A quantitative investigation of noncompetitive elution in affinity chromatography using bovine alpha-chymotrypsin as a model

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Title:
A quantitative investigation of noncompetitive elution in affinity chromatography using bovine alpha-chymotrypsin as a model
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A quantitative investigation of noncompetitive elution in affinity chromatography using bovine alpha-chymotrypsin as a model
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xi, 99 leaves : ill. ; 29 cm.
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Gilbert, William Arthur, 1953-
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Chromatography, Affinity   ( mesh )
Biochemistry and Molecular Biology thesis Ph.D   ( mesh )
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Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1978.
Bibliography:
Bibliography: leaves 95-96.
Statement of Responsibility:
by William Arthur Gilbert.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
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oclc - 25700987
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Full Text











A QUANTITATIVE INVESTIGATION OF NONCOMPETITIVE
ELUTION IN AFFINITY CHROMATOGRAPHY USING
BOVINE a-CHYMOTRYPSIN AS A MODEL


















BY
WILLIAM ARTHUR GILBERT


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





UNIVERSITY OF FLORIDA


1978















I dedicate this dissertation to my first grade teacher,

Dorothy Jo Gonser, who opened up the world to me through the

beauty of reading. Her enthusiasm at my early age prompted me

to reach for a dream and to get there.
















ACKNOWLEDGEMENTS


The author would like to express his deep appreciation to

Dr. Ben M. Dunn, his research director, for giving him the guidance

and freedom of thought during the completion of this work. Dr. Dunn's

open mindedness also allowed the author to develop skills outside of

the Department of Biochemistry., Wichout Dr. Dunn's cooperation this

author may not have been able to realize his full potential. The en-

vironment of Dr. Dunn's laboratory provided the author with the elements

required for productive work as well as for the development of deep

friendships.

Special thanks are given to the author'5 advisory committee and

fellow graduate students of the Department of Bioch.mistry and Molecular

Biology for their suggestions and criticism during the execution of this

research.

The author wishes to thank his parents for their years of under-

standing and for trust without which this work would have been impossible.




















TABLE OF CONTENTS


ACKNOWLEDGEMENTS .


LIST OF TABLES . . .


LIST OF FIGURES . .


KEY TO SYMBOLS NOT DEFINED IN TEXT .


ABSTRACT . . .


INTRODUCTION . . .


Background . .


Quantitive Affinity Chrocatography .


Affinity Chromatography of a-Chymotrypsin


MATERIALS AND METHODS ......


Chemicals .. . .


Equipment . ....


Spectroscopic Assay for a-Chymotrypsin .


Titrimetric Assay for a-Chymotrypsin .


Affinity Matrix Synthesi . .


Small Zone Elution Experijnents .


Large Zone Elution Experiments .


TPCK Inactivation of a-Chymotrypsin .

RESULTS . . .


Elution of the Affinity Matrix .


Soluble Ligand Experiments .

Determination of Dissociation Constants for
Soluble Complexes . .


. ..... vi




. ix


. x


. . 1


. . 1


. . .


. .14


. . .18


S. .18


. . .18


S. .19


. . .19


. . .20


. . .20


. .21


. . .22


. . .25


. . .25


. . .28


. . . .iii










Quantitation of Matrix Bound Ligand . 45

DISCUSSION . . . 51

ABENDIX . . . 90

BIBLIOGRAPHY . . . 95

BIOGRAPHICAL SKETCH ...... ....... ........ ..... 97















LIST OF TABLES


TABLE PAGE

I Dissociation constants for inhibitors of a-chymotrypsin
as determined from kinetics of the hydrolysis of BTEE .. 46

II Summary of predicted elution behavior in noncompetitive
affinity chromatography ... . ..... .. 62

III Comparison of soluble dissociation constants
obtained from affinity chromatography with
those obtained by solution kinetics ..... .. .. 78

















LIST OF FIGURES


FIGURE

1 Affinity Chromatography .


2 Competitive Binding Scheme for Affinity
Chromatography . . .

3 Small Zone Versus Large Zone Elutio .

4 a-Chymotrypsin Binding to Affinity Matrix .

5 TPCK-Chymorrypsin Elution on Affinity Matrix .

6 a-Chymotrypsin Elution from the Affinity
Matrix . . .

7 a-Chymotrypsin on an Unsubstituted Matrix .

8 Small Zone Experiments for
Carbobenzoxyplenylalanine . .

9 Small Zone Experiments for
N-Acetyl-L-Phenylalanine . .

10 Small Zone Experiments for
Carbobenzoxyphenyialanine . .....

11 Large Zone Experiments for
Carbobenzoxyphenylalanine . .

12 Ultraviolet Spectra of 4-Phenylbutylamine and
Supernatant from Solvolysis of Matrix .


PAGE


. 11

.. 24

. 27

. 30


. 32

. 34


. 37


. 39


. 41


. 44


. 48


13 Ultraviolet Spectra of Unsubstituted Matrix and
Affinity Matrix After Solvolysis .

14 Dissociation Constants Present in Competitive
Elution Scheme . .

15 Expected Results for Small Zone Experiments in a
Competitive Elution Affinity Column .

16 Noncompetitive Elution Scheme for Affinity
Chromatography .. ..... .


. 50


. 53


. 55


.... .. 58


vii











17 Simulation of Elution Behavior for
Noncompetitive Scheme . .... ... 61

18 Case I Type Noncompetitve Elution . 64

19 Case II Type Noncompetitive Elution .... 66

20 Case III Type Noncompetitive Elution .. 68

21 Linearized Form of the Case I Type Simulated
Results from Figure 18 . . 73

22 Results for Carbobenzoxy Deriviatives of the Aromatic
Amino Acids Plotted in Double Reciprocal Form .. 76

23 An Example of the Results Obtained from Solution
Kinetics Analyzed as a Double Reciprocal Plot
by the Method of Lineweaver and Burk ... .80

24 Linearized Form of the Case III Type Simulated
Results from Figure 20 . .. 82

25 Results for Carbobenzoxyalanylalanine Plotted
in Double Reciprocal Form . .... 85

26 The Structure of the a-Chymotrypsin Affinity
Matrix . . . 88


viii















KEY TO SYMBOLS NOT DEFINED IN TEXT


A280 -Light absorbance at 280 nm
280
CBZ -Carbobenzoxy (prefix)

TPCK l-tosylamido-2-phenylethyl
chloromethyl ketone

Tris tris(hydroxymethyl)aminomethane










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




A QUANTITATIVE INVESTIGATION OF NONCOMPETITIVE
ELUTION IN AFFINITY CHROMATOGRAPHY USING
BOVINE a-CHYMOTRYPSIN AS A MODEL

BY

WILLIAM ARTHUR GILBERT

June, 1978

Chairman: -Ben M. Dunn
Major Department: Biochemistry and Molecular Biology

Affinity chromatography has become a widely accepted and powerful

technique in protein purification. However, the method was developed in

a shroud of empiricism based on unproven assumptions. Most often, an

investigator would opt for the highest substitution obtainable and

evaluate the affinity matrix only on an operational basis. There existed

no unambiguous way to quantitatively evaluate a newly synthesized affinity

matrix.

This work presents a review of recent attempts in the development

of quantitative affinity chromatography and describes a widely accepted

method of zonal elution for determining intrinsic binding constants.

This method was applied to a system in which 4-phenylbutylamine was

immobilized to form an affinity matrix for a-chymotrypsin. a-Chymotrypsin

was passed through the column in the presence of varying concentrations

of known binding ligands. The results obtained were inconsistent with

those predicted for a simple competitive model.








A noncompetitive model involving two unique yet Interacting binding

sites was proposed. This model allows the enzyme to associate with the

column in either the free form, E, or as the complex, E-I.

Three types of elution behavior were predicted from the ratio of

column binding constants. Case I type of noncompetitive elution in-

volved soluble ligand retarded enzyme elation; case II type elution was

the degenerate case in which soluble ligand did not affect enzyme elution.

Finally, case III type elution involves soluble ligand facilitated enzyme

elution.

The experimental data obtained were found to fit Lhis model very

closely. Further, all three types of elution behavior were observed

and fit results predicted from the noncompetitive elution model.

This work is presented as a demonstration of the applicability of

quantitative affinity chromatography in examining sophisticated binding

phenomena.

This work is directly applicable to studies involving solid state

substrates. Examples of this are found in cell membrane, organelle and

biopolymer work.



Ben M. Dunn
Supervisory Chairman
















INTRODUCTION


Background

Since the beginnings of the science we now know as biochemistry,

investigators have been concerned with the purity of the material with

which they were working. Methods for protein purification governed the

rate at which this science developed. The separation techniques developed

were based on the physical properties of the proteins. The differences

in these physical properties were exploited to separate proteins into

classes depending on ionic charge, solubility, molecular size and a

variety of other parameters.

It is interesting to note that all of these separations were done

using properties which are secondary to the uniqueness of a protein. The

distinguishing feature of any protein is its specificity in the binding of

various ligands. Functions such as transport, catalysis, antibody recog-

nition and receptor binding all depend on ligands binding specifically to

the protein surface. This property was successfully used by uatCrecasas,

Wilchek and Anfinsen in 1968 to develop a method referred to as affinity

chromatography. The concept is realized by covalently attaching the ligand

to an insoluble matrix and packing the support into a chromatographic

column. Those macromolecules that display an affinity for the immobilized

ligand will be retained by the matrix, while those with little or no af-

finity will pass through unretardud. This is shown schematically in figure i,

where M is the insoluble matrix, L is the immobilized ligand which is

attached to the matrix, M, via a spacer arm assembly, S. This ligand marrix





































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conjugate has the element of specificity which would be unique for

the macromolecule to be isolated. The species which had been adsorbed

can then be desorbed by a change in the composition of the eluting buffer

to favor dissociation. These changes usually involve pH, ionic strength

or temperature.

This potentially easy, rapid and virtually limitless application of

affinity chromatography has promoted much growth in biochemistry over the

last decade. Wilchek and Jakoby (1974) list over 120 unique purification

applications. This technique can also be used to resolve and purify cells

and viruses (:urrr. t a i. 1972), chemically modified proteins (Wilkersor.

Mariano aRd~~S&76) and has potential use in the exploration of binding

sites, topographies and kinetic mechanisms (MareiniAzy et .1". 97r).

However, recent experience with affinity chromatography has given in-

vestigators a more cautious approach to this technique. An earl-y paper-by

Steers et al. (1971) describing the purif:c;tl n of 3-galactcsidase on

immobilized galactoside was later shown by, O'Carra. e al. (1973, 1974), to

be largely non-specific interactions with the spacer arm assembly. Such

phenomena can severely interfere with the true biospecific behavior and lead

to artifactual observations, such as electrostactic effects which may

attract non-specific proteins or repel the specific binding proteins. Care-

ful consideration must therefore be given to the nature of the matrix and

restrictions imposed by the immobilization of a ligand.

The selection of a matrix support is constrained by several factors,

The matrix should be composed of uniform spherical beads which are hydro-

philic, mechanically and biologically stable, and possess chemical groups

that can be easily modified without altering any of those properties.









Cuatrecasas and Anfinsen (1971) have reviewed the relative advantages

of cellulose, agarose, dextran, glass and polyacrylamide as affinity

matrices.

The activation of hydrophilic polysaccharides by cyanogen bromide

is currently the most commonly employed procedure for the immobilization

of ligands having primary amino functions (Axmn -et nr---C96-4. This

procedure although widely accepted is not without its disadvantages. The

activation procedure introduces an isourea group as the link between the

ligand and the saccharide matrix. This group has a pKa of 10.4 which

gives a positively charged matrix at neutral pH. The resulting matrices

could exhibit an electrostatic interaction cfr the protein which may

alter or obliterate the specific interaction by the immobilized ligand

(Nishikawa & Bailon 1975). Wilchek (1973) has proposed that the charge

introduced by cyanogen bromide coupling of primary amines may be extin-

guished by the use of acrylhydrazides which yield a linkage to Agarose

which has a pKa of 4.0 and thus is uncharged at neutral pH. Tesser et al.

(1974) have pointed out another failing of the cyanogen bromide coupling

technique. They suggest that a small but continuous solvolysis of the

linkage between the matrix and the ligand is present at pH's above 5.

Wilchek (1973) has described the preparation of stable agarose derivatives

by coupling multivalent spacer molecules such as polylysine, polyornithine

or polyvinylalanine to Agarose. This polymer which serves as a spacer

arm is attached to Agarose by multiple isourea linkages. Thus, as these

linkages break down. the polymer will remain attached to the Agarose bead

through other linkages. Attachment of ligands to this spacer can be

achieved through the unreacted amino functions on the polymer. Wilchek

and Miron (1974) later combined the advantages of multipoint attachment









of spacer arms and the coupling of acylhydrazides to Agarose. His new

matrix synthesis method involved the coupling of polyacrylic hydrazide to

Agarose. The unr-acted hydrazide groups could be used to directly couple

carboxyl containing ligand or could be modified to accept ligands con-

taining other types of reactive groups. The most widely used modification

involves succinylation of the hydrazide moiety giving a carboxyl group

which could be used to couple ligands containing primary amines.

It seems therefore, that while cyanogen bromide activation is a very

simple and rapid procedure, it has a number of serious shortcomings for

use in affinity chromatography. However, these shortcomings have stimu-

lated the development of sophisticated methodologies in the design and

synthesis of affinity matrices.

A number of considerations govern the selection of the ligand for

affinity chromatography. The ligand to be coupled to the solid support

or spacer must display a unique affinity for the macromolecule to be puri-

fied. It can be a substrate, inhibitor, allosteric effector, cofactor,

hormone, antigen or any species that displays the required specificity.

Further, the ligand must possess functional groups that can be modified

for attachment to the solid support without blocking its recognition by

the complementary macromolecule. Most investigators have relied on em-

pirical guidelines -which had developed during the first half-decade of

affinity chromatography. These guidelines did not give investigators an

unambiguous method for quantitatively evaluating a newly developed chroma-

tographic matrix. This lack of information on the binding strength be-

tween the immobilized ligand and macromolecule often precluded any rational

design strategy and the highest attainable ligand concentration was

generally opted for. Further, a column's effectiveness was measured solely









on whether it worked and not on any quantitative criteria which could

be used by other investigators working on similar systems for comparison.



Quantitative Affinity Chromatography

In 1971, Akanuma et al. studied the affinity chromatography of

carboxypeptidase 3 on columns of immobilized basic amino acid and aromatic

amino acid derivatives. They were interested in the elution of the enzyme

from the column in a concentration gradient of eluting ligand. They

observed two interesting effects, first, carboxypeptidase B was eluted

from a column of immobilized e-aminocaproyl-D-arginine by an increasing

gradient of e-aminocaproic acid; secondly carboxypeptidase B was eluted

from a column of immobilized c-aminocaproyl-D-phenylalanine by a decreasing

gradient of e-aminocaproic acid. These two observations demonstrated two

binding schemes which could exist in affinity chromatography. The former

was a ligand-inhibited binding and the latter a ligand-facilitated binding.

Akanuma et al., however, made no attempt to quantitate these two binding

phenomena.

In this context, several attempts were made to devise a method for

the measuring of the parameters surrounding this technique. Andrews et al.

(1973) provided the first example of such a quantitative approach, using

immobilized a-lactalbumin to determine the equilibrium constant between

galactosyltransferase and glucose, Their simplified approach was only

applicable to a ligand facilitated binding system. Their experiments con-

sisted of immobilizing a-lactalburin to Sepharose and placing the modified

gel in a chromatographic column. The column was then pre-equilibrated with

a buffer containing a desired concentration of glucose. Galactosyitransferase,

in a small volume of the pre-equilibrating medium, was applied to the column:









fractions were collected and assayed for the appropriate activity. By

varying the concentration of glucose in the equilibrating buffer, a

family of elution volumes were generated for the protein. They observed

that by plotting the data as the reciprocal of the difference between

the observed elution volume, Ve, and Vo, the elution for the enzyme in the

absence of glucose, versus the reciprocal of the glucose concentration,

a straight line was obtained. Further, they showed that by dividing the

ordinate intercept by the slope, a value was obtained which was very close

to the known dissociation constant for the glucose.galactosyltransferase

complex. They repeated these experiments using N-acetylglucosamine as the

soluble ligand. Again, results fitting a straight line were obtained and

a value for the dissociation constant cf the N-acetylglucosamine-galacto-

syltransferase complex was obtained which matched the known value. Andrews

et al. made no mention of the relationship between the elution volume to

the degree of substitution of ligand on the matrix.

Nichol et al. (1974) expanded the theory of this quantitative approach

to encompass many possible equilibria involving an immobilized ligand,

soluble protein and soluble ligand(s). Nichol's equations provided a

complete theoretical consideration for affinity chromatography; however,

his derivation was also very cumbersome and lacked the necessary simplifi-

cations which would make its application experimentally feasible.

Dunn and Chaiken (1974) were working on the problem of quantitating

these parameters at the time of Nichol's derivations. Dunn and Chaiken,

however, had a technique which was better suited to an experimental

evaluation. Their column system consisted of an Agarose matrix of im-

mobilized thymidine-3'-aminophenylphosphate-5'-phosphate. The enzyme









staphylococcal nuclease was known to bind specifically with this matrix

(Cautrecasas, Wilchek and Anfinsen 1968). Their experimental approach

was to pre-equilibrate a column of the affinity matrix with a known con-

centration of soluble thymidine-3',5'-bisphosphate. This soluble ligand

would compete for the binding site on staphylococcal nuclease and pre-

vent interaction with the immobilized ligand. This scheme is shown in

figure 2 where E, I and =EF are the free enzyme, soluble ligand and im-

mobilized ligand, respectively; E-I and EtL-T are the possible binary

complexes formed in this system with KI and K]pg as their dissociation

constants. At low concentrations of soluble ligand the enzyme would

spend more of its time in the stationary phase, this would lead to re-

tardation of the enzyme by the column and a large elution volume for

the enzyme. On the other hand, at high concentrations of soluble ligand,

the enzyme would not be bound tc the immobilized ligand as much as to the

ubiquitous soluble ligand. This would lead to a situation in which the

enzyme would spend most of its time in the mobile phase and be eluted from

the column at a small elution volume. By carefully choosing the concen-

tration of soluble ligand they were able to establish an equilibrium for

the enzyme between the immobilized ligand (stationary phase) and the soluble

ligand (mobile phase) as it passed down the column.

They presented a derivation in which their final equation was re-

presented in experimentally obtainable values.



1 KT [T
Ve-V (V + [ -)( V -)
-e o o m) KI (Vo Vm)[L-7M




























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ca $ 0
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00
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I
-J

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-J
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Where 1/(Ve Vo) is plotted versus [I] in which Ve, Vo and Vm are the

elution volume for the enzyme at a soluble ligand concentration, the

column void volume and volume of the mobile phase, respectively. K7--F,

KI, L- and I are as described. Here, the ordinate intercept divided by

the slope gives KI; K -= can be obtained from the ordinate intercept by

substitution of [L-M], Vo and Vm. A dissociation value for the nuclease

thymidine-3',5'-bisphosphate complex was obtained as well as a value for

the dissociation of the enzyme from the immobilized ligand. The measure-

ment of this latter value is very significant in that it could provide a

means of quantitatively expressing the ability of the column to bind pro-

tein. Further, the measurement of this value indicates that in an affinity

chromatographic system the enzyme is bound reversibly to the matrix and

exists in a dynamic equilibrium between the mobile phase and the stationary

phase. Thus in any affinity chromatographic system the protein will elute

at a finite volume, this volume being a function of the dissociation con-

stant between the enzyme, immobilized ligand, the concentration of im-

mobilized ligand, and the physical dimensions of the column.

This is somewhat different from the current attitude towards affinity

chromatography in which the adsorption process is maximized and not applied

quantitatively to the investigator's advantage. By the proper measurement

of the dissociation constant for the enzyme matrix interaction one could

rationally predict the behavior of the column and/or modify the ligand to

obtain the optimum behavior. The concept of a dynamic existence of the

enzyme between the two phases is extremely important and cannot be over-

emphasized when dealing with affinity chromatography.

Chaiken and Taylor (1976) used the analytical method of Dunn and

Chaiken to look at the behavior of ribonuclease A analogs. These analogs








were structurally competent complexes formed by allowing the cleaved S

peptide from ribonuclease A to reassociate with the remaining protein to

form a noncovalent complex which resembled the original enzyme. This

established a working base for determining binding constants for inactive

ribonuclease S, although catalytically nonfunctional, could recognize

substrates and substrate analogs.

Other attempts were made at developing quantitative affinity

chromatography. Kasai and Ishii (1975) did quantitative affinity chroma-

tography with a trypsin-glycylglycyl-L-arginine Agarose system; using

various concentrations of derivatives of L-arginine as the soluble ligand,

they generated a series of elution volumes for trypsin. Their results

fit the competitive binding scheme of Dunn and Chaiken. From these ex-

periments they obtained values for the Kl's of various inhibitors of trypsin.

Brinkworth et al. (1975) used quantitative affinity chromatography to

simultaneously determine the K,'s for NADH for the five isozymes of lactate

dehydrogenase. Their experiments point out the power an affinity system

would have when the intrinsic binding constants are known. By adjusting

the concentration of soluble ligand they were able to achieve maximum

separation of these isozymes from a tissue extract.

Finally, Brodelius and Mosbach (1976) developed a system in which

they could obtain the relative Km's for enzymes which bound to the same

affinity matrix. By applying all the enzymes to a column of immobilized

AMP, followed by an increasing gradient of soluble NADH, the bound en-

zymes were eluted as a function of their Km for NADH. The enzymes with

higher dissociation constants were found to elute first while the enzymes

with lower dissociation constants were eluted later.









They observed a linear relationship between the known Km's and con-

centration of NADH in the volume at which they eluted. From these re-

sults they were able to establish a calibration curve for their column.

They applied lactate dehydrogenase isozymes from various animal sources

and measured the eluting concentration of NADH against their calibration

curve.

Although this provided an empirical method for the determination of

soluble Km's, Brodelius and Mosbach did not derive a mathematical rela-

tionship for their calibration curve.



Affinity Chromatography of a-Chymotrypsin

Chymotrypsin is the product of tryptic activation of its inactive

precursor, chymotrypsinogen, which is produced by the pancreas. The iso-

lation of bovine chymotrypsinogen and its activation by trypsin were first

reported by Northrop and Kunitz (1932). The initial step involves the

splitting of the Arg(15) Ile(16) peptide bond leading to the formation

of catalytically active r-chymotrypsin. Subsequent autocatalytic events

give rise to S-, K-, and y-chymotrypsin as intermediates in the formation

of a-chymotrypsin (Wright et al. 1968; Miller er al. 1971). The final

species is the one most commonly encountered. All active forms of the

enzyme are known to exhibit a marked preference in catalyzing the hydrolysis

of peptide bonds on the carboxyl side of aromatic amino acid residues.

While the procedures for the isolation of chymotrypsin and chymotryp-

sinogen in a highly homogeneous state have been developed (Keller et al.

1958), the preparation of chymotrypsinogen devoid of chymotrypsin is dif-

ficult to achieve, even after repeated purification procedures. Cuatrecasas

et al. (1968) approached this purification through the use of their newly









developed affinity chromatography technique. They were able to selectively

adsorb chymotrypsin to Sepharose gels containing covalently bound e-amino-

n-caporyl-D-tryptophan methyl ester. Enzyme was adsorbed at pH 8.0 when

applied to this column. This adsorption was selective in that chymotryp-

sinogen, subtilisin, diisopropyl-phosphoryl-trypsin and pancreatic ribo-

nuclease were not bound. The a-chymotrypsin was not ad:orbed by a column

of unsubstituted Sepharose.

The possibility of coupling inhibitors other than E-amino-n-caporyl-

D-tryptophan methyl ester to Sepharose was considered by Stevenson and

Landman (1971), who studied the properties of Sepharose-4-phenylbutyl-

anine columns. Their choice of this molecule was based on the known binding

of the hydrophobic phenyl moiety to chymotrypsin and on minimization of

steric effects by virtue of the bucyl spacer arm in the molecule. Their

studies with Sepharose-4-phenylbutylamine were in accordance with these

expectations. The column was capable of adsorbing a-chymotrypsin from

solutions at pH 8.0 with recovery by elution at pH 3.0. The selectivity

of the adsorbent was further confirmed by its inability to adsorb porcine

trypsin, bovine trypsinogen and a-chymotrypsin inhibited with tosylpheny-

lalanine chloromethyl ketone (TPCK). The Sephiarose-4-phenylbutylaminne

columns were found to be effective in the isolation of chymotrypsin-like

proteases from extracts of moose (Alces alces) pancreas. The use of

4-phenylbutylamine is advantageous over that of e-amino-n-caproyl-D-

tryptophan methyl ester in that the synthesis of the later is a time-

consuming and tedious synthesis. Further, once synthesized it is subject

to degradation by light.

Hofatree (1973) investigated non-specific binding of negatively charged

proteins to column of Sepharose substituted with n-alkylamine or with









4-phenylbutylamine (PBA). Of the proteins tested, the only exception to

nonspecific binding was a-chymotrypsin which showed a strong affinity for

PBA-substituted material even in the presence of reagents which disrupted

the non-specific binding. By contrast, biidirg did not occur when the

ligand was an n-alkylamine. He concluded that although many of the proteins

he was testing were binding because of bydrophobic and/or electrostatic

forces that the binding of ut-chymotrypsin was a specific event involving

the complementarity of the contours (i.e., fit) of the interacting mole-

cules.

There is also much known about the kinetics and mechanism of a-

chymotrypsin (Boyer, the Enzymes, 1970). Work with synthetic inhibitors

by Morihara and Oka (1977) shows that the active center of the enzyme can

accommodate four to five amino acid residues along with the aromatic resi-

due which fits near the catalytic site. This is supported by X-ray

crystallographic work which shows the binding site in chymotrypsin as an

elongated groove capable of binding several amino acid residues (Segal

et al. 1971; Robertus et al. 1972).

The amount of information available on the affinity chromatography

of a-chymotrypsin with immobilized 4-phenylbutylamine makes this system

a logical choice in which to further investigate the binding constants

involved in affinity chromatography. The system has been shown to be both

specific and devoid of any interfering interactions. Further, the amount

of information available on a-chymotrypsin and its inhibitors encourages

the choice of this system for quantitative study. By further developing

the technique of quantitative affinity chromatography through the use of

an established system, we hope to extend this method to systems in which





17



the phenomena of immobilized binding are not well understood. This in-

volves enzymes whose substrates are actually substructures of very large

macromolecules such as cell walls or ribosomes. Most important of all

we hope to remove some of empiricism which surrounds affinity chromatography

and provide some rational approach in evaluating an affinity system.















MATERIALS AND METHODS

Chemicals

Bovine a-chymotrypsin was obtained from Sigma Chemical Company as

a three times crystallized and lyophilized solid with a specific acti-

vity of 43 Units per milligram. N-Benzoyl-L-tyrosine ethyl ester, car-

bobenzoxy (CBZ) amino acid derivatives and N-acetyl amino acid deriva-

tives were also obtained through Sigma Chemical Company. 4-Phenylbuty-

amine and 4-phenylbutyric acid were obtained from Aldrich Chemical Com-

pany. Succinylated polyacrylic hydrazide Agarose was obtained through

Miles-Yeda, Ltd. (51 pmole succinyl groups per ml, Lot No. SPAH3). All

other chemicals used were reagent grade and purchased from common suppliers.



Equipment

The column used in these experiments was a Chromatoflo Series A

borosilicate glass type (0.9 x 25cm) purchased from Pierce Chemical

Company. A sample injection port and adjustable plungers for variable

bed volumes were also obtained from Pierce. An LKB 2120 Varioperpex

peristaltic pump was used to maintain a uniform eluant flow through the

column. Fractions were collected using an LKB Redirac Fraction Collector.

The eluant from the column was passed through a ISCO Model 226 Absorbance

Monitor for recording the amount of 280 nanometer absorbing material.

Chromatograms were recorded using a Model 1310 strip recorder from Bio-Rad

Laboratories.









Spectrophotometric activity assays were performed using a Gilford

Model 250 spectrophotometer. Titrometric activity assays and syntheses

involving titration were performed using a Radiometer TTT60 Autotitrator.



Spectroscopic Assay for a-Chymotrypsin

Fractions were assayed for a-chymotrypsin activity by the method of

Hummel (1959). N-benzoyl-L-tyrosine ethyl ester (6.7 mg) was dissolved

in 20 milliliters of methanol. Twenty-two milliliters of 0.08 M tris.HCl-

0.1 M calcium chloride (pH 7.8) were then added and mixed. Nine-hundred

microliters of this assay buffer was placed in a one milliliter cuvette and

the absorbance of the substrate at 256 nanometers electronically subtracted.

The assay is started by adding 100 microliters of test solution (0.1 to

2.0 micrograms a-chymotrypsin) to the cuvette and mixing the contents. The

rate of enzymatic hydrolysis of N-benzyol-L-tyrosine ethyl ester is mea-

sured by a linear increase in absorbance at 256 nanometers. One unit of

enzyme activity is equivalent to one micromole of substrate hydrolysed per

minute at pH 7.8 and 250C.



Titrimetric Assay for a-Chymotrypsin

An alternative method for determining the activity of a-chymotrypsin

has been described by Morihara and Oka (1977) which involves measuring

the proton release, uptake of base, by a pH stat method. Reactions were

carried out in 2 mM N-benzoyl-L-tyrosine ethyl ester, 0.1 M in calcium

chloride with 0.01 N sodium hydroxide as titrate. A pH of 7.8 was main-

tained during the hydrolysis of substrate by the addition of sodium hy-

droxide. The rate at which titrant was added to the test solution is

measured on a servorecorder. One unit of enzyme activity is equivalent

to one micromole of substrate hydrolysed per minute at pH 7.8.








Affinity Matrix Synthesis

The affinity matrix used in this study was based on the immobiliza-

tion of 4-phenylbutylamine according to Stevenson and Landman (1971).

Twenty milliliters of settled succinylated polyacrylic hydrazide Agarose

(51 micromoles succinyl groups per milliliter, 1.02 mmoles total) was

washed with water to remove the storage buffer and taken up in dioxane/

water (4:1). 4-phenylbutylamine (15.3 mile, 2.28 g) was added and the

pH adjusted to 6.0 with 1 M hydrochloric acid (HC1). The coupling rea-

gent, l-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide (4 g), was added in

0.2 gram units into the stirred Agarose. The pH is maintained at 6.0

during these additions by dropwise titration with 1 M HC1. The reaction

was allowed to proceed for 10 hours with gentle mixing by using a stirring

bar and motor. At no time during the reaction should the gel be stirred

violently as this leads to a mechanical shearing of the gel particles,

thus reducing their porosity. The coupling was terminated by washing the

gel sequentially with water, 0.1 M sodium carbonate (pH 9.5), 0.1 M sodium

acetate and finally 40 mM tris.HC1-50 mM calcium chloride (pH 7.8); all

of the washing buffers contained 25% (v/v) methanol to aid in solubilizing

residual 4-phenylbutylamine.



Small Zone Elution Experiments

A solution of a known amount of ligand was prepared by first dis-

solving the ligand in about one milliliter of methanol. This was necessary

because of relative insolubility of the ligand in water. This was then

added to 40 mM tris-HC1-50 mM calcium chloride (pHI 7.8) 25% (v/v) methanol

and brought to a final volume of 200 milliliters. The solution was degassed

under vacuum and the affinity column pre-equilibrated with this buffer.









The eluant was monitored spectrophotometrically at 280 nm for the emergence

of ligand through the column. A stepped increase in the absorbance of

eluant was taken to indicate equilibration of the column. To insure full

equilibration no less than four column volumes were run after this initial

detection of ligand. An enzyme solution (1 mg/ml) was prepared in which

the ligand concentration had been adjusted to be the same as that of the

equilibrated column. One hundred microliters of this enzyme solution was

applied to the column via the sample injection port. The fraction col-

lector, peristaltic pump and strip chart recorder were started simultan-

eously; one minute fractions containing 0.74 milliliters were collected.

A run was terminated by the detection of enzyme elution from the eluant

monitor. The column was washed with the methanolic tris-Ca++ buffer to

remove soluble ligand and to prepare the column for the next determination.

The activity in the fractions was determined by using the spectrophotometric

assay as described above. One hundred microliters was taken from each

fraction and added to 900 microliters of substrate was sufficient to over-

come the effect of inhibition by that ligand. Results of activity assays

and A280 tracings were plotted and the elution volume defined as the volume

at which the enzyme is half-eluted, that is, the top of the eluting peak.



Large Zone Elution Experiments

In large zone experiments, the column was pre-equilibrated as in the

small zone experiments. The enzyme solution was prepared by first dis-

solving (37.5 mg) a-chymotrypsin into one milliliter of 0.001 M hydrochloric

acid to aid initial solubilization. This was then brought to a final

volume of 150 milliliters with the pre-equilibration buffer to give a final

concentration of 0.25 mg/ml c-chymotrypsin. This solution was passed through









the column, fractions were collected and the eluant monitored as in small

zone experiments. The enzyme was eluted as a front of increasing absorbancy

and reached a plateau corresponding to 0.25 mg/ml enzyme. The column was

then washed with methanolic-tris Ca++ buffer as in small zone experiments.

Activity assays were performed on each fraction as previously described.

The results were plotted along with the A280 tracings and the elution

volume was taken as the volume at which the activity is one-half that of

the plateau, or the centroid of the enzyme front.

Figure 3 gives a comparison of the expected elution profiles for

small zone and large zone experiments.



TPCK Inactivation of a-Chymotrypsin

a-chymotrypsin was inactivated by the method of Stevenson and Smillie

(1968). Enzyme (200 mg, 8 moles) was dissolved in 250 ml of 0.1 M tris-HCl-

50 mM CaC12 (pH 7.5). After removing 1 ml of solution to serve as a refer-

ence, 7.5 ml of ethanol were slowly introduced followed by the addition of

28 mg (80 moles) of tosylphenylalanine chloromethyl ketone (TPCK) in 5.0

ml of ethanol to give a final molar ratio of TPCK/enzyme of 10/1. The

solution was stirred at room temperature for 2.5 hr and assayed for re-

sidual activity. The enzyme preparation contained iess than 5% of the

initial activity. The reaction mixture was freeze-dried and passed over

a Sephadex G-25 column to remove the excess reagents and salts.





































0
M I -*
0 0 4




0 0
C a) ri 4-


> 0 0 0
0 *4 *- b C





N 0 0 O0

00 0 O
u0 &Co
0 N*HO


0 0 4-i 0



N n) en o o



S a o0
a < Xo M >
M )'Q)r
g, Pc L1
T^ c
arCPi nE




























o Dm




CL ^^ C



Q Y opz















RESULTS

Evaluation of the Affinity Matrix

The primary concern in using a newly synthesized affinity matrix is

to test for non-specific binding effects. A column was poured with fresh

4-phenylbutylamine-polyacrylhydrazide Agarose and washed with methanolic

tris.Ca+ buffer until a stable baseline was obtained on the eluant moni-

tor. This was to remove any 4-phenylbutylamine which may have leaked

from the column during storage. One hundred microliters of an a-chymo-

trypsin solution (1.0 mg/ml) were added to the column via the injection

port to give a small zone of enzyme passing through the column. An initial

peak of A280 absorbing material was observed on the eluant monitor. Sub-

sequent activity assays gave no indication of active enzyme in these

fractions. This is consistent with observations by Wilkerson, Mariano

and Glover (1976) who showed that this initial peak corresponds to low

molecular weight autolysis products present in the enzyme preparation.

The serendipitous presence of this peak provided us with an excellent

internal marker for column experiments. After the elution of this initial

peak the eluting buffer was changed to 0.1 M acetic acid, pH 3.0, resulting

in elution a larger peak of A280 absorbing material which was shown by

activity assay to be active enzyme (Figure 4).

A sample of a-chymotrypsin which had been inactivated by TPCK provided

us with an enzyme containing a blocked active site. This was applied to

the affinity column which had been shown to bind active ac-chymotrypsin.






























to








,CC

0





c0 ,-4


to




1. 0-0 0 c a O
4-1 4J J
1 '1j3 M1










. o s- T











r 0 14 1C 0
0 < r C0 *
CO l.


C O (0 *C
I *" 03 >C












*r 00 0
I 3 -J3 4J
CQ UQl rt J U


0a 0 0 t
N> ua-
Ce a n
'I-I 3 a -
o Gz 14







(Q--.) irOls! u

Q Q


-
". ...


I







t
I






0
I
r




t.




o i ci 0 i
O.
o o o o
(__)09


00
E



0

0u U
L-
u-

1"O


It

0o


.O
mS









In this experiment a large peak of A280 absorbing material was obtained

as an initial peak. We were not able to show that this was a-chymotrypsin

by activity assay but did show that it corresponded to all of the A280

absorbing material which was applied. A change in pH after this initial

peak gave a very small peak (less than 5%) of active a-chymotrypsin

(Figure 5). We assumed this to be unreacted c'-chymotrypsin. These results

are consistent with Stevenson and Landman (1971).

One hundred microliters of a 1.0 mg/ml c-chymotrypsin solution were

applied to the affinity column. After the initial peak of inactive material

the buffer was not changed to 0.1 M acetic acid but allowed to continue.

This will lead to the eventual elution of the sample as broad, shallow

peak. This is shown in Figure 6. The elution volume for enzyme through

the matrix under these conditions was used as a measure of the relative

amount of immobilized ligand present. Throughout the course of this project

the method of Figure 6 was used to monitor changes in the column due to

ligand leakage and to provide a reference for experiments involving soluble

ligands. The elution profiles determined from A280 tracings and activity

assays show good correspondence to each other. Both methods were used to

evaluate the peak position in subsequent experiments. This was done to

give a cross check on the two procedures.

A final experiment involved passing a-chymotrypsin over a column of

unreacted succinyl polyacrylic hydrazide Agarose. Active enzyme was

shown to elute in the column void volume (Figure 7).



Soluble Ligand Experiments

The column was pre-equilibrated as previously described with solu-

tions containing a known amount of carbobenzuxy (CBZ) phenylalanine.
































0
o C





4JC
V C C .4








o c* 0)
SC rl Q) 4J




H co 4
PN U 43






<0 C) 0






0 4 d 41
4J 44 4 r. C
- o o 0d
04 0 a)C 0

*| p, c a -4




a -' co 1


0 d Cd 4 4)
3o oU (0 (
a) 6

ft r-l 0 t-1




l> 4>134








OL/si!un


Q o o
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LO ,
I
I
I
I f
/


.-./
-

I








(-) 08 v


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(--~
































4i




-4









0
'-I
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0.





$4




4-1








c
CO







I

a to


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,C



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4a) 4-1


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44a) a.0
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,-I 0
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onlr









0 ca r-i 0









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0






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00









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ma


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00 0
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~cC r)
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.Z I
I


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*1
t

00
l<


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L

o _n


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-J


c
c


(_-) v


(..-..) IrOL/s.!un









Small zone experiments were performed using 100 microliters of a 1 mg/ml

a-chymotrypsin solution. This solution contained the same concentration

of CBZ-phenylalanine as the pre-equilibrated affinity column. The re-

sults for various concentrations of this soluble ligand and for enzyme

in the absence of ligand are shown in Figure 8.

Small zone analyses were also done using CBZ-tyrosine and CBZ-trypto-

phan. These soluble ligands gave elution patterns similar to CBZ-phenyla-

lanine. Although the general trend of increasing elution volume with

increasing soluble ligand concentration was maintained, the amount of

retardation for a given concentration of soluble ligand varied with the

ligand used. Small zone experiments were done in triplicate for each

concentration of each inhibitor. Values obtained within a set of triplicates

did not differ more than 5 percent from the average of the set of triplicates.

Small zone analyses were done using N-acetyl-L-phenylalanine as

the soluble ligand. For concentrations of ligand from 0.5 mM to 8 mM each

experiment gave an elution profile identical to that obtained for zero

concentration of inhibitor. A composite of the A280 tracings are shown

in Figure 9 along with a zero concentration experiment.

N-Acetyl-D-phenylalanine, N-acetyl-L-tyrosine and N-acetyl-L-phenylala-

nine were also used as soluble ligand for small zone experiments. Over

the concentration range tested (0.5 mM to 10 mM) the elution volumes did

not differ from those obtained for enzyme at zero concentration of ligand.

Soluble 4-phenylbutylamine and phenylbutyric acid were tested over

a concentration range of zero to 50 mM; again the elution volumes obtained

corresponded to those obtained for enzyme in absence of inhibitor.

Carbobenzoxyalanylalanine was used as a soluble inhibitor over a

concentration from 0.5 to 8.0 mM. The results shown in Figure 10 show





























04J0)


C
*M c0

0 B
ra) *c 0





4 Cd 0S
P- 1-1 4-1 UE-


0 EU 0 C




0CU 00V
r*j 410 P, l
0. 0


N c00 O C


0 CM C







cU0 cO
aU u


m a) 0
cflO
0ie 0





0-4 0 r 04
W w 0






CBZ-Phe


1.65 mM


1.10 mM


0.73 mM


0.36mM


No Inhibitor


20 40 60


80


VOLUME, ml


0
04


I ILf-IY~I.




























*rn

m 0 '
v4




*-I c i -

O 4-1*

0O C.

I 1
I co 0 U
3 CJ Uc
S00w
I 41 c
C.) c oJ


0e O


r-G( c0 *r
w (3 10




O *r4 X
0 H U-4



Lo o





C 4LI

S0 H- 0






N-acetyl-Phe


8.13 mM


4.01 mM


0 1,9




0.



No Int



20 40 60 80
VOLUME E mi


18 rnM


S2mM


hibitor





























uC

co c





Hd H
C n *h



0 0 CO

H c *H*
SM4U

CN 4M1 1
CC
(s a) o
0 01 0n,

$4 00



0 E- 0 W
OH a c.)
iH 0 u
'd Oi





H a c p.


41 C tv w
r-! OT-I 4>
w ww0C






CBZ ALA ALA


8.12 mM


4.18 mM


C0o 2.C




1.



No In



20 40 60 80
VOLUME,ml


4 mM


01 mM


iibitor









that with increasing concentration of soluble ligand there is a decrease

in the elution volume for the enzyme. It is also noted that for con-

centrations of CBZ-alanylalanine greater than 2.0 mM the elution volume

remains constant at a position between the column void volume and the

elution volume of enzyme with no inhibitor. Note that the column void

volume is represented in each experiment by the peak of inactive autoly-

sis products endogenous to enzyme preparation.

Large zone analysis was done using CBZ-phenylalanine over the same

ligand concentration range as for the small zone experiments done with

this inhibitor. These results are shown in Figure 11 as a composite of

the elution fronts obtained from this type of analysis. In agreement

with the small zone experiments the elution volume for the centroid po-

sition of the enzyme front increased with increasing concentration of

soluble CBZ-phenylalanine.



Determination of Dissociation Constants for Soluble Complexes

Dissociation constants for the soluble ligands and a-chymotrypsin

were determined by the method of Lineweaver and Burk (1934). Titra-

metric assays were done using a varying concentration of benzoyl-L-

tyrosine ethyl ester as substrate and a constant concentration of soluble

ligand. The experiments were repeated at two other concentrations of

soluble ligand to establish the type of inhibition involved. From a plot

of the reciprocal of the activity (velocity) versus the reciprocal of

soluble ligand concentration, three straight lines were obtained each

corresponding to a constant soluble ligand concentration. From the resulting

plot it was seen that the three lines intersected on the ordinate axis































0
,a u
c4-
o










CN
0




.4 0
I o











- O
0o 4-


t0






w w a)































- EE E

O LO o 0


0
Kr
T-~


o
O('


0
0


E
LLJ

D
0


0



0
N


0 ZV









indicating competitive inhibition. The dissociation constant (KI) for

enzyme-ligand complex could be graphically obtained. These results are

shown in Table I.



Quantitation of Matrix Bound Ligand

The immobilized 4-phenylbutylamine was removed from the matrix by

solvolysis over a period of 5 months at 40C. The supernatant was removed

from the matrix by filtration followed by washing of the matrix with

methanolic tris.Ca+ buffer. The washings were combined and an absorption

spectra taken. Figure 12 shows a composite spectra of pure 4-phenylbutyl-

amine and the supernatant from the matrix. Figure 13 shows composite

spectra of matrices suspended in buffer. The break at 270 nm is where

the scan was stopped and the matrix resuspended by mixing. The results

show the successful solvolysis of ligand from matrix thus providing a

method for determining the degree of substitution of the matrix.



























0 0C0 0 0 0 0 c
c4 r- oD rr. Co r- U- r

i CS r-l r-t C, 6 CM




0 *

r 0

HO


*& $% 4
0 4-H .
44I Z 0 .,
wIri T L4


-0 0 a)


.0


4 N1d I I
0 .0
Si m m O
0 J a a z
(f4 4-1 -H W

< z a- 1 w <

4 >- U>0 1 Z -

0 0. I- z r


o 4 0 >-0




B < rI I I Z


































't


L .
( x
7X M
41 4-1

rt






04- 0
ri 0





41
o 1
I v3
r-c





041
4 I
U 0



a*e

-> fl
4J &.
.-I


(J

.o
041
















,-4 r4
P a


" r




Go







CN
r4 -1
4) 0


3







r.



S*8
0 pO



0

41
a>)"-



































240


260


wavelength, nr


w
U
z
m
0
V)


0.7



0.6


0.5



0.4


0.3



0.2


0.1


280


___ __ __ __ -- -- I



































4

d




0







4-
3 *












,-I
-i 4O











n- ,-4


:: .04
c^-?








0.7


0.6


0.5

U

S0.43
m





0.2


0.1
B



240 260 280
wavelength, nm















DISCUSSION

The rationale involved in choosing the system was based on its es-

tablished performance in other laboratories. The matrix synthesis was

relatively simple and methods involved in testing for specific binding

had been established and were easily applied to this system with satis-

factory results. It was anticipated that by using the approach of Dunn

and Chaiken for obtaining column binding constants this system would be

easily quantitated. The information obtained will add both to the under-

standing of the 4-phenylbutylamine column and the applicability of

quantitative affinity chromatography for evaluating affinity matrices.

Dunn and Chaiken proposed the scheme shown in Figure 2, in which E,

I and L-R are the enzyme, soluble ligand and the immobilized ligand,

respectively. E-I and ERL-M are the binary complexes formed where KI and

KL-H are their dissociation constants as defined in Figure 14. Using

their experimental approach of pre-equilibrating the column followed by

passing a small zone of enzyme through this column, one would expect the

results shown in Figure 15. At low concentrations of I the enzyme would

exist predominantly as the free form, E; this free form would be able to

interact with the immobilized ligand. Through its binding to the im-

mobilized ligand, its passage down the column would be retarded giving a

large elution volume for the enzyme. At high concentrations of I, rela-

tive to KI, the enzyme would exist predominantly as the E-I complex,

this complex would be unable to bind to the immobilized ligand and pass

through the column very quickly. For this situation the elution volume

would be equivalent to the column void volume, Ve.

51






































0
41

04

*-






0
0
T.l






4J





to
o
CO


0
o
o4





m5
o =




o o
co W
(0 3
Tl r
Q Ml


00




to







04
N








0 0
C> a
rl-0
0 *

00










-40 0
0
t4 t



a )

Cw 0
*M *1


(B u

N .





aaI f
5 f +


















I-I
O -s
iI- I


Lihl
ki'
Ltm


I!
1..1


2I.


































a,

0)

S0
0.
0
U

















'.4
C












H-
a,
o





NA

U
O H


C- 0
*4

C3



S0


U )


ri
0

*ra



0

01 0
0 41






f4
C

0a)




0 Q)




>
H
*rd
00











0
,c ,C



o






> 0








r
t-1
I
...


~LU
w
/'Ca-


\_
3




----->3]^


[3]


C-









Thus, in a competitive elution scheme the observed elution volume

will vary as a function of the inverse concentration of soluble ligand

and at saturating concentrations of soluble ligand the elution volume

will be approach the column void volume.

The results obtained from the small zone experiments with the

4-phenylbutylamine column (Figures 8, 9 and 10) do not agree with the

competitive binding scheme presented by Dunn and Chaiken. The results

are, however, consistent with the early observations of Akanuma et al.

(1971) with carboxypeptidase B on columns of immobilized e-aminocaproyl-

D-phenylalanine in which they demonstrated the ligand-enhanced binding

of carboxypeptidase B to the column in the presence of e-aminocaproic

acid.

Based on the assumption by Akanuma et al., this type of elution

behavior was due to the presence of two unique, yet interacting sites on

the enzyme, the binding scheme shown in Figure 16 was hypothesized to

explain the results obtained. We shall refer to this type of binding

scheme as non-competitive elution as compared with the nomenclature of

Dunn and Chaiken of competitive elution for their scheme. This scheme

is similar to the one involving a competitive site, with the addition

of the E-I complex binding to the immobilized matrix with dissociation

constant Ky'g for the resulting complex. A mathematical evaluation of

these schemes is given in the Appendix. This derivation was based on the

starting assumptions presented by Dunn and Chaiken (1974). Their starting

equations were used only with the addition of I.E.L-M complex and appro-

priate substitutions involving Kf~r.
























*1-





0
co a












n41


, d 0
Ja


2 0
S441


U r4 1





(0 0
wu
Q*p
















o o

0 *


l4 r4 C
r(3 d f
tf E3 d













_.I
It


IQ
t--0
w~ ~~
+a
Hr


i
yL


I


I
_J
+

Lu


*








Equation 14 of the Appendix gives the elution volumes, binding

ratios and ligand concentrations in terms of dimensionless ratios.





Ve- Vo= K=7 1 [I] > 0
1 + 1 + KI
1+- I+KI



This equation was used to generate a family of curves shown in Figure

17. The elution volume is corrected for the column void volume and ex-

pressed relative to VT=-, the volume at which an enzyme would elute in

the absence of soluble ligand after correction for the column void volume.

The concentration of soluble ligand is expressed relative to KI. The

two column binding constants are expressed as a ratio of each other.

The point at which all of the lines intersect on the volume axis is that

point at which the concentration of soluble ligand is zero. For ratios

of KL./K 4I=z greater than one the elution volume will increase with in-

creasing ligand concentration; for KgL-/K=- equal to one there will be

no effect on the elution volume by ligand; finally, when K-r=/Kgr is

less than one the elution volume will decrease with increasing ligand

concentration (Table II). Figures 18 through 20 illustrate these three

types of elution patterns expected from this scheme for typical small

zone experiments. We have chosen to refer to them as Case I, Case II

and Case III for column binding ratios greater than, equal to and less

than one, respectively.

Let us examine each case in detail from the standpoint of the en-

zyme species. First, Case i (Figure 18) describes the situation in






















41

0t
4r4

41











0
4J

m





0 Q
4J







.C



0o
Tl
(A



00
co

00
0

04.



0











c'J
II


II


WA -^ 0


0
(0)









Qi














0
O






































0&
C,,

U
















4o
0
.00







(U -U



0


0


LU.
Q_
;5


0
Ll

LU
c-)
O
Z





LU


0
0
1-4






LU.












A
:14


-J

-J
0
C,
U-

u.

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which the binding of E-I to the matrix is tighter than the binding of

free E to the matrix, that is, KIr- > K=-. At high concentrations of

soluble ligand the enzyme will exist predominantly as the E*I complex.

As this complex, the enzyme will bind tighter to the immobilized ligand

than in the free form. Further, as saturating concentrations of I are

approached the elution volume will approach a finite value determined

by the dissociation constant KI-R. This is similar to the passage of

free E through a column in the absence of soluble ligand. This elution

is determined solely by the column dissociation constant KL-=, the nature

of the affinity matrix and the column dimensions. By causing the enzyme

to spend more time in the stationary phase, this will enhance the re-

tardation through the column, subsequently increasing the elution volume.

At concentrations near the KI of the enzyme the elution volume will vary

as a function of the concentration of soluble ligand.

Case II (Figure 19) presents us with some rather uninteresting data.

We find that the elution volume is independent of the concentration of

soluble ligand. In this case it does not matter which form the enzyme

is in. With KL-= equal to K=_p the free form of the enzyme will bind to

the column with the same affinity as the E.I complex.

It is important to nota that these results would be obtained for

soluble ligands which do not form binary complexes with the enzyme. In

these types of experiments the enzyme would exist in the free form giving

an elution volume corresponding to Vo. One would have to determine by

an independent method, such as inhibition assays that the binary complex

was formed with a finite value of KI before these results could be pro-

perly interpreted as Case II.









Finally, Case III non-competitive elution patterns (Figure 20) will

be obtained when KL=_ < Kj~R. As the concentration of soluble ligand is

increased in the pre-equilibrating buffer the enzyme will again exist

predominantly as the binary complex, E-I. The column will not bind this

complex as tightly as the enzyme in the free form. This will cause the

enzyme to spend more of its time in the mobile phase and will facilitate

elution. The enzyme will appear in the eluting stream at a volume less

than Vo but greater than Vo. Here we find that the elution volume will

vary inversely with the concentration of soluble ligand.

Although the results which would be obtained for Case III elution

appear to resemble the elution patterns obtained for competitive elu-

tion, a very subtle difference exists. In competitive eiution, when the

concentration of soluble ligand is saturating with respect to the KI, the

E.I complex will not interact with the matrix and the elution volume will

correspond to the column void volume. At saturating concentrations of

soluble ligand in non-competitive elution the E-I complex will interact

with the matrix and elute at a finite elution volume determined by the

column dissociation constant KL-M.

For Case III elution behavior it is interesting to note that as

K "- approaches infinity, i.e. no binding, the results predicted will

approximate those for competitive elution. Thus, competitive elution

can be shown to be a subset predicted from the noncompetitive model.

To be useful in obtaining quantitative results equation 14 must be

manipulated into a linear form. This leads to equation 16 of the Appendix



Ve- Vo 1 K [I]
1 = +
Vz-=R LIj + KI








which can be converted to a straight line equation having the form

y = mx + b by taking a double reciprocal to give:


1 + K
i = +
V, Vo K_-1~ KE--=
-1 1 [I]
VL-M K_= KC


This equation will give a straight line when the left side of the equa-

tion is plotted with respect to the reciprocal of the concentration of

soluble ligand, I. This is shown for theoretical data in Figure 21

where [I] and KI would have the same units. From the above linear equation

a line would have a slope of KI/((K -=/K-M)-I) and an ordinate intercept

of 1/((KI=-/K=-)-I). The quotient from the slope divided by the inter-

cept would give the value for KI, the dissociation constant for the E-I

complex.

Equation 16 from above can be expressed in terms of observed elu-

tion volumes by substituting in the definition, Vo = V-E= + Vo.

This substitution yields equation 19,


i1 [1]
Ve Vo = VL- [I + KI



This is similar to equation 16 in which the volume term is slightly re-

arranged to give a value which can be expressed directly from experimen-

tally obtained values; Ve is the elution volume at for a finite concen-

tration of I and Vo is the elution volume in the absence of I.

Equation 16 can be linearized by taking a double reciprocal to give,


S K + 1
Ve Vo K i Kr-1
VLM K VL.M K

































40








E:



0
0)
0j

000


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to
u<










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-H




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4 1







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mi'
II

it


0
II


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fi'
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1-.0 _


9





ciJ








Oj


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0


J ----'' -- ----









This equation will yield a straight line when elution volumes are plot-

ted as l/(Ve V') versus the reciprocal of the concentration of soluble

ligand. The values for the intercept and slope will be 1/V-r(KL-I/KR_')-I)

and K!/V-=V ((K-z/K='q)-l, respectively. Again the quotient from the slope

divided by the intercept will give KI, the dissociation constant for the

E.I complex.

Figure 22 shows the data for the elution volumes obtained from small

zone experiments with the carbobenzoxy (CBZ) derivatives of phenylala-

nine, tyrosine and tryptophan plotted in this manner.

The data shown represents the triplicate values obtained for each

concentration of I with multiple points shown where the data from the

triplicates varied.

The value for KL=-/K=~y could be determined from the intercept by

substituting in the value of V'=-, which was shown to be 1.49 for each

of the CBZ-derivatives shown. From equation 13 in the Appendix, where

[I] = 0 the elution volume is,


(Vo Vm)[ =]
Ve V =



Vo and Vo can be determined from the passage of enzyme through the column

in the absence of soluble ligand. The column void volume, Vo, would be

the elution volume for the autolysis products. Vm is volume of mobile

phase of the column and is determined at the volume at which a very large

molecular weight marker, such as Blue Dextran 2000, will elute. The im-

mobilized ligand concentration was obtained from extended solvolysis of

matrix, representing the amount of ligand physically substituted to the

matrix.





























0






0
*J



0


-4
0









--4
0
r-
oa














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o




o 4
*1 *r
z cd
a.













4J
rx1 n
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r-1 0
M (U









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a 0
00


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0 0


L_'u
0 0
vuir [ -


0
(0







t c





I
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0
I-









For the column used to analyze the CBZ-derivatives Vo, Vm, [-M]

and Ve were 17.3 ml, 3.7 ml, 30.4 mM and 65.5 ml, respectively. These

data give a value of 8.57 mM for the column dissociation constant,

Ki-. From the data giving the ratios of the column binding constant a

value of 5.75 mM was obtained for KM- using each of the CBZ-derivatives.

The linearity of the fit for these data suggest that the model im-

plemented to account for these data is correct. Further, the Kl's ob-

tained from the chromatography experiments agree with those obtained

through inhibition kinetics. These are compared in Table III.

Data obtained for N-acetyl-D-phenylalanine, N-acetyl-L-phenylala-

nine, N-acetyl-L-tyrosine, 4-phenylbutylamine and phenylbutyric acid as

soluble inhibitors cannot be analyzed graphically since no difference

between Ve and Vo is obtained. However, inhibition kinetics showed a bi-

nary complex between the enzyme and the ligand is formed at concentrations

used in our elution experiments. Figure 23 shows representative results

obtained with CBZ-phenylalanine as the soluble ligand. On the basis of

this determination, these ligands could be said to give authentic Case II

non-competitive elution in the immobilized 4-phenylbutylamine system.

Referring again to the linearized form of equation 15, Figure 24

shows simulated results for Case III behavior in which the column binding

constant, KL-= is less than the constant K]y. These results are charac-

terized by a leftward shift in the elution volume with increasing soluble

ligand concentration.

The values obtained for ordinate axis will have a negative sign.

This is because the results are normalized to give VL-= a value of unity.

When elution volumes less than VTL-_ are considered, this results in a































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fraction which when subtracted by one gives a negative value. The slope

and ordinate intercept will, however, have the same definition as before.

Further, K-=/K -= and KI are determined in the same way as for Case I

plots. The only difference here is that the values which are obtained

directly from the graph are negative. This is analogous to Lineweaver-

Burk plots in which the intercept on the 1/[S] axis is negative. Proper

substitution of these values will give rational results.

The results for a-chymotrypsin elution in the presence of CBZ-

alanylalanine are shown in Figure 25 plotted as the reciprocal of the

difference between Ve, observed elution volume, and Vo, versus the re-

ciprocal of CBZ-alanylalanine. The results are consistent with those

predicted in Figure 24. Note that the ordinate intercept and the slope

are both negative values.

From an intercept value of -.072 which is equal to an ordinate in-

tercept of l/VL=_((Ky-I/K=P-)-l), where Vyr- is equal to 31 ml, the

value of KI-E/K'^ is 0.55. This value is less than 1.0 which explicitly

demonstrates Case III noncompetitive elution. By substitution of the

previously determined value for KL-= equal to 8.57 mM, we find that Ki='

is 15.6 mM. Finally, the soluble dissociation constant for CBZ-alanyla-

lanine can be obtained by dividing the slope by the ordinate intercept.

This gives 2.5 mM which compares well with the value obtained from in-

hibition kinetics of 2.3 mM.

A model has been presented which satisfies all of the observations

obtained in this work. The data have been shown to fit closely the be-

havior predicted by this model and constants for the soluble binary com-

plexes were obtained which are in agreement with results from an independent





































0


0
r-4
",.






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r-4









0
0l


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00



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u

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o




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method. However, the initial assumptions made about the a-chymotrypsin--

4-phenylbutylamine Agarose system do not lead a prior to this model.

Soluble 4-phenylbutylamine has been shown by Stevenson and Landman (1971)

and by our work that it binds as a competitive inhibitor. However, when

this molecule is immobilized to succinylated polyacrylic hydrazine Agarose

it takes on new properties. It has been shown by our work with soluble

4-phenylbutylamine that the immobilized ligand and the soluble ligand bind

in such a way as not to affect the elution volume, i.e., Case IT results.

What evidence then would support this model and the observation

that 4-phenylbutylamine behaves differently when immobilized through its

amino function?

Morihara and Oka (1977) have shown that the active center in

a-chymotrypsin corresponds to at least five or six amino acid residues.

Their results showed that CBZ-glycylprolylphenylalanine (KI = 0.044 mM)

bound much tighter than acetyl-L-phenylalanine (KI = 0.57 mM). These re-

sults indicate the presence of multiple binding sites at the active center

of a-chymotrypsin other than the hydrophobic center long thought to be

responsible for binding. Evidence from X-ray crystallographic work by

Robertus et al. (1972) shows the active center to be an elongated groove

of anti-parallel B-sheets. Further, this active center appears to contain

charged groups for ionic interaction and small hydrophobic regions which

would provide ample opportunity of the binding of multiple species.

In free solution 4-phenylbutylamine exists as a positively charged

molecule with a hydrophobic tail. When immobilized to succinylated poly-

acrylic-hydrazide Agarose the molecule has the structure shown in Figure

26. The positively charged amine has now become a structure analogous




























S>
M 0

0


0 0


Ca


*rdd





S4*
41 U


4C CO


a i

QI O


4
a 10

Coo


00
3 N













r44
(U1H -
Z43

















-r
z
0


0


0




I


I
z
I:


II I II
O='U I U=O I U=O
i I I
' I I j


Z Z
z 1 z
Z Z=
I T
6 o
I _


z
I
z'=U
0
a:


-r
GDL








to a peptide bond. Also there exist other chemical functions capable of

binding to the active center groove. Finally, the phenylbutylamine molecule

could be held in such a position by the matrix that it can only orient one

way into the active center. These factors could contribute to the observed

behavior of immobilized 4-phenylbutylamine with respect to a-chymotrypsin.

Further, soluble ligands could bind to the enzyme and enhance the inter-

action between the matrix and enzyme. This was seen with the CBZ-derivatives

of the aromatic amino acids. Alternatively, the soluble ligand cculd dis-

turb that interaction as was seen with CBZ-alanylalanine. The soluble

ligand could bind and not affect the column interaction as was seen with

the n-acetyl derivatives and the phenylbutyl derivatives.