0
00
80
60
40
20
0
100
60
60
40
20
0
156
■ + A—
10 15 20 25
Time (min)
5

157
Profiles of the time-related pH changes among these C02-treated PPO
were similar (Figures 33a, 33b, and 33c). A sharp drop in pH from 8.5 to
5.3 occurred after the PPO solutions were bubbled with C02 for 1 min. The
pH remained constant at around 5.4 for the duration of the experiment.
The pKa value for the equilibrium between dissolved C02 and H+ and
HC03” is 6.1. (Montgomery and Swenson, 1969). According to the Henderson-
Hasselbalch equation, the molar concentration of HC03~ to C02 in solution
was reduced from 251 to 0.16 when the pH of the solution dropped from 8.5
to 5.3. This study showed that exposure to C02 yielded a lower pH
environment and resulted in a rapid acidification (Aickin and Thomas,
1975; Thomas and Ellis, 1976).
When the change in enzyme activity was compared to the change in pH
among the C02-treated PPO solutions, it was noted that PPO activities
decreased with an increase in C02 treatment time. The treatment of PPO
solution with C02 caused an instant drop in pH and it then remained
constant at 5.3 after 1 min. Using the pH control study, it was found
that enzymes under an environmental pH of 5.3 still had 66, 60, and 35% of
original activity after heating at 33°, 38°, and 43°C, respectively, for
30 min. Results from this study thus demonstrate that the loss in
activity was not due entirely to pH changes.
Effect of N; on PPO Activity
Nitrogen gas did not inactivate PPO activity. In contrast, the
relative activity of N2-treated PPO increased with time (Table 8). Such
significant (P < 0.05) increase in enzyme activity accompanied by a
decrease in volume and thus an increase in protein concentration were

158
Table 8. Effect of Nitrogen on Florida Spiny Lobster Polyphenol Oxidase
Activity
% Relative
activity
(«475 Jm'
n)#*
Heating
time (min)
Temperature (°C)
5
10
20
30
33
99a
100a
110a
129a
38
107b
129b
232b
378b
43
107b
-Q
00
C\J
r-H
176°
317°
* The reaction mixture contained 0.84 mL of 10 mM DL-DOPA in 0.05 M sodium
phosphate buffer (pH 6.5) and the assay was monitored at 475 nm (25°C)
for 10 min; the maximal initial rate for the control of this study was
0.059.
There are no significant differences (P > 0.05) among the treatments
within the same column with the same superscript letter.

159
observed for PPO samples heated at 38° and 43°C after 20 min (Table 8).
Thus, the loss in enzyme activity of C02-treated PPO was not due to the
purging effect from the bubbling gas.
Effect of High Pressure CO. on PPO Activity
Heating of lobster and brown shrimp PPO at 43°C for 30 min caused
some loss of enzyme activity (Figures 34 and 35). Such treatment,
however, caused only 5% loss of potato PPO activity (Figure 36). No
protein precipitation occurred in treated samples.
Treatment of these PPO with high pressure (58 atm) C02 at 43°C,
however, caused a dramatic loss of enzyme activity (Figures 34, 35 and
36). Lobster, brown shrimp, and potato PPO, after treatment for 1 min,
retained only 2 (AA475nm/rnin = 0.001 vs. 0.083), 22 (0.010 vs. 0.046), and
45% (0.240 vs. 0.540), of the original enzyme activity, respectively.
Extended treatment of lobster and brown shrimp PPO for more than one min
caused a complete loss of enzyme activity. For these two PPOs, the
treatment for 10 and 15 min respectively caused protein precipitation.
High pressure C02 treatment thus caused PPO denaturation and the loss of
lobster and brown shrimp enzyme activity. The results also showed that
brown shrimp PPO was slightly more resistant than lobster PPO to high
pressure C02 treatment at 43°C, and potato PPO was the most resistant of
the three. Potato PPO eventually lost 91% of its original activity after
treatment for 30 min (Figure 36). Florida spiny lobster PPO was more
susceptible to high pressure C02 than atmospheric C02; treatment of this
PPO for 20 min with atmospheric C02 (1 atm) at 43°C did not cause complete
loss of the enzyme activity (Figure 33c). In comparison with the studies

Figure 34. Effect of High Pressure (58 atm) Carbon Dioxide on the Change in pH (•) and Enzyme Activity
(o) of Florida Spiny Lobster PPO Incubated at 43°C; (□) Represents the Activity of PPO
Incubated at 43°C in the Absence of C02.

Time (min)
T9T
43 °C

Figure 35. Effect of High Pressure (58 atm) Carbon Dioxide on the Change in pH (•) and Enzyme Activity
(o) of Brown Shrimp PPO Incubated at 43°C; (□) Represents the Aactivity of PPO Incubated at
43°C in the Absence of C02.

Time (min)
% Relative activity
ro -tfc CD 03
o o o o o
E9T
100

Figure 36. Effect of High Pressure (58 atm) Carbon Dioxide on the Change in pH (•) and Enzyme Activity
(o) of Potato PPO Incubated at 43°C; (□) Represents the Activity of PPO Incubated at 43°C in
the Absence of C02.

Time (min)
o
% Relative activity
A
O
05
O
00
O
991

166
of Christianson et al. (1984) and Taniguchi et al. (1987), it also appears
that lobster PPO was more vulnerable to high pressure C02 treatment than
corn germ peroxidase, a-amylase, glucoamylase, /5-galactosidase, glucose
oxidase, glucose isomerase, lipase, thermolysin, alcohol dehydrogenase,
and catalase.
High pressure C02 treatment at 43°C of the three PPO systems for 1
min caused a sharp drop in pH from 9.1 to 5.4 for lobster, 6.5 to 4.8 for
brown shrimp, and 6.1 to 4.2 for potato (Figures 34, 35, and 36). The pH
of the treated lobster, brown shrimp, and potato PPO systems remained
constant at 5.3, 4.5, and 4.1, respectively, throughout the experiment.
Overall, there was no difference in the profiles of the time-related pH
change between the high pressure and atmospheric C02-treated PPO's (Figures
33a, 33b, and 33c).
Kinetics of PPO Inactivation
Kinetic parameters for PPO inactivation by C02 (1 atm) are given in
Table 9. The reaction constants for lobster C02-treated PPO at various
temperatures were comparatively higher than those for PPO with no C02
treatment. PPO exposed to C02 showed a higher activation energy (Ea = 39.7
KJ/mole) for inactivation than control (Ea = 26.6 KJ/mole) for the DOPA
reaction. The D values of PPO under C02 treatment are lower than those of
temperature controls indicating that at the temperature range used, it is
much easier to inactivate PPO by C02 and heat than by heat alone. The
smaller z values obtained for C02-treated samples also implies that the
enzyme is more sensitive to elevated temperatures when in a C02
environment. The reaction constants (k) for brown shrimp and potato PPO

167
Table 9. Kinetic Parameters of Florida Spiny Lobster Polyphenol Oxidase Inactivation by Carbon Dioxide
(1 atm) at Various Temperatures
Temperature (°C)
D (min)
Z (°C)
k (min *)
Ea (KJ/mole)
Control
33
320
7.2 x 10'3
38
314
69.1
7.3 x 10'3
26.6
43
229
1.0 x 10~2
CO.-treatment
33
17.4
1.2 x 10_1
38
13.1
43.3
1.6 x 10_1
43
10.2
1.9 x 10'1
39.7

168
at 43°C were determined to be 1.6 x 10~2, 9.4 x 10~3, and 2.5 x 10-3 min-1,
respectively. Under similar heating conditions in the presence of C02 (58
atm), the k values for brown shrimp and potato PPO were shifted to 0.98
and 6.9 x loomin'1. Potato PPO was more resistant to the treatments than
lobster and brown shrimp PPO. This study also demonstrates that PPO was
more susceptible to the combined treatment of high pressure C02 and heat
than by heat alone.
Polyacrylamide Gel Electrophoresis of C02 (1 atm)-treated PPO
Only one single protein band was observed for the control and en¬
treated PPO on the nondenaturing PAGE gel (Figure 37). No difference in
protein band position on the gel occurred among the control and C02-treated
groups. The molecular weight of this protein band was assessed as 255 kD
which is close to the total molecular weights (277 kD) of three isoforms
previously reported for spiny lobster PPO (Chen et al., 1991a). In
addition, it is apparent that no alteration of the intact protein molecule
occurred when subjected to C02 treatment.
Mass Balance of High Pressure C02-treated and Non-CO^treated PPO
Protein precipitates occurred after lobster and shrimp PPO were
subjected to high pressure C02 treatment. The combined protein contents
in the supernatant and pellet portions of the treated samples were close
to that of their respective untreated control (Table 10). Since C02-
treated potato PPO did not have a precipitate, no protein content was
detected in the pellet portion after centrifugation (Table 10).

Figure 37. Nondenaturing Polyacrylamide Gel Electrophoresis (PAGE, 7.5% Gel) Profile of Carbon Dioxide
(1 atm)-treated Florida Spiny Lobster (FSL) PPO; FSL (C) Represents Non-CO,-treated PPO,
While FSL (I), FSL (II), and FSL (III) Represent the PPO Heated at 33°, 38é, and 43°C,
Respectively, in the Presence of C02. Numerical Designations Represent Molecular Masses of
the Standard Proteins.

Protein
Standard
FSL
(C)
FSL FSL
(I) (ID
FSL
(HI)
^1
o

171
Table 10. Mass Balance of Protein Contents of High Pressure C02-treated
and Nontreated PPO
Protein
Content (/zg)
C02-treated
PPO
Nontreated*
Supernatant
Pel let
Lobster
464+4®
132+7
332±5
Brown
shrimp
507+5
148+3
359+6
Potato
140+2
133+4
ND#
*The total
protein content
based on a 1.5
mL of PPO
solution was quantitated using the Bio-Rad Protein
Assay kit.
®Mean value + standard deviation
#ND, No detection

172
Protein patterns of C02-treated and nontreated lobster PPO from
acrylamide gel also verified the mass balance (Figure 38). For lobster
PPO, the combined protein patterns of the supernatant and the pellet
portions matched those of the non-treated PPO. These results indirectly
suggest that high pressure C02 treatment could bring about precipitation
of protein molecules and thus causes PPO inactivation.
Polyacrylamide Gel Isoelectric Focusing of C02-treated PPO
The IEF gel patterns showed that the protein band of nontreated and
C02 (1 atm)-treated PPO groups were at the same position (Figure 39) and
the pi value was determined as 6.0. Thus, C02 (1 atm) treatment does not
alter the electrical properties of the PPO molecule.
Untreated lobster, brown shrimp, and potato PPO only showed one
protein band with an isoelectric point (pi) of 6.0 on the focused gel.
Upon treatment with high pressure C02, the lobster, brown shrimp, and
potato PPO showed several protein bands on IEF gel including one with a pi
of 6.2 (Figure 40). Therefore, high pressure C02 treatment might cause
disintegration of the PPO molecule.
Spectropolarimetric Analysis of High Pressure C02-treated PPO
CD spectra at the far UV range for control and high pressure C02-
treated lobster, brown shrimp, and potato PPO are given in Figures 41, 42,
and 43, respectively. The negative ellipticity between 207 and 220 nm of
controls was quite different from those of C02-treated PPO. C02-treatment
caused changes in the secondary structures (a-helix, /J-sheet, /J-turn, and
random coil) (Table 11). Lobster and brown shrimp PPO showed the most

Figure 38. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) Profile of High Pressure (58 atm) CO -
treated Florida Spiny Lobster (FSL) PPO; FSL (C) is Untreated
PPO, While FSL (S) and FSL (P) Respectively Represent the
Supernatant and Pellet Portions of High Pressure (58 atm) C02-
treated PPO Following Centrifugation.

174
205 kD
116 kD
97 kD
66 kD
45 kD
29 kD
FSL
(S)
SDS-6H
FSL
(C)
FSL
(P)

Figure 39. Polyacrylamide Gel Isoelectric Focusing (IEF, 5% Gel) Profile
of Carbon Dioxide (1 atm)-treated Florida Spiny Lobster (FSL)
PPO; FSL (C) Represents Non-CCL-treated PPO, While FSL (I), FSL
(II), FSL (III) Represent the PPO Heated at 33°, 38°, and 43°C,
Respectively, in the Presence of C02. Numerical Designations
Represent Isoelectric Points (pi) of the Standard Proteins.

176
i
8.15
7.35
6.85
6.55
5.85
5.2
4.5
3.6
FSL FSL FSL FSL
(III) (II) (I)
Std

Figure 40. Polyacrylamide Gel Isoelectric Focusing (IEF, 5% Gel) Profile
of High Pressure (58 atm) Carbon Dioxide-treated Florida Spiny
Lobster (FSL), Brown Shrimp (BS), and Potato (P) Polyphenol
Oxidase (PPO). FSL (C), BS (C), and P (C) Represent Non-C02-
treated PPO, Respectively, While FSL (T), BS (T), and P (T)
Are PPO Subjected to High Pressure Treatment at 43°C.
Numerical Designations Represent Isoelectric Point (pi) of the
Protein Standards.

178
- 8.15
- 7.35
- 6.85
- 6.55
- 5.85
- 5.2
- 4.5
- 3.6
P P
(T) (C)
BS FSL FSL
(C) (T) (C)
BS
(T)
Std

Figure 41. Comparison of Far UV Circular Dichroic Spectra of Nontreated Control ( ) and High Pressure
(58 atm) Carbon Dioxide-treated ( ) Florida Spiny Lobster PPO; Four-mL PPO (10 /xg/mL) in
0.05 M Sodium Phosphate Buffer (pH 6.5) Was Analyzed at Ambient Temperature.

o
-121—
200
210
1
220 230 240 250
Wavelength (nm)
180

Figure 42. Comparison of Far UV Circular Dichroic Spectra of Nontreated Control ( ) and High Pressure
(58 atm) Carbon Dioxide-treated ( ) Brown Shrimp PPO; Four-mL PPO (10 /xg/mL) in 0.05 M
Sodium Phosphate Buffer (pH 6.5) Was Analyzed at Ambient Temperature.

182

Figure 43.
Comparison of Far UV Circular Dichroic Spectra of Nontreated Control ( ) and High Pressure
(58 atm) Carbon Dioxide-treated ( ) Potato PPO; Four-mL PPO (10 /xg/mL) in 0.05 M Sodium
i jo a Liny Lai uuii uiuaiuw v / -
Phosphate Buffer (pH 6.5) Was Analyzed at Ambient Temperature.

[0] x lO^degres cmVdmole

185
Table 11. Secondary Structure Estimates of Nontreated Control and High
Pressure C02-treated Florida Spiny Lobster, Brown Shrimp, and
Potato Polyphenol Oxidases (PPOs) from Far UV Circular Dichroic
Spectra
% of secondary structure
PPO
a-helix
/3-sheet
0-turn
random
Lobster
Control
24.4
26.2
21.4
29.9
C02-treated
19.7
25.9
15.2
39.3
Brown
Control
20.1
22.3
15.2
42.4
shrimp
C02-treated
29.6
18.9
18.2
33.3
Potato
Control
14.8
34.6
28.4
21.2
C02-treated
17.8
35.9
25.9
20.4
The circular dichroic spectra of PPO was scanned at the far U.V. (250 -
200 nm) range. Four-mL PPO (10 /xg/mL) in 0.05 mM sodium phosphate buffer
(pH 6.5) was analyzed at ambient temperature.

186
noticeable alterations in the composition of a-helix and random coil. In
contrast, only minor alteration in secondary structures was observed in
high pressure C02-treated potato PPO (Table 11). These results thus
verified the previous finding that potato PPO was more resistant to high
pressure C02 than lobster and brown shrimp PPO (Figures 34, 35, and 36),
possibly due to its less responsiveness at altering the secondary
structure. The pH change of the system and the possible bubbling effect
due to C02, again were not completely responsible for the loss of PPO
activity. This was in agreement with previous findings of Miller et al.
(1981) who proposed that the pressure-induced effect from SC-C02 treatment
could cause changes in protein backbone structure and subunit dissociation
and thus inactivated the enzyme.
Restorative Ability of CO.-treated PPO Activity
Changes in PPO activity and pH after C02 (1 atm) treatment and during
frozen-storage are given in Figures 44a, 44b, 44c, and 44d. Non-C02-
treated controls gradually lost PPO activity as time proceeded. Nearly
50% of the original activity (AA475nm/rnin = 0.059) was lost after storage
over 5 weeks (Fig. 45a). For C02 (1 atm)-treated-PPO, only PPO heated at
33°C was restored by 10% of its original enzyme activity (AA475nm/rnin =
0.006) during the first week (Fig. 44b). After one week storage, the PPO
activity decreased as storage time increased. C02 (1 atm)-treated PPOs
heated at both 38° and 43°C showed no restoration in activities (Figures
44c and 44d). Unlike the pH change in non-C02-treated PPO which showed a
slight decrease in pH with increased storage time (Figure 44a), those
subjected to C02 (1 atm) treatment showed a rapid increase in pH after one

Figure 44. The Restorative Ability of Carbon Dioxide (1 atm)-treated
Florida Spiny Lobster PPO Activity (â–¡) and the Pertinent
Environmental pH Changes (a) during Frozen-storage. The C02-
treated PPO Was Heated at 33° (b), 38° (c), or 43°C (d), and
the Non-C02-treated PPO (a) Was Run as a Control.

100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
i
0;
9
8
7
6
-B-
- 7
- 6
^5 X
19 Q-
1 2 3 4 5
Time (week)
-¿5
6
188

189
week of storage (Figure 44b). The pH rose from 5.4 to 8.0 for those
treated-PPO at 33° and 38°C, but only to 7.2 for that heated at 43°C.
The profiles of the restorative ability of high pressure C02-treated
lobster, brown shrimp, and potato PPOs during a 6-week frozen storage are
shown in Figures 45, 46, and 47. After 6 weeks storage, the pH of high
pressure C02-treated lobster PPO returned to 9.1 from 5.3, whereas there
was no restoration of enzyme activity (Figure 45). Similarly, the pH of
brown shrimp PPO rose from 4.5 to 6.50 and there was also no restoration
of enzyme activity (Figure 46). For potato PPO, the pH change followed
the trend similar to those observed for the previous two PPOs, climbing
from 4.08 to 6.07. It was noted that 28% of the original activity
(AA475nn/min = 0.151 vs. 0.540) was restored for the C02-treated PPO during
the first two weeks of storage (Figure 47). After this period, the PPO,
however, gradually lost its activity as the storage time increased. This
result was similar to the previous observation for PPO subjected to
atmospheric (1 atm) C02 at 33°C. The treated sample restored 15% of its
original enzyme activity during the first week of storage (Figure 44b).
Conclusion
Lobster PPO exposed to C02 (1 atm) at 33°, 38°, and 43°C showed a
decline in enzyme activity with time. Inactivation kinetics study
revealed that lobster PPO was more labile to the combined treatment of C02
and heat than to heat alone. The activities of lobster, brown shrimp, and
potato PPOs followed trends similar to that of atmospheric C02 when these
enzymes were subjected to the high pressure (58 atm) C02 treatment at 43°C.
Results indicate PPOs were more susceptible to high pressure C02 than

Figure 45.
The Restorative Ability of High Pressure (58 atm) Carbon Dioxide-treated Florida Spiny
Lobster PPO Activity (â–¡) and the Pertinent Environmental pH (â– ) Changes during Frozen-storage

% Relativa activity
CL
191

Figure 46. The Restorative Ability of High Pressure (58 atm) Carbon Dioxide-treated Brown Shrimp PPO
Activity (â–¡) and the Pertinent Environmental pH (â– ) Changes during Frozen-storage

100
80
60
40
20
% Relative activity
—B—
é é é &
12 3 4
Time (week)
7
6
CL
5
193

Figure 47. The Restorative Ability of High Pressure (58 atm) Carbon Dioxide-treated Potato PPO Activity
(â–¡) and the Pertinent Environmental pH (â– ) Changes during Frozen-storage

Time (week)
195

196
atmospheric C02; potato PPO was more resistant to high pressure C02 than
lobster and shrimp PPOs. C02 (1 atm)-treated and untreated PPO did not
show any differences in protein patterns and isoelectric profiles, the
high pressure C02 treatment however affected the protein patterns and
isoelectric profiles as well as secondary structures of the treated
sample.

CONCLUSIONS
Polyphenol oxidase (PPO) from various sources vary with respect to
their substrate specificity, kinetic properties, isoforms, molecular
weights, activation energy (Ea), isoelectric point (pi) and activity and
stability to pH and temperature effects. Tyrosine, phenylalanine, DOPA,
and tyramine have been identified as predominant phenolic substrates of
crustacean (lobster, crab, and shrimp) PPO. For mushroom and other plant
PPOs, the substrate, however, varied with the sources of enzyme.
Crustacean PPO had a narrow range of optimum pH between 6 to 8 compared
with the broad one (4 to 7) observed for mushroom and plant PPOs.
Regarding temperature optimum, crustacean PPOs had greater ranges than
plant PPOs. Using immunological techniques, plant (potato and apple),
mushroom, and crustacean (Florida spiny lobster, white shrimp, and brown
shrimp) PPOs were shown to share similar antigenic determinants. Varied
compositional secondary structures (a-helix, /?-sheet, /?-turn, and random
coil) as revealed by spectropolarimetric analysis however existed among
these different PPOs.
Kojic acid was shown to inhibit PPO activity from the various
sources. It was a competitive inhibitor for the oxidation of chlorogenic
acid and catechol by potato PPO and 4-methyl catechol and chlorogenic acid
by apple PPO. This compound showed a mixed-type inhibition for lobster,
grass prawn, and white shrimp PPO when £-3,4-dihydroxyphenylalanine (DOPA)
and catechol were used as substrates, but a mixed-type and a competitive
197

198
inhibition for mushroom PPO when DOPA and L-tyrosine were used,
respectively. Significant inhibitory effects with different types of
inhibition mechanisms were observed with the various PPO activities when
DOPA was used as substrate. In addition to mechanisms described
previously, kojic acid was shown to inhibit melanosis by interfering with
the uptake of oxygen required for enzymatic browning. This compound was
capable of reducing o-quinones to o-diphenols; this result suggests that
kojic acid could be potentially used as an inhibitor in the prevention of
melanosis in plant and seafood products.
When exposed to C02 (1 atm) at 33°, 38°, or 43°C, lobster PPO showed
a decline in enzyme activity with heating time. When these enzymes were
subjected to high pressure (58 atm) C02 at 43°C, lobster, brown shrimp, and
potato PPOs followed trends similar to that of atmospheric C02.
Inactivation kinetics revealed lobster PPO was more labile to C02 and heat
than heat alone. Studies showed PPOs were more susceptible to high
pressure C02 than atmospheric C02; potato PPO was more resistant than
lobster and shrimp PPO to the high pressure C02. Studies employing
polyacrylamide gel electrophoresis showed there were no differences in the
protein patterns and isoelectric profiles between the nontreated and C02
(1 atm)-treated PPO. Differences in secondary structures, protein
patterns, and isoelectric profiles however were observed between the high
pressure (58 atm)-treated and untreated PPO. Results from the studies
suggest that low temperature (< 4°C) storage of foodstuffs that have
previously been treated with C02 could possibly be used as a processing aid
to enhance the inactivation of PPO and thus prevent enzymatic browning.

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BIOGRAPHICAL SKETCH
Jon-Shang Chen was born in Taiwan, Republic of China, on February
20, 1958. He attended the Chinese Culture University at Taipei City,
Taiwan, ROC, where he was awarded the degree of Bachelor of Science from
the Department of Food Science and Nutrition in June 1981. In January
1987, he received a Master of Science degree in food science from the
Department of Food Science and Nutrition at the University of Rhode
Island, Kingston, RI. In August 1987, he began his studies of food
science towards the degree of Doctor of Philosophy in the Food Science and
Human Nutrition Department at the University of Florida, Gainesville, FL,
and expects to graduate in December 1991.
216

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of Doctor
of Philosophy.
Marty R. Marshall, Chair
Professor of Food Science and Human
Nutrition
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of Doctor
of Philosophy.
Cheng-I Wei, Cqchair
Professor of Food Science and Human
Nutrition
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of Doctor
of Philosophy. /
W. Steven Otwell
Pi^fessor of Food Science and Human
Nutrition
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of Doctor
of Philosophy.
Associate Professor of Food Science
and Human Nutrition
Murat frrtalaban

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of Doctor
of Philosophy.
James F. Preston
Professor of Microbiology and Cell
Science
This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
December 1991 ^ ^
^
Dean, /College of Agriculture
Dean, Graduate School





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