• TABLE OF CONTENTS
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 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Prologue
 Introduction
 Materials and methods
 Results
 Discussion
 Introduction
 Materials and methods
 Results
 Discussion
 Summary
 Literature cited
 Biographical sketch
 Copyright














Title: Hydroxyproline biosynthesis in higher plants
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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
        Page iv
        Page v
    List of Tables
        Page vi
    List of Figures
        Page vii
        Page viii
    Prologue
        Page 1
        Page 2
    Introduction
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
    Materials and methods
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
    Results
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
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        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
    Discussion
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
    Introduction
        Page 38
        Page 39
        Page 40
    Materials and methods
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
    Results
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
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        Page 88
        Page 89
        Page 90
        Page 91
        Page 92
        Page 93
        Page 94
        Page 95
    Discussion
        Page 96
        Page 97
        Page 98
        Page 99
        Page 100
        Page 101
        Page 102
    Summary
        Page 103
        Page 104
    Literature cited
        Page 105
        Page 106
        Page 107
        Page 108
        Page 109
        Page 110
        Page 111
        Page 112
        Page 113
    Biographical sketch
        Page 114
        Page 115
    Copyright
        Copyright
Full Text












HYDROXYPROLINE BIOSYNTHESIS

IN HIGHER PLANTS


















By
ERNEST RAY STOUT










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











UNIVERSITY OF FLORIDA
April, 1965















ACKNOWLEDGMENTS


The author wishes to thank Dr. G. J. Fritz, chairman of the

supervisory committee, for his expert guidance, helpful suggestions

and incisive criticisms in the conduct of the research and in the

preparation of the manuscript.

Appreciation is extended to the committee members, Dr. T. E.

Humphreys, Dr. D. S. Anthony and Dr. W. B. Dempsey, who gave of their

time and advice. Dr. G. R. Noggle provided helpful guidance during

the early part of the graduate studies.

The United States Department of Health, Education and Welfare

is thanked for a National Defense Education Act, Title IV graduate

fellowship as well as the Department of Botany, University of Florida

for an assistantship.

My wife, Kaye Stout, assisted immeasurably in the preparation

and typing of the manuscript. Her help and confidence is gratefully

acknowledged.
















TABLE OF CONTENTS


Page


ACKNOWLEDGEMENTS . . . . . . . . . . .

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

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

PROLOGUE . . . . . . . . . . . .


PART I. HYDROXYPROLINE CONTENT OF ETIOLATED MAIZE
AND SOYBEAN SEEDLINGS . . . . .

INTRODUCTION . . . . . . . . .

MATERIALS AND METHODS . . . . . . .
Plant Material . . . . . . . .
Acid Hydrolysis . . . . . . .
Isolation of Amino Acids . . . . .
Fractionation of Tissues . . . . .
Colorimetric Determination of Hydroxyproline


RESULTS . . . . . . . . . . . 13
Plant Material . . . . . . . . 13
Estimation of Hydroxyproline . . . . . . 16
Hydroxyproline Content of Various Species of
Etiolated Seedlings . . . . . . . 18
Hydroxyproline Content of Maize and Soybean
Seedlings as a Function of Age . . . . 18
Hydroxyproline Content of Seedlings Parts as
a Function of Age . . . . . . . . 25
Hydroxyproline of Subcellular Fractions . . . 30

DISCUSSION . . . . . . . . . . 33

PART II. BIOSYNTHESIS OF HYDROXYPROLINE BY
OXYGEN FIXATION . . . . . . . . 38

INTRODUCTION . . . . . . . . . . 38

MATERIALS AND METHODS . . . . . . . . 41
Plant Material . . . . . . . . 41


vii









TABLE OF CONTENTS--Continued


Preparation of Oxygen-18 Gas . . . . .
Application of Oxygen-18 Gas to Seedlings . .
Application of Water Labeled with Oxygen-18
to Seedlings . . . . . . . .
Isolation of Proline and Hydroxyproline
from Seedlings . . . . . . . .
Preparation of Pyrolysis Tubes for Oxygen-18
Analysis . . . . . . . . .
Pyrolysis of Organic Material to Carbon Dioxide
Mass Spectrometric Analysis of Carbon Dioxide
and Oxygen . . . . . . . .
Exchange of Hydroxyproline Oxygen with Water
Oxygen . . . . . . . .

RESULTS . . . . . . . . . . .
Plant Material . . . . . . . ..
Column Chromatography . . . . . .
Thin-layer Chromatography . . . . .
Pyrolysis of Organic Material . . . . .
Exchange of Hydroxyproline Oxygen with
Water Oxygen . . . . . . . .
Source of the Hydroxyl Oxygen of Hydroxyproline
in Seedlings . . . . . . . .


Page

42
44


. 44

* 45


S. 54

. 55


. 65

. 66


DISCUSSION . . . . . . . . . . 71

PART III. BIOSYNTHESIS OF HYDROXYPROLINE FROM PROLINE . 79

INTRODUCTION . . . . . . . . . . 79

MATERIALS AND METHODS . . . . . . . . 82
Plant Material . . . . . . . . . 82
Application of Radioactive Proline to Seedlings . 83
Isolation of Proline and Hydroxyproline from
Seedlings . . . . . . . . .. 84
Extraction of Ribonucleic Acid . . . . . 84
Liberation of Ribonucleic Acid-bound Proline
and Hydroxyproline . . . . . . . 86
Counting of Radioactive Samples . . . . . 88


RESULTS . . . . . . . . . .
Conversion of Proline to Hydroxyproline in
Etiolated Seedlings . . . . .
Ribonucleic Acid Content of Maize Shoots .
Proline-C-14 and Hydroxyproline-C-14 in
Extracted Ribonucleic Acid . . . .


. . 88


* . 0


. . 89









TABLE OF CONTENTS--Continued


Page


Effect of Ascorbic Acid on the Activity of
Proline and Hydroxyproline Separated from
Extracted Ribonucleic Acid . . . . . . 94

DISCUSSION . . . . . . . . . . 96

SUMMARY . . . . . . . . . . . . . 103

LITERATURE CITED . . . . . . . .... 105













LIST OF TABLES


Table


1. Color yield of amino acids in the Neuman and
Logan determination of hydroxyproline. . . .

2. Hydroxyproline content of various species of
etiolated seedlings at five and ten days of age.

3. Distribution of hydroxyproline among cell wall,
protein and supernatant fractions of etiolated
maize seedlings. . . ... . . . ...

4. Distribution of hydroxyproline among cell wall,
protein and supernatant fractions of etiolated
soybean seedlings. . . . . . . .

5. Comparison of seven-day-old etiolated maize and
soybean seedlings grown in open and closed
atmospheres. . . . ........ ..

6. Recovery of hydroxyproline from cation exchange
columns. s . . . .*

7. Recovery of hydroxyproline from thin-layer
chromatography plates. . . . ... . .

8. Effect of increasing amounts of mercuric cyanide
on the estimated atom per cent 018 of hydroxy-
proline, as determined by the pyrolytic method
of Lee.. . . . . . . . . . .

9. Incorporation of 0218 into hydroxyproline of
maize and soybean. . . . . . . .


. 17


. 21



* 31



32



* 58


* 59


* 62




. 64


. 68


Incorporation of H2018 into hydroxyproline of
maize and soybean. . . . . . . . . .

Conversion of proline-C-14 to hydroxyproline-C-14
by maize and soybean seedlings . . . . . .

Ribonucleic acid content of seven-day-old maize
shoots . . . . . . . . . . . .

Radioactivity of proline and hydroxyproline
separated from ribonucleic acid which was
extracted from maize shoots. . . . . . .


Page














LIST OF FIGURES


Figure Page

1. Naturally occurring forms of hydroxy-L-proline. . 4

2. The variation in fresh and dry weight of etiolated
maize seedlings with increasing age . . . . 14

3. The variation in fresh and dry weight of etiolated
soybean seedlings with increasing age . . . .. 15

4. The absorption spectrum produced by hydroxyproline
in the Neuman and Logan method . . .. . 19

5. Standard curve relating absorbance and hydroxy-
proline concentration . . . . . . . . 20

6. The variation in hydroxyproline content of etiolated'
maize and soybean seedlings with increasing seedling
age. (The hydroxyproline content is expressed in
micrograms per seedling.) . . . . . . . 23

7. The variation in hydroxyproline content of etiolated
maize and soybean seedlings with increasing seedling
age. (The hydroxyproline content is expressed in
micrograms per gram of fresh weight.) . . . . 24

8. The variation in hydroxyproline content in several
organs of etiolated maize seedlings. (The hydroxy-
proline content is expressed in micrograms per
organ.) . . . . . . . . . . . 26

9. The variation in hydroxyproline content in several
organs of etiolated maize seedlings. (The hydroxy-
proline content is expressed in micrograms per
gram of fresh weight.). . . . . . . . 27

10. The variation in hydroxyproline content in several
organs of etiolated soybean seedlings. (The
hydroxyproline content is expressed in micrograms
per organ.) . . o . . . . . . 28

11. The variation in hydroxyproline content in several
organs of etiolated soybean seedlings. (The
hydroxyproline content is expressed in micrograms
per gram of fresh weight.). . . . . . . 29









LIST OF FIGURES--Continued


Figure Page

12. Flow sheet for the isolation of proline
and hydroxyproline. . . . . . . . ... 49

13. A series of diagrams illustrating the sequence
of steps in the preparation of pyrolysis tubes. . 51

14. Pattern of elution of hydroxyproline and proline
from a 50 cm Dowex 50W column . . . . . 60

15. Absorption spectra of RNA preparations:
(A) "original extract" and (B) ethanol
precipitation . . . . . . . . . 91

16. Possible loci for the hydroxylation of pro' 99

17. Diagrammatic scheme for hydroxyproline
biosynthesis . . . . . . .. 102


viii















PROLOGUE


The imino acid hydroxyproline (4-hydroxypyrrolidine-2-carboxylic

acid) was discovered in a gelatin hydrolysate (17). The configuration

has been established to be trans-4-hydroxy-L-proline (31, 65). Hydroxy-

proline differs from other amino acids in that it is found in only a few

proteins, particularly the proteins which are involved in structural roles,

whereas other amino acid constituents of proteins are present in almost

all proteins. Thus, for example, hydroxyproline in animal tissues is

found largely in collagen; in plant cells, hydroxyproline is generally

reported to occur in the primary cell wall. Also, hydroxyproline is

seldom found in the free state, whereas other amino acid constituents of

protein are always present in the cell in the uncombined form. Hydroxy-

proline also differs from the other normally-occurring protein amino acids

in that it occurs in plants and animals, but it has not been reported in

protein of bacteria.

It is widely recognized that cellular hydroxyproline is not meta-

bolically derived from free proline. Nor does the application of free

hydroxyproline to cells and tissues result in the incorporation of this

amino acid into protein. Instead, the hydroxyproline in proteins is

derived from "bound" or "activated" proline. This unique method of

biosynthesis of a protein amino acid has prompted considerable research

into the biochemistry and physiology of hydroxyproline and is the subject

of the investigations reported in this dissertation.









Most of the work which has been reported in the literature con-

cerning hydroxyproline biosynthesis has been done with animal tissues.

One of the few investigations concerning hydroxyproline biosynthesis in

plants is that of Lamport (44), who reported that the hydroxyproline of

sycamore cell suspensions is synthesized by the incorporation of molecular

oxygen. Similar reports are available concerning hydroxyproline bio-

synthesis in chick embryo.

The primary objective of the present investigations was the study

of the mechanism of hydroxyproline biosynthesis in seedlings of maize and

soybean. In Part I, a study was made of the hydroxyproline content,

variation and distribution in maize and soybean seedlings. The partici-

pation of molecular oxygen in the biosynthesis of hydroxyproline is

reported in Part II. Finally, in Part III, studies concerning the locus

of the hydroxylation reaction are reported.















PART I. HYDROXYPROLINE CONTENT OF ETIOLATED
MAIZE AND SOYBEAN SEEDLINGS


INTRODUCTION



Hydroxyproline occurs in appreciable amounts in only a few pro-

teins of animal origin, notably gelatin and collagen, where it comprises

13 to 14 per cent of the total amino acids (3, 112) and in elastin where

it occurs to the extent of 2 per cent (95). Attention was drawn to the

existence of hydroxyproline in higher plants by Steward and associates

(98, 99) during their early work with paper chromatography of amino acids.

They reported that the hydroxyproline of plants appeared only in the

hydrolysate of the water-, alcohol- and acid-insoluble fractions. Hydroxy-

proline has since been reported to occur in protein hydrolysates from a

wide variety of higher plants (47, 94, 108, 109). It is also reported

to occur in hydrolyzed ethanol extracts of Chlorella (91).

The bulk of protein hydroxyproline in plants and animals is trans-

4-hydroxy-L-proline (figure 1) (31, 108). The cis isomer (allohydroxy-

proline) of 4-hydroxy-L-proline (figure 1) was found to be widely dis-

tributed in several species of the plant genus Santalum, existing largely

in the free state in this particular genus (21, 79, 80); in Santalum

album L., the highest concentration was found in apical leaves, buds,

flowers and young fruit pericarp (79, 80). The concentration of cis-4-

hydroxy-L-proline in the young fruit pericarp of Santalum reached the











C OH H C
H C /COOH
I I
H H
TRANS-4-HYDROXY-L-PROLINE




H
N H

C H OHC
H\- 1 /COOH
HC- C C


TRANS-3-HYDROXY-L-PROLINE


H N H

H HC
H C'OOH
C-C
OH H
CIS-4-HYDROXY-L-PROLINE




H
N
H H
C H H C
Hl C /COOH
I I
H OH


CIS-3-HYDROXY-L-PROLINE


Figure 1. -- Naturally occurring forms of hydroxy-L-proline.









remarkedly high concentration of 10 per cent of the dry weight. Within

the past few years, trans-3-hydroxy-L-proline (figure 1) has also been

found (68); this isomer occurs to the extent of 0.26 per cent in bovine

collagen (68) and also occurs in the collagen of rat skin, sponge (32)

and shark (51). Cis-3-hydroxy-L-proline (figure 1) as well as trans-3-

hydroxy-L-proline have been found in the antibiotic telomycin (32, 90).

Only the trans isomers, trans-4-hydroxy-L-proline and trans-3-hydroxy-L-

proline, have been reported to be protein components.

The localization of hydroxyproline within plant cells has been a

subject of intensive investigation within recent years. Pollard and

Steward (74) reported that the hydroxyproline-containing protein of

tissue cultures of carrot root phloem and potato tuber occurs mainly in

the cytoplasm of the cell rather than in the particulate inclusions, but

in the light of later work by other investigators (and by Steward), this

conclusion regarding the localization of hydroxyproline within the cell

appears to be questionable. Preston (75) postulated that the plant

proteins which contain hydroxyproline might be closely associated with

the developing cell wall. Lamport and Northcote (40, 41) examined the

distribution of hydroxyproline between the cell wall and cell contents

of sycamore suspension cultures and found almost all of it was in the

cell wall fraction; less than 0.1 per cent of the total hydroxyproline

appeared within other portions of the cell contents. Amino acid analysis

of the cell wall hydrolysate revealed that hydroxyproline was a major

component of the cell wall protein, comprising 13 per cent of the amino

acids (40, 41). Using tobacco callus tissue cultures, Dougall and

Shimbayashi (14) also found most of the hydroxyproline of the cell in

the cell wall hydrolysate. In this case, hydroxyproline was 18 per










cent of the amino acids in the cell wall hydrolysate (14). The high

content of hydroxyproline in the cell wall protein is similar to the

condition which exists in collagen. However, cell wall protein differs

from collagen in having low levels of glycine and alanine and a high

level of lysine (14, 40). The finding that the bulk of the hydroxyproline

of plant cells is associated with the cell wall has been confirmed by

other workers (7, 16, 69). In more recent work, Steward and associates

(53) found that over prolonged periods of growth, the bulk of the protein-

bound hydroxyproline was found in portions of the cell which were obtained

by low-speed centrifugation of disintegrated cells, and thus was found

together with the cell wall fragments.

However, the question concerning the localization of the

hydroxyproline-containing protein in plants is not completely resolved.

Thus, Steward and Chang (103) separated the soluble proteins from carrot

explants and found not just one but eight proteins which contained hydroxy-

proline. However, both Dougall and Shimbayashi (14), working with cell

cultures of tobacco callus tissue, and Lamport and Northcote (40), work-

ing with cell cultures of sycamore, considered the possibility of adsorp-

tion by a cytoplasmic hydroxyproline-rich protein component to the cell

wall during isolation. Both of these groups of investigators examined

the cell wall hydroxyproline content after isolating the cell walls under

extreme conditions, including solutions ranging in pH from 2 to 10 and

solutions ranging in tonicity from distilled water to 40 per cent sucrose;

the hydroxyproline content of the cell wall remained essentially the same

in all cases. In the light of the findings of Lamport and Northcote (40)

and of Dougall and Shimbayashi (14), it may be assumed that the soluble

hydroxyproline-rich proteins found by Steward and Chang (103) are









newly-synthesized cell wall proteins that have not been firmly bound to

the cell wall. Lamport (42) observed traces of soluble acid-precipitable

hydroxyproline, and Olson (69) reported that a portion of the cell wall

hydroxyproline could be extracted by sodium hydroxide, indicating that

some of the cell wall protein was less securely anchored within the cell

wall than were other portions of the hydroxyproline-rich protein. In

summary, while most of the hydroxyproline (90 per cent or more) of the

plant cell is probably associated with the primary cell wall, there is

a small amount associated with the soluble protein of the cell. Perhaps

this soluble protein hydroxyproline is newly-synthesized and is under-

going transportation to the cell wall.

The plant protein fraction that contains hydroxyproline appears

to be metabolically inert and does not turn over its carbon. Steward and

associates reported that a portion of the proteins of carrot explants is

remarkedly stable and free from protein turnover, and that this portion

of the proteins included the combined hydroxyproline (101). Steward et

al. (74, 104) also observed that once incorporated into protein, hydroxy-

proline appears to undergo little change and does not contribute appreci-

ably to the carbon dioxide which is respired. The inert nature of plant

proteins that contain hydroxyproline is similar to collagen of animals,

in which protein turnover is very slow (92).

The objective of the work reported in this part of the disser-

tation was the estimation of the hydroxyproline content of etiolated

maize and soybean seedlings. This work was a necessary prelude to the

study of hydroxyproline biosynthesis (see Part II). The hydroxyproline

content of tissues, and variation and changes in hydroxyproline content








with increasing age of seedlings, as well as the distribution of hydroxy-

proline anong organs and also among subcellular fractions was investigated,

and the results are reported.



MATERIALS AND METHODS


Plant Material

The plant materials used in this investigation were etiolated

seedlings of maize (Zea mays L., Wf9 x 38-11, fertile version) and soybean

(Glycine max [L.] Merr., Hardee variety). The seeds were soaked for 6

hours under a stream of tap water after which they were sown in vermicu-

lite in aluminum trays (24 x 13 x 7 cm); the vermiculite was thoroughly

wetted with tap water. About 40 seeds were sown in each aluminum tray.

The seedlings were grown in the dark, except for occasional short exposures

to white light, in a temperature-controlled incubator at 25 C. The plants

were watered daily with tap water. Age of seedlings was calculated from

the time of immersion of the seeds in water.

At the time of harvest, a sample of seedlings which had attained

an average size was selected. On the basis of results from preliminary

experiments, it was found convenient to harvest 8 to 10 g of maize and 5

to 7 g of soybeans; therefore, the number of seedlings in a sample varied

with the age of the seedlings. These amounts of plant material contained

from about 0.5 to 1.5 mg of hydroxyproline. The seedlings were removed

carefully from the vermiculite so as not to sever any roots. The vermicu-

lite adhering to the roots was washed off under a stream of tap water.

The endosperm of the maize seed and the seed coat of the soybean was re-

moved and discarded. The seedlings were then blotted with paper toweling

to remove surface water, and the fresh weight was recorded immediately.









In some cases, the dry weight of seedlings was determined. Five

average seedlings were chopped with a razor blade and placed in a tared

weighing bottle. The plant material was dried to constant weight in an

oven at 95 C, after which the dry weight was recorded; on the basis of

preliminary experiments, it was found that 48 hours was a sufficient

drying period.



Acid Hydrolysis

The weighed fresh plant samples were chopped finely with a razor

blade and ground at room temperature in deionized water with mortar and

pestle. After 1 to 2 minutes of grinding, the slurry was poured into a

glass boiling flask. In order to produce a 6 N hydrochloric acid solution,

an amount of concentrated hydrochloric acid equal to the amount of water

used in the grinding process (20 to 25 ml) was added. Enough 6 N hydro-

chloric acid was then added to produce a volume equal to ten times the

fresh weight of the plant sample. The mixture was boiled under a water-

cooled reflux condenser for 24 hours. The temperature of the boiling

mixture was approximately 108 C.



Isolation of Amino Acids

After cooling, the acid hydrolysate was filtered through Whatman

No. 1 filter paper on a Buchner funnel. The hydrochloric acid was evapo-

rated in a Buchler rotary evaporator which was held at 50 C and evacuated

with a water aspirator. The resulting residue was dissolved in 40 to 50

ml of deionized water and stirred with 2 g of activated carbon for 1 hour

on a magnetic stirrer to remove humin and pigments. The carbon was then

removed by filtration through Whatman No. 2 filter paper on a Buchner


funnel.









The decolorized hydrolysate containing the amino acids was added

to the top of a 20 x 1.5 cm column filled with Amberlite IR-120(11+) medium

porosity cation exchange resin. The column was then washed with deionized

water until the effluent was neutral to pH paper. The amino acids were

then eluted from the column with 2 N ammonium hydroxide solution until

the effluent was very basic to pH paper; the ammonium hydroxide solution

was followed by deionized water until the effluent was slightly basic to

pH paper. These two eluants were combined and contained all of the amino

acids. A flow rate of approximately 1 ml/minute was maintained through-

out. The column was operated at room temperature.

The resin was regenerated to the hydrogen form by washing with

1.5 N hydrochloric acid until the effluent was very acid to p1H paper; the

hydrochloric acid was followed by deionized water until the effluent was

free of chloride ions; freedom from chloride ions was demonstrated by

the use of 1 per cent silver nitrate solution.

The solution containing the amino acids was evaporated to dryness

on the rotary evaporator operated at 50 C. The residue was dissolved in

either 10 or 25 ml of 0.05 N hydrochloric acid. The solution containing

the amino acids was stored in the refrigerator at 5 C until hydroxyproline

determinations were performed.



Fractionation of Tissues

In order to determine the amount of hydroxyproline in various

cellular fractions, some seedlings were fractionated into "cell wall,"

"protein" and supernatantt" fractions. The seedlings were homogenized

for 2 minutes with ice-cold deionized water in a Waring blendor, follow-

ed by 2 minutes of grinding in a motor-driven homogenizer equipped with a









Teflon tip. The "cell wall" fraction was isolated by filtration through

Miracloth (Chicopee Manufacturing Corp., Milltown, N. J.) (69, 87)

followed by three washings with 5 ml portions of ice-cold water. To

obtain the "protein" fraction an equal volume of 10 per cent trichloro-

acetic acid was added to the filtrate, which was permitted to stand at

room temperature for 30 minutes; the precipitate was collected by centri-

fugation at 10,000 x g for 10 minutes at 0 C and was designated "protein."

The "cell wall" and "protein" fractions were hydrolyzed, and the amino

acids were isolated as previously described. The supernatantt" fraction

was treated with activated charcoal, and the amino acids were isolated

but without acid hydrolysis.



Colorimetric Determination of Hydroxyproline

Hydroxyproline was estimated in amino acid mixtures by the colori-

metric method of Neuman and Logan (66) as modified by Leach (48). The

method involves the oxidation of hydroxyproline with hydrogen peroxide in

the presence of alkaline copper sulfate. The oxidized product is then

reacted with p-dimethylaminobenzaldehyde (Ehrlich's reagent) to produce

a red chromophore with an absorption maximum at 556 mp. The identity

of the oxidation product of hydroxyproline has not been definitely

determined. When the oxidation was performed by chloramine T, Bergman

and Loxley (2) determined that the product was neither pyrrole, pyrrole-

2-carboxylic acid nor A' pyrroline-4-hydroxy-2-carboxylic acid. Also,

Blomfield and Farrar (4) oxidized hydroxyproline with hydrogen peroxide

and concluded that the oxidation product was not pyrrole-2-carboxylic

acid, but a substance readily converted to it.









The presence of salt depresses the absorbance of the colored end-

product (4). In order to compensate for this depression, all solutions

which were analyzed were of the same concentration of hydrochloric acid.

Solutions of amino acids were stored in dilute acid to prevent microbial

decomposition.

A stock solution of 100 )ig/ml of hydroxyproline was prepared by

dissolving 50 mg of crystalline hydroxyproline in 0.5 N hydrochloric acid

in a final volume of 500 ml. This solution could be stored for several

months at 5 C without detectable loss. Standard solutions of 5, 10 and

15 Pg/ml of hydroxyproline were prepared from the stock solution by

dilution with 0.05 N hydrochloric acid.

Unknown solutions which were to be analyzed were diluted to insure

a concentration of 15 )g/ml or less of hydroxyproline. Analysis of un-

known solutions as well as standards were run in triplicate. The average

reading for the three determinations was used in the calculations.

One ml of each of the hydroxyproline standard solutions as well

as 1 ml of each of the unknown solutions were pipetted into separate 25

x 150 mm test tubes. A tube containing 1 ml of 0.05 N hydrochloric acid

was carried through the procedure as a reagent blank. One ml of 0.05 N

copper sulfate and 1 ml of 2.5 N sodium hydroxide were added to the

hydroxyproline solutions in that order. The test tubes were placed in

a 40 C water bath for 10 minutes. One ml of 6 per cent (v/v) hydrogen

peroxide, prepared immediately before using, was then added to each tube.

The contents were thoroughly mixed by swirling the test tubes. After

heating in the water bath for an additional 10 minutes, the test tubes

were cooled in tap water. Four ml of 3.0 N sulfuric acid was then added,

followed by 2 ml of 5 per cent (w/w) p-dimethylaminobenzaldehyde in









1-propanol solution. (The p-dimethylaminobenzaldehyde solution was

freshly prepared each day.) The test tubes were lightly corked and heated

in a 70 C water bath for 16 minutes to develop the chromophore; then

the tubes were cooled immediately in tap water, and the optical absorbance

was read in 1 cm cuvettes against the reagent blank at 556 qi in a Beckman

Model DB spectrophotometer.

A standard curve (figure 5) of hydroxyproline concentration

against absorbance at 556 mp was constructed. In order to minimize a

slight deviation from Beer's law, a straight line was drawn between

successive pairs of points instead of fitting a straight line to all of

the points. Also, a slight variation of the standard curve from day to

day was eliminated by constructing a standard curve for each analysis.



RESULTS



Plant Material

Maize and soybean seedlings could be grown in the etiolated

condition at 25 C for about 14 days before visual signs of senescence

and death began to appear. The fresh weight of maize seedlings increased

rapidly from about 0.3 g/seedling at 1 day of age to a maximum of about

1.5 g/seedling at 12 days of age (figure 2). The dry weight of maize

was approximately 0.2 g/seedling at 1 day and decreased to 0.1 g/seedling

at 14 days of age (figure 2). The fresh weight of soybean seedlings

increased from about 0.3 g/seedling at 1 day to approximately 1.2 g/

seedling at 12 days of age while the dry weight decreased from about

0.12 g/seedling to about 0.08 g/seedling at 14 days of age (figure 3).













Fresh Weight
Dry Weight


0 2 4 6 8 10 12 14

AGE (DAYS)


Figure 2. -- The variation in fresh and dry weight of
etiolated maize seedlings with increasing age.
















CO



0-
0






LU


i.6



1.2



0.8



0.4


Fresh \!c"hI
Dry Wckt


2 4 6 8 10 12


AGE (DAYS)


Figure 3. -- The variation in fresh and dry weight of
etiolated soybean seedlings with increasing age.


2.0









Estimation of Hydroxyproline

The proteins of the plant material were hydrolyzed in 6 N hydro-

chloric acid (see page 9). To test whether hydrolysis by refluxing

destroyed any of the hydroxyproline, one sample of plant material was

hydrolyzed by refluxing at 108 C for 24 hours; a second equivalent

sample was hydrolyzed in a sealed tube at 108 C for 24 hours, and a third

equivalent sample was hydrolyzed in a sealed tube at 140 C for 4 hours.

No differences in the hydroxyproline content of the three plant samples

were found. Since the refluxing method was more convenient, it was used

routinely.

The Neuman and Logan method for the determination of hydroxy-

proline may be used on crude hydrolysates. However, the oxidation step

by hydrogen peroxide is difficult to control, and varible results are

sometimes obtained from crude hydrolysates. In order to eliminate as much

variation as possible, the amino acids in the acid hydrolysate were isolat-

ed by cation exchange chromatography. The recovery of hydroxyproline

from Amberlite IR-120(H+) columns was consistently 90 per cent or higher.

The Neuman and Logan method is not completely specific for hydroxy-

proline. Tyrosine was reported to yield 1.5 to 2.0 per cent as much color

as hydroxyproline while tryptophan yielded 0.7 to 1.3 per cent as much

(49, 62, 66). The color yield of equal molar quantities of the twenty

normally-occurring protein amino acids in addition to hydroxyproline is

shown in table 1. Tryptophan gave 1.1 per cent as much color as hydroxy-

proline, and tyrosine gave 1.5 per cent as much. The other amino acids

tested did not yield measurable color. Samples of tryptophan and tyrosine

tested after treatment with activated carbon did not yield a measurable

amount of color. Since all acid hydrolysates were treated with activated






17
Table 1. -- Color yield of amino acids in the Neuman and Logan
determination of hydroxyproline.



Per cent of
Amino acid A(556) hydroxyproline


Hydroxyproline 0.226 100

Alanine 0.000 0.0

Arginine 0.000 0.0

Asparagine 0.000 0.0

Aspartic acid 0.000 0.0

Cysteine 0.000 0.0

Glutamic acid 0.000 0.0

Glutamine 0.000 0.0

Glycine 0.000 0.0

Histidine 0.000 0.0

Isoleucine 0.000 0.0

Leucine 0.000 0.0

Lysine 0.000 0.0

Methionine 0.000 0.0

Phenylalanine 0.000 0.0

Proline 0.000 0.0

Serine 0.000 0.0

Threonine 0.000 0.0

Tryptophan 0.003 1.1
carbon treated 0.000 0.0

Tyrosine 0.004 1.5
carbon treated 0.000 0.0

Valine 0.000 0.0









carbon before analyzing for hydroxyproline, it is assumed that the method

as used here was specific for hydroxyproline. The absorption spectrum of

the chromatophore produced by 15 pg of hydroxyproline is given in figure

4; the maximum absorption was at 556 mp. A typical standard curve of

hydroxyproline concentration against absorbance at 556 mp is plotted in

figure 5.



Hydroxyproline Content of Various Species of Etiolated Seedlings

Etiolated maize seedlings were selected as the plant material for

this investigation because of previous work with this species (18). In

addition to maize, it was of interest for comparative purposes to in-

vestigate hydroxyproline biosynthesis in a dicotyledonous species. The

hydroxyproline content of soybean, pea, peanut and sunflower as well as

maize are compared in table 2. Both soybean and pea have a high content

of hydroxyproline and also show an increase of hydroxyproline on a fresh

weight basis between 5 and 10 days of age, whereas sunflower showed only

a slight increase, and peanut showed a decrease with increasing age.

Since the study of oxygen fixation into hydroxyproline was a major

objective of this work, it was desirable to select a species which would

produce the maximal amount of hydroxyproline; therefore, the choice of

a dicotyledonous species was between soybean and pea. Because soybean

seedlings appeared to have a more uniform growth pattern in the etiolated

condition than pea, soybean was selected as the dicotyledonous species

to be used in subsequent experiments.


Hydroxyproline Content of Maize and Soybean Seedlings as a Function of Age

In order to determine the amount and variation of hydroxyproline

in maize and soybean seedlings and also possibly the period of greatest





















0.4-
0 -





0.2


0.1-




460 500 540 580 620

WAVE LENGTH (mp,)


Figure 4. -- The absorption spectrum produced by hydroxyproline
in the Neuman and Logan method.














0.32

0.28

0.24


0.20


0.16 -


0.12-

0.08 -

0.04-

I I f I I I I I
5 10 15

HYDROXYPROLINE (pLg/ml)


Figure 5. -- Standard curve relating absorbance and
hydroxyproline concentration.









Table 2. -- Hydroxyproline content of various species of etiolated
seedlings at five and ten days of age.




Hydroxyproline ug/g fresh weight

5 days 10 days




Maize 55 52


Alaska pea 148 275


Peanut 430 185


Soybean 135 225


Sunflower 48 62


__









hydroxyproline synthesis, the amount of hydroxyproline in maize and soybean

seedlings was determined from 0.25 day to 15 days of age. The 0.25 day

samples were taken after the 6 hour soaking period. The hydroxyproline

variation of maize and soybean expressed on a seedling basis is shown in

figure 6. The hydroxyproline of maize seedlings at 0.25 day is 8 ,"g/

seedling and increases to 80 pg/seedling at 12 days of age. After 12 days

of age, hydroxyproline begins to slowly decrease in the maize. Soybean

seedlings, on the other hand, contain 35 pg/seedling at 0.25 day of age

and increases to 300 ;ig/seedling at 14 days of age and then begins to

decrease. The maximal amount of hydroxyproline (300 pg) in soybean

seedlings is approximately four times the maximal amount found in maize

(80 pig) when expressed on a seedling basis.

The differences between maize and soybean were also very striking

when hydroxyproline was expressed on a fresh weight basis (figure 7).

Both maize and soybean had 140 pg/g at 0.25 day. The hydroxyproline of

maize rapidly decreased to 50 Pg/g at 5 days of age and remained at that

level until the end of the sampling period, which was 15 days of age.

The hydroxyproline of soybean also initially decreased from 140 pg/g to

125u g/g at 3 days of age, but then increased to a maximum of 260 pg/g

at 13 days of age. The amount of hydroxyproline in soybean seedlings at

13 days of age (260 ug/g) was approximately five times the amount in

maize at the same age (50 1g/g).

The initial decrease of hydroxyproline in both maize and soybean

when expressed on a fresh weight basis was probably a reflection of the

change in fresh weight of the seedlings rather than a change in the

hydroxyproline content. As shown in figure 6, the amount of hydroxy-

proline per seedling actually increased somewhat during the first few














300- 0--D Soybecn
0----o aize e

250-


200 -


150-


100 -


50



0 2 4 6 8 10 12 14

AGE (DAYS)


Figure 6. -- The variation in hydroxyproline content of
etiolated maize and soybean seedlings with increasing seedling age.
(The hydroxyproline content is expressed in micrograms per seedling.)














Soybean


Maize


S250


. 200- 0






0 0
S100-
00






0 2 4 6 8 10 12 I

AGE (DAYS)



Figure 7. -- The variation in hydroxyproline content of
etiolated maize and soybean seedlings with increasing seedling age.
(The hydroxyproline content is expressed in micrograms per gram of
fresh weight.)
fresh weight. )









days, but this small increase was more than offset by a very large in-

crease in fresh weight figures2 and 3); the net effect was that of a

decrease in hydroxyproline on a fresh weight basis.



Hydroxyproline Content of Seedling Parts as a Function of Ago

It was of interest to determine the distribution of hydroxyproline

in the cotyledons, roots and shoots of etiolated maize and soybean seed-

lings. The hydroxyproline content of the cotyledon (scutellun) of maize

was constant throughout the life of the plant, and was approximately 8

)jg/seedling (figure 8) and 140 ug/g of fresh weight (figure 9). The

amount of hydroxyproline in both maize roots and shoots increased almost

identically from 4 pug at 3 days of age to 40 ,g at 14 days of age (figure

8). When plotted on a fresh weight basis, the maize shoot was found to

contain 60 pg/g and the root had approximately 45 ,g/g from 3 days to

14 days of age (figure 9).

The cotyledons of soybean showed a slight increase in hydroxy-

proline content during the period examined, beginning at 40 jug at 1 day

of age and increasing to 45 pg at 14 days of age (figure 10). An increase

was also noted in soybean cotyledons when the hydroxyproline was plotted

on a fresh weight basis, increasing from 140 jg/g at 1 day of age to

160 ig/g at 14 days of age. The hydroxyproline of soybean roots and

shoots was almost identical when plotted on an organ basis (figure 11);

hydroxyproline increased from 20 pig at 3 days of age to 160 ug at 14 days

of age. However, when plotted on a fresh weight basis, the soybean roots

and shoots are quite dissimilar (figure 11) with respect to hydroxyproline

content. Beginning at 3 days of age, the hydroxyproline of roots rose

rapidly from 150 ag/g to 425 ug/g at 14 days. On the other hand, the












Cotyledons
Shoot
Root


I I I I 1 I I I I I 1 1
2 4 6 8 10 12


AGE (DAYS)


Figure 8. -- The variation in hydroxyproline content in several
organs of etiolated maize seedlings. (The hydroxyproline content is
expressed in micrograms per organ.)


50h


c:
0


U)






0
C
cr


40-


301


20-


IOh


0













Cotyledon
Shoot
Root


(, n 0 0 0 0
0 0
0
-OoOO-- a < --- --- O O
O


A A
AA A A -- / I'


I I I I I I I I I I I I I 14
2 4 6 8 10 12 14


AGE (DAYS)



Figure 9. -- The variation in hydroxyproline content in
several organs of etiolated maize seedlings. (The hydroxyproline
content is expressed in micrograms per gram of fresh weight.)


200k


150


l00-


50












Cotyledons
Shoot
Root


50h


I I I I I I I I I I I I I I


2 4 6


8 10


AGE (DAYS)


Figure 10. -- The variation in hydroxyproline content in several
organs of etiolated soybean seedlings. '(The hydroxyproline content is
expressed in micrograms per organ.)


200


150




100












o--o Cotyledons
--o Shoot
-- Root'


1 1 I I I I I I I I I I I 1


6 8 10


14


AGE (DAYS)


Figure 11. -- The variation in hydroxyproline content in several
organs of etiolated soybean seedlings. (The hydroxyproline content is
expressed in micrograms per gram of fresh weight.)


450-

400-

450-

300-

250-


200

150

100

50


0 2









hydroxyproline content of shoots dropped from 80 pg/g at 3 days to 60

)lg/g at 6 days after which it increased to 210 ug/g at 14 days of age.


Hydroxyproline of Subcellular Fractions

It has been reported that 90 per cent or more of the cellular

hydroxyproline of plants is associated with the cell wall (see Introduction

to Part I). Because almost all of this reported work was done with cells

in tissue culture, it was of some interest to examine maize and soybean

seedlings in this respect. When seedlings were fractionated as described

in the Materials and Methods, the bulk of the hydroxyproline was found

with the cell wall fraction. Maize seedlings of several ages were analyzed

and the results recorded as the per cent of the total hydroxyproline in

each fraction (table 3). The per cent of the hydroxyproline in the cell

wall of maize was more or less constant at approximately 75 per cent of

the total. The protein fraction contained 18 to 20 per cent while the

supernatant fraction had 5 to 6 per cent. No real differences were noted

with increasing age of seedlings. For soybean seedlings, the cell wall

contained approximately 80 per cent of the total hydroxyproline at 3 days

of age, which increased to 93 per cent at 14 days of age (table 4). At

the same time, the per cent of the total hydroxyproline in the protein

fraction was decreasing from 18 per cent at 3 days of age to 5 per cent

at 14 days of age. The supernatant fraction was constant at about 2

per cent throughout.









Table 3. -- Distribution of hydroxyproline among cell wall, protein
and supernatant fractions of etiolated maize seedlings.




Age of seedlings Per cent of total hydroxyproline
(days)
cell wall protein supernatant


77.4


76.2


75.1

69.8


69.6


77.0


78.7


83.3


17.1

16.7


18.1


21.1


21.8


16.9


15.2

11.1


75.8 18.2


5.5


7.1

6.8


9.1

8.6


6.1


6.1


5.6

6.0









Table 4. -- Distribution of hydroxyproline among cell wall, protein
and supernatant fractions of etiolated soybean seedlings.


Age of seedlings
(days)


Per cent of total hydroxyproline

cell wall protein supernatant


79.8


79.4


81.0


84.0


89.1


92.2


92.0


90.3


92.3


93.0


17.9


18.0


17.1


13.9


9.4


5.9

6.0


7.7


5.6


5.1


2.3


2.6


1.9


2.1


1.5


1.9


2.0


2.0


2.1


1.9










DISCUSSION



The data reported here show that the hydroxyproline content of

individual seedlings of maize and soybean increased with increasing age

of the seedlings (figure 6). As the plant grows and produces new cells,

a greater quantity of all the normal constituents would be expected to

be elaborated; thus, the results appear to be reasonable and are those

which might be expected. However, when the hydroxyproline content was

plotted on a fresh weight basis (figure 7), there was an initial decrease

of hydroxyproline in both maize and soybean, probably due to the rela-

tively large uptake of water during the first few days of germination.

After this initial decrease, the hydroxyproline content of maize re-

mained at a constant level, whereas hydroxyproline content of soybean

increased markedly (figure 7). It appears that after the initial period

of rapid water uptake following germination, hydroxyproline synthesis

occurred in maize seedlings at about the same rate as the fresh weight

increased, whereas in soybean seedlings, hydroxyproline was synthesized

at a rate which was much more rapid than the rate of fresh weight in-

crease. At 13 days of age, soybean contained five-fold more hydroxy-

proline than maize seedlings on a fresh weight basis. It is not easy to

assign a reason to such a large difference in the hydroxyproline content

of maize and soybean. In his review, Lamport (47) pointed out that the

high hydroxyproline content of young herbaceous dicotyledons, particular

legumes, contrasts sharply with the relatively low hydroxyproline content

in monocotyledons, particular the grasses, at least for those species

which have been examined. Therefore, the large differences observed here










between maize monocott) and soybean dicott) lend support to Lamport's

(47) observations.

Another point that warrants discussion concerns the observed

changes in hydroxyproline content in different tissue types (organs) of

maize and soybean. The cotyledon (scutellum) of maize had a constant

amount of hydroxyproline throughout the sampling period, 1 day to 14 days

of age (figure 9); on the other hand, the cotyledons of soybean showed a

slight increase in hydroxyproline content with increasing age (figures

10 and 11). This increase in hydroxyproline of soybean cotyledons is

surprising because cotyledons are considered to be storage organs and

may be presumed not to be actively growing nor synthesizing new protein.

However, Lamport (45) also observed that traces of hydroxyproline were

synthesized in the cotyledons of germinating peas.

With respect to maize roots and shoots, the hydroxyproline con-

tent reflects that situation found in the intact plant, namely that there

was a constant amount of hydroxyproline on a fresh weight basis, at least

after the period of relatively large uptake of water during the first few

days of germination (compare figure 7 with figure 9). On the other hand,

the situation found in the roots and shoots of soybean is different from

that in maize. When expressed on a fresh weight basis, the roots of the

soybean seedling contain two-fold or more hydroxyproline than the shoots.

A possible explanation for this large difference between these tissues

is that the shoots of etiolated soybean represent predominantly hypocotyl

material; the epicotyl does not begin to elongate appreciably until 10

days of age. Because the hypocotyl of soybean seedlings elongates very

rapidly in the etiolated condition, it is possible that the synthesis of

hydroxyproline does not keep pace with the rapid fresh weight increase.









Lamport (47) reported that young pea roots are much richer in hydroxy-

proline than are young pea epicotyls. These differences in hydroxyproline

content between shoot and root tissue may be a reflection of different

types of cell wall structures in these different tissues.

With respect to localization of hydroxyproline within the cell,

the major portion of the hydroxyproline of etiolated maize and soybean

seedlings was found in the cell wall fraction (tables 3 and 4). However,

some hydroxyproline was also always found to be present in fractions which

were not associated with cell wall material (tables 3 and 4). The hydroxy-

proline which is found in the cytoplasmic protein fraction may represent

newly-synthesized protein which is not securely anchored within the cell

wall. The very small amount of hydroxyproline which is present in the

supernatant fraction may have originated from a hydroxyproline inter-

mediate in protein synthesis (see Part III) or from degradation of the

protein which contains hydroxyproline. Contrary to the situation found

by Steward (see Introduction to Part I) in carrot explants, the data

found here may be interpreted to indicate that there may be some turnover

of the protein which contains hydroxyproline in an intact plant. Because

much cell differentiation and cell death is occurring in an intact plant,

it is possible that some of the cell wall protein may be metabolized and

may release free hydroxyproline.

The existence of a hydroxyproline-rich protein associated with the

primary cell wall has prompted speculation as to its possible function.

Steward et al. (100) found that rapidly dividing cells in potato tuber

and carrot root explants of cell cultures in the presence of coconut milk

contain several-fold more hydroxyproline than non-growing cells of the

same tissues. Also, Newcomb (67) commented that it is not clear why









hydroxyproline should be relatively low in tissues taken from intact

plants, when it is such a prominent constituent of cells grown in tissue

culture. Newcomb (67) suggested that differences in chemical composition,

physical structure, or mode of formation of cell walls might underlie the

large differences observed in wall protein between tissue cultures and

tissues of an intact plant. Steward et al. (100) proposed that plant

proteins which contain hydroxyproline must in some way be characteristic

of cells in the randomly-dividing state. Lyndon and Steward (53) specu-

lated that the protein which contains hydroxyproline in the growing carrot

and potato explants may be associated with the phragmoplast (the primitive

cell wall produced during telophase at the equator of the dividing cells)

and with the synthesis of new cell wall material which must go on at a

rapid rate to keep pace with the division and growth of the cells. Lamport

(44) has given the name extension to the hydroxyproline-rich protein of

the primary cell wall. He proposed that this specific protein is directly

involved in cell wall structure as well as in the regulation and control

of cell wall extension by providing a network of labile cross-linkages

between the cellulose microfibrils. Lamport (47) reasoned that cell walls

containing such a protein-polysaccharide complex would have a greater

tensile strength than cell walls not having such a complex. Thus, the

protein which contains hydroxyproline may be considered to be a "struc-

tural" protein because it would be contributing to the strength and

rigidity of the cell wall. In another aspect of Lamport's hypothesis,

the protein-polysaccharide cross-linkages may sometimes be broken, and

when this happens, then the cell wall would have less strength and rigidity

and be more able to expand or extend, thus allowing growth to occur. In









this sense, the hydroxyproline-rich cell wall protein could be considered

a "regulatory" protein.

Assuming that the protein which contains hydroxyproline in the

plant cell wall has a structural function, it is tempting to speculate

whether there is a correlation between the morphological and anatomical

differences of maize and soybean and the hydroxyproline content of these

plants. In this connection, the contributions of Schwendener (89) are

pertinent. Schwendener analyzed the mechanical means of support of

plant tissues; he considered the monocotyledons to be a more advanced

group of plants than dicotyledons, not only in terms of the distribution

and arrangement of sclerenchyma and vascular elements within specific

tissues but also in terms of the contributions of these specialized cells

to mechanical support of the tissues. Schwendener considered that

herbaceous dicotyledons, on the other hand, rely more on cell turgidity

than on specialized cell structures for mechanical support. Assuming

Schwendener was correct, then one might expect to find chemical differences

in cell wall composition of monocotyledons and dicotyledons. To pursue

this line of reasoning, if dicotyledons lack an extensive system of

specialized cells for mechanical support, then the cell walls of dicoty-

ledons may require greater strength and ridigity to support the plant than

would be needed if an extensive system of specialized cells contributing

to mechanical support were present. Thus, if a structural role is assigned

to the hydroxyproline-rich protein of the plant cell wall, then it is not

surprising to find more hydroxyproline in the cell walls of dicotyledons

than in the cell walls of monocotyledons.















PART II. BIOSYNTHESIS OF HYDROXYPROLINE
BY OXYGEN FIXATION



INTRODUCTION



Prior to 1955, the only known biological oxidations in which

molecular oxygen had a direct role were those involving the reduction of

the oxygen to either water or hydrogen peroxide; enzymes which catalyze

these reactions are known as oxidases or oxidoreductases (82). In his

famous book, On the Mechanism of Oxidation, Wieland stated (111):

Limiting ourselves to the chief energy-supplying
foods, we have in this class carbohydrates, amino acids,
the higher fatty acids, and glycerol. There is no known
example among them of an unsaturated compound in the
case of which it is necessary to assume direct addition
of oxygen, that is, additive oxidation.

In 1955, Mason et al. (58) with the use of isotopic oxygen-18

demonstrated that during the oxidation of 3, 4-dimethylphenol to 4, 5-

dimethylcatechol by the phenolase complex from mushrooms that the oxygen

atom incorporated into the substrate was derived exclusively from mole-

cular oxygen. Also in 1955, Hayaishi and associates (28) were able to

show that the oxygen introduced into the substrate during the oxidation

of catechol to cis-cis-muconic acid by pyrocatechase from Pseudomonas

came from oxygen gas and not from water. These findings were in direct

contrast with the accepted view that the sole function of molecular

oxygen was that of an electron acceptor in terminal oxidation. Thus,









the phenomenon of "oxygen fixation" or the direct addition of molecular

oxygen to organic substrate became known.

As a result of recent intensive investigations into the mechanism

of oxygen fixation, it has become apparent that there are two types of

additive oxidation reactions catalyzed by two kinds of enzymes: (1)

enzymes that add both atoms of an oxygen molecule to a molecule of sub-

strate:
S + 02 ___. S302

(2) enzymes that incorporate one atom of an oxygen molecule into a sub-

strate while the other oxygen atom is reduced to water in the presence

of an appropriate electron donor:

S + 02 + AH2 SO + H120 + A

Enzymes of the first type are called oxygenases (29, 82). The second

class of enzymes have been termed mixed function oxidases (57) or mixed

function oxygenases (29) in reference to their dual role of oxygenation

and oxidation. The more concise term hydroxylase (56) is now preferred

for enzymes of the second type (82). Most oxygenases and hydroxylases

are similar to oxidases in that they are metallo-proteins, containing

iron or in some cases copper (27).

Since 1955, many enzymes from microbial, plant and animal sources

have been classified as oxygenases and hydroxylases. Many of the reactions

catalyzed by these enzymes have been studied and characterized by experi-

ments -with oxygen-18. The properties of oxygenases and hydroxylases have

recently been extensively reviewed (26, 27, 56).

Hydroxylase enzymes exhibit somewhat paradoxical requirements (27):

oxygen is necessary as the specific oxidizing agent and an electron donor

is needed as a reductant. In most hydroxylation reactions, NADPH serves









as the electron donor, but in many microbial systems, NADH is utilized

(26, 27). Other known electron donors include ascorbic acid, dihydroxy-

fumaric acid and tetrahydropteridine (26, 39). In the case of lactic

oxidative decarboxylase, which catalyzes the oxidative decarboxylation

of lactate to acetate, the substrate itself serves as the electron donor

(30); thus, the substrate, L-lactate accepts one atom of oxygen from

oxygen gas and simultaneously provides two hydrogen atoms to reduce the

other atom of oxygen gas to water.

The amino acid hydroxyproline is probably synthesized in cells

by enzymes having hydroxylase activity. The earliest report that mole-

cular oxygen has a direct role in hydroxyproline biosynthesis was that of

Scharpenseel and Wolf (88). While studying the conversion of proline-C-14

to hydroxyproline-C-14 in fetal rat and chick skin preparations, they

observed that the transition was dependent upon a supply of molecular

oxygen. Later, Fujimoto and Tamiya (19) incubated chick embryos in an

atmosphere enriched with oxygen-18 and concluded that 40 to 60 per cent

of the hydroxyl oxygen in hydroxyproline was derived from atmospheric

oxygen. In more refined experiments (20), Fujimoto and Tamiya were able

to increase their estimation of molecular oxygen incorporation into hydroxy-

proline to 80 to 93 per cent. Also in 1962 and 1963, Prockop et al. (76,

77) incubated chick embryos in oxygen gas enriched with oxygen-18 and in

water labeled with oxygen-18 and found that the hydroxyl oxygen of hydroxy-

proline was derived exclusively from molecular oxygen. The only work to

date concerning the biosynthesis of hydroxyproline by molecular oxygen

in higher plants is that of Lamport, who grew cell suspensions of sycamore

(Acer pseudoplatamus L.) in an atmosphere enriched with oxygen-18 (44).









He interpreted his results to indicate that all of the hydroxyl oxygen

of primary cell wall hydroxyproline came from molecular oxygen.

The work undertaken in this part of the dissertation has as its

objective the demonstration that the hydroxyl oxygen of hydroxyproline in

etiolated maize and soybean seedlings originates in molecular oxygen. The

techniques used to fulfill this objective involved growing etiolated seed-

lings of maize and soybean in an atmosphere enriched with oxygen-18. The

hydroxyproline in the seedlings was then isolated and the oxygen-18 en-

richment determined by mass spectrometry.



MATERIALS AND METHODS



Plant Material

Maize and soybean seeds which had been soaked for 6 hours in tap

water were sown in vermiculite in 250 ml Erlenmeyer flasks. Twelve seeds

were usually placed in each flask. The seedlings were grown at 25 C in

a temperature-controlled incubator in the dark except for occasional

short exposures to white light. The age of seedlings was calculated from

the time of immersion of the seeds in water.

Since the atmospheres in which the seedlings were grown were en-

riched at intervals with oxygen gas labeled with oxygen-18, it was neces-

sary to seal the Erlenmeyer flasks (see page 44) and to change the com-

position of the atmospheres as desired. The necessity of using closed

containers required at the outset a determination of the conditions under

which the seedlings could be grown satisfactorily. It was found that

normal growth of etiolated seedlings could be obtained in small closed

chambers if they were supplied with sufficient water at the beginning of









the experimental period and if an efficient carbon dioxide absorbent was

placed in the chamber. A ratio of 10 g of dry vermiculite to 50 ml of

water was found to give good seedling growth. This amount of water was

sufficient to enable the 10 to 12 seedlings in each flask to grow to 7

or 8 days of age but was not so much as to impair root growth.

In addition to the need for an adequate supply of water, it was

necessary to remove respiratory carbon dioxide so as to prevent deleterious

effects to the seedlings. A further advantage to the continuous removal of

respired carbon dioxide during the experiment is the production of a

partial vacuum in the sealed Erlenmeyer flask, a condition which permitted

the addition of an equal volume of oxygen gas at regular intervals. The

carbon dioxide absorbent was placed in a small vial, and the vial was

placed in an upright position inside the Erlenmeyer flask at the beginning

of each experiment. A 40 per cent solution of potassium hydroxide was

found to absorb the respired carbon dioxide quite satisfactorily. When

a vial containing 5 ml of this solution together with a short fluted

filter paper wick --- one end of which was dipped into the potassium

hydroxide solution --- was placed into the seedling flasks, then the

seedlings grew quite well. A disadvantage to the use of this strong

alkali solution was the danger of spillage. However, if the vial which

was used was long enough (about 7 cm) to reach the neck of the Erlenmeyer

flask, the danger of spillage was eliminated for all practical purposes.



Preparation of Oxygen-18 Gas

Water containing approximately 10 atom per cent oxygen-18 was

purchased from YEDA Research and Development Co., Ltd., Rehovoth, Israel.










Four ml of labeled water were pipetted into the electrolysis cell and

enough concentrated sulfuric acid was added to make a 5 N solution.

The electrolysis cell was fitted to a mercury regulator and a

drying chamber containing magnesium perchlorate desiccant and soda lime;

the drying chamber was attached to a Toepler pump on a vacuum manifold

evacuated to 1 p of mercury or less. Also, a 1 liter gas storage flask

was fitted to the Toepler pump. By applying a slight vacuum to the water

during electrolysis, the oxygen gas was collected in the gas storage

flask; the evolved hydrogen gas escaped into the room. The mercury regu-

lator served to stabilize and regulate the flow rate of the oxygen gas.

The purpose of the drying chamber was two-fold: (1) the magnesium per-

chlorate was used to dry the oxygen gas, and (2) the soda lime was used

to decompose the ozone which was produced in the electrolysis process;

that is, ozone was reduced to oxygen in the presence of soda lime (6,

34, 81).

During operation, a direct current of 2 amp was applied to the

platinum electrodes of the electrolysis cell. After 5 minutes of flush-

ing the system with the oxygen gas produced by electrolysis, the stop-

cock to the vacuum pump was closed. The oxygen gas was collected by the

downward displacement of mercury in the Toepler pump. The electrolysis

cell was immersed in an ice-water bath during the electrolysis process.

Approximately 3 hours were required to generate 1 liter of gas.

The labeled oxygen gas was stored over a solution called the

displacing fluid which was contained in a separatory funnel and fitted

to the gas storage flask. The displacing fluid consisted of a solution

of 20 per cent sodium sulfate in 5 per cent sulfuric acid solution. Its

purpose was the prevention of the loss of oxygen gas by dissolution in

water.









Mixtures of approximately 80 per cent nitrogen gas and 20 per

cent oxygen gas were prepared with the use of the vacuum manifold and a

mercury manometer. To determine the exact composition of the gas mixtures,

an aliquot was analyzed with a Beckman E2 Oxygen Analyzer. The atom per

cent oxygen-18 of enriched gas mixtures was determined by analyzing an

aliquot with the mass spectrometer.



Application of Oxygen-18 Gas to Seedlings

To apply an oxygen-18 enriched atmosphere to seedlings, the flasks

containing the seedlings were fitted with one-hole rubber stoppers into

which a short piece of glass tubing was inserted. A short piece of rubber

tubing equipped with a screw clamp was connected to the glass tubing.

The flask containing the seedlings was connected to the gas storage flask

and to water aspirator vacuum by a three-way stopcock. The seedling

flask and the stopcock assembly were evacuated for 2 minutes with a water

aspirator at 22 mm of mercury. The stopcock to the vacuum line was then

closed, and the stopcock of the gas storage flask was opened, allowing

the displacing fluid to push the gas mixture (20 per cent oxygen and 80

per cent nitrogen) into the seedling flask until 1 atm of pressure was

reached. Oxygen consumed by the seedlings was replaced with 100 per cent

oxygen gas of the same oxygen-18 content every 12 hours by the same pro-

cedure.


Application of Water Labeled with Oxygen-18 to Seedlings

In some experiments, seedlings were grown in water labeled with

oxygen-18. Seeds which had been soaked in tap water were sown in 10 g

of dry vermiculite in 250 ml Erlenmeyer flasks, and 50 ml of water









containing 1.52 or 1.33 atom per cent excess oxygen-18 was then added to

the vermiculite. Water labeled with oxygen-18 was purchased from Bio-Rad

Laboratories, Richmond, California. The flasks were handled as described

in the previous section, except that the seedlings were grown in normal

(0.204 atom per cent) oxygen gas instead of in oxygen gas enriched with

oxygen-18.



Isolation of Proline and Hydroxyproline from Seedlings

After the seedlings had been exposed to an oxygen-18 atmosphere

for 4 to 8 days, the seedlings were removed from the flasks. A sufficient

number of seedlings were taken so as to provide 1.0 to 1.5 mg of hydroxy-

proline; the exact number of seedlings which was needed was determined

from previous experiments (figure 6); seedlings were taken from more than

one Erlenmeyer flask if necessary. The seedlings were washed under a

stream of running tap water, blotted with paper toweling, and weighed.

The samples were then finely chopped with a razor blade and dried under

vacuum lyophilizedd) for 6 hours; liquid nitrogen was used in the cold

trap. Six hours was determined to be sufficient time to dry the seedlings

because further lyophilization did not decrease the weight of the sample

measurably.

Acid hydrolysis and isolation of the amino acid fraction were per-

formed as previously described (page 9). After isolating the amino acids

from the acid hydrolysate with a 20 x 1.5 cm of Amberlite IR-120(11+)

column, the primary 6 amino acids were deaminated to the corresponding

hydroxy acids with nitrous acid. The nitrous acid solution was pre-

pared by mixing one part of a 30 per cent sodium nitrite solution with

two parts of 9 N hydrochloric acid and was freshly prepared immediately









prior to use. After the vigorous reaction which accompanied the mixing

process had subsided, the nitrous acid solution was decanted into a

round-bottom flask which contained the dry amino acids oluted from the

cation exchange column. One hundred ml of the nitrous acid solution were

used per gram of dry plant material which was originally hydrolyzed.

The amino acid and nitrous acid mixture was allowed to stand at

laboratory temperature for 1 hour after which the flask was fitted to a

reflux condenser and boiled for 1 hour. After the boiling, the mixture

was concentrated on the rotary evaporator, and most of the sodium chloride

was precipitated. The sodium chloride was removed by filtration on a

Buchner funnel using Whatman No. 50 paper. The precipitate was washed

with two 5 ml portions of cold 6 N hydrochloric acid. The remainder of

the acid was evaporated on the rotary evaporator.

The procedure of Hamilton and Ortiz (25) was used to extract the

Ct hydroxy acids resulting from the deamination process. In this pro-

cedure 1 ml of 3 N hydrochloric acid was added to wet the residue, and

the CZ hydroxy acids were extracted twice with 50 ml portions of diethyl

ether. The C6 hydroxy acids are soluble in ether; proline and hydroxy-

proline are not soluble in ether. The residue containing the proline and

hydroxyproline was dissolved in 20 ml of water and placed on the top of a

50 x 1 cm column of Dowex 50W-X8(H+) resin. The column was washed with

50 ml of deionized water and eluted with 1.5 N hydrochloric acid (37).

In preliminary experiments, the eluant was collected in 10 ml fractions.

In order to determine the elution pattern of hydroxyproline and proline

from the column, each 10 ml fraction was analyzed for proline and hydroxy-

proline. Hydroxyproline was estimated by the method of Leach (48) as

previously described (page 11). Proline was estimated by procedure B of









Piez et al. (73) by adding 1 ml of each eluted fraction to 7 ml of 0.15

per cent ninhydrin in glacial acetic acid solution to produce a red

chromophore with an absorbance maximum at 510 mp.

As a result of these preliminary determinations of proline and

hydroxyproline, it was found that most of the hydroxyproline was in the

50 ml to 150 ml fraction (figure 14); consequently it was possible in

subsequent work to discard the first 50 ml which was eluted from the

column and to collect the next 100 ml fraction. Since the primary ob-

jective of this work was the study of oxygen fixation into proline to

form hydroxyproline, it was desirable to recover hydroxyproline quanti-

tatively. On the other hand, no effort was made to recover the proline

quantitatively and only an amount (approximately 1 mg) sufficiently large

enough for mass spectrometric analysis was needed. The collected fraction

(50 ml to 150 ml) from the column was evaporated to dryness on the rotary

evaporator. The residue was then dissolved in 10 ml of water and chromato-

graphed on an Amberlite IR-120(H+) 10 x 1 cm column;1 this column was used

to free the imino acids from the chloride. The imino acids hydroxyprolinee

and proline) were eluted from the column with 2 N ammonium hydroxide

solution. The eluant was evaporated to about 10 ml in a 100 ml round-

bottom flask, and then transferred to a 25 ml conical flask and evaporated

to dryness. The residue was dissolved in 1 ml of 70 per cent ethanol.



1
It should be noted that three cation exchange columns were
used in the isolation and purification of hydroxyproline and proline.
(1) An Amberlite IR-120(H+) 20 x 1.5 cm column was used to isolate
the amino acids from the hydrochloric acid hydrolysate. (2) A Dowex
50W-X8(H+) 50 x 1 cm column was employed in the partial separation
of hydroxyproline from proline. (3) Finally, a 10 x 1 cm Amberlite
IR-120(H+) column was used to purify hydroxyproline and proline
after separation by thin-layer chromatography.










Hydroxyproline and proline were further separated and further

purified by preparative (5) thin-layer chromatography on a cellulose

absorbent. The method of Myhill and Jackson (63) was used. Twenty g of

cellulose powder (MN-300, Brinkmann Instruments, Inc., Greak Neck, New

York) without binder were boiled in about 400 ml of deionized water for

10 minutes and filtered, so as to remove any soluble components. The

washed cellulose was then made into a fine slurry with deionized water

and poured into a spreader which gave a standard 250 p layer. After

spreading, the plates were air dried and stored in a desiccator. Marker

spots of reagent grade proline and hydroxyproline were spotted on the

thin-layer plates. The imino acids prolinee and hydroxyproline) isolated

from plant material as described above were spotted on the plate with a

micropipette. Two plates were generally used for each 1 ml sample with

fifteen spots per plate. About 30 ul were required for each spot in

order to chromatograph the 1 ml sample. The warm air from a laboratory

air gun rapidly evaporated the solvent. The chromatograms were developed

by ascending chromatography for 5 hours in a solvent consisting of 63

per cent n-butanol, 27 per cent glacial acetic acid and 10 per cent water.

After removing the. chromatograms from the solvent tank, they were dried

in a forced air oven at 105 C for 15 minutes. When cool, the marker

spots of reagent proline and hydroxyproline were sprayed with a 0.5 per

cent solution of ninhydrin (1, 2, 3-triketohydrindene) in acetone. The

color was developed in the forced air oven at 90 C for 3 minutes. Strips

of cellulose opposite the proline and hydroxyproline markers were re-

moved from the thin-layer plates with a razor blade. Proline or hydroxy-

proline was extracted from the cellulose with 10 ml of water by heating

in a boiling water bath for 10 minutes. The suspension was centrifuged












column chromatography
(Amberlite IR-120)


Amino Acids
-----


Neutral and
Anionic Compounds


nitrous acid



Imino Acids and
L Hydroxy Acids



ether extraction


. IHydroxy Acids


Proline


purification by
column chromatography
(Amberlite IR-120)


I
Imino Acids


column chromatography
(Dowex 50W)
and
thin-layer chromatography


7oxyproline

purification by
column chromatography
(Amberlite IR-120)


Hydroxyproline


Figure 12. -- Flow sheet for the isolation of proline
and hydroxyproline.


Proline


F










at 10,000 x g for 5 minutes, and the supernatant was decanted. The

extraction was repeated a second time. The combined supernatants were

then filtered through washed Whatman No. 2 filter paper to remove any

cellulose particles that may have remained in suspension. The filtrate

was then chromatographed on an Amberlite IR-120(H+) 10 x 1 cm column to

remove any soluble components from the cellulose or remaining chromato-

graphic solvent. The column was eluted with 2 N ammonium hydroxide

solution, and the eluant was then evaporated to about 0.3 ml in a small

conical flask. To determine the purity of the eluted hydroxyproline, a

25 Pl sample was spotted on a 5 x 20 cm thin-layer plate of cellulose

and developed by ascending chromatography in 77 per cent ethanol (71).

If the eluted hydroxyproline was found to be chromatographically pure,

the remainder of the sample was pipetted into the pyrolysis tube. A

flow sheet for the isolation of proline and hydroxyproline is shown in

figure 12.



Preparation of Pyrolysis Tubes for Oxygen-18 Analysis

Pyrex glass tubing, 8 or 10 mm outside diameter, was cut into

30 cm lengths. The tubing was cleaned by immersion in a dichromate-

sulfuric acid solution overnight followed by a thorough rinsing with

deionized water. The pyrolysis tubes were prepared after the procedure

of Wiener (110). A length of the cleaned tubing was heated in a medium

hot methane-oxygen flame about 10 cm from one end. It was rotated until

the walls became thickened (figure 13a). While still hot, the tubing was

pulled to form a capillary about 1 mm thick (figure 13b). A very low

flame was directed to the capillary about 3 cm from the main part of the

combustion tube. When the glass had melted and the two sections had















C


d


Figure 13. -- A series of diagrams illustrating the sequence
of steps in the preparation of pyrolysis tubes.


iLI










separated, pressure was applied to the tube by blowing while the capillary

was still hot (figure 13c). The capillary was heated in a very low flame,

and a break-off tip was formed, with the aid of forceps, into a shape

similar to a question mark (figure 13d). A 14/35 ground glass outer

joint was sealed to the end of the tube opposite the break-off tip (figure

13e).

The tube was tested for leaks on the vacuum manifold, following

which it was heated in a muffle furnace at 500 C for 1 hour to dry and

to anneal it. After cooling, the tube was stored in a desiccator over

magnesium perchlorate desiccant.



Pyrolysis of Organic Material to Carbon Dioxide

Pyrolysis of organic samples was performed according to the pro-

cedure of Lee (50). In this method, the organic material is mixed with

five times its weight of mercuric cyanide and placed in a glass pyrolysis

tube. The tube is then sealed under vacuum, following which the mixture

is pyrolyzed at 500 C to 550 C for 1 hour. During this process, all of

the molecular bonds are broken; the mercuric cyanide acts as an oxidant.

The carbon dioxide formed in the process has the same distribution of

oxygen-18 as that of the original sample. The essential difference between

the method of Lee and the better known method of Rittenberg and Ponticorvo

(84) is that the Lee method uses mercuric cyanide as the oxidizing agent,

whereas Rittenberg and Ponticorvo used mercuric chloride. When mercuric

chloride is used, hydrogen chloride is produced which must be removed from

the mixture with quinoline or 5, 6-benzoquinoline. If mercuric cyanide

is used as the oxidizing agent, then the hydrogen cyanide which is produced

during pyrolysis may be removed by the use of a dry ice-acetone trap.










Mercuric cyanide was stored in a small vacuum desiccator over magnesium

perchlorate in order to keep it dry.

Hydroxyproline or proline samples from plant material purified as

previously described were pipetted in a minimal amount of water, about

0.3 ml, directly into the pyrolysis tube. The tube was attached to the

vacuum line, and the water was evaporated from the frozen state (lyo-

philized). The pyrolysis tube was then removed from the vacuum line

and mercuric cyanide was added to the proline or hydroxyproline; the

mercuric cyanide was added in the ratio of five parts mercuric cyanide

to one part proline or hydroxyproline. A medium hot flame was then

directed to a point about 3 cm from the ground glass joint of the pyrolysis

tube to thicken the walls. While still hot, the thickened portion was

pulled into a capillary of approximately 5 mm outside diameter (figure 13f).

The pyrolysis tube was again attached to the vacuum line and

evacuated to less than 1 p of mercury for 30 minutes. The pyrolysis tube

was warmed with the hot air from a laboratory air gun during the evacuation

so as to remove any moisture adhering to the inner walls. The removal of

water was necessary because it was found that when moisture was present

during the pyrolysis of mercuric cyanide alone, then small quantities of

carbon dioxide were produced; this carbon dioxide --- arising from the

reaction of mercuric cyanide with water --- would dilute any oxygen-18

enriched carbon dioxide which came from an organic sample.

When completely evacuated, the thick-walled capillary portion of

the pyrolysis tube was heated in a low flame, followed by a sharp hot

flame until the glass melted and sealed the evacuated tube. The sealed

pyrolysis tube was slowly pulled away from the joint end while the hot

flame was still being applied. The sealed end of the tube was then









annealed in an annealing flame. This procedure formed a sturdy thick-

walled seal (figure 13g). The sealed pyrolysis tubes were heated in a

muffle furnace at 500 C to 550 C for 90 minutes (77).



Mass Spectrometric Analyses of Carbon Dioxide and Oxygen

After pyrolysis, the sealed tubes were placed into a receiver

tube together with a small cylindrical steel magnet. The receiver tube

was attached to the vacuum line and evacuated to less than 1 of mercury

for 30 minutes. After evacuation, the stopcock to the vacuum line was

closed and the break-off tip of the pyrolysis tube was broken by raising

the magnet inside the receiver tube with a small horseshoe magnet applied

to the outside of the receiver tube and allowing it to drop on the break-

off tip. The receiver tube was then immersed in liquid nitrogen for 10

minutes. The stopcock to the vacuum line was opened and non-condensable

gases were pumped out. The stopcock was then closed and the liquid

nitrogen was replaced by a dry ice-acetone trap for 10 minutes to hold

the water and hydrogen cyanide. The carbon dioxide was collected by

transferring it first into the Toepler pump and then into the mass

spectrometer bulb.

The carbon dioxide was introduced into a Consolidated Electro-

dynamics Corporation type 21-130 mass spectrometer. The peak heights of

the masses were read from the recorder chart paper after a scan in the

appropriate mass region had been performed. The mass ratio, R, of mass

46 (C12 016 018) to mass 44 (C12 016 016) was determined from the re-

spective peak heights. The atom per cent oxygen-18 was calculated from

the formula (9, 22, 35, 59, 83):

atom per cent oxygen-18 = 100 R
2 _+R









The above formula neglects the very small contribution to mass 46 by

C13 016 017 and C12 017 017.

The atom per cent oxygen-18 of oxygen gas samples produced by

the electrolysis of water (page 42) was also determined mass spectro-

metrically from the ratio of the peak heights of mass 34 (016 018) to

mass 32 (016 016). The same formula was used in the calculations of the

atom per cent oxygen-18. Ozone was never detected in the oxygen gas.

A background scan was always run before each analysis and the

peak heights were corrected if necessary. A very small background peak

at mass 44 (presumably due to contamination of the inlet system of the

mass spectrometer with carbon dioxide) was usually found in the mass

spectra and was substracted from the results.



Exchange of Hydroxyproline Oxygen with Water Oxygen

In order to investigate the exchange of the oxygen atoms of

hydroxyproline with water oxygen under the hydrolytic conditions used

in this investigation, 20 ml of hydroxyproline were sealed in a glass

tube together with 4 ml of a 6 N hydrochloric acid solution containing

oxygen-18. The contents of the tube were heated at 108 C for 24 hours.

At the end of that time, the tube was opened, and the contents were

transferred to an inverted glass U-tube fitted with a ground glass

connection which was attached to the vacuum manifold.

To separate the hydroxyproline from the acid solution, the

mixture was frozen in liquid nitrogen, and the U-tube was evacuated.

The evacuated U-tube was sealed near the ground glass joint with a flame

so that subsequent operations could be performed in a closed system in

order to prevent dilution of the labeled water by atmospheric water.









The empty arm of the U-tube was then immersed in liquid nitrogen, and

the acid was distilled from the hydroxyproline into the empty arm. The

hydroxyproline which remained in the first arm of the U-tube was trans-

ferred to a pyrolysis tube and pyrolyzed as previously described.

The distilled hydrochloric acid was neutralized with metallic

sodium, and the water was separated from the salt by vacuum distillation

as above. A portion of the water was electrolyzed, and the atom per

cent oxygen-18 of the resulting oxygen gas was determined by mass

spectrometry.



RESULTS



Plant Material

Maize or soybean seedlings which were grown as described in the

section on Materials and Methods (page 41) appeared normal in all re-

spects when compared to etiolated seedlings grown in open trays. Some

of the growth parameters of seedlings grown in an open and in confined

atmospheres are compared in table 5. These results show that both maize

and soybean seedlings are similar with respect to shoot length, root

length, number of lateral roots, and fresh weight when they are grown in

an open tray or in a closed flask. The hydroxyproline content of seed-

lings grown in open trays and closed flasks is also quite similar, in-

dicating that the subatmospheric pressures developed in the flask due

to the utilization of oxygen gas and the removal of carbon dioxide did

not affect hydroxyproline synthesis. From these results, it can be

concluded that etiolated seedlings grown in small closed chambers are

not different from etiolated seedlings grown in open trays.









Column Chromatography

The recovery of known quantities of hydroxyproline from each of

the cation exchange columns is given in table 6. The recovery of hydroxy-

proline in all cases was 90 to 100 per cent. It was found that Dowex

50W-X8 in the 50 cm column gave a better separation of proline and

hydroxyproline than did Amberlite IR-120 when the same conditions were

used. The elution pattern of 1 mg each of hydroxyproline and proline is

shown in figure 14. The figure is a plot of the volume of eluting sol-

ution through the column against absorbance. In the case of hydroxy-

proline, absorbance was measured at 556 Wm, whereas proline was measured

at 510 mu. The figure shows that when 1 mg each of proline and hydroxy-

proline was chromatographed on the column and eluted with 1.5 N hydro-

chloric acid, most of the hydroxyproline was eluted in the 50'- 150 ml

fraction. The proline, however, was spread from the 50 ml to 400 ml.

Although the proline and hydroxyproline were not completely resolved

on the column, the hydroxyproline-rich fraction could be further chro-

matographed without difficulty by thin-layer chromatography.



Thin-layer Chromatography

Thin-layer chromatography, used as described in the section on

Materials and Methods (page 48), provided a convenient and rapid method

for separating hydroxyproline and proline which had been partially

separated by column chromatography. When developed in the n-butanol-

acetic acid-water solvent for 5 hours, the center of the hydroxyproline

spot moved 5 cm from the origin while the proline spot moved 10 cm.

(The term, Rf, usually employed for designating the position of a spot

on a chromatogram is not applicable here because the solvent front









Table 5. -- Comparison of seven-day-old etiolated maize and soybean
seedlings grown in open and closed atmospheres.




Maize Soybean

open closed open closed



Shoot length (cm) 8.5 8.9 12.7 11.8


Primary root length (cm) 24.2 21.9 5.7 7.1


Number of lateral roots 3.3 3.3 8 7

Fresh weight (g/seedling) 0.81 0.79 0.76 0.82


Hydroxyproline (pg/g) 43 46 165 161

Hydroxyproline (pg/seedling) 37 36 125 132









Table 6. -- Recovery of hydroxyproline from cation exchange
columns.




Material Hydroxyproline Per cent
Column added recovered (mg) recovered




1 mg hydroxyproline
Amberlite IR-120(H+) +1 mg each of 16
20 x 1.5 cm other amino acids 1.00 100


1 mg hydroxyproline 0.95 95



Amberlite IR-120(H+) 1 mg hydroxyproline
10 x 1 cm +1 mg proline 0.91 / 91


1 mg hydroxyproline 1.01 101



Dowex 50W-X8(H+) 1 mg hydroxyproline a
50 x 1 cm +1 mg proline 0.96 96




aRecovered from the 50 ml to 150 ml fraction.







Hydroxyproline


0---- Pro lin


50 100 150 200 250 300 350


ON







400


MILLILITERS


THROUGH


COLU1M.,N


Figure 14. -- Pattern of elution of hydroxyproline and proline from a 50 cm
Dowex 50W column.


0.2


0.1


0.31









reached the top of the thin-layer plates in less than 5 hours.) It was

necessary to use fresh solvent for each set of chromatograms because it

was found that the proline and hydroxyproline spots did not move as far

from the origin and did not separate as well if the same solvent was used

a second time. This failure of used solvent to provide good separation

was apparently due to the depletion of some component of the solvent,

probably water. A quantity as small as 10 ,ug each of proline or hydroxy-

proline was visible when sprayed with ninhydrin. On the other hand, an

amount as large as 1 mg each of these compounds could also be separated

from a mixture of the two compounds, but the spots were too close to-

gether to be removed conveniently. In order to insure complete separation,

quantities of 100 ,ug or less were always used. Recovery of hydroxyproline

from the thin-layer plates after chromatography was almost 100 per cent

for 100, 250 or 500 ug of hydroxyproline, as shown in table 7. These

results are better than those of Myhill and Jackson (63) who reported

only about 60 per cent recovery of hydroxyproline from thin-layer plates.



Pyrolysis of Organic Material

Lee (50) reported that a 60 minute pyrolysis of sugars at 500 C;

to 550 C in the presence of mercuric cyanide was sufficient time to con-

vert all of the oxygen-18 to carbon dioxide. However, Prockop et al.

(77) found it necessary to heat hydroxyproline samples 90 minutes at

these same temperatures to effect a complete conversion of the oxygen-18

to carbon dioxide by the method of Rittenberg and Ponticorvo (84), which

utilizes mercuric chloride instead of mercuric cyanide as the oxidant.

When these findings were considered, it was decided to pyrolyze for 90

minutes at 500 C to 550 C in the present investigation; as previously

stated, the method of Lee (50) was used.









Table 7. -- Recovery of hydroxyproline from thin-layer
chromatography plates.


Hydroxyproline Hydroxyproline Per cent
added (pg) recovered (pg) recovered


Mean


250


250


250


96


97


102


Mean


515


500


480


103


100


96


Mean 99.7


97.0
!


98.3









The average value of the oxygen-18 content of 16 samples of tank

carbon dioxide, tank oxygen and pyrolyzed reagent proline and hydroxy-

proline analyzed with the recorder by scanning was 0.193 0.006. This

value is somewhat less than the accepted value of 0.204 atom per cent

for the normal enrichment of oxygen-18 and the discrepency was due to a

maladjustment of the recorder in the mass spectrometer. In fact, the

accepted value for the atom per cent oxygen-18 (0.204) for carbon dioxide

samples was obtained on several occasions when the isotope ratio accessory

of the mass spectrometer was used; however, it was more convenient to

scan the samples and use the recorder. Since a proline sample from the

same plant material was always used as the base for calculating the atom

per cent excess oxygen-18 in the recovered hydroxyproline, the fact that

the measured normal enrichment differed slightly from the accepted value

of 0.204 atom per cent does not affect the results reported in this

dissertation.

To eliminate the possibility that decomposition products arising

from mercuric cyanide contributed to the peak heights of recovered carbon

dioxide, a 50 mg sample of mercuric cyanide was pyrolyzed and handled

as if it contained carbon dioxide. Mass spectrometric examination of

the products which resulted from the pyrolysis of mercuric cyanide showed

the presence of several atomic combinations of carbon and nitrogen; how-

ever, none of these peaks was in the carbon dioxide mass range.

Lee (50) suggested that an amount of mercuric cyanide 4 to 6

times the weight of organic material should be used for the pyrolysis of

sugars. Table 8 shows the effect of varying the amounts of mercuric

cyanide during the pyrolysis of reagent hydroxyproline on the atom per

cent oxygen-18 in the hydroxyproline. A ratio of 5 to 1 of mercuric









Table 8. -- Effect of increasing amounts of mercuric cyanide on the
estimated atom per cent 018 of hydroxyproline, as determined by the
pyrolytic method of Lee.




Hydroxyproline Mercuric cyanide Atom per cent Per cent of
(mg) (mg) 018 maximum



10 10 0.191 96


10 50 0.199 100


10 100 0.196 98


10 500 0.177 89









cyanide to hydroxyproline was found to give expected results, whereas

a ratio of 50 to 1 depressed the atom per cent oxygen-18 considerably.

Therefore, in accord with the results of table 8, five times the weight

of mercuric cyanide to the weight of hydroxyproline or proline was al-

ways used in the present investigation.



Exchange of Hydroxyproline Oxygen with Water Oxygen

In order to determine the source of any atom in a molecule, it

is necessary to know whether that atom is exchangeable with like atoms

of the solvent. If exchange does occur under the experimental conditions

employed, then the rate of exchange should be known. In general, the

oxygen of alcohols, esters and frequently acids do not exchange with the

oxygen of water; however, exchange with water often occurs in'aldehydes

and ketones (13). Also it is known that the exchange of carboxyl oxygen

of organic acids with water is dependent upon the pH of the solution.

For example, there was no exchange of the oxygen of glycine with water

after 72 hours at 100 C at neutral pH; however, after 24 hours at 100 C

and at pH 2, all of the oxygen of glycine had exchanged (13).

Based on these generalizations, one would expect the carboxyl

oxygens of hydroxyproline to exchange with water during hydrolysis in 6

N hydrochloric acid; on the other hand, the hydroxyl oxygen should not

exchange. Fujimoto and Tamiya (19) found that two of the three oxygens

of hydroxyproline had exchanged after 6 hours in 6 N hydrochloric acid

at 110 C; after 72 hours, the third atom remained unexchanged. Prockop

et al. (77) reported a slightly less than expected exchange after 3 days

in 4 N hydrochloric acid at 100 C. They attributed this finding to some

re-exchange of the carboxyl oxygen atoms during recrystallization of the

sample.









In order to confirm the findings of Fujimoto and Tamiya, and

those of Prockop et al., reagent hydroxyproline was dissolved in enriched

water. When 20 mg of hydroxyproline were equilibrated in a sealed tube

for 24 hours at 108 C in 4 ml of a water solution of 6 N hydrochloric

acid containing 1.15 atom per cent excess oxygen-18, the atom per cent

excess oxygen-18 in the recovered hydroxyproline was determined to be

0.796. The expected atom per cent excess oxygen-18 for an exchange of

two of the three oxygen atoms would be 1.15 x 2/3 = 0.767. The difference

between the expected (0.767) atom per cent excess oxygen-18 in the re-

covered hydroxyproline and the result which was actually found (0.796) is

considered negligible in view of the following circumstances: (1) a

dilution in the oxygen-18 of the water probably occurred when the water

was exposed to the atmosphere at various times during the purification

and electrolysis of the sample, and (2) since the oxygen-18 enrichment

of the water was determined at the end of the equilibration period, its

oxygen-18 content must be considered to be the absolute minimum enrich-

ment. In view of these results, it is concluded that under the conditions

of hydrolysis used here the carboxyl oxygen atoms of hydroxyproline ex-

changed with the oxygen of the water, and the hydroxyl oxygen atom did

not exchange.



Source of the Hydroxyl Oxygen of Hydroxyproline in Seedlings

When maize or soybean seedlings were grown in a atmosphere con-

taining molecular oxygen labeled with oxygen-18, the hydroxyproline re-

covered from these seedlings contained excess oxygen-18. Many trials

were performed to gain experience with the techniques which were in-

volved; the results of two complete experiments for maize seedlings and










also two complete experiments for soybean seedlings are shown in table

9. When maize seedlings were grown from seed to 8 days of age in an

atmosphere containing 9.9 atom per cent excess oxygen-18, the recovered

hydroxyproline contained 0.383 atom per cent excess oxygen-18 while the

proline recovered from the sameplant material was normal with respect

to oxygen-18. Similarly when maize was grown from 3 days of age to 7

days of age, a period of 4 days, in an atmosphere containing 10.4 atom

per cent excess oxygen-18 gas, the excess oxygen-18 in the recovered

hydroxyproline was determined to be 0.605. In like manner, the hydroxy-

proline from soybeans which were grown from seed to 8 days in an atmo-

sphere enriched with 10.3 atom per cent excess oxygen-18 contained 0.271

atom per cent excess oxygen-18. Similarly when soybeans were exposed

to a 10.4 atom per cent excess oxygen-18 atmosphere for 4 days (3 to 7

days of age), the recovered hydroxyproline was found to be labeled to

the extent of 0.222 atom per cent excess oxygen-18.

A possibility that must be considered in the interpretation of the

above results --- alternative to the direct addition of molecular oxygen

to organic substrate --- is that the oxygen-18 in the hydroxyproline

might have come from respiratory water produced by the reduction of

oxygen gas during respiration. This possibility may be considered in

further detail as follows. By knowing the volume of oxygen gas consumed

by the seedlings during the exposure periods, the atom per cent excess

oxygen-18 of the water may be calculated. The maximum volume of oxygen

gas utilized, even in an 8-day growth period, was estimated to be 300 cc

at standard pressure and room temperature. If 300 cc (0.013 moles) of

oxygen gas were completely reduced to water, then 0.026 moles or 0.47 g

of water would be produced. When this 0.47 g of respiratory water is








Table 9. -- Incorporation of 0218 into hydroxyproline of maize and soybean.


Age (days) of Total Total Atom %
seedlings at Total % fresh dry excess
beginning and 02 in Atom % Number weight (g) weight (g) 010 in
end of experi- atmos- excess of of of hydroxy-
ment phere 021 seedlings seedlings seedlings proline



Maize

1. 0 8 22.2 9.9 24 19.3 1.83 0.383


2. 3 7 24.0 10.4 24 23.0 2.20 0.605


Soybean

1. 0 8 23.4 10.3 9 7.6 1.19 0.271

2. 3 7 22.2 10.4 10 8.3 1.23 0.222










mixed with the 50 ml of water initially present in the seedling flask,

then the dilution factor is almost one hundred. Since the oxygen gas

contained 10 atom per cent excess oxygen-18, then the enrichment of the

water inside the flask would be a maximum of 0.1 (10 per cent x 1/100)

atom per cent excess oxygen-18. Therefore, it would appear from these

calculations that the oxygen-18 in hydroxyproline could not have come

from respiratory water because the oxygen-18 enrichment in hydroxyproline

was much higher (table 9) than 0.1 atom per cent excess oxygen-18. How-

ever, the possibility exists that respiratory water may be "caged" within

the plant cells and recycled without being in equilibrium with the extra-

cellular water. If this happened, then the oxygen-18 labeled water in-

side the cell could conceivably be high enough to produce the results

obtained in table 9 without involving the direct incorporation of oxygen

gas into organic substrate.

To eliminate the possibility presented by the above argument,

namely, that the oxygen-18 in hydroxyproline might have arisen from

respiratory water, it was necessary to grow seedlings in oxygen-18

labeled water and inan atmosphere of normal oxygen gas. As shown in

table 10, no oxygen-18 excess was found in the recovered hydroxyproline

when either maize or soybean seedlings were grown under these conditions.

When maize seedlings were grown from seed to 8 days of age in 50 ml of

water containing 1.52 atom per cent excess oxygen-18, the oxygen-18 con-

tent of both the recovered proline and hydroxyproline was normal. Similar

results were obtained when soybean seedlings were grown in water which

contained 1.33 atom per cent excess oxygen-18.

In summary, the results given in tables 9 and 10, when considered

together, show clearly that when seedlings were grown in an atmosphere








Table 10. -- Incorporation of H2018 into hydroxyproline of maize and soybean.


Age (days) of Total Total Atom %
seedlings at fresh dry excess
beginning and Volume Atom % Number weight (g) weight (g) 01d in
end of experi- (ml) of excess of of of hydroxy-
ment H2018 018 seedlings seedlings seedlings proline




Maize 0 8 50 1.52 22 23.2 2.18 0.00


Soybean 0 8 50 1.33 12 10.7 1.50 0.00









labeled with oxygen-18, then the oxygen gas was incorporated into

hydroxyproline; oxygen-18 was not incorporated into hydroxyproline when

seedlings were grown in water labeled with oxygen-18. The fact that

oxygen-18 was never found in proline recovered from plants which were

grown in either an oxygen-18 atmosphere or oxygen-18 water, together

with the exchange data (page 66), provide ample evidence for the de-

duction that the oxygen-18 excess found in the recovered hydroxyproline

in table 9 must be in the hydroxyl group. In view of these findings, it

is concluded that the biosynthesis of hydroxyproline by etiolated maize

and soybean seedlings is accomplished by the direct addition of mole-

cular oxygen to a precursor molecule. In view of the results reported

in Part III of this dissertation, it can be assumed that proline in

some bound or activated form is the substrate for the hydroxylation

reaction.



DISCUSSION



The results which have been presented show that the biosynthesis

of hydroxyproline in etiolated maize and soybean seedlings occurs by the

fixation of molecular oxygen (oxygen fixation) into organic substrate.

The basis for this conclusion may be summarized as follows. When

etiolated maize or soybean seedlings were grown in an oxygen-18 atmo-

sphere, the hydroxyproline extracted from these plants contained an

excess of oxygen-18. On the other hand, excess oxygen-18 was not found

in the hydroxyproline recovered from plants which were grown in water

labeled with oxygen-18. Moreover, the proline isolated from the









experimental plants --- regardless of the conditions under which they

were grown --- always contained the normal enrichment of oxygen-18.

It is concluded that the observed oxygen fixation occurred into

the hydroxyl group of hydroxyproline. This conclusion is supported by

the experiment in which reagent hydroxyproline was incubated with oxygen-

18 enriched water. The results of this experiment showed that the

carboxyl oxygen atoms of hydroxyproline exchange with water and that the

hydroxyl oxygen did not exchange. Also, another independent line of

reasoning may be advanced in support of the conclusion that oxygen

fixation occurred into the hydroxyl group of hydroxyproline. Since all

of the oxygen atoms in proline are in the carboxyl group and since proline

isolated from the seedlings was found always to contain the normal en-

richment of oxygen-18, it is apparent that the carboxyl group of proline

was always normal with respect to oxygen-18. If the plausible assumption

is made that the carboxyl group of hydroxyproline behaves in a manner

similar to that of proline, then it can be concluded that all of the

excess oxygen-18 found in hydroxyproline was in the hydroxyl group.

The results reported here confirm and extend the results of pre-

vious investigators which may be summarized as follows. Fujimoto and

Tamiya (19) used a chick embryo system and axi oxygen-18 atmosphere and

concluded that about 40 to 60 per cent of the hydroxyl oxygen of hydroxy-

proline was derived from molecular oxygen. In subsequent experiments

(20), they were able to increase their estimation of incorporation to

80 to 93 per cent. Prockop et al. (76, 77) also used chick embryos and

determined that the oxygen required for the hydroxylation of proline to

collagen hydroxyproline came from molecular oxygen and not from water.

Lamport (44) grew cell suspensions of sycamore (Acer pseudoplatamus L.)









in an oxygen-18 atmosphere. Although the results of only a single

experiment were reported, he was able to conclude that all of the

hydroxyl-oxygen of primary cell wall hydroxyproline was derived from

molecular oxygen. His actual results agreed well with his estimations

of expected incorporation of oxygen-18 into hydroxyproline under the

conditions of his experiments, but he did not rigorously eliminate

water as a possible source of hydroxyproline oxygen by means of

independent oxygen-18 water experiments, as was done in the present

investigation.

Not only do the present results show that molecular oxygen from

the atmosphere is the source of the hydroxyl oxygen of hydroxyproline of

etiolated maize and soybean seedlings, but for the first time, hydroxy-

proline biosynthesis by the direct addition of molecular oxygen to

organic substrate has been shown in an intact and growing species of a

monocotyledonous plant (Zea mays) as well as in a dicotyledonous plant

(Glycine max).

It is very likely that hydroxyproline biosynthesis is a result of

reactions catalyzed by hydroxylase enzymes (see Introduction to Part II).

The hydroxylase character of the enzyme or enzymes involved in hydroxy-

proline biosynthesis has been investigated with tritiated proline. Ebert

and Prockop (15, 78) exposed chick embryos to 3, 4-tritiated proline and

found that 25 per cent of the tritium was lost from the proline when it

was converted to hydroxyproline. If the tritium is randomly distributed

around carbon atoms three and four of 3, 4-tritiated proline, then a 25

per cent loss of tritium would be equivalent to the loss of one hydrogen

atom. The loss of one hydrogen atom would be consistent with a

cylase mechanism. In contrast, to these findings, Stone and Meister









(105, 106) exposed minced guinea pig granulomas to 3, 4-tritiated proline

and showed that an approximately equal distribution of tritium appeared

in the water and in hydroxyproline, corresponding to a 50 per cent loss

of hydrogen. A 50 per cent loss of hydrogen would mean that two hydrogen

atoms were lost from proline when the hydroxylation reaction occurred,

indicating that either a 4-keto intermediate or 3, 4-unsaturation was

produced. These two conflicting reports were apparently resolved by

Lamport (43, 46), who found that the tritium of 3, 4-tritiated proline

was predominantly in the trans positions of carbon atoms three and four

of proline and were not distributed randomly:

H
N
H H
C C
H\T T /600H
C C
I I
H H

Thus, when Lamport's sycamore cell cultures synthesized hydroxyproline

from 3, 4-tritiated proline, Lamport found a 50 per cent loss of tritium

which he rightly concluded to be the result of the loss of one hydrogen

atom. In summary, the conclusion to be drawn from these tritium studies

is consistent with a hydroxylase mechaniam: one hydrogen is lost from

proline in the hydroxylation reaction.

The nature of the enzyme or enzymes involved in the biosynthesis

of hydroxyproline, as well as the identity of the cofactors, still re-

mains unknown. Ascorbic acid has been implicated as a possible cofactor

in ascorbic acid deficient guinea pigs (106). Also, studies have been

carried out with 4-ketoproline reductase, an enzyme which catalyzes the

reduction of chemically-prepared 4-ketoproline (70) to hydroxyproline;

this enzyme has been partially purified from rat and rabbit kidney (61,










93). The purified enzyme has a specific requirement for NADH. Although

many aldehydes and ketones were tested as substrates for the enzyme, 4-

ketoproline was by far the best substrate found. The only known natural

source of 4-ketoproline is actinomycin V from Streptomyces (37). However,

the relation of this enzyme, 4-ketoproline reductase, to in vivo hydroxy-

proline biosynthesis is not clear. Still another possibility for hydroxy-

proline biosynthesis has been reported by Yip (113): he found that

proline can be hydroxylated in the presence of horseradish peroxidase,

hydrogen peroxide and ferrous ions. The isomeric and sterochemical form

of the product of the peroxidase reaction was not reported; therefore,

the specificity of the reaction is not known. At the present time, it

is impossible to evaluate the hydroxylation of proline by horseradish

peroxidase in terms of the in vivo reaction. However, it seems probable

that the peroxidase reaction reported by Yip is a non-specific hydrox-

ylation and bears no relation to the in vivo synthesis of hydroxyproline

because there is no known in vivo system which will convert free proline

to free.hydroxyproline.

An interesting facet of hydroxyproline biosynthesis that should

be considered involves the following question. Is the incorporation

of molecular oxygen into organic substrates necessary for life to a

higher plant? In other words, does oxygen have an obligatory function

other than that of a terminal oxidant? At the present time, in the

absence of direct information, one can only speculate. The existence

of hydroxyproline in higher plants is well documented (see Introduction

to Part I). The almost unanimous opinion of investigators is that the

bulk of hydroxyproline in plants is associated with the primary cell wall.

If a hydroxyproline-rich protein performs a regulatory and/or structural










role in the cell wall as suggested by Lamport (see Discussion, Part I),

then it may be concluded that hydroxyproline is most likely a necessary

complement of the cell machinery. In the absence of any evidence that

hydroxyproline can be biosynthesized in vivo by any mechanism other than

the hydroxylase reaction, it may be concluded that the incorporation of

molecular oxygen, at least into hydroxyproline, is a vital process for

the higher plant. However, a direct answer to the question regarding

the essentiality of oxygen fixation may be difficult to obtain experi-

mentally. As pointed out by Goldfine and Bloch (23), the tensions of

oxygen required for many biosynthetic purposes are very much lower than

those needed for respiration in aerobic organisms. In particular, this

has been found to be true for the synthesis of unsaturated fatty acids

and for squalene cyclization in yeast. It is difficult, therefore, to

separate the effects of low oxygen tension upon the oxygen incorporating

systems from the effects upon the very important energy-yielding

oxidations associated with the cytochrome system.

Another related question of interest in the present context

concerns the possibility of a dehydrogenation-hydration mechanism for

the biosynthesis of hydroxyproline from proline, instead of oxygen

fixation. In general, oxidation reactions are energy-yielding processes.

On the contrary, hydroxylase reactions represent a waste of energy from

the thermodynamic point of view (26, 27); that is, if a reduced cofactor

is used to accomplish the hydroxylation, then a source of potential

energy is consumed. (It should be pointed out that in a hydroxylase

reaction both the cofactor and the substrate are oxidized, and molecular

oxygen is reduced.) On the other hand, if hydroxylation reactions were

accomplished by dehydrogenation followed by the addition of water across










the .resulting double bond (for example, the succinate-fumarate-malate

transition), then the reduced cofactor resulting from the dehydrogenation

represents potential energy. In the case of the succinate-fumarate-

malate transition, the aliphatic carbon atoms of succinate are activated

by the presence of adjacent carboxyl groups. Fatty acids are also

oxidized to the /-hydroxy form by a dehydrogenation-hydration mechanism,

but in the case of fatty acids, the molecule must first be activated by

formation of the CoA derivative; the dehydrogenation then takes place

immediately adjacent to the activated carbon atom. From these examples,

it would appear that the dehydrogenation of an aliphatic chain has

specific requirements which are met in only a limited number of bio-

logical reactions. Unlike succinate or the fatty acids, there are no

active groups near the oxidation site of proline and there are no

adjacent carbon atoms capable of being activated; it may be impossible

for the plant to accomplish a dehydrogenation of proline at the appro-

priate position. Therefore, it appears then that oxygen fixation may

be a more feasible mechanism than a dehydrogenation-hydration for the

biological oxidation of proline to hydroxyproline. Yet in spite of the

reasons which have been advanced to support the view that hydroxyproline

is probably not biosynthesized by a dehydrogenation-hydration mechanism,

a pathway other than the hydroxylase mechanism by oxygen fixation cannot

be arbitrarily ruled out. The reason for this conclusion is obvious:

only four species of organisms (chick, sycamore, maize and soybean) have

been investigated to date; there may be other organisms which perform

the hydroxylation by a method other than oxygen fixation. In conclusion,

judging from the evidence presently available, derived from direct

experimentation as well as from the indirect considerations discussed









here, it appears reasonable to assume that hydroxyproline is the product

of an aerobic reaction or group of reactions associated with aerobic

plants and animals.

Finally, it is interesting to examine another case involving the

hydroxylation of an amino acid to yield a protein amino acid; this is

the hydroxylation of phenylalanine to tyrosine. Tyrosine is a universal

protein constituent,whereas hydroxyproline has a limited distribution.

Two pathways leading to the formation of tyrosine have been found: (1)

hydroxylation of phenylalanine by hydroxylase enzymes utilizing molecular

oxygen in mammalian tissues (38, 60, 107), and (2) a de novo synthesis

of tyrosine from shikimic acid without phenylalanine as an intermediate

(12) in anaerobic bacteria. In the latter case, none of the oxygen

atoms is derived from molecular oxygen. The shikimic acid pathway

apparently is the major route for tyrosine biosynthesis in anaerobic

bacteria (23). According to Neish (64), higher green plants apparently

synthesize tyrosine by the shikimic acid route, but critical oxygen-18

studies have not been reported. Thus, it seems that only animals have

adopted the aerobic pathway to tyrosine formation while other forms

(microorganisms and higher plants) probably continue to use the pre-

sumably more primitive anaerobic route.















PART III. BIOSYNTHESIS OF HYDROXYPROLINE
FROM PROLINE



INTRODUCTION



During their pioneering investigations of hydroxyproline bio-

synthesis in the rat, Stetten and Schoenheimer (97) and Stetten (96)

demonstrated that protein hydroxyproline is derived from proline and not

from hydroxyproline. Stetten (96) found that less than 0.1 per cent of

the hydroxyproline of the rat was derived from dietary hydroxyproline in.

3 days of feeding nitrogen-15 labeled hydroxyproline. Stetten (96) con-

cluded that . the hydroxyproline of the proteins is not derived to

any appreciable extent from dietary hydroxyproline but rather from the

oxidation of proline which is already bound, presumably in peptide

linkage. . According to recent reviews (24, 86), the precursor

of protein hydroxyproline has been confirmed to be free proline and

not free hydroxyproline in all plants and animals which have been in-

vestigated; the only reported instance of hydroxyproline being effec-

tively and directly incorporated into peptide linkage is into actinomycin

I by Streptomyces antibiotics (37). According to Allen et al. (1) a

hydroxyproline activating enzyme does not exist, at least in guinea pig

liver; the non-existence of this enzyme probably accounts for the failure

of free hydroxyproline to be incorporated into protein.









Not only does the available evidence indicate that protein

hydroxyproline is derived from proline, but also the presence of hydroxy-

proline in the growth media of plants has been shown to inhibit growth.

Steward and associates (74, 101, 102) found that hydroxyproline in con-

centrations as low as 10 parts per million in the growth medium of carrot

or potato explants inhibited growth induction and total protein synthesis

otherwise stimulated by the liquid endosperm of coconut; also hydroxy-D-

proline was found to be less inhibitory than hydroxy-L-proline. By

altering the structure of the hydroxyproline molecule before addition

to the explants, Steward et al. (102) demonstrated that the inhibition

of growth was reduced. Also it was found that the inhibition of growth

and protein synthesis in carrot and potato cultures by hydroxyproline

was specifically reversed by proline (101, 102). Cleland (8) 'reported

that the auxin-induced growth of Avena coleoptiles was severely inhibited

by 10-4 M hydroxyproline; the inhibition was completely reversed by

equimolar quantities of proline. From these several studies, it would

appear that free hydroxyproline inhibits growth by acting as an antago-

nist of proline metabolism.

The locus of the conversion of proline to hydroxyproline has also

been studied, and several investigators have concluded that hydroxy-

proline biosynthesis occurs after proline is incorporated into protein.

Steward and Pollard (101) concluded that proline was incorporated into

the protein of carrot explants and oxidized to hydroxyproline in situ

(74). Peterkofsky and Udenfriend (71) prepared a cell-free, microsome-

rich fraction from chick embryo that would incorporate proline into a

microsomal-bound protein containing hydroxyproline. They concluded that

the substrate for the hydroxylation of proline in this in vitro system









was a microsomal RNA-bound polypeptide of considerable size. Also, they

considered that their findings would rule out a sRNA-hydroxyproline

intermediate (72). Kao et al. (36) concluded that the collagen hydroxy-

proline of sponge biopsy connective tissue was derived from peptide

proline. Olson (69) worked with tobacco suspension cultures and con-

cluded that the hydroxylation of proline to hydroxyproline occurs after

proline is incorporated into a protoplasmic protein or polypeptide and

before or during its deposition in the cell walls. Olson, however,

pointed out that his results do not rule out the possibility of an

activated hydroxyproline intermediate.

In contrast to the view that proline hydroxylation occurs after

proline is bound in peptide linkage, evidence also exists which has been

interpreted to indicate that the hydroxylation may occur before proline

is bound into protein. Daughaday and Mariz (11) found that after in-

cubation of segments of costal cartilage from rapidly growing rats in

proline-C-14, radioactive hydroxyproline appeared free in the medium

as well as in the collagen. They found that the steroid hormone cortisol,

inhibited the incorporation of carbon-14 into cartilage hydroxyproline

but did not inhibit the labeling of free hydroxyproline in the medium.

They interpreted these results to mean that proline was hydroxylated

before incorporation into protein, possibly at the prolyl-adenylate

stage. Stone and Meister (106) suggested that the immediate precursor

of protein hydroxyproline might be hydroxyprolyl-RNA. Manner and Gould

(54, 55) attacked the problem directly by isolating the soluble-

ribonucleic acid of chick embryos after injection of proline-C-14.

They found a small amount of highly radioactive hydroxyproline associ-

ated with the sRNA. Coronado et al. (10) were also able to isolate










hydroxyprolyl-sRNA from chick embryo. The same results were also ob-

tained by Jackson et al. (33) from chick embryo and guinea pig wound

tissue. Lamport (47) reported that hydroxyproline-C-14 could be sepa-

rated from the sRNA of sycamore cell suspensions after an exposure to

proline-C-14. In view of the conflicting ideas regarding the locus of

proline hydroxylation, further study of this problem with higher plants

was considered desirable.

The objective of the investigations reported in this part of the

dissertation was the elucidation of the question regarding the locus of

the proline hydroxylation reaction in maize seedlings, whether before

or after the incorporation of proline into protein. In order to arrive

at a decision regarding this question, it was first necessary to estab-

lish that externally applied proline could serve as the precursor of

protein hydroxyproline in maize seedlings. After it was shown that

proline absorbed by seedling tissue was metabolized to hydroxyproline,

then experiments were performed to determine whether RNA-bound hydroxy-

proline could be detected in maize seedlings which had absorbed radio-

active proline. These experiments showed that RNA-bound hydroxyproline

did indeed exist, indicating that proline hydroxylation occurred prior

to the incorporation of proline into protein.


MATERIALS AND METHODS


Plant Material

The plant materials used in the experiments described in this

section of the dissertation were 7-day-old maize and soybean seedlings.

These seedlings were grown as previously described (page 8).









Application of Radioactive Proline to Seedlings

In order to demonstrate that exogenous proline could be converted

in vivo to protein hydroxyproline, radioactive proline was applied to

maize and soybean seedlings. For this purpose the roots of 7-day-old

etiolated maize and soybean seedlings were placed in 10 ml of deionized

water containing 1.0 pc of DL-proline-5-C-14 (6.9 mc/mmole, New England

Nuclear Corporation, Boston, Massachusetts) in a 100 ml beaker. The

number of seedlings varied with each experiment and are indicated in

table 11. The roots of the seedlings were exposed to the labeled pro-

line solution for 8 hours in the dark at 25 C. The seedlings were then

washed in tap water for 10 minutes to remove surface radioactivity from

the roots; proline and hydroxyproline were isolated from the seedlings

(see page 45).

In other experiments in which ribonucleic acid (RNA) was ex-

tracted from seedlings which had been exposed to radioactive proline,

the excised ends of 40 to 50 7-day-old maize shoots were dipped into

1 ml of water in a 50 ml beaker containing either 1.0 or 2.5 1c of L-

proline-C-14 (uniformly-labeled, 205 mc/mmole, purity greater than 99.7

per cent, New England Nuclear Corporation). (Uniformly-labeled proline

was used in the RNA experiments because it had a higher specific activity

than the DL-proline-5-C-14 which was available.) In some experiments,

10-2 M or 10-5 M ascorbic acid was added to the bathing solution. The

beaker which contained the shoots and the bathing solution was placed

in a small vacuum desiccator. The desiccator was evacuated with a water

aspirator; then the vacuum was suddenly released so that atmospheric

pressure forced the solution into the shoots. The vacuum treatment was










repeated two additional times. After vacuum infiltration, the beaker

containing the shoots and the radioactive proline was placed in the

dark for either 2, 3 or 6 hours (see Results).



Isolation of Proline and Hydroxyproline from Seedlings

From seedlings whose roots had been exposed to radioactive pro-

line for 8 hours, proline and hydroxyproline were isolated by hydrolyzing

the seedlings and recovering the amino acids by column chromatography,

as described on page 9. Proline and hydroxyproline were then separated

and purified by thin-layer chromatography following deamination of

primary aL amino acids by nitrous acid as previously described (page 45)

with the exception that a preliminary partial separation on the Dowex

50W column was omitted; it was possible to omit this step because much

smaller amounts of plant material were used in these experiments than in

the experiments reported in Part II of this dissertation. Another reason

for omitting the preliminary partial separation on the Dowex 50W column

was that it was desirable to recover all of the proline.



Extraction of Ribonucleic Acid

If the hydroxylation of proline occurs before incorporation into

protein --- that is, during or prior to the time that proline is bound

to sRNA --- then it should be possible to isolate hydroxyproline from

RNA. Therefore, RNA was extracted from maize shoots which had absorbed

radioactive proline of high specific activity. The excised ends of the

shoots which had been exposed to the radioactive solution were washed in

a small amount of water so as to remove any labeled proline adhering to

the shoot surface. Ribonucleic acid was extracted from these shoots









after the method of Rosenbaum and Brown (85). This method utilizes a

phenol-buffer mixture to extract RNA; the RNA is extracted into the

aqueous phase while the protein is extracted into the phenol. In order

to prevent possible enzymic destruction of RNA, it is necessary to

inhibit ribonuclease; the use of phenol as a component of the extracting

mixture provided a convenient method for preventing the enzymic de-

struction of RNA as well as for separating the protein from the RNA.

The shoots were chopped with a razor blade and dropped into a

cold mortar containing liquid nitrogen; the material was frozen to pre-

vent enzyme activity during the grinding operation. The plant material

was ground in the frozen state to a very fine powder; liquid nitrogen

was added as necessary to keep the plant material frozen. After approxi-

mately three minutes of grinding, the powder was recovered and added to

a mixture of phenol, buffer and ethylenediamine tetraacetate. For each

10 g of fresh plant material, 10 ml of water-saturated phenol, 10 ml of

0.14 M sodium chloride-0.01 M phosphate buffer at pH 7 and 0.1 ml of 1

per cent enthylenediamine tetraacetate solution were used. The mixture

was stirred vigorously as the powdered plant material was added, and

stirring was continued for 6 additional minutes. The mixture was centri-

fuged for 10 minutes at 1,000 x g to separate the phenol and aqueous

phases. The aqueous layer containing the RNA was decanted into an

equal volume of water-saturated phenol (88 per cent phenol), so as to

remove possible traces of protein; the extraction was performed three

times by hand shaking for 2 minutes each time. The phases were sepa-

rated after each extraction by centrifugation as above; the aqueous

phase containing the RNA was removed from the centrifuge tube by

suction after each centrifugation. Traces of phenol were then removed









from the aqueous phase by five successive extractions with equal volumes

of diethyl ether in a separatory funnel. Residual ether in the aqueous

phase was removed by bubbling nitrogen through the solution for 1 hour.

All operations subsequent to the grinding were performed at 0 C to 5 C.

The recovered aqueous solution containing the RNA was designated as the

"original extract," and the optical absorbance in the ultraviolet region

was measured against a buffer blank in a Beckman DB spectrophotometer.

In order further to purify the RNA, it was precipitated by the

addition of two volumes of ethanol to the "original extract," and the

mixture was permitted to stand for 12 hours at 0 C. The precipitated

RNA was collected by centrifugation at 20,000 x g for 20 minutes. The

precipitate was dissolved in the sodium chloride-phosphate buffer

solution, and the absorbance in the ultraviolet region was again

measured. In order still further to purify the RNA, ethanol was again

added to the solution, and the RNA was precipitated a second time from

ethanol and recovered by centrifugation.

The amount of RNA in a sample was estimated from a standard

curve prepared by plotting the concentration of reagent RNA against the

optical absorbance at 260 nu.



Liberation of Ribonucleic Acid-bound Proline and Hydroxyproline

Since the amino acyl-sRNA bond is very labile in basic solution

(114), amino acids are usually stripped from sRNA by base hydrolysis.

However, acid hydrolysis was used here --- with the exception of one

experiment noted below --- because it was desirable to deaminate the

primary -amino acids with nitrous acid.









The amino acids bound to RNA were liberated by adding 100 ,g

each of unlabeled carrier proline and hydroxyproline to the second

ethanol precipitate of RNA and by hydrolyzing the alcohol precipitate

in 1.0 N hydrochloric acid for 2 hours (52) at 100 C. The acid hydro-

lysate was then stirred with 1 g of activated carbon for 30 minutes and

filtered, so as to remove nitrogenous bases and nucleosides resulting

from the hydrolysis of RNA. Primary C amino acids in the solution were

deaminated by the addition of 1 g of sodium nitrite. After standing

for 30 minutes, the acid mixture was evaporated on the rotary evaporator.

In order to separate proline and hydroxyproline from the 0- hydroxy acids

resulting from the deamination process, the residue was dissolved in 20 ml

of deionized water and chromatographed on a 10 x 1 cm Amberlite IR-120(H+)

column. The column was eluted with 2 N ammonium hydroxide, and the eluant

was evaporated to dryness on the rotary evaporator. The resulting resi-

due was dissolved in 0.3 ml of 70 per cent ethanol; 0.2 ml of this sol-

ution was spotted on cellulose covered thin-layer chromatography plates.

This procedure left much of the salt behind. Proline and hydroxyproline

were separated by thin-layer chromatography as described previously (page

48).

In order to determine whether the amino acids were completely

liberated from RNA by acid hydrolysis, RNA was subjected to base hydrolysis

in one experiment. The RNA from a second ethanol precipitation was heated

for 1 hour at 37 C in 0.1 M sodium carbonate solution adjusted to pH 10

with sodium hydroxide (114). The solution was then neutralized with

hydrochloric acid, and the RNA was precipitated with 2 volumes of ethanol.

The ethanol supernatant fraction containing the amino acids was evapo-

rated and chromatographed by thin-layer chromatography.









Counting of Radioactive Samples

Proline and hydroxyproline were eluted from the cellulose

absorbent following thin-layer chromatography as described on page 48.

The solution containing either proline or hydroxyproline was dried on

1.25-inch copper planchets under an infra-red lamp. Samples were counted

in a Nuclear-Chicago model D-47 gas flow counter for a sufficient time

so that at least 1,000 counts were recorded. The efficiency of the

counting system for carbon-14 was 30 per cent. All results are reported

as counts/minute (cpm) above background.



RESULTS



Conversion of Proline to Hydroxyproline in Etiolated Seedlings

When the roots of maize seedlings were placed in a solution of

proline-C-14 for 8 hours, radioactivity was found in both the proline

and hydroxyproline which was subsequently isolated (table 11). The

isolated proline had 1,071 to 1,383 cpm/g fresh weight of plant material

while the isolated hydroxyproline contained about one-tenth as much

radioactivity as the proline, or 96 to 140 cpm/g of fresh weight. When

soybean seedlings were treated in a similar manner, the proline which

was isolated had 1,776 to 2,012 cpm/g fresh weight of plant material

(table 11), and the hydroxyproline contained 181 to 196 cpm/g;this was

about one-tenth the activity of the proline, and was similar to the

results found with maize seedlings. Therefore, it could be concluded

that etiolated maize and soybean seedlings absorbed proline and converted

a portion of it to hydroxyproline.









Ribonucleic Acid Content of Maize Shoots

The ultraviolet absorption spectrum of an "original extract"

from etiolated maize shoots is shown in figure 15A. Two peaks are pre-

sent in the spectrum, one at 265 mu and the other at 310 mp. Apparently

much of the 265 Amj peak and all of the 310 mu peak resulted from soluble

mucleotides and other impurities in the extract, because the first

ethanol precipitation gave only one peak at 260 nu (figure 15B); this

peak is typical for nucleic acid (52). Therefore, the material obtained

after ethanol precipitation was assumed to be ribonucleic acid.

The amount of RNA extracted from 7-day-old etiolated maize shoots

was determined by reference to the standard curve (page 86) and varied

from 0.19 mg/g to 0.36 mg/g of fresh plant material (table 12). The

average amount of RNA from nine extractions was 0.26 mg/g. /



Proline-C-14 and Hydroxyproline-C-14 in Extracted Ribonucleic Acid

When maize shoots were exposed as previously described to a

solution containing 2.5 pc of uniformly-labeled proline-C-14 for 6 hours,

the proline which was separated from the extracted RNA contained 18 cpm/mg

RNA, and the hydroxyproline contained 50 cpm/mg RNA (table 13). Shoots

which had been exposed to 1.0 pc for 1 hour contained 15 cpm/mg RNA in

the proline and 11 cpm/mg RNA in the hydroxyproline. Shoots which had

been exposed to a solution containing 1.0 pc of proline-C-14 for 3 hours

contained 14 cpm/mg RNA in the proline and 33 cpm/mg RNA in the hydroxy-

proline liberated from RNA. From these results, it could be concluded

that the conversion of proline to hydroxyproline occurred before the

incorporation of proline into protein in maize seedlings.









Table 11. -- Conversion of proline-C-14 to hydroxyproline-C-14
by maize and soybean seedlings.




counts/minute/g
Experi- Number of total counts/minute fresh weight
ment seedlings proline hye p e
proline hydroxyproline proline hydroxyproline


Maize


5,300


4,282


10 11,340


642


521


787


1,150


1,071


1,383


Soybean


6,440


2,012


196


5,328 543


(2) 4


1,776 181












0.6E


0.5- v/


U,
o 0.4 A


S0 .




0.2



0.1



240 280 320 360 400

WAVE LENGTH (mp.)


Figure 15. -- Absorption spectra of RNA preparations:
(A) "original extract" and (B) ethanol precipitation.









The possibility exists that the radioactivity found in proline

and hydroxyproline came from contamination of the extracted RNA by free

proline and free hydroxyproline and did not arise from proline and

hydroxyproline which was bound to RNA. However, this possibility of

contamination of extracted RNA was eliminated by the following experi-

ment. Instead of exposing the shoots to radioactive proline as de-

scribed on page 83, 1.0 ,c of proline-C-14 was added directly to the

ground plant material together with the phenol-buffer extractant. After

RNA was extracted and the imino acids were isolated, it was found that

neither proline nor hydroxyproline contained radioactivity. Thus, it

could be concluded that contamination of the RNA by free proline and

free hydroxyproline was not the source of the radioactivity reported

in table 13.

When the amino acids bound to RNA were liberated by base hydro-

lysis instead of acid hydrolysis, the proline contained 22 cpm/mg RNA

and the hydroxyproline had an activity of 35 cpm/mg RNA (table 13). It

is evident from table 13 that the proline liberated by base hydrolysis

contained more (approximately 50 per cent) activity than proline liberated

by acid hydrolysis, while the activity of the isolated hydroxyproline

was about the same as for acid hydrolysis. Two explanations may be

offered to account for the greater radioactivity in proline liberated

by base hydrolysis: (1) acid hydrolysis did not completely liberate all

of the amino acids bound to RNA, and (2) during the time when the shoots

were exposed to radioactive proline, some of the absorbed proline-C-14

was probably converted to other amino acids which chromatographed with

proline. This latter explanation is possible because when base hydro-

lysis was used, the primary a amino acids were not deaminated by the




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