Affinity Chromatography (Journal of chromatography library)

Journal of Chromatography Library - Volume 12

AFFINITY CHROMATOGRAPHY

JOURNAL O F CHROMATOGRAPHY LIBRARY Valume 1 Chromatography of Antibiotics by G.H. Wagman and M.J. Weinstein Volume 2 Extraction Chromatography edited by T. Braun and G . Ghersini Volume 3 Liquid Column Chromatography. A Survey of Modern Techniques and Applications edited by 2.Deyl, K. Macek and J. Janak Volume 4 Detectors in Gas Chromatography by J. SevZik Volume 5 Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods by N.A. Parris Volume 6 Isotachophoresis. Theory, Instrumentation and Applications by F.M. Everaerts, J.L. Beckers and Th.P.E.M. Verheggen Volume 7 Chemical Derivatization in Liquid Chromatography by J.F. Lawrence and R.W. Frei Volume 8 Chromatography of Steroids by E.. Heftmann Volume 9 HPTLC - High Performance Thin-Layer Chromatography edited by A. Zlatkis and R.E. Kaiser Volume 10 Gas Chromatography of Polymers by V.G. Berezkin, V.R. Alishoyev and I.B. Nemirovskaya Volume 1 1 Liquid Chromatography Detectors by R.P.W. Scott Volume 12 Affinity Chromatography by J. Turkova Volume 13 Instrumentation for High-Performance Liquid Chromatography edited by J.F.K. Huber Volume 14 Radiochromatography. The Chromatography and Electrophoresis of Radiolabelled Compounds by T.R. Roberts

Journal of Chromatography Library - Volume 12

AFFINITY CHROMATOGRAPHY

Jaroslava Turkov6 Institute of Organic Chemistry and Biochemistry, CzechoslovakAcademy of Sciences, Prague

ELSEVIER SCIENTIFICPUBLISHING COMPANY AMSTERDAM - OXFORD - NEW YORK 1978

ELSEVIER SCIENTIFIC PUBLISHING COMPANY 335 Jan van Galenstraat P.O. Box 211, Amsterdam, The Netherlands

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?urkovL, : a r o s h v s . A f f i n i t y -nromatography, (Jc,urndl 0 5 2hrm.ato:raphy l i b r a r y ; v . D1 Includes hiblio,graphical references. 1. A f f i n i t y chromatography. I . T i t l e . 11.

W519.9.A35T8?

ISBX O-lr44-LlGC5 45

547' .349'2

Seriss.

78 -815

1SBN:O-444-41605-6 (V01.12) 1SBN:O-444-41616-1 (Series) Elsevier Scientific Publishing Company, 1978 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Scientific Publishing Company, P.O. Box 330, Amsterdam, The Netherlands @I

Printed in The Netherlands

Contents Acknowledgements

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1 Introduction . References . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The principle. history and use of affinity chromatography . References . . . . . . . . . . . . . . . . . 3. Theory of affiity chromatography . . . . . . . .

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. . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Theoretical guidelines deduced on the basis of the equilibrium model . . . . . . 3.1.1 Equilibrium model for adsorption with a fixed binding constant . . . . . . 3.1.2 Equilibrium model for elution by a change in KL . . . . . . . . . . . . . . . . . 3.1.3 Equilibrium model for elution by a competitive inhibitor 3.1.4 Simulation of column chromatographic results . . . . . . . . . . . 3.1.5 Conclusion . . . . . . . . . . . . . . . . . . . . . 3.1.6 List of symbols used . . . . . . . . . . . . . . . . . . 3.2 Theory of cooperative bonding within the plate theory . . . . . . . . . . 3.2.1 Isotherm of binding of oligoadenylic acid to polyuridylic acid . . . . . . 3.2.2 Cooperative adsorption column chromatography . . . . . . . . . . 3.2.3 Characteristic features of cooperative adsorption chromatograms . . . . . 3.2.4 List of symbols used . . . . . . . . . . . . . . . . . . 3.3 Statistical theory of chromatography applied to affinity chromatography . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Application of affiity chromatography to the quantitative evaluation of specific complexes 4.1 Determination of dissociation constants by elution analysis . . . . . . . . . 4.2 Determination of dissociation constants by frontal analysis . . . . . . . . . 4.3 Cooperative elution of oligoadenylic acid in immobilized polyuridylic acid chromatography . . . . . . . . . . . . . . . . . . . . .

IX 1 4

1 10 13 13 13 19 22 24 24 26 27 21 29 31 33 33 33 35 35 41 44

4.3.1 List of symbols used . . . . . . . . . . . . . . . . . . 46 4.4 Other methods for the quantitative evaluation of interactions with immobilized affinity ligands 47 References . . . . . . . . . . . . . . . . . . . . . . . . . 49

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5 General considerations on affinant-sorbent bonding 5.1 Steric accessibility 5.2 Conformation of attached affiant 5.3 Concentration of the affinant on the matrix 5.4 Concentration of proteins. equilibration time and flow-rate . . . . . . . . . 5.5 Effect of temperature . . . . . . . . . . . . . . . . . . . . 5.6 Effect of pH and ionic strength 5.7 Elution with competitive affinity ligands . . . . . . . . . . . . . . 5.8 Non-specific effects . . . . . . . . . . . . . . . . . . . . 5.8.1 Effect of ionic strength on non-specific sorption . . . . . . . . . . 5.8.2 Extended Lkbye-Hiickel theory applied to the study of the dependence of the ionic strength on the adsorption equilibrium constant and the rate of desorption of the enzyme from the substituted gels 5.8.2.1 List of symbols used . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

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51 53 60 62 67 71

75 17 80 80

81 86 87

VI

CONTENTS

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6 . Choice of affinity hgands for attachment 6.1 Highly specific and groupspedfic matrices . 6.2 Isolation of enzymes. inhibitors and cofactors . 6.3 Immunoaffinitychromatography . . . . . . 6.4 Isolation of lectins. glycoproteins and saccharides 6.5 Isolation of receptors. binding and transport proteinLS 6.6 Isolation of -SH proteins and peptides . . . . 6.7 Isolation of specific peptides . . . . . . . . 6.8 Isolation of nucleic acids and nucleotides 6.9 Isolation of lipids. hormones and other substances . 6.10 Isolation of cells and viruses . . . . . . . 6.1 1 Commercially available insoluble affmants . . References . . . . . . . . . . . . . . .

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89 89 92 . 95 99 . 103 . 106 -108 . 111 . 114 . 116 . 118 . 127

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7 . Hydrophobic chromatography. covalent affinity chromatography. affiity elution and related methods . . . . . . . . . . . . . . . . . . . . . . . 7.1 Hydrophobic chromatography . . . . . . . . . . . . . . . . . 7.2 Covalent affinity chromatography . . . . . . . . . . . . . . . . 7.3 Affinity elution . . . . . . . . . . . . . . . . . . . . . 7.4 Affmity density perturbation . . . . . . . . . . . . . . . . . 7.5 Affinity electrophoresis . . . . . . . . . . . . . . . . . . . 7.6 Metal chelate affinity chromatography . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

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8 . Solid matrix supports and the most used methods of binding 8.1 Required characteristics . . . . . . . . . . . . . . . . . . 8.2 Survey of the most common solid supports and coupling procedures 8.2.1 Cellulose and its derivatives . . . . . . . . . . . . . . . 8.2.2 Dialdehyde starch-methylenedianiline (S-MDA) . . . . . . . . . 8.2.3 Dextran gels . . . . . . . . . . . . . . . . . . . . . 8.2.4 Agarose and its derivatives . . . . . . . . . . . . . . . 8.2.5 Copolymer of ethylene and maleic anhydride 8.2.6 Polyacrylamide supports and their derivatives 8.2.7 Hydroxyalkyl methacrylate gels . . . . . . . . . . . . . . 8.2.8 Glass and its derivatives 8.2.9 Other supports . . . . . . . . . . . . . . . . . . . . 8.3 Spacers . . . . . . . . . . . . . . . . . . . . . . . . 8.4 Blocking of unreacted groups . . . . . . . . . . . . . . . . 8.5 Leakage of the coupled affinant . . . . . . . . . . . . . . . . 8.6 General considerations in the choice of sorbents, spacers and coupling and blocking procedures References . . . . . . . . . . . . . . . . . . . . . . . . .

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9 . ~aracteruationofsupportsandimmobilizedaffmityligands . . 9.1 Methodsfor thedeterminationof non-specificsorption . . . . . . . . . . 9.1.1 Determination of adsorption capacity . . . . . . . . . . . 9.1.2 Determination of residual negatively charged groups . . . . . . . . . 9.2 Determination of activatable and active groups . . . . . . . . . . . . 9.2.1 Determination of wboxyl,. hydrazide and amino groups on the basis of acid -base titration 9.2.1.1 Dry weight determination 9.2.1.2 Determination of carboxyl groups 9.2.1.3 Determination of hydrazide groups . . . . . . . . . 9.2.1.4 Determination of aliphatic amino groups 9.2.2 Determination of the content of free carboxyl groups . .

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131 131 137 139 140 144 147 149 151 151 153 153 157 158 159 173 174 178 180 181 182 187 189 195 201

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203 203 204 204 204

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204 204 205 205 205 206

CONTENTS

VII

Determination of free amino groups in polymers on the basis of the condensation reaction with 2-hydroxy-1-naphthaldehyde 9.2.4 Procedure for azide assay . . . . . . . . . . . . . . . 9.2.5 The sodium 2.4.6.trinitrobenzenesulphonate colour test . . . . . . 9.2.6 Fluorescamine test for the rapid detection of trace amounts of amino groups 9.2.7 Determination of oxirane groups 9.2.8 Determination of the capacity of p-nitrophenol ester derivatives of hydroxyalkyl methacrylate (NPAC) gels 9.2.9 Determination of the degree of substitution of benzylated dibromopropanol c r o s s l i e d Sepharose 9.2.10 Determination of vinyl groups 9.2.1 1 Determination of sulphydryl groups 9.3 Methods for the determination of immobilized affinity ligands . . . . . . 9.3.1 Difference analysis 9.3.2 Spectroscopic methods 9.3.3 Determination by means of acid-base titration . . . . . . . . . 9.3.4 Determination of immobilized proteins. peptides. amino acids, nucleotides, carbohydrates and other substances after liberation by acid. alkaline or enzymatic hydrolysis . . . . . . . . . . . . . . . . . 9.3.4.1 Determination of immobilized amino acids, peptides and proteins 9.3.4.2 Determination of nucleotides . . . . . . . . . . . . 9.3.4.3 Determination of carbohydrate 9.3.5 Determination of the amount of bound affinant on the basis of elemental analysis . . . . . . . . . . . . . . . . . . . . . . 9.3.6 Determination of labelled affinity ligands . . . . . . . . . . . 9.3.7 Determination of immobilized diaminodipropylamine by ninhydrin colorimetry 9.3.8 Determination of immobilized proteins on the basis of tryptophan content . 9.4 Active-site titration of immobilized proteases 9.5 Study of conformational changes of immobilized proteins . . . . . . . . . 9.6 Studies of the distribution of proteins bound to solid supports References . . . . . . . . . . . . . . . . . . . . . . . . .

9.2.3

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.206 .206 .201 . 201 .208 .209 .209 .209 .209 .210 .210 .210 .212 .213 213 .213 .214

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214

. 215 215 . 215 . . . . . . . . . . . . 216 . 219 . . . . . . . 221

10. General considerations on sorption. elution and non-specific binding . 10.1 Sorption conditions . . . . . . . . . . . . . 10.1.1 Effect of temperature. pH and salts . . . . . . . 10.1.2 Practice of sorption . . . . . . . . . . . 10.2 Conditions for elution . . . . . . . . . . . . . 10.2.1 Practice of desorption . . . . . . . . . . . 10.2.2 Effect of the heterogeneity of the immobilized affiants . 10.2.3 Establishment of optimal conditions and saturation effect 10.3 Non-specific sorption . . . . . . . . . . . . . 10.4 Regeneration and storage of affiity columns . . . . . . References

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225 225 225 230 232 233 237 240 241 243 243

1 1 . Examples of the use of affinity chromatography . . . . . . . . . . . . . 2 4 5 11.1 Isolation of biologically active substances . . . . . . . . . . . . * 2 4 5 11.2 Resolution of DL-tryptophan by affinity chromatography on bovine serum albumin . agarose column . . . . . . . . . . . . . . . . . . . . *319 11.3 Semi-synthetic nuclease and complementary interaction of nuclease fragments . . . 319 11.4 Study of interactions of biologically active substances . . . . . . . . . * 3 2 4 11.5 Study of the mechanism of enzymatic action . . . . . . . . . . . 1 3 2 7 11.6 Molecular structure of fibroblast and leucocyte interferons investigated with lectin and hydrophobic chromatography . . . . . . . . . . . . . . * 3 2 9 11.7 Immunoassay . . . . . . . . . . . . . . . . . . . . * 332

CONTENTS

VIlI

11.7.1 Solid-phase radioimmunoassay . . . . . . . . . . . . . . 332 . . . . . . . . . 333 11.7.2 Enzyme-linked immunosorbent assay (ELISA) 11.7.3 Microfluorimetric immunoassay . . . . . . . . . . . . . . 334 11.8 Specific removal of bovine serum albumin (BSA) antibodies in vivo by extra-corporeal . . . . . . . . 334 circulation over BSA immobilized onnylon microcapsules References . . . . . . . . . . . . . . . . . . . . . . . . . 336

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12 Immobilized enzymes . . . . . . . . . . . . . . . . . . . 12.1 Classification of immobilized enzymes . . . . . . . . . . . . 12.2 Attachment of enzymes to solid supports and activity of immobilized enzymes 12.3 Stability of immobilized enzymes . . . . . . . . . . . . . . 12.3.1 Stability during storage . . . . . . . . . . . . . . 12.3.2 Dependence of stability on pH . . . . . . . . . . . . 12.3.3 Thermal stability . . . . . . . . . . . . . . . . 12.3.4 Stability against denaturing agents . . . . . . . . . . . 12.3.5 lncrease of stability . . . . . . . . . . . . . . . 12.4 Application of immobilized enzymes . . . . . . . . . . . . . 12.4.1 Affinity ligands . . . . . . . . . . . . . . . . . 12.4.2 Study of stabilized enzyme molecules and of their subunits . 12.4.3 Models of biological systems . . . . . . . . . . . . . 12.4.4 Application of immobilized enzymes . . . . . . . . . . 12.5 “Synthetic biochemistry” . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

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Subject index

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List of compounds chromatographed

. 365 . 365 . 366 . 374 . 374 . 375 . 376 . 371 . 378 . 379 . 379 . 379 *

380 382 383 384 381

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IX

Acknowledgements I should like t o express my deepest gratitude to Associate Professor Karel Macek of the 3rd Medical Department of the Medical Faculty, Charles University, Prague, for his constant interest during the writing of this book, and also for his valuable advice and remarks which were a great help to me. My thanks are also due to Associate Professor Karel Bliiha of the Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, for his kind revision of the manuscript and many valuable suggestions. To Associate Professor David J. Graves of the University of Pennsylvania, Philadelphia, and to Professor Akioshi Wada of the University of Tokyo I am indebted for reading the chapter on the theory of affinity chromatography and for their very useful remarks. I thank Dr. 2elimir Prochizka for the translation of the manuscript. I am also grateful to Mr. Vladimir Mafik for re-drawing the figures. Finally, my thanks are also due to my mother, husband and daughters for the understanding they have shown during the period when this book was being written.

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1

Chapter 1

Introduction Macromolecules such as proteins, polysaccharides and nucleic acids differ only negligibly in their physico-chemical properties within the individual groups, and their isolation on the basis of these differences, for example by ion-exchange chromatography, gel filtration or electrophoresis, is therefore difficult and time consuming. Consequently, considerable decreases in their activity occur during the isolation procedure, owing to denaturation, cleavage, enzymatic hydrolysis, etc. One of the most characteristic properties of these biological macromolecules is their ability to bind other molecules reversibly. For example, enzymes form complexes with substrates or inhibitors, antibodies bind antigens against which they were prepared, and nucleic acids such as messenger RNA hybridize with complementary DNA, etc. The formation of specific dissociable complexes of biological macromolecules can serve as a basis for their purification by the method known as affinity chromatography. The term affinity chromatography, however, raised (and still raises) many objections. Endeavours have been made to replace it, for example with the more accurate term “biospecific adsorption” or “bioaffinity chromatography” (O’Carra et al. ;Porath), especially when it was found that a series of adsorbents, mainly those with synthetic inhibitors bound by hydrophobic hydrocarbon chains, can sorb macromolecules rather on the basis of hydrophobic interactions (O’Carra). The exploitation of the formation of complexes of biological macromolecules on the basis of hydrophobic bonds gave rise to the so-called hydrophobic chromatography (Shaltiel). However, the differentiation between a biospecific complex and a complex formed on the basis of non-specific hydrophobic forces is not so simple, as could be observed in many instances. Often one substance bonded to a carrier may form biospecific complexes with one group of macromolecules, with another it may undergo complex formation on the basis of non-specific hydrophobic interactions exclusively, and in bond formation with a further group of macromolecules both types of bonds may take part. Hexamethylenediamine can be mentioned as an example. This compound, when bound to Sepharose, was used by Henderson et al. and Jakubowski and Pawdkiewicz as a sorbent in the hydrophobic chromatography of aminoacyl-transfer RNA synthetases or L-histidinol phosphate aminotranspherase. Toraya et al. suggested it as a biospecific sorbent for aminooxidase from Aspergillus niger, while Vosbeck et al., when using the same sorbent for the isolation of aminopeptidases from Streptumyces griseus, reached no definite conclusion about which type of bond is operative during specific sorption. Therefore, it seems a logical consequence that Jakoby and Wilchek classified not only affinity chromatography among the affinity methods in the sense of biospecific adsorption, but also hydrophobic chromatography (Shaltiel), as also have been covalent chromatography (Blumberg and Strominger; Brocklehurst et al), affinity elution (Von der Haar), affinity density perturbation (Wallach) and affinity electrophoresis (HorejSi and Kocourek). Recently, the terminology of affinity chromatography was further extended by the concept of metal chelate affinity chromatography (Porath et al., 1975), and for the study of interactions on carriers with bound oligo-

2

INTRODUCTION

nucleotides Schott et al. used the name template chromatography. From the above, it is evident that today affinity chromatography no longer utilizes the formation of complexes on the basis of a narrow biospecific interaction alone. On the contrary, this term is also used for the isolation of biological macromolecules by simple sorption on a specific sorbent, which, moreover, is often carried out batchwise. Evidently this term is incorrect, but today it is in general use. The idea of basing protein separation methods on the molecular affinity found in biological systems has been known for several decades, as has the binding of enzymes to solid carriers. The preparation of insoluble heterogeneous catalysts has many advantages, such as easy separation from the reaction mixture, the possibility of continuous catalysis and an increase in the stability of enzymes. Nevertheless, the full development of both affinity chromatography and the binding of enzymes to solid carriers has taken place only in recent years, for the same reason. The development started with the use of highly porous hydrophilic carriers, mainly agarose, after the working out of suitable binding methods (Axdn et al.; Porath et al., 1967). The fundamental principle of affinity chromatography consists in the utilization of the exceptional property of biologically active substances to form stable, specific and reversible complexes. If one of the components of the complex is immobilized, a specific sorbent is formed for the second component of the complex, with the assumption, of course, that all of the conditions necessary for the formation of this complex are maintained. The binding sites of the immobilized substances must retain good steric accessibility even after their binding to the solid carrier, and they must not be deformed. The first examples of specific sorbents prepared by covalent bonding to a solid support were immobilized antigens (Campbell et d.).The methods developed for the attachment of antigens and antibodies to solid supports were used immediately for the preparation of immobilized enzymes; at the same time, the earlier method of binding enzymes to cellulose by means of an azide bond (Micheel and Ewers) began to be used for the preparation of immunosorbents. The parallel development of the two branches based on the use of the bonds of biologically active substances to solid carriers is best evident from the first review papers: “Reactive Polymers and Their Use for the Preparation of Antibody and Enzyme Resins” by Manecke, “Water-insoluble Derivatives of Enzymes, Antigens and Antibodies” by Silman and Katchalski and “The Chemistry and Use of Cellulose Derivatives for the Study of Biological Systems” by Weliky and Weetall. This simultaneous development of the two branches took place after the discovery of better carriers and the elaboration of methods of bonding that permitted the preservation of those properties which the immobilized substances possessed in solutions. Their common development also contributed substantially to the introduction of a new area of specialization - enzyme engineering. According to Wingard, this new specialization consists in the production, isolation, purification, immobilization and utilization of enzymes in various types of reactors. The endeavour to make practical use of enzymes is a logical continuation of the development of enzymology in recent years, during which the structures and the mechanisms of action of a number of enzymes have been elucidated, because after the solution of the questions of what an enzyme is and how it acts, the question necessarily arises of how to exploit this knowledge in practice. Many possibilities exist in analysis, medicine and industry for the practical application of immobilized

INTRODUCTION

3

enzymes. The simplified isolation of enzymes by affinity chromatography promises the preparation of required amounts of enzymes at lower cost. The immobilization of enzymes on suitable carriers considerably increases the versatility of their use and thus also their economic availability. The study of various types of enzymic reactors creates conditions for their practical application. However, much additional effort will be required before the hope of a number of enzymologists that enzymes, as highly specific catalysts, will penetrate into the structure of production process could become a reality; however, there is some scepticism about the possibility of achieving this aim at all. However, enzymes bound to solid supports are important not only from the practical point of view. From the theoretical point of view, enzymes bound to well characterized surfaces of solid supports represent simple models for the study of the effect of microenvironments on the binding of substrate, and also of the general course of catalysis. As most enzymes in vivo are bound to membranes or occur in the form of some other complex of the native environment, the possibility of such studies is undoubtedly important. It is in the study of the effect of the micro-environment on the specific interactions that the method of affinity chromatography begins to play an important role. As will be described in detail in Chapter 4, it is possible, for example, to use a column of a support with a bonded inhibitor and to displace the specifically sorbed enzyme with solutions of its inhibitors of various concentrations. On the basis of the elution volumes, the dissociation constants of the enzyme with both the bonded and the dissolved inhlbitor can be determined. When the same inhibitor is used for the binding on a solid support and for the elution of the specifically sorbed enzyme, information can be obtained on the effect of the environment on complex formation from the differences, if any, in the determined dissociation constants. As specific interactions play a very important role in most of the processes that take place in nature, the development of a simple method for the determination of dissociation constants of complexes is undoubtedly of great importance. Many review papers have already been written on affinity chromatography and the binding of enzymes to solid supports (Brummer; Crook et al. ;Cuatrecasas; Cuatrecasas and Anfinsen, 1971a,b; Falb; Feinstein; Friedberg; Goldman et a l ; Goldstein; Gryszkiewicz; Guilford; Mosbach, 1971;Orth and Brummer; Porath; Porath and Kristiansen; Reiner and Walch; Turkova’, 1974,1975; Weetall). Further, the proceedings of a symposium have appeared (Dunlap) and two large volumes of Methods in Enzymology, “Affinity Methods’’ (Jakoby and Wdchek) and “Immobilized Enzymes’’ (Mosbach, 1977). Two monographs have also been devoted to immobilized enzymes (Messing; Salmona et aL) and one t o affinity chromatography (Lowe and Dean). The aim of this book is to present a bibliographic review of the use of affinity chromatography, extended by characteristic data such as the supports used, the affinity ligands and the spacers. Further, it gives examples of enzymes bound covalently to solid supports, with special reference to their use in affinity chromatography, either for the isolation of their inhibitors or cofactors, or in the study of the mechanism of their enzymatic effect or participation in metabolic pathways; it also summarizes briefly the principles that must be observed for the successful use of affinity chromatography, mainly with reference t o the latest knowledge. Particular attention will be focused on the solid supports used both in affinity chromatography and for the binding of enzymes, and

4

INTRODUCTION

on the use of affinity chromatography in the quantitative evaluation of complex formation and the effects of the carrier.

REFERENCES Axin, R., Porath, J . and Ernback, S., Nature (LondonJ, 214 (1967) 1302-1304. Blumberg, P.M. and Strominger, J.L., Methods Enzymol., 34 (1974) 401 -405. Brocklehurst, K., Carlsson, J., Kierstan, M.P.J. and Crook, E.M., Methods Enzymol., 34 (1974) 531544. Briimmer, W., Kontakte (Merck), 1/74 (1974) 23-29; 2/74 (1974) 3-13. Campbell, D.H., Luescher, E.L. and Lerrnan, L.S.,Proc Not. Acad. Sci U.S., 37 (1951) 575-578. Crook, E.M., Brockiehurst, K. and Wharton, C.W., Methods EnzymoL, 19 (1970) 963-978. Cuatrecasas, P., Advan. Enzymol, 36 (1972) 29-89. Cuatrecasas, P. and Anfmsen, C.B., Methods Enzymol., 22 (1971a) 345-378. Cuatrecasas, P. and Anfmsen, C.B., Annu Rev. Biochem., 40 (1971b) 259-275. Dunlap, R.B. (Editor), Advan. Exp. Med Biol., 42 (1974) 1- 377. Falb, R.D., Biotechnol. Bioeng., S3 (1972) 177-184. Feinstein, G., Naturwissenschaften, 58 (1971) 389-396. Friedberg, F., Chromarogr. Rev., 14 (1971) 121-131. Goldman, R., Goldstein, L. and Katchalski, E., in G.R. Stark (Editor), Biochemical Applications of Reactions on Solid Supports, Academic Press, New York, 1971, pp. 1-78. Goldstein, L., Methods Enzymol., 19 (1970) 935-962. Gryszkiewicz, J . , Folk Biol., 19 (1971) 119-150. Guilford, H., Chem. SOC.Rev., 2 (1973) 249-270. Henderson, G.B., Shaltiel, S. and Snell, E.E., Biochemistry, 13 (1974) 4335-4338. HofejXi, V. and Kocourek, J.,Methods Enzymol., 34 (1974) 178-181. Jakoby, W.B. and Wilchek, M. (Editors), Methods Enzymol., 34 (1974) 1-810. Jakubowski, H. and Pawdciewicz, J., FEBSLett., 34 (1973) 150-154. Lowe, C.R. and Dean, P.D.G., Affinity Chromatography, Wiley, London, 1974, pp. 272. Manecke, G., Pure Appl. Chem., 4 (1962) 507-520. Messing, R.A. (Editor), Immobilized Enzymes for Industrial Reactors, Academic Press, New York, 1975, pp. 232. Micheel, F. and Ewers, J., Makromol. Chem., 3 (1949) 200-209. Mosbach, K., Scient. Arner., 224 (1971) 26-33. Mosbach, K. (Editor), Methods Enzymol., 44 (1977) 1-999. O’Carra, P., Biochem SOC. Trans., 2 (1974) 1289-1293. OCarra, P., Barry, S. and Griffin, T.,Methods Enzymol., 34 (1974) 108-126. Orth, H.D. and Briimmer, W., Angew. Chem., 84 (1972) 319-330. Porath, J., Biochemie, 55 (1973) 943-951. Porath, J., A&, R. and Emback, S.,Nature (London), 215 (1967) 1491-1492. Porath, J., Carlsson, J., Olsson, I. and Belfrage, G., Nature (LondonJ,258 (1975) 598-599. Porath, J. and Kristiansen, T., in H. Neurath and R.L. Hill (Editors), The Proteins, Academic Press, New York, 3rd ed., 1975, pp.95-178. Reiner, R.H. and Walch, A., Chromarographia, 4 (1971) 578-587. Salmona, M., Saronio, C. and Garattini, S. (Editors), Insolubilized Enzymes, Raven Press, New York, 1974, pp. 226. Schott, H., Eckstein, H. and Bayer, E.,J. Chromatogr., 99 (1974) 31-34. Shaltiel, S., Methods Enzymol., 34 (1974) 126-140. Silman, I.H. and Katchalski, E., Annu. Rev. Biochem., 35 (1966) 873-908. Toraya, T.,Fujimura, M., Ikeda, S., Fukui, S., Yamada, H. and Kumagai, H., Biochim. Biophys. Acta, 420 (1976) 316-322.

REFERENCES

Turkovi, J., J. Chromatogr., 91 (1974) 267-291. Turkovi, I., in Z. Deyl, K . Macek and J. Jan& (Editors), Liquid Column Chromatography, Elsevier, Amsterdam, Oxford, New York, 1975, pp. 89-97, 215-231 and 369-376. Von der Haar, F.,Methods Enzymol., 34 (1974) 163-171. Vosbeck, K.D., Chow, K.F. and Awad, Jr., W.M.,J. BioL Chem., 248 (1973) 6029-6034. Wallach, D.F.H.,Methods Enzymol., 34 (1974) 171-177. Weetall, H.H., Separ. &rif Methods, 2 (1973) 199-229. Weliky, N. and Weetall, H.H., Zmmunochemistry, 2 (1965) 293-322. Wingard, Jr., L.B., Biotechnol. Bioeng., 53 (1972) 3-13.

5

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Chapter 2

The principle, history and use of affinity chromatography Affinity chromatography (or, more exactly, bioaffinity or biospecific affinity chromatography) is based on the exceptional ability of biologically active substances to bind specifically and reversibly other substances, generally called ligands or affinity ligands (Lowe and Dean) or simply affinants (Reiner and Walch). If an insoluble affinant is prepared, usually by covalent coupling to a solid support, and a solution containing the biologically active products to be isolated is passed through a column of this affinant, then all compounds which, under the given experimental conditions, have no affinity for the affinant, will pass through unretarded; in contrast, products that show an affinity for the insoluble affinity ligand are sorbed on the column. They can be released later from the complex with the attached affinant, e.g., with a solution of a soluble affinant or by a changing the solvent composition. The dissociation of the complex can often be achieved by changing the pH, ionic strength or temperature, or alternatively with dissociating agents, as will be shown later. According to O’Carra et al., the biospecific sorption and desorption can be represented, in contrast to non-biospecific desorption, by the so-called “deforming buffers”, as shown schematically in Fig. 2.1. In the history of affinity chromatography, the isolation of &-amylaseby means of an insoluble substrate (starch) should be mentioned first; it was described in 1910 by Starkenstein. The principle of affinity chromatography, using affinants covalently bonded to a solid matrix, has been known for more than 20 years. Campbell et al. were the first to use this principle, in 1951, for the isolation of antibodies on a column of cellulose with covalently attached antigen. Affinity chromatography was first used in the isolation of enzymes in 1953 by Lerman, who isolated tyrosinase on a column of cellulose with

-DEFORMING B

/ /

U

F

F

E

T

cl

I/

8

PRINCIPLE, HISTORY A N D USE OF AFFINITY CHROMATOGRAPHY

ethereally bound resorcinol residues. In subsequent years affinity chromatography was employed only rarely, the reason obviously being the character of the insoluble supports which did not offer sufficient possibilities for complex formation between the product to be isolated and the attached affmant. Non-specific adsorption was often observed when supports with hydrophobic or ionogenic groups were used. The last few years, however, have witnessed an extensive development of this method. A milestone in this development was the method of attachment of affinant t o agarose activated with cyanogen bromide, developed by Porath and co-workers (Axtn and Emback; Axe‘n et a2.; Porath et al.). Cuatrecasas and Anfinsen have shown that agarose (most often the commercial product Sepharose) possesses almost all of the characteristics of an ideal support. In 1968, Cuatrecasas et al. successfully employed affinity chromatography for the isolation of nuclease, chymotrypsin and carboxypeptidase A. This study, in which the term affinity chromatography was used for the first time, stimulated the extensive use of this method in the isolation of enzymes, their inhibitors, antibodies and antigens, nucleic acids, transport and repressor proteins, hormones and their receptors, and of many other products, as evidenced by Table 1 1.I in Chapter 1 1. However, the use of affinity chromatography is not limited to the isolation of biologically active substances. As early as 1960 Yagi et al. described a quantitative determination of small amounts of antibodies by means of solid carriers with bonded antigens. The use of solid carriers in radioimmunoassays is discussed in detail in Section 11.7. Immobilized oligomers of polythymidylic acid were used by Edmonds et al. for the quantitative determination of polyadenylic acid. The use of affinity gel filtration as a microscale method for rapid determinations of apparent molecular weights of dehydrogenases, based on their exclusion from gel filtration medium of various pore sizes, was described by Lowe and Dean. By its nature, affinity chromatography is ideal for the study of interactions in biochemical processes. Immobilized leucyl-tRNA synthetase was used not only for the isolation of isoleucyl-tRNA, but also for the study of protein interactions with nucleic acid (Denburg and De Luca). Interactions of peptides with proteins (Gawronski and Wold) and of nucleotides with amino acids and peptides (Schott et a/.)have also been studied. Further applications of this method are the study of the mechanism of enzymatic processes and the elucidation of molecular structures. Akanuma et al. employed this method for the study of the binding site of bovine carboxypeptidase B on the basis of complex formation with immobilized substrate analogues of basic and aromatic amino acids. Using affinity chromatography, Delaney and O’Carra showed that oxaloacetate inhibits lactate dehydrogenase by forming a “dead-end” complex with enzyme-NAD’ complex rather than with enzyme-NADH complex, as was proposed originally. Analytical affinity chromatography has greatly contributed to the elucidation of trypsinogen activation kinetics (Kasche). The molecular structures of human fibroblasts and leucocyte interpherons were studied by means of affinity chromatography by Jankowski et al. For the separation of isoenzymes of lactate dehydrogenase, Brodelius and Mosbach (1973) used Sepharose with an attached AMP analogue; five separated peaks of isoenzymes could be eluted by increasing the NADH concentration, as shown in Fig. 2.2. The separation has been interpreted as a result of the differences in dissociation constants (Kdiss) for the binary enzyme-NADH complex. Brodelius and Mosbach (1974) subse-

9

PRINCIPLE, HISTORY AND USE OF AFFINITY CHROMATOGRAPHY 0.6

0.6,

R

50

100

150

ELUTION VOLUME, rnl

Fig. 2.2. Elution of lactate dehydrogenase isoenzymes with a concave gradient of NADH. Protein (0.2 mg) in 0.2 ml of 0.1 M sodium phosphate buffer (pH 7.0), 1 mMp-mercaptoethanol and 1 M sodium chloride was applied to an AMP-analogue-Sepharose column (140 X 6 mm, containing 2.5 g of wet gel) equilibrated with 0.1 M sodium phosphate buffer (pH 7.5). The column was washed with 10 ml of the latter buffer, then the isoenzymes were eluted with a concave gradient of 0.0-0.5 mM NADH in the same buffer, containing 1 mM P-mercaptoethanol. Fractions of 1 ml weIe collected at the rate of 3.4 ml/h. Reproduced with permission from P. Brodelius and K. Mosbach, FEBS Lett., 35 (1973)223-226.

quently chromatographed, on the same support and in an analogous manner, a series of lactate dehydrogenases from various sources, the dissociation constants of which were known. Fig. 2.3 shows a direct proportionality between these Kdiss values and the elution concentration of NADH. The linearity indicates that in the given case the dissociation constants for the enzyme-NADH complex play a greater role than those for the complex between the enzyme and the immobilized affinity ligand (AMP). Hence, it is possible to determine the dissociation constants for binary complexes between the enzyme and NADH on the basis of the determination of elution concentrations of NADH. No differences in Kdiss values were observed if a crystalline or crude preparation was used. Other proteins present in crude preparations, even when bound in the column, do not affect the elution pattern. This is a great advantage of this determination in comparison with the conventional methods for the determination of dissociation constants, which require not only pure enzymes but also homogeneous isoenzymes. In addition to the advantage of using affinity chromatography for the determination of the dissociation constants of crude preparations, a further advantage is that it is very rapid and requires only a very small amount of enzyme for each determination. The use of affinity chromatography for the determination, for example, of the inhibition constants of enzymes seems to have good prospects. On the basis of the elution volumes of the enzyme eluted from the column with immobilized inhibitor using various concentrations of soluble inhibitor, the inhibition constants can be determined both with

PRINCIPLE, HISTORY AND USE OF AFFINITY CHROMATOGRAPHY

10 4

3 -

I

4 m

e

2' 1 -

I

I

I

0.1

02

0.3

n 0

0.4

ELUTING CONCENTRATION OF N A D H , m M

Fig. 2.3. Dissociation constant for the binary complex between enzyme and NADH as a function of eluting concentration of NADH. Reproduced with permission from P. Brodelius and K. Mosbach, Biochem. SOC.Trans., 2 (1974) 1308-1310.

bound inhibitors and with the soluble inhibitors employed. This method is discussed in greater detail in Chapter 4.The great advantage of this method is that when using the same inhibitor for the immobilization and the elution, direct conclusions can be drawn about the effects of the bonds and the support on the interaction being studied, from the agreement between or the difference in the dissociation constants determined. Hence the method of affinity chromatography opens up new possibilities, not only for the study of the interactions of biologically active substances, but also in the future for the elucidation of the effect of the micro-environment on the formation of these complexes.

REFERENCES Akanuma, H., Kasuga, A., Akanuma, T. and Yamasaki, M., Biochem. Biophys. Res. Commun., 45 (1971) 27-33.

Axin, R. and Ernback, S., Eur, J. Biochem., 18 (1971) 351-360. Axin, R., Porath, J . and Ernback, S., Nature (London), 214 (1967) 1302-1304. Brodelius, P. and Mosbach, K., FEBS Lett., 35 (1973) 223-226. Brodelius, P. and Mosbach, K., Biochern. Soe. Trons., 2 (1974) 1308-1310. Campbell, D.H., Luescher, E.L. and Lerman, L.S.,Proc. Nut. Acad Sci. US., 37 (1951) 575-578. Cuatrecasas, P. and Anfinsen, C.B., Methods Enzymol., 22 (1971) 345-378. Cuatrecasas, P., Wifchek, M. and Anfmsen, C.B.,Proc. Nut. Acad. Sci. US.,61 (1968) 636-643. Delaney, M. and O'Carra, P., Biochem Soc. Trans., 2 (1974) 1311. Denburg, 1. and De Luca, M.,Proc. Nut. Acad. Sci. U S . , 67 (1970) 1057-1062. Edrnonds, M., Vaughan, M.H. and Nakazato. H.,Proc. Nut. Acad. Sci. U S . , 68 (1971) 1336-1340. Gawronski, T.H. and Wold, F., Biochemistry, 11 (1972) 442-448. Jankowski, W.J., Davey, M.W., O'Malley, J.A., Sulkowski, E. and Carter, W.A., J. Virol., 16 (1975) 1124-1 130.

REFERENCES

Kasche, V., Arch. Biochem. Biophys., 173 (1976) 269-272. Lerman, L.S., Proc. Nat. Acad. Sci. US.,39 (1953) 232-236. Lowe, C.R. and Dean, P.D.F., FEBS Lett., 18 (1971) 31-34. O’Carra, P., Barry, S. and Griffin, T., Methods Enzymol., 34 (1974) 108-126. Porath, J., A x h , R. and Ernback, S., Nature (London), 215 (1967) 1491-1492. Reiner, R.H. and Walch, A., Chromatographia, 4 (1971) 578-587. Schott, H., Eckstein, H., Gatfield, I. and Bayer, E.,Biochernistry, 14 (1975) 5541-5548. Starkenstein, E., Biochem. Z., 24 (1910) 210-218. Yagi, Y., Engel, K. and Pressman, D.,J. Immunol., 85 (1960) 375-386.

11

This Page Intentionally Left Blank

13

Chapter 3

Theory of affinity chromatography 3.1 THEORETICAL GUIDELINES DEDUCED ON THE BASIS OF THE EQUILIBRIUM MODEL* In contrast to the considerable number of papers in which the most varied factors affecting the results of affinity chromatography are treated empirically, theoretical guidelines based on physico-chemical properties and relationships have very rarely been presented (Kasche; Lowe et al., 1974b; Nishikawa et al.; Porath; Porath and Kristiansen; Reiner and Walch). Among them, the elaboration by Graves and Wu of simple kinetic and equilibrium models of affinity sorption and desorption are most instructive. They analysed the adsorption and desorption phases of affinity chromatography separately, and in each treatment they first considered only the limitations based on equilibrium relationships and did not take into consideration rate processes (diffusion and reaction). As it is much easier to analyse the results of adsorption or desorption in a batchwise arrangement, they deduced the main relationships in a simple model consisting of batch adsorption, washing and elution, under the assumption that equilibrium is attained in each step. During the deduction an enzyme was considered as the substance being isolated, but the same relationships are, of course, also valid for any other substance.

3.1.1 Equilibrium model for adsorption with a fured binding constant The main prerequisite for affinity chromatography is the formation of a specific complex (EL) between the isolated enzyme (E) and the affinity ligand (L) bound on a solid carrier. Hence the following equations apply: E t L-

kl k-l

EL .

and (3.2)

where K L is the equilibrium constant for ligand-enzyme interaction during adsorption steps. Turkovi et al. studied the attainment of equilibria during the sorption of chymoas a function of trypsin on to N-benzyloxycarbonylglycyl-D-phenylalanyl-NH2-Spheron pH and ionic strength in a batchwise manner (Fig. 3.1). It is evident that in all instances equilibrium was attained within 1 h. Porath and Kristiansen indicated the dependence of the rate of sorption of trypsin on the concentration of the enzyme sorbed on to immobilized soybean trypsin inhibitor, which is discussed in detail in Chapter 10 (see Fig. *For list of symbols see p. 26.

14

THEORY OF AFFINITY CHROMATOGRAPHY

1

f 6

0

20

I

60

40

min

0

20

60

40

rnin

Fig. 3.1. Adsorption of chymotrypsih (A) To ZGly-D-Phe-NH,Spheron as a function of pH: (1) 0.1 M Tris-HC1 buffer, pH 8.0; (2) 0.1 M Tris-HCl buffer, pH 7.2; (3) 0.1 MTris-maleate buffer, pH 6.0. (B) To ZGly-D-Phe-NH,Spheron as a function of ionic strength, with Tris-HC1 buffers, pH 8.0, of the following concentrations: (1) 0.05 M; (2) 0.1 M; (3) 0.25 M; (4) 0.35 M, (5) 0.5 M,(6) 1 M. Data from J. Turkovi et at., Biochim. Biophys. Acta, 427 (1976) 586-593.

10.4). Lowe et QI. (1974a) compared the amount of lactate dehydrogenase sorbed on to N6(6-aminohexyl)-5’-AMP-Sepharose both in a column and in a batchwise arrangement. While in the first instance 100%of the enzyme was bound within 1 h, in the batchwise arrangement 16 h were necessary for 100% binding. The batchwise adsorption of , lactate dehydrogenase on to N6(6-aminohexy1)-S’-AMP-Sepharose (Lowe ef ~ l .1974b) in the presence of various concentrations of the competitive inhibitor NADH is shown in Fig. 3.2. From Figs. 3.1 and 3.2, it is evident how a decrease in pH, an increase in the ionic strength or the presence of a competitive inhibitor shifts the equilibrium in favour of the free enzyme in the examples mentioned. In order to derive further relationships from eqn. 3.2 Graves and Wu made the following assumptions. The gel volume (u’) includes a smaller volume (u) of the solution within the network composed of the polymeric molecules of the gel. Before the addition of an enzyme solution, the affinity ligand bound in the gel has a concentration Lo (moles per volume u) and no enzyme is present in u. Only after the addition of an enzyme solution of volume V and concentration Eo is equilibrium attained. The concentration of the bound enzyme at equilibrium, [EL], can be expressed by the equation

.

LO [EL] = - [ B ] (1 --) 2

(3.3)

15

EQUILIBRIUM MODEL

Assuming that A is much smaller than 1, the relationship can be simplified:

-+

1 -(A/2)

(3.6)

and eqn. 3.3 reduces to the expression

The error originating from this approximation is about 2.6% for A = 0.1, 5.2%for A = 0.2 and 17.1% for A = 0.5. The quantity B defined by eqn. 3.4 usually has a value close to unity and, if it is assumed that it is equal to unity, then eqn. 3.5 shows that A is the four-fold ratio of the number of moles of the enzyme to the number of moles of the ligand. Under normal conditions, the concentration of the enzyme in solution is usually lo-’ M or less and a typical concentration of the ligand in the gel phase is usually 10-2M.

N

t,

40

W W

-

U

LL

20-

0

0

I

I

40

80

120

160

INCUBATION TIME , mh

Fig. 3.2. Batchwise adsorption of lactate dehydrogenase to N6-(6-aminohexyl)-5’-AMP-Sepharosein the presence of NADH. A glass tube (14 mm diameter), covered at one end with a double thickness of fine gauze (100pm mesh), contained 0.5 g of N6-(6-aminohexyl)-5’-AMP-Sepharose(1.5 pmol/ml of 5’-AMP) layered on the fine gauze. The tube was suspended in a gently agitated incubation solution (10 ml) and at the time intervals indicated aliquots (50 pl) of the incubation solution were assayed for enzymic activity. The enzyme was dialysed overnight against the appropriate incubation solution prior to experimentation. The basic incubation solution (O),10 mMKH,PO,-KOH, pH 7.5, containing 0.02% of NaN,, was supplemented with 2 mM NADH (0)and 5 mM NADH (A). Reproduced with permission from C.R. Lowe et al., Eur. J. Biochem., 42 (1974)1-6.

16

THEORY OF AFFINITY CHROMATOGRAPHY

Therefore, at a reasonable ratio of the volume of the solution V to the gel volume, the error introduced by the approximation is very small. When these approximations are used, the fraction of the bound enzyme (with respect to the total amount of enzyme) at equilibrium can be defined as bound enzyme - [EL] v total enzyme

EoV

LOV . KL(V+v)+Lov+EoV

1 .

In most instances Eo V is much less than the other two expressions and can be neglected in eqn. 3.8. Eqn. 3.8 can then be used to express the effectiveness of the binding for the given parameters K L , Lo, Eo, V and v. When Graves and Wu considered the enzyme entrapped non-specifically in the gel, in addition to the enzyme molecules bound in a complex with the ligand, they obtained the following expression: bound t entrapped enzyme - V(Eo- E ) LOV t total enzyme VEO . KL(VtV)+L()V ~

This fraction represents the separation achieved simply by the separation of two phases, and the difference between eqns. 3.9 and 3.8 represents the enzyme entrapped in the gel in addition to the complex with the ligand. From eqn. 3.9, it can be deduced that free t entrapped enzyme bound enzyme

(V+M (EO)V-E(V+V)

7 .

V

-.Eo+3

LOV

LO

(F)

(3.10)

The left-hand side of eqn. 3.10 can be plotted on the ordinate against Eo on the abscissa. A straight line is obtained, the slope of which is V/(Lov),and from this relationship Lo can be determined. The intersection with the ordinate gives the term KL( Vtv)/(Lov), from which K L can be determined. Returning to eqn. 3.8 and again using the limitation that the total number of moles of enzyme, Eo V, is much smaller than the total number of moles of the ligand, Lov, and now introducing the further limitation that the volume V is much smaller than the volume Y,Eo, V and Y are eliminated and the following simple relationship is obtained: bound enzyme -. - LO total enzyme KL +Lo

(3.1 1)

From t h s relationship, the effects of various values of K L and Lo are evident; their mutual dependence is illustrated in Fig. 3.3. The curves obtained are rectangular hyperbolas similar to Langmuir’s adsorption isotherm or to the Michaelis-Menten equation. It is evident from Fig. 3.3, for example, that at the commonly occurring concentration of the ligand, 10 mM, effective binding of the enzyme will occur at K L values of 10-3M and lower, while at higher K L values ineffective retention will take place. Similar conclusions

EQUILIBRIUM MODEL

5D

17

l o

09

:

08

$

07

06

$

05

8

04

Z

03

PIu

02

a

01

u

o

U

0

I

2

3

4

5

6

7

[TOTAL

8

9

10

11

LIGAND

I3

12

LJ

,

14

15

16

17

18

19

20

m M

Fig. 3.3. Fractional enzyme binding for low enzyme concentrations as represented by eqn. 3.11. This general hyperbolic graph can also be used with the more correct eqn. 3.8 and even to estimate the fractional ligand saturation. Reproduced with permission from D.J. Graves and Y.-T.Wu, Methods Enzyrnol., 34 (1974) 140-163.

have also been reached, for example, by Steers et al. on the basis of experimental results. However, it should be stressed that the K L values given are considered for the complex of the enzyme with a ligand covalently bound to a solid support, and hence it need not agree with the equilibrium constants determined in solution. Fig. 3.4 shows the dependence of the amount of bound lactate dehydrogenase from rabbit skeletal muscle, hog heart muscle and glycerokinase on the concentration of N6-(6-aminohexyl)J’-AMP bound t o Sepharose, as determined by Harvey et al. Whether the differences in the courses of the curves correspond to the differences in the equilibrium constants of particular complexes is unfortunately not known to the author of this book, but it can be judged, on the basis of the concentration of potassium chloride solution necessary for the elution of single enzymes from the nucleotide carrier mentioned, that the bond of glycerokinase is weaker (see Table 4.2). The fraction of the total amount of the ligand groups which, under given conditions, are saturated with enzyme molecules is given by the term ELILO.This term can be obtained when both sides of eqn. 3.7 are divided by Lo. If, in Fig. 3.3, Lo on the abscissa is replaced with Eo V and instead of K L the expression KL( V t v) -+ Lov is used as a variable parameter, the fraction ELILO can be measured simply on the ordinate. In Table 3.1, a series of values for percentage ligand saturation under various conditions are given, assuming that Lo is 0.01 M and o is the ratio of V to v. It follows that the saturation of the ligand molecules with the enzyme is very low. Hence, in chromatography on affinity sorbents it can be expected that the enzyme will begin to flow out of the column much sooner than at the time when all of the reactive groups in the gel have become saturated. The true capacity of affinity sorbents is far from corresponding to the amount of bound ligand in the gel, which is determined, for example, in the case of a protein or peptide material, on the basis of the amount of amino acids determined in the hydrolysate. Turkovri et a1 found, for example, that only 0.3%of the molecules of N-benzyloxycarbonylglycyl-D-phenylalaninebound to NH2-Spheronare saturated with

THEORY OF AFFINITY CHROMATOGRAPHY

18

0

250

500

750

[LIGAND] , nmol/ml

Fig. 3.4. Capacity of immobilized nucleotide adsorbent [N6-(6-aminohexyl)-5'-AMP-Sepharose]in relation to ligand concentration. The sample (100pl), enzyme ( 5 U) and bovine serum albumin (1.5 mg) were applied to a column (50 X 5 mm) containing 1.0 g of the affinity adsorbent diluted to the appropriate ligand concentration with Sepharose 4B.4 Lactate dehydrogenase (rabbit skeletal muscle); 0, lactate dehydrogenase (pig heart muscle); Q glycerokinase. Reproduced with permission from M.J.Harvey el al., Eur. J. Biochem., 41 (1974)335-340.

TABLE 3.1 PERCENTAGE AFFINITY LIGAND SATURATION UNDER VARIOUS CONDITIONS (FOR Lo = 0.01 M)

J%

KL

(M)

(M)

ELILO(%I w=l

-

10-4

10-3

lo-* 10-5 lo-"

10-5

10-~ 10-4 lo-*

lo-* 10-6

10-3

10-5 lo-6 10-7

10.~ 10-4

10-5 10-

-

0.8264 0.9709 0.9881 0.9899 0.0833 0.0979 0.0997 0.0999 0.0083 0.0098 0.0100 0.0100 0.0008 0.0010 0.0010 0.0010

w=lO

w=100

~~

4.5455 8.2645 9.0009 9.0818 0.4739 0.8929 0.9794 0.9890 0.0476 0.0900 0.0988 0.0998 0.0048 0.0090 0.0099 0.0100

8.2645 33.2226 47.5964 49.7488 0.8929 4.7393 8.3264 9.0082 0.0900 0.4950 0.9001 0.9803 0.0090 0.0497 0.0907 0.0989

19

EQUILIBRIUM MODEL

chymotrypsin. Lowe et al. (1974b) found for each binding of lactate dehydrogenase to N6(6-aminohexy1)-5’-AMP-Sepharose a 0.76% capacity of the adsorbent for the batchwise arrangement and <0.1% for frontal analysis chromatography. Similarly, Hierowski and Brodersen determined that only about one tenth of the bound molecules of bilirubin are capable of binding serum albumin under the given experimental conditions. 3.1.2 Equilibrium model for elution by a change in KL As discussed in Chapter 2 (see Fig. 2.1), the specifically sorbed isolated enzyme can be liberated from the complex with bound affinity ligand either with a solution of a competitive inhibitor (biospecific elution) or by changing the medium. The latter method is far more common and a change in pH or ionic strength is most commonly employed. Although the effects of these changes can be complicated, Graves and Wu, when deriving the relationship for the liberation of the enzyme from the affinity adsorbent, made use of a further apprbximation. They assumed that a change in ionic environment results only in an increase in KL. They again considered the batchwise process, which they divided into six stages: (1) gel volume v is equilibrated with the enzyme volume V, with equilibrium constant K L ;(2) V is separated from v ; (3) the volume of the (non-eluting) wash solution w is added to v and equilibrium is attained (KL does not change); (4) w is separated from v, eliminating the fraction of contaminating proteins which were sorbed in the gel (a partial loss of the bound and the entrapped enzyme also takes place). (4A) steps 3 and 4 are repeated as often as necessary to decrease contaminants to a low level; ( 5 ) the volume V’ of the eluent is added to v and equilibrium is attained at a higher equilibrium constant K’L;(6) V’ is separated from v and pure enzyme is obtained. Steps 5 and 6 can be repeated in order to increase the yield of the enzyme, but in practice multiple elution is not used.

Elution of the enzyme without washing out The deduction of the relationships that describe the elution process is similar to that for adsorption processes. However, as the complexity of the processes has increased, Graves and Wu employed in their further deduction the relationships based only on the approximation in eqn. 3.6. On the basis of eqn. 3.9, Graves and Wu derived an expression for the sum of the enzyme lost in V, the enzyme obtained in V‘, and the enzyme entrapped in v: entrapped t V and V’ fractions of enzyme total enzyme

.

f 1-

LOWLY + L o 4 [KL( Vtv) + L O Y ]

-

[K)L( V ’ t v )+Lev]

(3.12)

For the enzyme fraction obtained by elution from the affinity sorbent, they derived the expression:

enzyme in V’ total enzyme

.

LOV(KLV t Lov)

-

[KL( Vtv)t Lov] [K’L( V’ t v) t Lov]

(3.13)

20

THEORY OF AFFINITY CHROMATOGRAPHY

This deduction is useful for the understanding of the whole process. It is worth noting that the recovery of the enzyme is strongly affected by the ratio of I/’ to v, and less by thc ratio of V to v. Eqn. 3.13 is expressed graphically as an asymptotic limit for the obtainment of the enzyme (Fig. 3.5A), even for an enzyme with very high K L values. From the graph, the unfavourable nature of K L values higher than 10-3Mis again evident, but it also indicates the difficulties that arise when K L has an excessively low value, because then part of the enzymes may remain bound in the gel phase after elution. It further follows clearly from Fig. 3.5A that the larger the difference between the K L and K i values, the more easily the enzyme is obtained. Fig. 3.5A also gives the purification factor (Pf), which is defined as the ratio of the resulting enzyme t o the total protein

10

’

10

*

10

10

*

10

’

10

K: CMOLES/L) f-’ig. 3.5. (A) Fractional recovery and purification factor for no washing steps, a 1 : 1 volume ratio of sample to gel and a 10:1 volume ratio of eluting solution to gel. (B) Recovery and purification factor for 10 washing steps. Reproduced with permission from D.J. Graves and Y.-T.Wu, Methods Enzymol., 34 (1974) 140-163.

divided by the initial ratio. In this arrangement, in which washing is not carried out, Pfis very low. It is obvious that without the washing steps the purification will not be effective, even with favourably low K L values and sufficiently high K i values, because the enzyme obtained will be contaminated with proteins. The evaluation of the efficiency of subsequent washing out steps, leading to a reduction in contamination, is simple:

EQUILIBRIUM MODEL

21

contaminant protein in elutant vv' total contaminant protein [ ( V t V)(V't

v,l (5) "

(3.14)

It is better to wash more often with smaller volumes than once with a large volume. This also indicates the advantage of column chromatography, because a gradual passage of the eluent through a fixed bed of material in fact brings about an elution with a large number of washing steps (n). Elution of the enzyme following multiple washes The calculation of the amount of enzyme obtained is complicated by the fact that the enzyme is simultaneously bound by the ligand and also physically entrapped within the gel. For the total enzyme fraction which remains bound after n washing steps, Graves and Wu derived the expression fraction of enzyme bound total enzyme

=

f&

L OV(KLv t Lo v)" [ K L ( V t V ) tLOV] [KL(w+v)+LoVln

(3.15)

For the fraction of the enzyme remaining bound after the elution step, they deduced the equation

L Ov(KLv t Lov)n+ 1 fEE

+

[KL( V t v) t Lov] [KL( v ' + v) + Lov] [KL(W + v) + Lov] "

(3.16)

Taking eqns. 3.8 and 3.12 as a basis, they derived the following expression for the enzyme fraction obtained in the eluate:

(-)V V'' t v

recovered enzyme = fR total enzyme

(1-fEf3-z n fi) i= 0

(3.17)

where fo represents the fraction of the initial amount of the enzyme which is lost in the discarded V. The fractions cfi) are related through relatively simple recursion relationships:

fo

fi

+(-)V Vt v

* (Lw) + v W

fn

.G-

wtv

(1 -

KL( vt v) t Lov

(I-fEl-fa)

(3.18)

(3.19)

n-1

(1 - f E n -

i=O f i )

(3.20)

The expressions for single fractions become increasingly more complex because each new expression contains the expression from preceding fractions. Hence the expression for the fraction from the initial amount of the enzyme cfn), which is lost in discarded washing volumes w after an n-fold washing, is

22

THEORY OF AFFINITY CHROMATOGRAPHY

(3.21) With increasing number of washings, the purification factor, Pf,also increases considerably. For example, when a single wash with a relatively large volume has been employed (w/v= lo), Pf increases from 2 to 22. The decrease in the percentage of the enzyme obtained is also dependent on K L . For example, for K L = lO-’M the percentage of the enzyme obtained decreases only negligibly, for K L = 10-4M the recovery at high K L values decreases from 90%t o 8276, and for K L = loA3M it decreases as much as from 83%to 44%. Fig. 3.5B shows the dependences of the percentages of the obtained enzyme on K after a 10-fold wash, when w/v= 1. For K L = lO-’M a very high degree of purification is already achieved (Pf= 2000), while the percentage of the enzyme obtained is also relatively high.

3.1.3 Equilibrium model for elution by a competitive inhibitor The second general method for the setting free the enzyme from its complex with a bound affinity ligand consists in washing it out with a solution of a soluble competitive inhibitor. In t h s instance the main prerequisite is that K L should not change during the elution process. To eqns. 3.1 and 3.2, expressing the attainment of equilibrium between the enzyme and the bound affinity ligand, the following equations should be added: k

E+I&EI

(3.22)

k-2

and (3.23) which relate to the formation of the enzyme-soluble inhibitor complex. All symbols used so far have the same meaning, but V ‘ now means the volume of the inhibitor solution. In addition, the term Zo is required, denoting the initial concentration of the inlubitor, and also [I] for the final equilibrium concentration of the inhibitor, [EI] for the equilibrium level of the complexed enzyme in solution and VELfor the number moles of the enzyme present in the gel before the addition of the inhibitor solution. Furthermore, all the calculations just given on of the effects of washing, preceding elution analysis, can still be applied. The expression for the bound plus entrapped enzyme (remaining in the gel after n washings) is then

5 (L)(l -fBn VEo w+v *

n-1

-

fi) i=O

fBn

(3.24)

23

EQUILIBRIUM MODEL

wherefB, can be taken from eqn. 3.15 andfi from eqn. 3.18, through eqns. 3.20 and 3.21. Combining eqns. 3.2 and 3.23 and using a suitable material balance, Graves and Wu made permissible simplifications and derived for the equilibrium concentration of the complex of the enzyme with the soluble inhibitor the following expression: [EI]

VVY0EL + v(V’+ V)(~-KI/KL)EL+V’(V’+v)Zo+KI(V’+v)2 + KILOV(V’+v)/KL

(3.25)

It follows logically (and can even be proved) that in the presence of two inhibitors, one in solution and the other as an immobilized affinity ligand, only a negligible amount of free enzyme, [El, is present after equilibration, although a considerable amount of the enzyme can be in solution (as [EI]). In most instances we can use the expression V‘[EI] for the total amount of enzyme in solution instead of V’( [EI] + [El) without introducing a serious error. With such a simplification and using eqn. 3.25 and the expression p = V’/v, Graves and Wu deduced the following equation for the fraction of the enzyme obtained: recovered enzyme

V*[EI]

total enzyme in washed gel

VEL

PI0

K.

l(3.26)

I

‘r ’” ~ ~ L + p ~ ~ f ~ I ( l + p ) + ~ ( L o - E L ) KL

I

As shown earlier, Lo is normally much larger than EL and therefore the following expression can be used in most instances:

(3.27) In order to simplify eqn. 3.27, it is useful to consider as a first approximation the case when LO/KL is much higher than p and the term KI( 1 t p ) is consequently neglected. The realistic maximum value for p is about 10 because higher values would lead to excessive dilution of the isolated enzyme. When 1 t p is neglected and when K L = 10-3M, the error introduced is about lo%,and at lower K L values it is much lower. Therefore, instead of K I and KL only their ratio can be considered:

(3.28) Eqn. 3.28 is illustrated graphically for Lo = 10 mM and for p = 1 and 10 in Fig. 3.6. A very important parameter here is the ratio of the equilibrium constants for the free and the bound inhibitor, K I / K L .If the ratio is unity then the concentration of the inhibitor in the eluent necessary for an effective elution of the enzyme is 10-100 mM(assuming, as has been done throughout, that the ligand concentration in the gel is 10 mM). The necessary concentration of the inhibitor and the ratio of the equilibrium constants, KI/KL, are interdependent. If the soluble inhibitor is about 10 times stronger than the ligand, then its necessary concentration in the solution decreases by about one order of magnitude, while if it is 10 times weaker the necessary concentration of the soluble inhibitor is 0.1-1 M.As the elution of the enzyme from the affinity sorbent with solutions of soluble inhibitor of various concentrations represents the basis of the presently

24

THEORY OF AFFINITY CHROMATOGRAPHY 100

I

I

I

, 20

0 10.‘

10.’

lo-’

10.’

10

Cl1,C MOLESIL) Fig. 3.6. Enzyme recovery for elution by the addition of a soluble inhibitor. The solid curves represent a V’ : v ratio of 10 and the broken curves a V’ :Y ratio of 1. No washing steps are assumed. Note that the elution volume ratio strongly influences the overall recovery that can be achieved. Reproduced with permission from D.J. Graves and Y.-T. Wu, Merhods Enzyrnol., 34 (1974) 140-163.

most commonly used method for the determination of equilibrium constants of the isolated enzyme with bound inhibitor, practical examples of this method are presented in detail in Chapter 4.

3.1.4 Simulation of column chromatographic results The sorption, washng and elution of enzymes by means of column affinity chromatography are represented on the basis of the equations derived for the batchwise model of adsorption and elution by changes in K L in Fig. 3.7. From Fig. 3.7A, for K L = I K 3 M, the considerable ioss of enzyme (61.6%)that occurs during the washing process is evident. Elution at K i values of 1.0,O.l and 0.01 M then removes virtually all sorbed enzyme (38.4%) free. In Fig. 3.7B, which illustrates the isolation of an enzyme with a K L value of M , completely different behaviour can be seen, only 10.3%of the enzyme being lost during the washing steps. In elutions where K i is 0.1 or 0.01 M,about 89.7% of the enzyme can be recovered. For K i = M the recovery is about 86.2% and for K i = M it is only 16%. Fig. 3.7C illustrates a very favourable case for K L = lo-’ M or less. In this instance virtually no enzyme is lost during the washing. Elution at K L values from lo-’ to M gives recoveries of 98.9,98.9,95.6,29.7 and 1.9% of the amount of the starting enzyme after 20 washes. High K L values are desirable, because they lead to much more concentrated solutions of the recovered enzyme. 3.1.5 Conclusion

Although many of the relationships that have been deduced will have to be checked

EQUILIBRIUM MODEL

25

BINDING

I r

WASnCS

ELUTIONS

I

~

>

U W

$

Y U

c

2

3

a

I

9

n

15

15

11

19

21

23

?a

11 =OM

TOTAL WASHES A N D ELUTIONS. cIv'+Iw)tv

Fig. 3.7. Simulated column results for (A) a poor binding case (KL = lo-' M) where elution is achieved by a change in KL t o KL; considerable enzyme loss occurs during washing; (B) moderately good binding (KL = lo-' M); little enzyme is lost during washing; (C) very good binding (KL < practically no enzyme is lost by washing. Reproduced with permission from D.J. Graves and Y.-T.Wu, Methods Enzymol., 34 (1974)140-163.

experimentally, they are already useful today in indicating the aspects on which the investigator should focus his attention if he wishes to obtain good results when using affinity chromatography. An affinity sorbent with a concentration of bound affinity ligand of about 10 mM should be used only for isolations of substances the equilibrium constants M or less. For systems with higher (less of which (with the attached affinant) will be

THEORY O F AFFINITY CHROMATOGRAPHY

26

favourable) equilibrium constants, sorbents should be used with a correspondingly higher content of the bound affinity ligand. Ths, however, has numerous practical limitations, which are discussed in Chapter 5. Another important property deduced is the low saturation of the immobilized ligand molecules by the isolated substance. In order t o increase this saturation, it is necessary to use as concentrated a solution as possible for adsorption. The practically determined low percentages of saturation should be explained not only by steric factors, but also on the basis of the equilibrium-based limitation. From the relationship between the washing and elution brought about by a change in K L , two important facts follow. For K L = lo-' M or less, the batchwise adsorption and elution may give the same results as, and sometimes even better than, those in column chromatography. At K L = 1O-j M only column chromatography can give good results. Further, the increase in K during the elution step should lead to a value of at least lo-* M,regardless of the K L value, in order for the enzyme to be removed from the gel effectively. The achievement of such high values sometimes limits the use of affinants with a too low K L value. The number of washing steps and the volume of each wash are decisive for the elimination of contaminating components and they therefore influence the purification factor strongly. If K L is less than M the losses will be negligible, even when the washing is carried out with a large volume of the washing solution. For elution with a solution of soluble inhibitor, the most important parameter is the ratio of the equilibrium constants, K I / K L .The lower this ratio, the lower is the concentration of the soluble inhibitor that can be used for elution. The basic relationships derived for the batchwise method of affinity chromatography also apply to column chromatography.

i

3.1.6 List of symbols used A

B [El EL EO

[ELI f BE fBn

fn

fo fR fR(m) 10 KL

Ki

A parameter defined by eqn. 3.5; A parameter defined by eqn. 3.4; Enzyme concentration at equilibrium; Pseudo-enzyme concentration representing ligand-bound and gel-entrapped enzyme following washing but prior t o elution (see eqn. 3.24); Initial enzyme concentration in solution phase before contact with gel phase; Concentration of enzyme-ligand complex at equilibrium; Fraction of initial enzyme remaining bound to ligand after n washes and one elution; Fraction of initial enzyme remaining bound t o ligand after n washing steps; Fraction of initial enzyme lost when volume w of wash n is discarded; Fraction of initial enzyme lost when Vis discarded; Fraction of initial enzyme recovered in V' after n washes and one elution; Fraction of initial enzyme recovered in V' with elution M after n washes; Initial concentration of inhibitor; Ligand-enzyme equilibrium constant during adsorption steps; defined by eqn. 3.2; Ligand-enzyme equilibrium constant during elution;

27

COOPERATIVE BONDING

R V U’

V V’ W

P 0

Equilibrium constant for enzyme-soluble inhibitor complex; Rate constant for enzyme-ligand combination; Rate constant for enzyme-ligand dissociation; Rate constant for the combination of enzyme with soluble inhibitor; Rate constant for the dissociation of enzyme-soluble inhibitor complex; Ligand concentration at equilibrium; Initial concentration of ligand in gel; Number of elution steps; Number of washing steps; Purification factor: final enzyme to total protein ratio divided by the initial ratio; Fraction enzyme recovery defined by eqn. 3.27 or 3.28; Volume of gel phase which is accessible to penetrating protein molecules; Actual volume of gel phase; Volume of enzyme solution phase initially added to the gel; Volume of each eluting solution; Volume of each wash solution; Ratio of the elution volume V‘ to the gel volume v; Ratio of the sample volume V to the gel volume v.

3.2 THEORY OF COOPERATIVE BONDING WITHIN THE PLATE THEORY* A mathematical deduction and a simulation of elution profiles was carried out by Okada et al. on the basis of a cooperative elution of oligoadenylic acid on agarose with attached polyuridylic acid. 3.2.1 Isotherm of binding of oligoadenylic acid to polyuridylic acid For a content of helical structure in polyuridylic acid the following equation (according to Damle) can be written:

eu

= ~c/c,f’(c)

(3.29)

or = CtCu/Ne,

(3.30)

The binding of polyuridylic acid t o agarose was found to occur at the 5’-terminal. On the basis of this finding and further characterization, it seems justified to propose that the immobilized polyuridylic acid has the same conformation as in solution, and the calculation of the partition functions of the system can then be carried out in the usual manner when the partition function of the non-bonded oligomer and the non-occupied binding site on the affinity adsorbent is unity. If cu >> c, the overlapping of two bound oligomers can be neglected. Okada e t al. limited the analysis t o short oligomers and were

*

For list of symbols see p. 33.

THEORY GF AFFINITY CHROMATOGRAPHY

28

able to assume an all-or-none binding of the oligomer on to polyuridylic acid. Under these conditions, the partition function of the system is given by the Nth power of the smallest root p of the secular equation (Damle; Magee et ul.): (1 -x)(l -x%J

(3.31)

= 0

-SIP+'

When the concentration of free oligomer is buffered, the helical content of polyuridylic acid is given by

eu

N

__

=

(3.32)

x s N F p N { [ u p ( 2-p)/(l -p)'] + 1 ) + N - 1 From eqn. 3.3 1 we have

(3.33)

,'.

i'

. ,','. 0.5

i

.'

..

I

.,

,"

,

I

$"

I 0

1

I

.-..

I .

om ai

0.5

1.0

5.0

1Qo

[FREE O L l G O M E R l (MOLES/LITER

10'1

Fig. 3.8. Binding isotherm for the formation of polyuridylic acid-oligoadenylic acid complex at 5"C, pH 7.0,0.033 M phosphate buffer, 1 M NaCl, 1 mM MgCl,. (A)In solution, polyuridylic acid concentration 3.7-10-' M.(B)In gel after washing at pH 10.0, polyuridylic acid concentration 4.9 lW4 M. (C)In gel before washing at pH 10.0, polyuridybc acid concentration l.2*lW4M.Broken line, theoretical curve. The ordinate indicates the helical content of polyuridylic acid in 1 : 2 stoichiometry of adenylic acid : uridylic acid. Reproduced with permission from S. Okada et al., Biopolymers, 14 (1975) 33-49.

-

COOPERATIVE BONDING

29

D

Fig. 3.9. Temperature dependence of elution profile in polyuridylic acid immobilized chromatography with oligoadenylic acid. Elution buffer, 1/30 Mphosphate buffer, 1 M NaCl, 1 mM MgCl,, pH 7.0; M, each volume, 0.1 ml; polyuridylic acid concentraapplied oligoadenylic acid concentration, 4.9 tion, 0.98010-~M. V,, = void volume; V f = bed volume. Reproduced with permission from S. Okada et al., Biopolyrners, 14 (1975) 33-49.

(3.34) The binding isotherm can be obtained from eqns. 3.33 and 3.34. The parameters xsz and u in the equations are determined from the free oligomer concentration and the slope at the mid-point of an experimental binding isotherm, respectively. The binding isotherm ( N = 2, xsz = 1.1 X lo5 M,u = 0.017) thus simulated is shown by the broken line in Fig. 3.8.

3.2.2 Cooperative adsorption column chromatography Fig. 3.9 shows typical elution profiles of oligoadenylic acid at various temperatures. The elution centre of the peak is shifted and the peak shape is distorted from a Gaussian type by a decrease in temperature from 15 to O°C. These results indicate that the binding of oligoadenylic acid to polyuridylic acid is stabilized by decreasing the temperature. Further, the distorted elution profiles indicate the cooperativity of the binding of oligoadenylic acid to polyuridylic acid. The binding isotherm shows that the binding of oligoadenylic acid takes place in a cooperative manner and so the apparent dissociation constant is not constant. In agreement with this type of binding, the elution profiles obtained at 5 and 10°C with different concentrations of oligoadenylic acid change as shown in Fig. 3.10. On the other hand, it is known that in non-cooperative binding the elution profile retains a Gaussian shape, the maximum of which does not change with concentration. Making use of the plate theory,

30

THEORY OF AFFINITY CHROMATOGRAPHY

ELUTION VOLUME,

ml

Fig. 3.10. Concentration dependence of elution profie in polyuridylic acid immobilized chromatography with otigoadenylic acid. Applied oligoadenylic acid concentrations, 9.8 * l W 3 ,4.9 o ~ U -2.5 ~, l.2*10-3,0.6.10-3M ;each volume, 0.1 ml. Temperature: (A) 10°C; (B) 5°C. V , = void volume; V , = bed volume. Reproduced with permission from S. Okada et al., Biopolymers, 14 (1975) 33-49.

Okada et al. studied cooperative adsorption chromatography and neglected the effect of gel fdtration because the oligomers are very small in comparison with the pore size of the gel. The first condition is that the concentration of the free oligomer should be the same both in the mobile phase and in the gel phase. The total concentration, Ckt, of the oligomer in the kth plate is then described by the concentration of the free oligomer, Ck, and the concentration of the bound oligomer, Ck*: (3.35) where q is the volume fraction of the gel phase. The second condition is that the binding equilibrium must be sufficiently rapid to be attained in each plate. Thus, using eqns. 3.33 and 3 3 4 , the relationship between ckb and ckf can be deduced: 4Ck

= f(Ckr) Ck

'

(3.36)

wherefis obtained from f by replacing c,, with QC,. Thus in the next elution step, I?k V oligomers move to the (k+l)th plate and QCkb V oligomers remain in the kth plate, where V is the volume of the plate (minusthe gel volume). In terms Of f ( C k r ) , indicating the fraction of oligomers that remains in the next elution step, the column equation, using total concentration as an independent variable, is given by

COOPERATIVE BONDING

31

ckf(i) = c k f ( i - l)f[ckf(i - l ) ] t ck-l '(i-

1){1 -f[ck-,'(i - l)] } (1

< k < i)

COt(i) = COt(i- l)f[cof(i - l)]

(3.37a) (3.37b)

where i denotes the step number (i.e,, time parameter). When the total number of the plates is km - 1, the elution profile is given by Ck,(i) with the initial condition ckt(0) = 0,

km

>k >

1

(3.38a) (3.38b)

= co

CO'(0)

The elution profiles obtained by digital computer simulation using eqn. 3.37 were in good agreement with the experimental results given in Fig. 3.10. The binding isotherm used in this calculation was deduced from eqns. 3.33 and 3.34 because their parameters best matched the experimental curve of equilibrium dialysis (see Fig. 3.8).

3.2.3 Characteristic features of cooperative adsorption chromatograms In order to find the general characteristics of non-linear adsorption chromatograms, eqn. 3.37 was re-written in the form of a partial differential equation:

(3.39) where c(x,t) is the total oligomer concentration at time t at a point x below the top of the column (x is already multiplied by the cross-section of the column) and u = dx/dt is the flow velocity. Eqn. 3.39 is transformed into

(3.40) using the free oligomer concentration as an independent variable and qcb =f
(3.41)

and gives the solution

c= x =

i(X) U t

1

-f[h(A)]

- (dfldlnq+j&)

t x

(3.42)

where X is a parameter. When ;(A) = Foe-h2/A,the elution profile is given in an implicit form:

(3.43)

32

THEORY OF AFFINITY CHROMATOGRAPHY

where Vr is the bed volume of column and Ve = u f is elution volume. The equation that gives the peak of the elution profile is

Vemm = [l +f(cmax)

i.

(df/dlnE)+Fmax] V,

(3.44)

This is the equation to give the “peak trajectory”, that is, the Vemax versus cam relationship. Fig. 3.1 1 shows some examples of the peak trajectory for (a) a cooperative binding isotherm without saturation of binding sites, (b) a non-cooperative binding isotherm with saturation (Langmuir-type isotherm), and (c) a cooperative binding isotherm with saturation.

Fig. 3.11. Some examples of peak trajectories: (a) cooperative binding isotherm without saturation; (b) non-cooperative binding isotherm with saturation, Ce., Langmuir-type isotherm; (c) cooperative binding isotherm with saturation. Abscissa, elution volume; ordinate, concentration of the solute at the exit monitoring point; KO= the concentration-independent term of the association constant. The broken curves demonstrate the leading form of cooperative chromatogram explained by the peak trajectory theorem. Reproduced with permission from S. Okada et al., Eiopolymers, 14 (1975) 33-49.

The intercept, Ve(O), of the peak trajectory on the Ve axis gives the association constant at infinite dilution, and the slope at that point, Ve‘(O), gives the first non-linear term off. The thermodynamic parameters for the oligoadenylic acid-polyuridylic acid system are given by this procedure as follows: (3.45) (3.46) As a conclusion to their deductions, Okada el al. published a theorem on peak trajectories: any elution profile crosses (or osculates) the peak trajectory at only one

REFERENCES

33

point (i.e., the peak). This theorem is easily derived from a lemma on the elution profile: the elution profiles for various input concentrations do not cross each other. This lemma is easily derived under the condition that the initial curve @) depends linearly on the input concentration. From this theorem, a schematic diagram of a class of elution profiles can be obtained, as is shown by the broken curves in Fig. 3.1 1 for the leading form. The nearly concurrent leading parts of the elution profiles is understandable from this theorem.

3.2.4 List of symbols used

N

Degree of polymerization of oligoadenylic acid; cu CU Concentration of total and free binding sites on the polyuridylic acid chain, respectively; C, c Concentration of total and free oligoadenylic acid, respectively; OU = (cu - Cu)/cu Helical content of polyuridylic acid; f’(c) = (c - 3 / c Adsorption coefficient of oligomer to polymer as a function of total oligomer concentration; S Partition function of a nucleotide pair in the bound state when the partition function in the free state is taken as unity; $1 Partition function of a bound oligomer having an adjacent bound oligomer and its complement; x = s l / ( s N F ) Correction factor for the end-effect of the oligomer; U Inter-oligomer cooperativity parameter = (partition function of an isolated bound oligomer and its complement)/sl. 3

3.3 STATISTICALTHEORY OF CHROMATOGRAPHY APPLIED TO AFFINITY CHROMATOGRAPHY An application of the statistical theory of chromatography elucidated by Giddings and Eyring (plate theory) to affinity chromatography was elaborated by Denizot and De Laage. They established by means of a convenient expression of moments the convergence towards a Laplace-Gauss distribution. The Gaussian character is not preserved if other causes of dispersion are taken into account, but expressions of moments can be obtained in a generalized form. They also deduced a simple procedure for expressing the fundamental constants of the model in terms of purely experimental quantities.

REFERENCES Damle, V.N., Biopolymers, 9 (1970) 353-372. Denizot, F.C. and De Laage, M.A., Proc. Nat. Acad. Sci. US.,72 (1975) 4840-4843. Graves, D.J. and Wu, Y.T., Methods Enzymol., 34 (1974) 140-163. Harvey, M.J., Lowe, C.R., Craven, D.B. and Dean, P.D.C., Eur. J. Biochem., 41 (1974) 335-340. Hierowski, M. and Brodersen, R., Biochim Biophys. Acta, 354 (1974) 121-129. Kasche, V., Biochem. Biophys. Res. Commun.,38 (1970) 875-881.

34

THEORY OF AFFINITY CHROMATOGRAPHY

Lowe,C.R., Harvey, M.J. and Dean,P.D.G., Eur. J. Biochem., 41 (1974a) 341-345. Lowe, C.R., Harvey, M.J. and Dean, P.D.G., Eur. J. Biochem.. 42 (1974b) 1-6. Magee, Jr., W.S., Gibbs, J.H. and Zimm, B.H.,Biopolymers, 1 (1963) 133-143. Nishikawa, A.H., Bailon, P. and Ramel, A.H., Advan. Exp. Med. Biol., 42 (1974) 33-42. Okada, S., Husimi, Y., Tanabe, S. and Wada, A., Biopolymers, 14 (1975) 33-49. Porath, J.,Methods Enzymol., 34 (1974) 13-30. Porath, J. and Kristiansen, T., in H. Neurath and R.L. Hill (Editors), The Proteins, Academic Press, New York, 3rd ed., 1975, pp.95-178. Reiner, R.H. and Walch, A., Chromarographia, 4 (1971) 578-581. Steers, E., Cuatrecasas, P. and Pollard,!., J. Bwl. Chem., 246 (1971) 196-200. TurkovB, J., Bliha, K., Valentovi, O., Coupek, J. and Seifertovi, A.,Eiochim. Biophys, Acta, 427 (1976) 586-593.

35

Chapter 4

Application of affinity chromatography to the quantitative evaluation of specific complexes Association processes undoubtedly play one of the most important roles in biochemical processes. The initial degree of bonding to a substrate has an important effect on the catalytic properties of an enzyme. Control processes in biology depend on the association of the repressor with the operon, hormones with specific receptors, etc. The formation of specific complexes of nucleic acid chains or of antibodies with antigens plays an important role. Methods in which the ligand-protein interaction is used for direct analysis, such as equilibrium dialysis, ultrafiltration, gel filtration, spectroscopic methods and steady-state kinetic analysis, have now been supplemented by a new technique for the determination of dissociation constants based on affinity chromatography (Andrews et al.; Brinkworth et al.; Chaiken and Taylor; Dunn and Chaiken, 1974,1975; Gawronski and Wold; Kasai and Ishii; Lowe et al.; Nichol et al.). The application of affinity chromatography to the study of interaction forces in biological complexes seems very promising. However, so far few papers describing quantitative determinations of interactions by affinity chromatography have been published and the results do not permit any general conclusions to be drawn. Therefore, the determination of dissociation constants of nuclease with its inhibitors by elution analysis will be described in detail here, together with a description of the application of this method to the determination of the dissociation constants of chymotrypsin complexes with synthetic low-molecular-weightas well as natural high-molecular-weightinhibitors (Section 4.1). Further, the determination of the dissociation constants of trypsin complexes with lowmolecular-weight inhibitors by frontal analysis (Section 4.2) is also considered. Section 4.3 discusses the co-operative elution of oligoadenylic acid when it is chromatographed on immobilized polyuridylic acid. In the last part of this chapter the direct titration of ribonuclease-S-protein with S-peptide is described, together with the determination of the bond strength between dehydrogenases and nucleotides based on the determination of the concentration of potassium chloride solution necessary for their elution, and the determination of the interaction of nucleotides bound with peptides on the basis of relative elution ratios.

4.1 DETERMINATION OF DISSOCIATION CONSTANTS BY ELUTION ANALYSIS The most commonly used method of quantitative affinity chromatography is based on the elution of a biological macromolecular substance from an affinity matrix with soluble affinant solutions of various concentrations (Dunn and Chaiken, 1975). The elution volume of a macromolecular substance is directly dependent on the concentration of the affinity ligand bound to a solid support if the concentration of the soluble affinity ligand is constant. Further, it is indirectly proportional to the concentration of the soluble affinant if the concentration of the immobilized affinant is constant. These dependences

QUANTITATIVE EVALUATION O F SPECIFIC COMPLEXES

36

can be expressed by the equation

where V = elution volume; Vo = volume at which the macromolecular substance is eluted if no interaction with the immobilized ligand takes place (for example, with an enzyme in the presence of a strong inhibitor); V , = void volume, determined, for example, by means of blue dextran elution; [L] = concentration of the immobilized ligand, determined on the basis of the operating capacity of the sorbent; K L = dissociation constant for the interaction of the macromolecular substance with the immobilized ligand; [I] = concentration of the soluble affinity ligand; and K I = dissociation constant for the soluble binary complex.

I

001

FRACTION

NUMBER

T 10

1

2

3

4

5

C LlxlOs,M

Fig. 4.1. (A) Composite plot of chromatography of nuclease on thymidine 3'-@-Sepharose-aminophenyl phosphate) S'-phosphate (pdTpAP-Sepharose) with various bound tigand concentrations ([L]). The affinity matrix was mixed with underivatized Sepharose 4B in the following proportions of millilitres of pdTpAP-Sepharose to millilitres of Sepharose 4B: 5.0 (0),2.5 : 2.5 (o),2 : 4 (O),1.5 : 4.5 (A), 1 :4 (o),0.5 :4.5 (=I. The mixtures were then used to prepare 5-ml columns of packed gel equilibrated with 5 M thymidine 3',5'-biphosphate (pdTp); 200-pg samples of nuclease were applied and eluted with 0.1 Mammonium acetate buffer containing 5 . 1 F 6 M pdTp. ~A,,,*min/ml is the activity in the hydrolysis of salmon sperm DNA. (B) Plot of elution volume versus concentration of immobilized affinity ligand. Reproduced with permission from B.M. Dunn and I.M. Chaiken, Biochemistry, 14 (1975) 2343-2349.

-

31

ELUTION ANALYSIS

The validity of this equation was checked by Dunn and Chaiken (1975) by examining the chromatography of staphylococcal nuclease on thymidine 3’+-Sepharose-aminophenyl phosphate) 5’-phosphate (abbreviation: pdTpAP-Sepharose) as a function of the concentration of both the immobilized nucleotide and the soluble nucleotide used for the elution of nuclease. Fig. 4.1 shows the effect of the dilution of the affinity matrix with unsubstituted Sepharose. The same amount of nuclease is always eluted with a solution of the inhibitor thymidine 3 I , 5‘-bisphosphate (pdTp) from the columns of pdTpAP-Sepharoses differing by the concentration of the immobilized thymidine 3’(p-aminophenyl phosphate) 5’-phosphate (pdTpAP) only. The elution volume of nuclease is directly proportional to the concentration of the immobilized nucleotide (Fig. 4.1 B). The determined dependence is in agreement with eqn. 4.1. Extrapolation of the plot of V versus [L] to [L] = 0 gives the value of V,. The value of V, obtained from Fig. 4.1 B was in excellent agreement with the value obtained experimentally by elution of phenol red from the same column, or by elution of nuclease from the same volume of unmodified Sepharose. On the basis of eqn. 4.1, the slope of the curve in Fig. 4.1B is given by the relationship

Hence, when one of the dissociation constants ( K L or KI) and the slope are known, the other constant can be calculated. Fig. 4.2 itlustrates the dependence of the elution volume of nuclease on the concentration of the soluble nucleotide. The same amount of nuclease was always introduced into columns of pdTpAP-Sepharose equilibrated with pdTpAP solutions of various concentrations, and eluted with a solution of pdTpAF’ of adequate concentration (Fig.4.2B). With decreasing concentration of the inhibitor, [I], the protein peak becomes broader when the retention increases. Eqn. 4.1 can be transformed into 1

-

1

+

[I1

(4.3)

In Fig.4.2B, -is plotted against [I], the latter being obtained on the basis of

v- v,

elution volumes from Fig. 4.2A: Slope = l/K1A

(4.4)

Intercept = 1/A

(4.5)

where A = ( VO- Vm) ( [L]/KL). From eqns. 4.4 and 4.5, it follows that the ratio intercept/slope is equal to KI. The expression for the intercept on the -axis (Fig.4.2B) contains KL and, if [L], Vo

v- v*

and Vm are known, K L can be calculated from that expression. In Table 4.1 the values for

QUANTITATIVE EVALUATION OF SPECIFIC COMPLEXES

38

KIand KL obtained by the method described are given in comparison with the values of KI obtained from kinetic measurements or from equilibrium dialysis. On the basis of the agreement between the given constants, it can be concluded that in this system the binding of nuclease on the affinity matrix is completely reversible and that the immobilization of the ligand does not limit the complex formation. On the basis of the determination of phosphate in the acid hydrolysate of pdTpAPM. Sepharose, the amount of the bound pdTpAP-Sepharose was determined as 2.1 However, when dissociation constants were calculated, the concentration of pdTpAP M.From which was determined from the operating capacity was used, which gave 5

FRACTION

I

I

1

1

2

NUMBER

, 3

4

5

CpdTpAPl x 10’,M

Fig. 4.2. (A) Composite plot of chromatography of nuclease on thymidine 3’-@-Sepharose-aminophenyl phosphate) 5‘-phosphate (pdTpAP-Sepharose) with various concentrations of soluble thymidine 3‘-(p-aminophenyl phosphate) S’-phosphate (pdTpAP) in the eluting buffer; 200-pg samples of nuclease were applied to columns equilibrated with 0.5 *1U-’ M (o), 0.75 *lU-’ M (o), 1.0 .lo-’ M (A), 2 .lo-’ M fe), 3-10-’ M(D) and 4.0*10-’ M(D) pdTpAP each in 0.1 Mammonium acetate solution, and eluted with these buffers. (B) Plot of 1/( V- V,,) versus [pdTpAP] according to eqn. 4.3, correlation coefficient r = 0.997. For the sake of clarity, the result obtained for (pdTpAP] = 5 .lo-‘ M is included in Fig. 4.2(B) but the elution profde is omitted from Fig. 4.2A. Reproduced with permission from B.M. Dunn and I.M. Chaiken, Biochemistry, 14 (1975) 2343-2349.

39

ELUTION ANALYSIS

TABLE 4.1 DISSOCIATION CONSTANTS FOR INTERACTION OF AFFINITY LIGAND WITH STAPHYLOCOCCAL NUCLEASE (pH 7.5, IN THE PRESENCE OF 10 mM CaCl,) Affinity ligand

Thymidine 3'-(paminophenyl phosphate) S'-monophosphate Thymidine 3',5'-bisphosphate Thymidine 5 'monophosphate Thymidine 3'-phosphate 5'-(p-nitrophenyl phosphate) Thymidine 3'-@-nitrophenyl phosphate) S'-phosphate Thymidine 3'-(p-nitrophenyl phosphate) None

KI (M) Chromatography*

Kinetics**

KL (M)***

2.3

2.5 .

1.1.10-6

2.5-10-' g 1.6*10-5 1.1 .lo-'

5.9-10-6 2.8 . l o - 5 6.3.10-6

1.0*10-6 o.9*10-6 0.6.10-6

4.1

3.5

4.3 .lo-'

''

2.6. 1.5 *lo-6 1.2.10-6

*Results (+ 30%) from competitive elution experiments. ** are KI values (+ 30%) determined from Dixon plots. ***Values Dissociation constant (t 50%) for binding of nuclease to thymidine 3'-(pSepharoseaminopheny1 hosphate) 5 '-phosphate. !value (-+ 25%) derived from equilibrium dialysis. IK, value determined for enzymatic release of p-nitrophenyl phosphate.

a comparison of these values it follows that only 24% of molecules of the insolubilized ligand are capable of binding nuclease. KL values were obtained independently by elution with six different affinity ligands and their values varied within the range 0.6-2.6010-~ M. The scatter in KL seems to be independent of the nature of the substituents bound to thymidine. The agreement of the KL values (within experimental error) shows that the nuclease-affinity matrix interaction is independent of the presence of another ligand. In contrast to the KL values, the values of KI are strongly dependent on the substitution of the ligand. Virtually identical KI values for pdTp and pdTpAP indicate that the aminophenyl group of the spacer arm has no effect on the interaction of nuclease with the immobilized nucleotide. Similarly, a comparison of KI (2.3010-~M) and KL (1.1 *loT6M) for pdTpAP shows that the binding of the nucleotide on a solid support does not affect the interaction of nuclease with pdTpAP. Hence, the same forces are operative in the binding both in solution and after the binding of the nucleotide on a solid support. Hence, this method serves not only for the determination of dissociation constants but also for the evaluation of the effect of immobilization on complex formation. Objections have been expressed by Nichol ef ~ lto. the equations derived by Dunn and Chaiken (1974). DUM and Chaiken (1975) restricted their validity to cases when [E]/KL is small, in which event a very low concentration of enzyme has to be used. A further restriction concerns the interactions with too low KL values. If the binding constant is M, for example, then the calculated rate constant for the dissociation of the complex EL will be very low, viz., about 0.042/sec (Dunn and Chaiken, 1975). The elution of the

QUANTITATIVE EVALUATION OF SPECIFIC COMPLEXES

40

protein will then be affected on the basis of the kinetic effect, and it will be unattainable in an experimentally feasible time. The addition of the dissolved ligand to the eluting solvent will not affect the elution of the protein because the dissociation is unimolecular. These kinetic effects explain the occasionally observed failure of some enzymes to be eluted from affinity sorbents with buffers that contain strong inhibitors. In some instances the protein peak is so broad that it is undetectable. The method is suitable mainly for cases when sorbents are avadable that contain ligands with a medium bonding

0.4

0

0

20

10

30

ELUTION VOLUME,ml

ot 0

I

I (12

1

I

I

0.4

I 0.6

I A N T I LY SlNE]x 10.',M

Fig. 4.3. (A). Affinity chromatography of chymotrypsin on N-benzyloxycarbonyl-glycyl-D-phenylalanyl-NH,-Spheron 300 (52 X 11 mm), eluted with solutions of various concentrations of antilysine. A 3.0-mgamount of chyrnotrypsin was applied in each run on a column equilibrated with antilysine solution of a corresponding concentration. The activity of the desoibed chymotrypsin was measured on an Opton recording spectrophotometer using N-benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester (.4.,,) as substrate. Concentration of antilysine in the eluent (when 0.05 MTris-HC1 buffer of pH 8.0 at 25°C was used): (0) 2.64.10-' M ( o ) 1.28.10-4M;(@ 0.66*10'4M;(e)0.33.10-4M, (0)0.13 .lo-" M ; (0) 0.07 .loa4M. (B) Plot of l / ( V - V,) versus molar concentration of antilysine.

FRONTAL ANALYSIS

41

strength. In such cases the system is completely reversible within the time period of the chromatographic experiment. In order to elucidate more completely the elution curves for enzymes, obtained on elution with solutions of high molecular weight inhibitors, Turkovil et al. applied the method of Dunn and Chaiken (1975) to the determination of the dissociation constant of chymotrypsin with antilysine. Antilysine is a polyvalent inhibitor of proteases isolated from lungs by the company LCEiva (Prague, Czechoslovakia), and is closely related to the pancreatic trypsin inhibitor. The sorption of chymotrypsin on to N-benzyloxycarbonylglycyl-D-phenylalanine-NH2-Spheron was carried out on the basis of the optimal conditions, taken from Fig. 3.1. Solutions of antilysine of various concentrations were used for elution. The concentration of the immobilized affinity ligand, [L], was determined on the basis of the working capacity of the column as 2.6*10-’ M. Vm was determined with Dextran 2000 as 2.1 ml. The dissociation constant of chymotrypsin-immobilized inhibitor complex, K L , was 4.5 M, and the dissociation constant of the chymotrypsin complex with antilysine, Kr, was 8.8*10-6 M (Fig.4.3A,B). The affinity chromatography of chymotrypsin was further carried out on Spheron with bonded antilysine. On the basis of the determined Vm = 4.0 ml and the concentration of the immobilized affinant [L] = 1.8*10-’ M ,the dissociation constant of the complex of chymotrypsin with bonded antilysine was calculated to be K L = 9.0*10-6 M and the dissociation constant with M. From the agreement of the KL and KI values it can soluble antilysine K I = 8 9 be concluded that the formation of the complex between chymotrypsin and antilysine is not affected by its covalent bond with Spheron. The dissociation constants for the complex of chymotrypsin and antilysine, either in solution or bound to a solid matrix, are in good agreement. Further, they also agree with the dissociation constant for the antilysine with chymotrypsin complex obtained by kinetic determination according to Dixon (9.8 - 1 Od6 M). From the same kinetic measurement using N-benzyloxycarbonyl-Ltyrosine-p-nitrophenyl ester, the Michaelis constant was determined as K,,, = 3.2-10-5 M, which agrees closely with the published value of 3 lo-’ M (Walsh and Wilcox).

4.2 DETERMINATION OF DISSOCIATION CONSTANTS BY FRONTAL ANALYSIS

The derivation of quantitative affinity chromatography by frontal analysis (Kasai and Ishii) is based on the assumption that if the total length of the agarose column is 1 and the total amount of the immobilized ligand in the adsorbent is Lt, then Lo = Lt/l, where [Lo] is the density of affinity ligands per unit length of the agarose column. When an affinity adsorbent with a weak affinity is used, enzyme solution of concentration [Eo] is applied on the column continuously, and the enzyme will be eluted from the column with a volume V . Then the following equation can be written: ( V - Vo) Wol = 1 [ELI

(4-6)

where V, is the elution volume if n o interaction takes place between the active site of the enzyme and the immobilized affinity ligand (for example, in the presence of a soluble, strong competitive inhibitor) and [EL] is the density of the enzyme-immobilized ligand

QUANTITATIVE EVALUATION OF SPECIFIC COMPLEXES

42

complex per unit length of the agarose column. During the complex formation, equilibrium is attained:

k+l EtL-EL k-1 If the concentration of free enzyme [El = [EO],the following equation can be written:

K L = -k-1 = k+i

[El [ L l f ~--

B'ol(W01

[ELlfEL

-

PLl1.f~

LEL1fEL

where KL is the dissociation constant of the enzyme-immobilized ligand complex, [L] is the density of the unoccupied ligand per unit length and f is the activity coefficient of the reactant, the mobility of which is restricted. If chromatography takes place under the conditions when [EO]<
KL = W L / ( ~I/O)~EL (4.8) If the enzyme is eluted with volumes Vl and V2 under two different conditions, we can write

where K L ( ~ and ) K L ( ~are ) the corresponding dissociation constants. Hence, eqn. 4.9 is also applicable to a comparison of different enzymes. If the enzyme is eluted in the presence of a competitive inhibitor of concentration [Zo] (where [Zo] >> [Eel) by a volume Vi, then the following equation can be deduced: (4.10) [ ~ o I / K I= (v- Vo)/(Vi- VO) where KI is the dissociation constant of the enzyme-soluble inhibitor complex, defined by the relationship KL(I)/KL = 1 +

(4.1 1) KL(I) is defined as an apparent dissociation constant of the enzyme-immobilized ligand

complex, in the presence of soluble inhibitor: KL(1) =

(IE]

[Ell) fLlfL/[ELlfEL

(4.12)

Eqn. 4.10 can be re-written as

(v- vi)/(v- VO) =

1/(1 +KI/[I,I)

(4.1 3)

It follows that ( V - Vi)/(V - Vo) is equal t o the ratio of the enzyme-soluble inhibitor complex concentration to the total enzyme concentration. The concentration of the

FRONTAL ANALYSIS

43

inhibitor at which ( V - Vj)/(V - Vo) = 0.5 gives the values for KI.Eqn. 4.13 can be written as Vi =

v,t KI( v- vj)/ [lo]

(4.14)

If Vi is plotted against ( V - Vi)/[lo], then KIand Vo can be calculated from the slope and the intercept on the ordinate. If Vo and Vare known, then the value of KI for any competitive inhibitor can be obtained from a single chromatographic run in the presence of the corresponding inhibitor. The chromatography of bovine trypsin on Sepharose with covalently bonded glycylglycyl-L-arginine (2.2 pmole of peptide per millilitre of Sepharose) is illustrated in Fig. 4.4A.On the affinity sorbent, equilibrated with the given concentrations of inhibitor of benzyloxycarbonylarginine, a solution of trypsin was always introduced, together with an inhibitor of corresponding concentration. From the plot of Vi versus ( V - Vj)/[lo](Fig. 4.4B),a straight line was obtained, confirming the validity of the equation derived. From

NUMBER

FRACTION

-

E >-

10

Fig. 4.4. (A) Chromatography of bovine trypsin on glycylglycyl-L-arginie-Sepharose (180 X 6 mm, 5.1 ml). A column was equilibrated with 0.1 M Tris-acid maleate-NaOH buffer, pH 6.2, containing the indicated concentration (mM) of benzyloxycarbonylarginine. Bovine ptrypsin solution in the same buffer (about 10 figglml, 5 .lo-' M) was applied continuously. The flow-rate was 3 ml/h and fractions of 1.1 ml were collected. Chromatography was carried out in a cold room (4°C). Elution of trypsin was monitored by measuring enzymatic activity using benzoyl-DL-arghine pnitroanilide as substrate. The elution volume was determined as the volume in which the enzyme concentration reached half that of the plateau. (B) Plot of Vj against ( V - Vi)/I,. Reproduced with permission from K. Kasai and S. Ishii, J. Biochem (Tokyo),77 (1975) 261-264.

44

QUANTITATIVE EVALUATION OF SPECIFIC COMPLEXES

the slope and the intercept on the ordinate, the values K1 = 0.29 mM and VO= 11.6 ml were obtained. The KI value for benzyloxycarbonylarginine, determined under the same conditions in the solution from the inhibition of the enzymatic hydrolysis of benzoyl-Larginine p-nitroanilide with trypsin according to Dixon, was 0.1 7 mM. The slight difference may be caused by using conditions that are optimal for sorption, but less suitable for kinetic analysis. Using the method described, Kasai and Ishii determined KI for various inhibitors. For a- and 0-trypsin and Streptomyces gri’seus trypsin they have determined KL using glycylglycyl-L-arginine bound to Sepharose. With @-trypsinthey also determined the effect of various concentrations of salts on the KL values. As nothing is known about the activity all fvalues were taken as unity. coefficient 0, An advantage of the method of Kasai and Ishii is its sensitivity and simplicity: it requires only a small amount of the protein being investigated; in comparison with the method of Dunn and Chaiken (1975), the enzyme concentration is negligibly low in comparison with the KL value; the use of frontal analysis simplifies the equation deduced; and the elution volumes can be determined more accurately because their dependence on concentration is negligible. When using this method for the determination of the dissociation constants of chymotrypsin on Spheron on which N-benzyloxycarbonylglycyl-Dphenylalanine was bound by means of hydrophobic hexamethylenediamine, Turkova et al. met with difficulties stemming, evidently, from non-specific sorption. The K j values of trypsin determined by this method on a Spheron column to which p-aminobenzamidine was coupled through hexamethylenediamine were as follows: Bz-L-arginine, 5.5 M; n-butylamine, 3.7-10-3 M ;benzylamine, 5.0010-~ M; benzamidine 5.3*10-’ M, and p-aminobenzamidine, 1.9*10-’ M. The Ki values of trypsin determined by the method of Dunn and Chaiken (1975) on the same column were 5.3010-~M for Bz-L-arginine, 1.7-10-3 M for n-butylamine, 3.1 M for benzylamine, 6.7*10-’ M for benzamidine and 1.9*10-’ M for p-aminobenzamidine. These values were in good agreement with those obtained by kinetic measurement. The KL value determined for immobilized p-aminobenzamidine was 3.7*10-6 M by the method of Kasai and Ishii and 1.9*10-6 M by the method of Dunn and Chaiken (1975). When I/( Vi - Vo)was plotted versus 1/Ki (determined by the method of Durn and Chalken, 1975) or Vj versus K j ( V - Vi) (determined by the method of Kasai and Ishii) for various inhibitors of identical concentration, linear profiles were obtained. Therefore, if a column of immobilized inhibitor is available and the slope of the curve has been determined from Kj values of known inhibitors, the Kj values of unknown inhibitors can be read from the diagram after their elution volumes have been measured (Turkova e t d.).

4.3 COOPERATIVE ELUTION OF OLIGOADENYLIC ACID IN IMMOBILIZED POLYURIDYLIC ACID CHROMATOGRAPHY* (Okada et al.) The analysis of the elution profile of a specifically sorbed macromolecule from an affinity sorbent is a potential analytical method for the study of the specific interactions *For list of symbols see p.46.

IMMOBILIZED POLYURIDY LIC ACID CHROMATOGRAPHY

45

of biopolymers. The elution profile is closely related to the bonding isotherm of the eluted substance and the immobilized affinant and, consequently, the thermodynamic parameters of molecular interactions can be deduced from the chromatogram. The method is equivalent to repeated dialysis and has many advantages in comparison with normal equilibrium dialysis. For example, it operates at much lower concentrations and with small amounts of sample, several molecular species can be applied at the same time and it is possible t o detect small differences in bonding abilities at a high resolving power. The method of analysis of elution profiles was applied in the column chromatography of oligoadenylic acid on agarose with bonded polyuridylic acid at several temperatures and concentrations of oligoadenylic acid. The theory of the cooperative bonding of oligonucleotide with polynucleotide has been extended so that this chromatographic system is included in the theoretical plates theory, as mentioned in Section 3.2. Hence the binding of oligoadenylic acid to polyuridylic acid takes place in a cooperative manner and the stability of the complex depends on the temperature and the concentration of the oligoadenylic acid. Therefore, the elution profiles of oligoadenylic acid chromatographed on agarose with immobilized polyuridylic acid depend on the concentration of oligoadenylic acid and on temperature, as is evident from Figs. 3.9 and 3.10. Several thermodynamic parameters have been derived from the experimental binding isotherm. (xs2)-' was determined from the free oligomer concentration at the mid-point of the binding isotherm, and (442du)-' was determined from the slope of the binding isotherm at the mid-point. These procedures are justified when o << 1. The self-consistent value u = 0.017 was obtained. Under experimental conditions, the complex of polyuridylic acid with oligoadenylic acid is in a triple-stranded helical state when in solution. Therefore, when the binding isotherm is calculated, the loop entropy of two strands of polyuridylic acid was taken into consideration. When using the polylogarithm Li&) from

-

c = [ x s N p N - ' ( p - bA)] c =

-'

(4.15) p-bA

c+cu P -k b "P/(1

-dP>i- 21og(l -dd -PI

i-

(N - 1) (P - bA)

(4.16)

where

A = 410g(l - 4 p )

-k

4Li2(dp)

i-

p

In the case when p is taken as a parameter, the binding isotherm can be obtained. If the excluded volume effect is taken into consideration, the parameter a can be determined as 2, which appears in a factor due to loops in the partition function, 2n~b(j+l)/(j+2)a. Taking the established value of the cooperative parameter b (0.1-0.001), the binding isotherm and the column elution profiles were simulated, but rectangular-type elution profiles were obtained because the binding isotherm tends to zero abruptly at some oligomer concentration, so that it reflects the loop entropy effect of two polymer chains. However, experimental elution profiles were not rectangular (Fig. 3.10) and the experimental binding isotherm did not tend to zero abruptly (Fig. 3.8). This inconsistency can be interpreted as follows. As the ratio of cu to c is 102-103 and the amount of diphosphate in polyuridylic acid is 102-103, the loop formation of poly-

QUANTITATIVE EVALUATION OF SPECIFIC COMPLEXES

46

uridylic acid with oligoadenylic acid as a clasp can be neglected (although it cannot be neglected for infinitely long polymers). Hence loops can be neglected in double- or triplestranded helices. Under our experimental conditions, polyuridylic acid forms at least a partial double helix. Therefore, the above-mentioned treatment, which is justified for the formation of a double helix, is also justified for the formation of a triple helix as cooperative adsorption on a linear chain. The chemical loosening of the binding of oligomer proceeds for about 10 msec, which is sufficiently rapid in comparison with the diffusion time into the gel, with a flow-rate 5-40 ml/h. The assumption of a chemical equilibration is thus substantiated. Although the flow-rate (20 ml/h) is not sufficiently slow in comparison with the diffusion process, the elution profiles do not change even after an eight-fold change in the flow-rate. Hence, the number of plates seems sufficiently large and the given analytical treatment is justified. On the basis of eqn. 3.46, derived in Section 3.2, thermodynamic parameters can be determined from the trajectory of the experimental peak. The intersection of the trajectory over the V, axis gives the partition function of the isolated bound oligomer: Ve(0) - vt

vt

= qs2qcu

(4.17)

and the slope at that point gives the partition function: (4.18)

Following this procedure with the values q = 0.55 and c, = 0.98010-~M, xs2 = 3.4*104/M +- 50% is obtained for 1 M sodium chloride solution at 10°C and xs2 = 8-10"/Mk 5% for 1 M sodium chloride solution at 5°C. Unfortunately, F, giving a good signal-to-noise ratio in an elution profile, is not small enough to permit a two-term approximation. As a result, the peak trajectory is concave and these two values cannot be determined precisely. However, these values agree roughly with the values obtained from the binding isotherm. 4.3.1 List of symbols used

Degree of polymerization of oligoadenylic acid; Concentration of total and free binding sites on the polyuridylic acid chain, respectively; Concentration of total and free oligoadenylic acid, respectively; Helical content of polyuridylic acid; Adsorption coefficient of oligomer to polymer as a function of total oligomer concentration; Partition function of a nucleotide pair in the bound state when the partition function in the free state is taken as unity; Partition function of a bound oligomer having an adjacent bound oligomer and its complement; Correction factor for the end-effect of the oligomer; Inter-oligomer cooperativity parameter = (partition function of an isolated bound oligomer and its complement)/sl.

OTHER IMMOBILIZED AFFINITY LIGANDS

47

4.4 OTHER METHODS FOR THE QUANTITATIVE EVALUATION OF INTERACTIONS WITH IMMOBILIZED AFFINITY LIGANDS The possibility of obtaining quantitative binding data for the interaction of proteins with immobilized peptide was investigated for the first time by Gawronski and Wold. M by direct titration of They determined the dissociation constant to be 2.5 k l.0-10-6 ribonuclease S-protein bound to S-peptide immobilized on agarose. Good titration curves were obtained if the concentration of S-protein was in the concentration range 10-7-10-5 M. As the concentration of the bound protein is equal to the concentration of the proteinpeptide complex, the interaction can be described by the relationship 1

-

[bound S-protein]

1

k=d

[free S-protein]

[total S-peptide]

t

1

[total S-peptide]

The plot of [bound S-protein]-’ versus [free S-protein]-’ ,illustrated in Fig. 4.5, shows a linear relationship. The intercepts on the abscissa and ordinate represent KL-’ and [total S-peptide] respectively. Of course, the validity of this deduction is limited to systems in which it is certain that no non-specific sorption of the protein being investigated takes place.

-’,

Kd 6-

0

=

20 x 10-a

When comparing the binding of various dehydrogenases and kinases on N6-(6-aminohexyl)-5’-AMP-Sepharose and P’-(6-aminohexyl)-Pz-(5’-adenosine)pyrophosphateSepharose, Harvey et al. expressed the strength of the interaction between the enzyme and the immobilized nucleotide by the so-called “binding” (fl). This term represents the concentration of potassium chloride (mM) in the centre of the enzyme peak when the enzymes are eluted with a linear potassium chloride gradient (Fig. 4.6 and Table 4.2). For the determination of the dissociation constant of the complex of lactate dehydrogenase

QUANTITATIVE EVALUATION OF SPECIFIC COMPLEXES

48

L

ELUTION W)LUME.ml

Fig. 4.6. Chromatography of a crude yeast extract on N6-(6-aminohexyl)-5’-AMP-Sepharose. A sample (100 pl) of a crude yeast extract was applied to a column (50 X 5 mm) containing 0.5 g of N6-(6-aminohexy1)-5’-AMP-Sepharose equilibrated with 10 mM KH,PO,-KOH buffer, pH 7.5. After washing through non-adsorbed proteins, enzymes were eluted with a linear salt gradient (0-1 MKC1; 20 ml total volume) at a flow-rate of 8 ml.h-’. Inert protein (-), glucose 6-phosphate dehydrogenase (O), glutathione reductase (O), malate dehydrogenase (0) and yeast alcohol dehydrogenase (A) were assayed in the effluent. Reproduced with permission from D.B.Craven e f ul., Eur. J. Biochem., 41 (1974) 329-333.

with N6-(6-aminohexyl)-5’-AMP bound to Sepharose, Lowe et al. used the linear dependence between the bound enzyme and the immobilized nucleotide concentration. The chromatography of peptides on poly(viny1 alcohol) substituted with oligodeoxythymidyiic acid and bound irreversibly on DEAE-cellulose by ionic bonds has been employed for the study of interactions of peptides with nucleotides (Schott et al.). For this type of chromatography, the term “template chromatography” has been introduced. The quantitative measure of the peptide-nucleotide interaction is the increase in the retention of a peptide on oligonucleotide-DEAE-cellulose in comparison with that on unmodified DEAE-cellulose. In order to eliminate possible effects of various column parameters, Schott et 02. expressed all elution volumes relative to the elution volume of alanine, which displays no measurable retention on these columns. The relative elution ratio (V,) is thus obtained as the ratio of the elution volume found for the investigated peptide to that for alanine (V, = Vobs/VAla). The peptide-oligonucleotide interaction is then evaluated on the basis of the difference in the relative elution volumes (AVr) obtained by chromatography on both columns. The determination of dissociation constants of the binary complex of dehydrogenases and NADH on the basis of the NADH concentration necessary for elution has already been discussed in Chapter 2 (see Fig. 2.3):

REFERENCES

49

TABLE 4.2 COMPARISON OF THE BINDING OF VARIOUS ENZYMES TO N6-(5-AMINOHEXYL)-5'-AMPSEPHAROSE AND Pi -(6-AMINOHEXYL~-Pf-15'-ADENOSINEbPYROPHOSPHATE-SEPHAROSE Enzyme

Source

Code number

Name

E.C. 1.1.1.27

Lactate dehydrogenase Lactate dehydrogenase Glucose 6-phosphate dehy drogenase Malate dehydrogenase Alcohol dehydrogenase pGlyceraldehyde 3-phosphate dehy drogenase 3-Phosphoglycerate kinase Pyruvate kinase Hexokinase Creatine kinase Myokinase Glycerokinase

E.C. 1.1.1.49 E.C. 1.1.1.37 E.C. 1.1.1.1 E.C. 1.2.1.12 E.C. E.C. E.C. E.C. E.C. E.C.

2.7.2.3 2.7.1.40 2.7.1.1 2.7.3.2 2.7.4.3 2.7.1.30

Binding (p)* I

I1

Pig heart Rabbit muscle Yeast

>1000** >1000** 0

>1000** >1000** 170

Pig heart Yeast Rabbit muscle

65 400 0

490 0 >1000**

Yeast Rabbit muscle Yeast Rabbit muscle Rabbit muscle Candida mycoderma

70 100 0 0 0 122

260 110 0 0 380 0

*Binding (p) refers to a measure of the strength of the enzymeimmobilized nucleotide interaction and is the KCI concentration (mM) at the centre of the enzyme peak when the enzyme is eluted with a linear gradient of KCl. 5 U enzyme applied to a column (50 X 5 mm) containing 1 g of the affinity adsorbent. (I) N6-(6-Aminohexyl)-5'-AMP-Sepharose(1.5 pmole/ml of AMP); (11) P' -(6-aminohexyl)-Pz-(5'aden0sine)pyrophosphate-Sepharose (6.0 pmole/ml of AMP). **Elution was effected by a 200-4 pulse of 5 mMNADH.

REFERENCES Andrews, P., Kitchen, B.J. and Winzor, D.J., Biochem. J., 135 (1973) 897-900. Brinkworth, R.I., Masters, C.J. and Winzor, D.J., Biochem J., 151 (1975) 631-636. Chaiken, I.M. and Taylor, H.C., J. Biol. Chem., 251 (1976) 2044-2048. Craven, D.B., Harvey, M.J., Lowe, C.R. and Dean, P.D.G.,Eur. J. Biochem., 41 (1974) 329-333. Dixon, M.,Biochern. J., 55 (1953) 170-171. Dunn, B.M. and Chaiken, I.M.,Proc. Nat. Acad. Sci. US.,71 (1974) 2382-2385. Dunn, B.M. and Chaiken, I.M., Biochemistry, 14 (1975) 2343-2349. Gawronski, T.H. and Wold, F.,Biochemisfry, 11 (1972) 442-448. Harvey, M.J., Lowe, C.R., Craven, D.B. and Dean, P.D.G., Eur. J. Biochem., 41 (1974) 335-340. Kasai, K. and Ishii, S., J. Biochem. [Tokyo), 7 1 (1975) 261-264. Lowe, C.R., Harvey, M.J. and Dean, P.D.G., Eur. J. Biochem., 42 (1974) 1-6. Nichol, L.W., Ogston, A.G., Winzor, D.J. and Sawyer, W.H., Biochem. J., 143 (1974) 435-443. Okada, S., Husimi, Y., Tanabe, S. and Wada, A.,Biopolymers, 14 (1975) 33-49. Schott, H., Eckstein, H., Gatfield, I. and Fayer, E., Biochemistry, 14 (1975) 5541-5548. Turkovi, J., Strinski, M., Bliha, K. and Coupek, J., unpublished work. Walsh, K.A. and Wilcox, P.E.,Methods Enzymol., 19 (1970) 31-41.

This Page Intentionally Left Blank

51

Chapter 5

General considerations on affinant-sorbent bonding The bonding between the monomeric affinant, for example the inhibitor I, and the enzyme E is expressed by the equilibrium constant of the reaction, K I , on the supposition that the enzyme exists in a single tertiary conformation:

kl EtI-EI k2

When the affinant is bound to the solid support, the equilibrium constant, K I , is affected to a certain extent. An increase in K I brings about a modification of the affinant by binding to the matrix, and the steric accessibility of the affmant is limited as a consequence of this binding. On the other hand, a decrease in K I causes non-specific adsorption of the enzyme to the solid support and to the molecules of the already adsorbed enzyme. Assuming that a single enzyme of the crude protein has an affinity for the matrix, the equilibrium between the bound affinity ligand L and the isolated enzyme E is given by the equation

The successful isolation of an enzyme by affinity chromatography requires a very small K I or K L for the desired enzyme. Both constants should be much smaller than any dissociation constant for adsorption between the protein and the matrix surface (ie.,nonspecific adsorption). The maximum K L can be estimated as follows. Starting from a mole/l concentration of inhibitor in the insoluble affmant and the requirement of 99% retention of the enzyme from the raw material which contains about mole/l of mole/l is enzyme in a three-fold volume of the insoluble affmant, a K I value of obtained as the upper limit for an effective affinant. In a 3% protein solution, where the active enzyme constitutes 10%of the total protein, which should have a molecular weight of lo5,about 10%of the capacity of the matrix is utilized under the above conditions. From this estimate, it further follows that in view of the bond that can be formed between the inhibitor and the enzyme, the whole purification process should be considered to be precipitation rather than chromatography. This can also be shown by means of the adsorption isotherm for affinity chromatography, shown in Fig. 5.1, from which it is evident that the gross adsorption isotherm (curve 3) can be defined as the sum of the specific (curve 1) and non-specific (curve 2) adsorption isotherms. The specific

AFFINANT-SORBENT BONDING

52

E“’

ENZYME CONCENTRATION,rns’rnl

Fig. 5.1. Adsorption isotherm for affinity chromatography. Adsorption isotherms: 1 , specific; 2, nonspecific; 3, gross.

adsorption isotherm characterizes the ideal specific adsorption when the adsorption energy, AG, for all adsorbed particles is constant and relatively large. Adsorption ceases when all accessible “affinant” sites are occupied. The non-specific adsorption isotherm characterizes the adsorption of proteins on non-specific sites of the matrix and on already adsorbed protein. The AGL value is the sum of ACI and AGnon-sp., where AG,on.sp is the reaction energy of non-specific complexing and hindrance. Taking a mean value of lo-’ molell for K1,a value of about 7 kcal/mole is obtained for A G I . The adsorption energy for non-specific adsorption, AGnon-sp., results from the hydrophobic, hydrophilic or even ionic interactions and is comparable to the adsorption energy in normal chromatography. It depends very much on the nature of the solid carrier and the protein. AGnon-sp. should be as low as possible because it also includes the adsorption of molecules that form non-specific complexes with the affinant. There are, however, instances in which the crude protein contains two or more enzymes that display affinity for the bound affinant. If the equilibrium constant of the reaction mole/l, then only minute amounts of of the second enzyme, KI(II), is greater than the second enzyme will be retained together with the enzyme sought. If KI(I1) is less than or equal to molell, then a mixture of both enzymes will be adsorbed, even though the KI value of the desired enzyme may be much less than KI(II). This follows from the specific form of the adsorption isotherm for affinity chromatography, because the heat of adsorption is extremely high under chromatographic conditions. If KI(II) differs from K I by more than 50-100, a separation can still be achieved if differential elution is applied, for example, or if the isolation i s carried out by a batch process, using an amount of the insoluble affinant that corresponds exactly to the more intimately binding enzyme, or if chromatography during which equilibrium between [E(II)*I] and [E(I)-I] must be attained is very slow. The difference between AGI and ACL is given by the change in steric accessibility of the affinity ligand after its immobilization, by its modification due to its binding on the carrier, by the nature of the solid support, etc.

STERIC ACCESSIBILITY

53

5.1 STERIC ACCESSIBILITY

The basic requirement for successful affinity chromatography is that the formation of the complex of the macromolecular substance with the affinant covalently bound to the solid support should correspond to the formation of their complex in solution. This requires, above all, sufficient space, especially if we have to deal with the interaction of substances of high molecular weight. For this reason, high porosity is one of the most important requirements of solid supports. The sorption of Pgalactosidase on to sorbents through a prepared by binding the inhibitor of p-aminophenyl-P-D-thiogalactopyranoside hydrocarbon arm both to the polydextran gel Sepharose and to the polyacrylamide gel Bio-Gel P-300 (Steers ef al.) is an example. The contents of the bound inhibitor were almost identical in the two instances, but the isolation of the 0-galactosidase by affinity chromatography was successful only with Sepharose. In spite of a high concentration of inhibitor (50 pmole/ml), the enzyme was not retained on Bio-Gel P-300, possibly owing to the excessively large volume of the tetramer of 0-galactosidase (molecular weight 540,000; Craven et al.), which could not enter the Bio-Gel pores. On the other hand, when nuclease of molecular weight 17,000 (Cuatrecasas) was isolated from staphylococci, BioGel P-300 appeared to be a suitable support. A high degree of porosity of the solid support is also necessary for the isolation of substances with relatively weak affinity for the bound affinant (dissociation constant 2 The concentration of the bound affinant that is freely accessible to the isolated substance should be very high in this instance, in order to achieve a strong interaction which would retain physically the isolated substances migrating through the column. The effect of gel porosity on the accessibility of the immobilized affinity ligand necessary for complex formation with the complementary macromolecule is shown in Fig. 5.2. Lowe and Dean (1971) demonstrated the effect of the degree of porosity of the Sephadex matrix on the binding of lactate dehydrogenase and malate dehydrogenase in a mixture with serum albumin on immobilized NAD'. On a column of a highly crosslinked Sephadex G-25 with bonded NAD', both dehydrogenases as well as serum albumin appear in the hold-up volume of the columns, because the immobilized ligand is inaccessible to enzymes. The NAD+-Sephadex G-100 complex had sorbed malate dehydrogenase, while most of the lactate dehydrogenase of higher molecular weight passed through with the serum albumin. Both dehydrogenases were then sorbed on NAD+-Sephadex G-200. The force of interaction between the enzyme and the immobilized NAD' increases with increasing gel porosity, as is evident from the potassium chloride concentration necessary for the elution of enzymes when a linear potassium chloride gradient was used. The exclusion of dehydrogenases from affinity sorbents prepared from gels of various pore sizes was used by Lowe and Dean (197 1) as a micro-scale method for the rapid determination of apparent molecular weights by so-called affinity gel filtration. Another example of the substantial effect of gel porosity on the capacity of the affinity sorbent is the finding that the amount of sorbed trypsin on 6% and 4% agarose substituted with rn-aminobenzamidine

54

AFFINANT-SORBENT BONDING

is dependent on the concentration of trypsin (Nishikawa et d.).Assuming a modest binding constant, Nishikawa et at. deduced, on the basis of adsorption theory, a hypothetical system in which they plotted the amount of the enzyme-ligand complex formed, [EL], as a function of the concentration of enzymes in the sorption solution, [El, for three fixed values of the ligand concentration, Lo, as shown in Fig. 5.3A. By plotting [El on a logarithmic scale they obtained a sigmoidal pattern of binding. They made the following assumptions: (1) the ligand in the gel behaves in the same manner as a corresponding, freely dissolved, molecule; in fact the immobilization of the affinity ligand causes a loss of at least one degree of freedom in translation entropy; ( 2 ) the concentration of the gel Iigand, EL], closely approximates to the concentration measured per unit volume of gel; (3) the enzyme interacts freely with all accessible ligands, while the inaccessible ligands have no influence on the binding potential of the enzyme; and (4) the solid support has no effect on the enzyme-ligand bond, except for steric exclusion of some ligands. Fig. 5.3B shows that the amounts of the bound trypsin established experimentally

L2

-0.2

1.1

-0.1

)

-0

:-

0

m w. w

-

uz "

I

I

-

s

- = m u g s u

c n m a

t

ELUTION V O L U M E , mi

Fig. 5.2. Affinity gel filtration of synthetic mixtures of (A) lactate dehydrogenase (LDH), and (B) malate dehydrogenase (MDH), with bovine serum albumin (BSA) on NAD-Sephadex of various pore sizes. A 50-wl sample containing 1.85 units of LDH (or 0.335 units of MDH) and 0.8 mg of BSA was applied to a 20 X 5 mm column of the appropriate NAD-Sephadex equilibrated with 10 mM phosphate buffer, pH 7.5. Non-absorbed protein was washed off with the same buffer and the column was eluted with a 0-0.5 M KCl gradient in 10 mM phosphate buffer, pH 7.5; 20 ml total. LDH (a), MDH (0) and BSA (-1 were assayed in the effluent. Reproduced with permission from C.R. Lowe and P.D.G. Dean, FEBS Lett., 18 (1971) 31-34.

STERIC ACCESSIBILITY

55

IL$ 50mM

t -. >’ .-

3

’

D

--z I

g0.4B -

I

I

1

I

I I Ill1

I

I

I 1 1 1 1 1 )

I

I

I l l

E -3 .3

m

-5 $02

-

-

-

I

I

I

I

,

I

,

[ E N Z Y M E 1 ,M

Fig. 5.3. (A) Ideal enzyme binding plot; (B) trypsin binding to different gels. Reproduced with permission from A.H. Nishikawa et al., Advan. Exp. Med. Biol., 42 (1974) 33-42.

during the equilibrium binding studies are dependent on the concentration of the starting trypsin solution (in 0.05 M bicin, pH 8.15, and 0.25 M potassium chloride solution; 4 h at 4OC). From this figure it is evident that only the affinity adsorbent prepared from 4% agarose gel containing 19.2 pequix/ml of m-aminobenzamidine approaches ideal behaviour. This carrier had a substantially higher saturation capacity than the affinity adsorbent prepared from 6%agarose, containing 22.9 pequiv./ml of rn-aminobenzamidine; at higher concentrations of trypsin it was similar to a 6%gel containing 48.9 pequiv./ml. In Fig. 5.3B,the saturation capacity of a high ligand gel (Lo = 48.9 pequiv./ml) which was diluted with the same volume of non-modified 6% agarose, is also given. The resulting concentration of the ligand in the gel is thus Lo = 24.45 pequiv./ml. However, the binding curve is substantially lower than in 6% gel with Lo = 22.9 pequiv./ml. The properties of affinity sorbents diluted with unmodified gels are discussed in Section 5.3.

AFFINANT-SORBENT BONDING

56

Of course, there are instances where the gel porosity does not have any effect on the interaction of the macromolecules with an immobilized affmant, such as in systems with d high affinity or with extremely large macromolecules, where only affinity ligands immobilized on the surface of the beads take part in complex formation. An example is the affinity chromatography of polysomes, ribosomes, intact cells, organelles or membrane fractions. In these instances penetration in the bead pores can hardly be expected. However, for the attainment of good accessibility of the immobilized affinity ligands and the binding sites of biological macromolecules, even a high porosity of the solid support does not suffice. The chemical groups of the affmant, participating in the interaction with the macromolecular substance, must also be sufficiently remote from the surface of the solid matrix in order to avoid steric hindrance. The importance of the spacing of the low-molecular-weight inhibitor from the surface of the rigid matrix as regards the course of affinity chromatography was illustrated by Cuatrecasas et at. in one of the first successful applications of this method in the isolation of enzymes. Fig. 5.4 represents the affinity chromatography of a-chymotrypsin, both on Sepharose coupled with E-aminocaproyl-D-tryptophan methyl ester (A) and on Sepharose coupled with D-tryptophan methyl ester (B), in comparison with chromatography on unsubstituted Sepharose (C). In the first instance (A), the bonded inhibitor has a high

rn

-___--

20c

- - --

132)

3

ELUTION VOLUME rnl

Fig. 5.4. Affinity chromatography of a-chymotrypsin on inhibitor Sepharose columns. The columns (50 x 5 mm) were equilibrated and run with 0.05 MTris-hydrochloric acid buffer of pH 8.0. Each sample (2.5 mg) was applied in 0.5 ml of the same buffer. The columns were run at room temperature with a flow-rate about 40 ml/h and fractions containing 1 ml were collected. The arrows indicate a change of elution buffer (0.1 M acetic acid, pH 3.0). (A) Sepharose coupled with e-aminocaproyl-Dtryptophan methyl ester; (B) Sepharose coupled with D-tryptophan methyl ester; (C) unsubstituted Sepharose. The first peaks in A and B were devoid of enzyme activity. Reproduced with permission from P. Cuatrecasas et al., Proc. Nar. Acud. Sci. U.S., 61 (1968) 636-643.

STERIC ACCESSIBILITY

51

affinity for a-chymotrypsin and the enzyme can be released from the complex only by decreasing the pH of the eluting buffer. By using 0.1 M acetic acid of pH 3.0, the chymotrypsin fraction is eluted as a sharp peak and the volume of the eluted chymotrypsin does not depend on the volume of the sample applied to the column. In the second instance (B), the inhibitor coupled directly on Sepharose has a much lower affinity for the isolated a-chymotrypsin, owing to steric hindrance. In this instance a change of buffer is not necessary for enzyme elution and, as can be seen from the graph, the enzyme is eluted in a much larger volume closely after the inactive material. In order to verify that non-specific adsorption on the carrier did not take place under the given experimental conditions, the chromatography of a-chymotrypsin on an unsubstituted carrier was carried out (C). However, as was observed, the described chromatography of chymotrypsin on unsubstituted Sepharose does not provide sufficient proof of non-specific sorption. On the contrary, Hofstee demonstrated for Sepharose with bonded e-aminocaproyl-D-tryptophan methyl ester that it sorbs, for example, serum albumin or y-globulin completely nonspecifically. Thus, it was found that a series of substances, both enzymes and substances such as immunoglobulin, serum albumin and ovalbumin, contain hydrophobic regions on the surface of their molecules, by which they are capable of being bound to hydrophobic spacers, such as hexamethylenediamine or e-aminocaproic acid. The utilization of this phenomenon for the separation of a number of biological macromolecules gave rise to a new technique, the so-called hydrophobic (affinity) chromatography, which is dealt with in greater detail in Chapter 7. In view of the different structures of the substances isolated, no general rule exists on the minimum distance between the affinant and the surface of the solid support. However, the affinant should be located at such a distance from the carrier surface that the bond does not require the deformation of the isolated substance. The effect of the distance of the affinant 3'-(4-aminophenylphosphoryl)deoxythymidine-5'-phosphate from the solid support surface (both Sepharose 4B and Bio-Gel P-300) on the capacity of the gel in the chromatography of staphylococcal nuclease (Cuatrecasas) is shown in Table 5.1. In type A, the inhibitor is bound directly to the matrix, and in other types a chain of varying length is inserted between it and the carrier surface. Hipwell et al. carried out the chromatography of several dehydrogenases on N 6 - o aminoalkyl-AMP-Sepharose. From the concentration of potassium chloride necessary for the release of dehydrogenase from its complex with nucleotide, the strength of interaction [binding (p)] can be derived. The effect of the length of the spacer arm on the binding (p) of several dehydrogenases on N6-w-aminoalkyl-Ah4P-Sepharoseis evident from Fig. 5.5. The binding (0) of two isoenzymes of lactate dehydrogenases increases rapidly from n = 2 to n = 5 . When the spacer arm is further lengthened, the elution must be enhanced by a pulse produced by the addition of a small amount of NADH. The binding of malate dehydrogenase, D -glucose-6-phosphatedehydrogenase and D-glyceraldehyde-3-phosphate dehydrogenase is significantly weaker than that of lactate dehydrogenase. Nonetheless, in polymers where the number of CH2 groups k > 7, it does not seem that the binding of enzyme would change substantially. It is further evident from Fig. 5.5 that for lactate dehydrogenase at least four methylene groups are necessary in order to achieve binding

AFFINANT-SORBENT BONDING

58

TABLE 5.1 CAPACITY OF INSOLUBLE AFFINANTS PREPARED BY BINDING 3‘-(4-AMINOPHENYLPHOSPH0RYL)DEOXYTHYMIDINE-5‘-PHOSPHATE ON SEPHAROSE 4B AND BIOGEL P-300 DERIVATIVES IN THE AFFINITY CHROMATOGRAPHY OF STAPHYLOCOCCAL NUCLEASE Structure Capacity (mg of Type of nuclease per ml of gel) inhibitor bound on derivative of on matrix Sepharose BioGel 4B P-300

z iii

g04

2

0.6

8

2

8

3

I NUMBER OFCH, G R O U P S IN SPACER A R M

Fig. 5.5. Effect of spacer arm length on the binding of several dehydrogenases to N6-w-aminoalkyl-AMPSepharose. Columns of the modified gels (50 X 5 mm) were equilibrated at 4°C in 10 mMKH,PO,KOH at pH 7.5 containing 0.02% sodium azide. The enzyme-protein sample was run into a moist bed of each polymer and developed by washing with several bed volumes of equilibration buffer, a linear l of 5 mM NADH applied gradient of KCl(0 to 1.0 M; 20.0 ml total volume) followed by a 2 0 0 - ~ “pulse” to the column in the same way as the enzyme-protein mixture. The column flow-rate was maintained at 8.0-10.0 ml/h and 1.4-mlfractions were collected. Binding ((3) represents the concentration of KC1 (20°C) required to elute the enzyme. Lactate dehydrogenase-H, (4 U, m); lactate dehydrogenase-M, (4 U, 0); D-glucose-6-phosphate dehydrogenase (2 U, 0);malate dehydrogenase (4 U, 0);and Dglyceraldehyde-3-phosphate dehydrogenase (2.5 U, Reproduced with permission from M.C. Hipwell et al., FEBS Lett., 42 (1974) 355-359.

n.

STERIC ACCESSIBILITY

59

of enzyme on the immobilized nucleotide. It is considered that the use of an extension arm at least 0.5 nm in length enables the nucleotide to traverse the barrier imposed by the micro-environment associated with the hydrophilic polymer (Lowe et al., 1973). This can be caused by the ordered layer of water molecules surrounding the matrix backbone and limiting diffusion in this region, or by the vibrational movement of the lattice. In any event, the region of the solvent in close proximity to the surface of the solid support represents a real barrier for the interaction of the macromolecule with the complementary affinity ligand, especially if an interaction with weak affinity is concerned. However, from the experiments mentioned, it does not yet follow that the distance of the affinant from the matrix alone is decisive for the bond strength. In connection with hydrophobic spacers, O'Carra et al. (1973, 1974a, b) showed that in many instances the sorption is much more influenced by the hydrophobic binding of the isolated substance to the hydrophobic chain than by the formation of the biospecific complex. As an example one of the most often quoted papers on spacers, dealing with the affinity chromatography of P-galactosidase(Steers et al.) on carriers, can be mentioned:

OH

dH

'

The affinity sorbent prepared by binding the inhibitor of p-aminophenyl-PD-thiogalactopyranose directly to Sepharose (derivative A) did not either bind or retard the enzyme to a detectable extent. Attachment of the inhibitor over a short arm (about 10 A) gave a sorbent (derivative B) that slightly retarded the enzyme during its passage through the column. In order to free the enzyme from the complex there was no need to change the composition of the buffer and the enzyme left the column immediately after the inactive protein. Only when a long arm (about 21 A) was inserted between the inhibitor and the solid carrier surface was a sorbent obtained (derivative C) that firmly bound Pgalactosidase from various bacterial sources. Elution of the enzyme took place only after a change of buffer. This affinity chromatography has often been quoted as an example of how a very effective specific sorbent can be prepared even from an inhibitor with a relatively high inhibition constant ( 5 M) and a relatively low concentration of the affinant (0.6 mM). O'Carra et al. (1974a) prepared analogous sorbents of derivative C in which they replaced the specific inhibitor, P-thiogalactoside, with the non-specific a-glucoside or N-phenylglycine. Both sorbents retained a strong affinity for p-galactosidase. However, when they

-

60

AFFINANT-SORBENT BONDING

used a hydrophilic chain

as a spacer for the binding of 0-thiogalactoside, this sorbent no longer adsorbed 3-galactosidase strongly. Hence, the adsorption of fl-galactosidaseto derivative C arises mainly as a result of the hydrophobic interaction with the spacer arm. In addition to the hydrocarbon nature of this spacer, the fact that high concentrations of salts have little effect on sorption also supports this view. The interfering effect of nonspecific adsorption in bioaffinity chromatography is discussed in greater detail in Sections 5.8 and 10.3. This effect is best avoided if hydrophilic spacers are used, and the preparation of these is described in Section 8.3. The use of hydrophilic spacers also prevents a further undesirable possibility that can occur when a hydrophobic affinant is bonded to a long, flexible hydrophobic chain. This can then itself interact with the spacer and become masked or occluded. Such a “conformational occlusion” can be the reason for the inaccessibility of the immobilized affinant for complex formation with the substance isolated. The affinity chromatography of Fans-N-deoxyribosylase(Holguin and Cardinaud) is a practical example. When using Sepharose-N6-p-amino-n-hexyladenine, they assumed that under the effect of the flexible chain the active part of the ligand came into close proximity of the solid support and that therefore an effective interaction with the transfer enzyme could not take place.

5.2 CONFORMATION OF ATTACHED AFFINANT

The main principle of specific interactions of biological macromolecules is the complementarity of the binding sites. For example, the high reactivity of specific substrates follows from the perfect interaction of configurationally and conformationally oriented groups of the substrate with the complementarily located groups or sites of the enzyme. The interaction of the substrate and the inhibitor with the enzyme is thus the greater, the greater is the complementarity of the binding sites. This is true not only with respect to the spacial arrangement, but also with respect to the nature of the complementing parts of the molecules. However, the means of bonding the affinity ligand to the solid support is thus considerably limited, because the ligand must be bound to that part of the molecule which does not participate in the binding. In addition, the immobilization of the affinant should not cause a change in conformation or affect the nature of its binding sites. The effectiveness of the affinity adsorbent depends on the extent to which this is achieved. The basic importance of the attachment of nucleotides to a solid support as regards the efficiency of the affinity chromatography of kinases and dehydrogenases depending on pyridine nucleotide has been demonstrated by the studies of Harvey et al. (1974a). The adsorbent N6-($-aminohexyl)-BMP-Se~liarosecontains AMP bound to Sspharose by means of the N6-adenine part:

61

ATTACHED AFFINANT

while in the sorbent P1-(6-aminohexyl)-P2-(5'-adenosine)pyrophosphate-Sepharose AMP is linked by 5'-phosphate:

OH OH

The linking of various dehydrogenases and kinases on to these two adsorbents is mentioned in the preceding chapter in Table 4.2. Glucose-6-phosphatedehydrogenase, D-glyceraldehyde 3-phosphate dehydrogenase and myokinase were bound to P1-(6-aminohexyl)-P2-(5'-adenosine)pyrophosphate-Sepharose only, while alcohol dehydrogenase and glycerokinase were bound only to N6-(6-aminohexy1)d'-AMP-Sepharose. Lactate dehydrogenase, malate dehydrogenase, 3-phosphoglyceratekinase and pyruvate kinase were bound to both sorbents, while hexokinase and creatine kinase were bound to neither of them. These results reflect the nature of the enzyme-nucleotide interactions and it can be concluded that while the free 5'-phosphate group is essential for the binding, for example, to alcohol dehydrogenase or glycerokinase, it has a completely different role in the interaction of glyceraldehyde 3-phosphate dehydrogenase. In this instance the decisive role is played by the adenosine part of the affmant. The much stricter binding requirements with hexokinase and creatine kinase evidently result in these enzymes not being attached to either of the adsorbents. High-molecular-weightaffinity ligands usually offer more possibilities for the preparation of affinity adsorbents. A series of very active affinity adsorbents has been prepared by direct attachment of the protein to a solid support, and many examples are given in Chapter 11 in Table 11.l. However, a very important condition in this instance is that the attachment to the solid support should not cause a change in its native conformation. For illustration, an example from immunoadsorption is given here. Cuatrecasas isolated insulin on columns of Sepharose with an antibody against hog insulin bound at both pH 6.5 and 9.5. As is shown in Section 8.2.4, protein is bound on cyanogen bromideactivated Sepharose by its non-protonated forms of amino groups. On decreasing the pH, a decrease in the number of binding groups also takes place. The result of the pH difference was that the first derivative was able to bind almost 80%of the theoretically possible amount of insulin, while the second derivative, prepared by binding at pH 9.5, took only 7%of the capacity for insulin. As the total content of the bound affinant was

AFFINANT-SORBENT BONDING

62

identical in both instances, the second derivative must have contained immunoglobulin, which is unable to bind antigen effectively. In the case of a large number of bound amino groups, disturbance of the native tertiary structure evidently occurred. Even at a low pH, adsorbents can be obtained that contain a large amount of active protein attached on Sepharose if the amount of cyanogen bromide is increased during the activation and the amount of protein during the binding.

5.3 CONCENTRATION OF THE AFFINANT ON THE MATRIX The theoretical deduction of mutual relationships between the amount of sorbed enzyme, the concentration of the affinity ligand and ligand-enzyme equilibrium constants has been mentioned in Chapter 3. It is evident from Fig. 3.3 that for interacting systems of low affinity ( K L= M) the concentration of the bound affinity ligand represents a critical parameter in the preparation of an effective adsorbent. In Fig. 5.6, the affinity chromatography of glucokinase on 2-amino-2-deoxy-D-glucopyranose-N-(6-amino-

{

0.8

JO

FRACTION NUMBER

Fig. 5.6. Effect of ligand concentration on the elution of glucokinase by glucose from Sepharose-N(6-aminohexanoyl)-2-amino-2deoxy-D-glucopyranose. Each column (1 00 X 8 mm) was equilibrated with the buffer 20 mM triethanolamine-HCl, pH 7.0, containing 10 mMKC1,4 mM EDTA, 7.5 mM M a , 1 mM dithiothreitol and 5% (v/v) glycerol and operated at 20 ml/h; 3.0-ml fractions were collected. Glucokinase (2 ml, 2 unitslml) purified by DEAE-cellulose chromatography was applied to each column followed by 25 ml of the equiliiration buffer. At (a), the columns were developed with a linear gradient formed from 75 ml of the column buffer and 75 ml of 1 M glucose dissolved in this buffer. At (b), 0.5 M K C l was included in the buffer. A, 1.2 @moleof glucosamine derivative coupled per gram (wet weight) of packed gel; B, 3.75 Nmolelg; C, 6.0 fimolelg; D, 10 rmolelg. The fmal ligand concentrations were achieved by diluting the 10 I.rmole/g material with unsubstituted Sepharose. 0,A zso; a, glucokinase activity; - - -,glucose concentration. Reproduced with permission from M.J. Holroydeetal., Biochem. J., 153 (1976) 351-361.

- -

AFFINANT ON THE MATRIX

63

hexanoy1)-Sepharose is represented at four concentrations of the bound affinant (Holroyde et a/.). It is evident that an optimal concentration of the affinity ligand is 3.75 pmole/g (Fig. 5.6B): at lower concentrations the enzyme does not separate from the inactive material, while at higher concentrations the glucose concentration in the eluent also should be increased, while the elution of glucokinase takes place with a larger volume of the eluent. At a concentration of 10 pmolelg, glucokinase cannot be eluted even with high concentrations of glucose, and it can be displaced only by the addition of 0.5 M potassium chloride. Another example of the dependence on the concentration of the affinity ligand is the affinity chromatography of a mixture of lactate dehydrogenase and serum albumin on N6-(6-aminohexyl)-5‘-AMP-Sepharose(Harvey et ul., 1974a). At a high ligand concentration (1.5 pmole of 5’AMP per ml) a pulse of NADH was necessary for the elution of the enzyme. At a lower concentration (0.125 m o l e of 5’-AMP per ml) the desorption of the enzyme was achieved by a mere 0-1 M potassium chloride gradient. A further reduction in the amount of the attached ligand (0.025 pmole of 5’-AIvlP per ml) resulted in a progressive increase in the proportion of lactate dehydrogenase eluted by the equilibrium buffer even before the application of the linear gradient of salts. At the same time, the enzymatic activity was weakly retarded with respect to the bovine serum albumin. At a 0.125 pmole per ml ligand concentration of S’-AMP, when lactate dehydrogenase was eluted quantitatively with a salt gradient, 1 g of column packing adsorbed 0.1 nmole of enzyme. From this result, it follows that only 0.1% of the total amount of the attached affinity ligand was utilized for the binding of lactate dehydrogenase. A similar value was also obtained by frontal chromatographic analysis. At a lower ligand concentration this proportion decreased to 0.025%.As is evident from Fig. 5.7A, the capacity of N6-(6-aminohexy1)-5 ’-AMP-Sepharose for lactate dehydrogenase plotted against the concentration of ligand (uniformly distributed in the particle) gives a sigmoidal response (Dean et ul.). From this, it can be deduced that for the retardation of lactate dehydrogenase during chromatography the juxtaposition of more than one molecule of the immobilized nucleotide is necessary (Harvey et al., 1974a). The sigmoidal nature of the curve could mean a cooperative interaction of the enzyme with the immobilized ligand. This supports the idea that higher concentrations of ligand are maintained by the enzyme in bound form by limitation of its subsequent dissociation from the matrix, in agreement with the kinetics for a reversible equilibrium. The sigmoidal shape of the curve differs from the dependence of the amount of the bound enzyme on the ligand concentration, given in Fig. 5.7B, when a different concentration of ligand in the gel was achieved by dilution of the gel with unmodified Sepharose. Similarly, Nishikawa et al. compared gels with different concentrations of the affinant in terms of its distribution, and differentiated the enzyme capacity per unit of gel and the affinity with which this gel binds enzymes. The concentration of ligands in the gel affects both properties, but not in an identical manner. Gel beads with a high concentration of affinant possess a high affinity and a correspondingly high capacity of the bed. If this gel is diluted with unmodified agarose, the intra-gel ligand concentration in the modified beads is still high and the gel retains its high affinity. However, not all of the gel beads are capable of binding enzyme and therefore the bed capacity is lower. Finally, if the gel is uniformly derivatized with ligand at a low concentration, it has both a low affinity and a low capacity of the bed.

AFFINANT-SORBENT BONDING

64 I

loop-----.

I

50

[LIGAND].

1

,

150

1

2:

mp moles/ rnl

Fig. 5.7. Effect of affinity ligand distribution on the capacity of N6-(6-aminohexy1)-5'-AMP-Sepharose for lactate dehydrogenase. (A) Ligand uniformly coupled to Sepharose; (B) ligand diluted with unsubstituted Sepharose. Reproduced with permission from P.D.G. Dean et al., Advan. Exp. Med. Biol., 42

(1974)99-121.

TABLE 5.2 INTERACTION O F MYOKINASE WITH P'-(6-AMINOHEXY L)-Pz-(5'-ADENOSlNE)PY ROPHOSPH ATE-SEPHAROSE The enzyme sample (100pl), containing myokinase (4 v) and bovine serum albumin (1.5 mg), was applied to a column (50 X 5 mm) containing 1 g of P1-(6-aminohexyl)-P '-(5'-adenosine)pyrophosphateSepharose diluted to the appropriate concentration of ligand with unmodified Sepharose 4-3.Binding (0) is defined in Fig. 5.5. Ligand concentration (rcmole/ml of 5'-AMP)

6.00 3.00 1.00

Amount bound from % to 5'-AMP

0.05

400 315 200 185 140 95 70

0.03

50

0.50

0.30 0.10

%

U per gram of adsorbent

U per pmole of 5'-AMP

100 100 97.4 90.4 83.3 42.5 27.0 14.7

4.00 4.00 3.90 3.62 3.33 1.69 1.08 0.59

0.67 1.33 3.90 7.24 11.10 16.80 21.55 19.60

AFFINANT ON THE MATRIX

65

The difference between the affinity and the capacity of P1-(6-aminohexyl)-Pz-(5’aden0sine)pyrophosphate-Sepharose diluted with various amounts unmodified Sepharose during the interaction with myokinase is clearly evident from Table 5.2 (Harvey et al., 1974a). The dilution of the affinity adsorbent leads to a decrease in binding (p) values (expressed by the potassium chloride concentration necessary for the elution of the enzyme). The capacity of the adsorbent, expressed in terms of units of enzyme sorbed on 1 pmole of nucleotide, is increased at low concentrations of nucleotide, although the absolute capacity per gram of column material decreases with dilution. The dependences of the binding (p) of lactate dehydrogenase on the concentration of the ligand on N6-(6-aminohexyl)-5’-AMP-Sepharose diluted with unsubstituted Sepharose to the required concentration of the ligand, and on the same concentration of N6-(6-aminohexyl)-5’-AMP uniformly bound to Sepharose, bad similar courses (Harvey eta)., 1974a). The difference, manifested in a stronger binding in the case of diluted affinity adsorbent, was attributed to the presence of gel beads in which the concentration of ligand remains identical with that in the original undiluted gel. In gels that contained a uniform distribution of ligand, the force of interaction (0)for lactate dehydrogenase, malate dehydrogenase and glycerokinase increased linearly with increasing concentration of N 6-(6-aminohexyl)5’-AMPligand attached to Sepharose, as shown in Fig. 5.8 (Harvey et al., 1974a). 1

‘i

[L IGAND],pmol/ml

Fig. 5.8. Binding (0) of malate dehydrogenase (O),lactate dehydrogenase (pig heart muscle) (A)and glycerokinase (0)to N6-(6-aminohexyl)-S’-AMP-Sepharosein relation to affinity ligand concentration. The sample (100 pl) of enzyme (5 U) was applied to the columns (SO X 5 mm) containing 0.S g of Sepharose 4B to which N6-(6-aminohexyl)-S1-AMP had been coupled at the indicated ligand concentrations. The binding (p) is defined in Fig. 5.5. Reproduced with permission from M.J. Harvey et al., Eur. J. Biochem., 41 (1974) 335-340.

66

AFFINANT-SORBENT BONDING

When both the concentration of the ligand [N!-(6-aminohexyl)-5’-AMPimmobilized on Sepharose] and the total amount of ligand were left constant and the length of gel bed was changed by changing the column diameter, the strength of the interaction (p) of both alcohol dehydrogenase and glycerokinase increased with increase in the column length (Lowe et al., 1974a). When the total amount of affinant was constant at the same column diameter, the binding (0) of lactate dehydrogenase and glycerokinase on to N6-(6-aminohexyl)-5’-AMP-Sepharose was directly proportional to the concentration of ligand and hence, in t h s instance, indirectly dependent on the column length, in proportion to the decreasing ligand concentration. However, when the column length was changed while the concentration of the affinant and the column diameter were kept constant in such a manner that the total amount of affinity ligand was proportional to the column length, the binding (p) of lactate dehydrogenase and glycerokinase increased linearly. The capacity of the column, under identical conditions ( i e . , at constant concentration of ligand and constant diameter), is again linearly dependent on the column length and thus also on the total amount of ligand in the bed. The effect of the geometry of the columns, the concentration of the affinity ligand and its total amount are the three basic parameters that determine both the force of interaction and the capacity. For systems with a high affinity, when using affinity adsorbents that contain a high concentration of ligand, the column length is of little importance, the binding being dependent on the concentration of the affinant and not on the column length. A practical consequence is that columns that contain a high concentration of ligand can be used for the concentration of dilute solutions of enzymes. Further, in systems with a high affinity, in which the elution of the adsorbent macromolecule is difficult without denaturation, dilution of the affinity adsorbent with unsubstituted gel or reduction in the ligand concentration may result in a much easier elution under milder conditions. In some instances, of course, the enzyme differentiates only the ligand concentrations in beads of modified gel, which does not, however, change with dilution. In such instances, the difficulties with the elution persist even after dilution of the gel (&son and Nishikawa). For interacting systems with a low affinity, the column geometry will be an important parameter. In order to differentiate specifically adsorbed from nonadsorbed proteins the use of long columns will be more advantageous than the use of short columns. Of course, an affinity sorbent with a high concentration of ligand is not desirable in all instances. Fig. 5.9 shows the affinity chromatography of acetylcholine receptor from the electric organ of Torpedo culifomicu (Schmidt and Raftery) on Sepharose containing M (Fig. 5.9A) and NH(CH2)5CONH(CH2)3N+(CH3)3at concentrations of 2 . 0.4 M (Fig. 5.9B). It is evident that the decrease in concentration converted the weak, non-specific ion exchanger into an affmity adsorbent of high selectivity. The latter was then used with success for the purification of the basic a-bugarotoxin-binding membrane component on a large scale. The decrease in selectivity for isolated macromolecules after the critical ligand concentration (when the adsorbent begins to act as an unspecified ion exchanger) had been exceeded was described for the first time by Kalderon et al. During the isolation of acetylcholinesterase the affinity adsorbent lost its specificity when the concentration of ligand t o 1.6.10-3 M.In both instances the critical concentration was increased from 0.15

67

PROTEINS, EQUILIBRATION TIME AND FLOW-RATE I

lA

I

FRACTION

I

1

NUMBER

Fig. 5.9. Chromatography of crude membrane extract from the electric organ of Torpedo californica on Sepharose 2B with NH(CH,),CONH(CH,),N+(CH,), at different concentrations of ligand: (A) 2 .lo-, M and (B) 0.4 .lo-’ M.Affinity sorbents were packed in Pasteur pipettes (bed volume ca. 1.5-2 ml) and equilibrated with starting buffer (10 &sodium phosphate, pff 7.4-0.1% Emulphogene). Small aliquots of crude extract (2-6 units; 0.6-3.0 mg) were washed in with starting buffer. The arrow indicates the start of a linear salt gradient (total volume, 100 ml; final NaCl concentration, 1M). Fractions of ca. 1.6 ml were collected and assayed for protein and toxin-binding activity (cpm). Reproduced with permission from J. Schmidt and M.A. Raftery, Biochemistry, 12 (1973) 852-856.

M,which corresponds to a nearest neighbour distance of ligand is thus approximately of about 100 A, assuming that the molecules of ligand are distributed evenly throughout the gel and located at the intersections of a cubic lattice. It is conceivable that at concentrations below M the ligands are distributed at sufficiently large distances to prevent the non-specific proteins from interacting with more than one charged group at a time. On such a loosely distributed ligand with charged groups, only molecules with choline recognition sites rather than non-specific negatively charged functions would then be sorbed. Another undesirable phenomenon in some adsorbents with high concentration of affinity ligand is too strong an interaction with the isolated substance, which causes difficulties with its elution. Thus, for example, careful control of the ligand concentration is crucial for the isolation of glucokinase on agarose with bonded N-(6-aminohexyl)-2-amino2-deoxy-D-glucopyranose(Holroyde and Trayer), as shown in Fig. 5.6. 5.4 CONCENTRATION OF PROTEINS, EQUILIBRATIONTIME AND FLOW-RATE The concentration of the solution of the substance to be isolated acts in various ways if the process represents affinity separation in a column or batchwise arrangement. Another important factor is the level of affinity of the interacting complementary sites of the isolated substance and the affinant. The equilibration time and the flow-rate are related to this factor.

TABLE 5.3 EFFECT OF EQUILIBRATION TIME ON THE EFFICIENCY OF A COLUMN OF N6-(6-AMINOHEXYL)-5’-AMP-SEYHAROSE The enzyme sample (100 gl), containing enzyme (5 U) and bovine serum albumin (1.5 mg), was applied and washed into a column (34 X 5 mm) of N6-(6-aminohexyl)-5’-AMP-Sepharose (0.95 g moist weight, 0.125 gmole/ml of 5’-AMP). HETP = column length/l6 ( Ve/w)2,where Ve is the elution volume, measured from the start of the KCl gradient t o the centre of the enzyme peak, and w is the peak width, determined at the base of the enzyme peak. Binding (0) is defined in Fig. 5.5. Equilibration time (h)

1 20 67

Glycerokinase

Lactate dehydrogenase (pig heart muscle)

Amount bound (%)

Binding (0) (MKCI)

Peak width (ml)

HETP (mm)

Amount bound (%)

Binding (0) (mM KCl)

Peak width (ml)

HETP (mm)

26

20 50 150

7.0 6.0 5.6

0.42 0.25 0.17

100 100 100

360

18.5 11.2 13.8

1.50 0.52 0.76

51

59

355

490

PROTEINS, EQUILIBRATION TIME AND FLOW-RATE

69

In column affinity chromatography, a sufficielitly strong interaction of the isolated substances with the immobilized affinity ligand’causes the substance to be concentrated and slows its migration down the column. This process is dependent on the concentration of ligand and almost independent of the starting concentration of the free macromolecule. For example, in the affinity chromatography of glycerokinase or lactate dehydrogenase on N6-(6-aminohexyl)-5’-AMP-Sepharoseno effect of the concentration of the enzyme on the capacity was observed when the concentration of enzymes was either directly or indirectly proportional to the concentration of nucleotide (Lowe et al., 1974a). The restrictions that a sub-saturation amount of the complementary macromolecule should be applied on to the column and that the flow-rate should be adjusted adequately apply here (Lowe and Dean, 1974). There may be two reasons for the appearance of the isolated protein in the void volume of the eluate at high flow-rates applied to highly overloaded columns. The first concerns a process known as secondary exclusion. The diffusion rate of the molecules into the gel does not depend only on the pore size, but also on the relationship between the molecular size and the pore volume. When a concentrated solution of large macromolecules is applied on to the gel, some molecules will diffuse into the accessible pores, but later incoming molecules will find many pores occupied and their probability of diffusion into the occupied pores will be reduced, depending on the reduction in the accessible pore volume. This leads to exclusion from the pores, which is the origin of immobilized ligands whose accessibility is changed. The second reason is the steric hindrance to subsequent molecules by the molecules of the protein that have already been adsorbed, A globular protein of medium size, such as a haemoglobin, covers an area of about 2500 A2 (Lowe and Dean, 1974), which itself produces considerable steric hindrance. As the interaction of macromolecules with an immobilized affinant is a time-dependent process, it is affected by the flow-rate and the equilibration time. In many instances adsorption equilibrium is attained very slowly. For the interaction of a macromolecule with an immobilized affinant, mere collision of appropriate molecules does not suffice, because a correct orientation of binding sites, or their conformational adaptation, is also necessary. Table 5.3 shows the influence of the equilibration time on the efficiency of a column of N6-(6-aminohexyl)-5’-AMP-Sepharose(Lowe et al., 1974a). When glycerokinase and lactate dehydrogenase are equilibrated with the adsorbent, both the column efficiency expressed as the height equivalent to a theoretical plate (HETP) and the strength of the enzymatic interaction (p) increased with time up to the 67th hour. In general, the lower the HETP value the greater is the effect of the adsorbent (Lowe and Dean, 1974). With glycerokinase, the percentage of the bound enzyme also increased. An important practical consequence ensues from the comparison of the HETPs, viz., that not only the strength of the bond increases, but also the affinity adsorbent displays a better resolution ability. This evidently led Ohlsson et al. to interrupt the flow through the column for several hours before the specific elution. The equilibration time had no effect on the bonding of lactate dehydrogenase on to immobilized NAD’, or on the bonding of glycerokinase on immobilized ATP when the enzymes were left in contact with the immobilized nucleotides for 1 , 5 and 20 h before elution (Lowe and Dean, 1974). This observation may be useful for the storage of enzymes the stability of which depends on the presence of substrates or cofactors.

70

AFFINANT-SORBENT BONDING

The attachment of the ligand and the limitation of the static film on the surface and inside the pores of the carrier cause diffusion to influence the overall kinetics of the reaction. The limitation of the diffusion, imposed by the nature and the mechanism of :he chromatographic processes, can be classified into three types (Lowe and Dean, 1974): (1) Longitudinal diffusion, which represents the classical Fickian molecular diffusion caused by the concentration gradient, which may act both radially and axially. In normal affmity chromatography it is of no great importance, but it could become more important at slow flow-rates in weakly interacting systems. ( 2 ) Eddy diffusion, which may be a very important factor at high flow-rates. It is caused by irregularities in flow, produced by the gel particles in the bed. If the rate of attainment of equilibrium is very slow, then the dissolved molecules moving with the rapid stream will be less likely to interact with the immobilized affinant than in a slow stream path. (3) Restricted diffusion appears when the molecular diffusion within reach of the gel matrix pores is limited, because the latter might seriously hinder the correct approach of the macromolecule to the attached affinity ligand. The contribution of this diffusion can be determined in practice only with difficulty, but this difficulty can be minimized by using a very porous carrier for affinity chromatography. From the practical point of view, in order t o achieve equilibrium conditions it is desirable that the flow-rate should be as low as possible. For example, when Cuatrecasas et al. used a flow-rate of 400 ml/h for the isolation of staphylococcal nuclease on a 20-ml column, a small amount of nuclease appeared in the first peak together with protein impurities, especially when the total concentration of the proteins in the sample was also high (20-30 mg/ml). However, even at such a high flow-rate nuclease was completely sorbed if a less concentrated sample was applied. The dependence of the binding of lactate dehydrogenase on N6-(6-aminohexyl)-S'-AMP-Sepharose on flow-rate was investigated by Lowe et al. (1974a), who found that an increase in the flow-rate had a relatively small influence. The column efficiency (HETP) decreased at high flow-rates, as did the strength of the interaction (0). The effect of the flow-rate was more pronounced on small columns, with which some of the enzymatic activity is eluted with the void volume. No effect of the concentration of the inert protein (bovine serum albumin) was observed at high-flow rates (67 d / h ) either. In the batchwise arrangement of the sorption on an affinity adsorbent, the enzyme concentration in the sorption solution plays an important role. In Table 3.1 the percentage occupation of the molecules of the immobilized affinant by the sorbed enzyme at various concentrations of the starting solution of the enzyme is given (Graves and Wu). From this table, it is evident that a direct proportionality is obtained. The same dependence is also evident in Fig. 5.3, where both the derived and the experimentally determined dependence of the amount of the enzyme-immobilized ligand complex formed on the enzyme concentration are shown for affinity adsorbents with various contents of ligand. For successful sorption, higher concentrations of enzymes are necessary mainly with sorbents with a low content of the attached affinity ligand. Fig. 5.10A shows the effect of the lactate dehydrogenase and glycerokinase concentrations on the capacity of N6-(6-aminohexyl)-5'-AMP-Sepharose in the batchwise arrangement (Lowe et al., 1974a). The percentage of the bound enzyme increases with enzyme concentration, which is mainly operative at concentrations of up to 2 units per millilitre.

TEMPERATURE

71

""f z

* 25

0

5

10

15

CE N Z Y M Elunlts/ml

0

1

2

INCUBATION T 1 M E . h

Fig. 5.10. Effect of enzyme concentration (A) and incubation time (B) on the capacity of N6-(6-aminohexy1)d'-AMP-Sepharose for lactate dehydrogenase (0) and glycerokinase (0) under batchwise conditions. The enzyme, diluted with equilibration buffer (10 nMKH,PO,-KOH, pH 7.5) to the appropriate concentration (A) or 10 U of lactate dehydrogenase suspended in 100 ml of equilibrated buffer (B), was incubated with N6-(6aminohexy1)-5'-Sepharose (0.5 g moist weight, 1.5 pmole/ml of 5'-AMP) on a Coulter mixer at 4.5"C. After 30 min (A) or at suitable time intervals (B), the incubation was stopped by allowing the adsorbent to settle (15 min) and the supernatant volume was then removed. Using this procedure, with a 5-min incubation period, the adsorbent was washed three times with 5 ml of equilibration buffer prior to re-suspension in 2.5 ml of equilibration buffer and packing in a column (50 X 5 mm). The column was eluted with 11.0 ml of equilibration buffer and then with a linear gradient of KCl(0 to 1.0 M,20 ml total volume). Enzymes were assayed in the washes and column effluent. Reproduced with permission from C.R. Lowe er al., Eur. J. Biochem., 41 (1974) 341-345.

Although the affinity (0) of both enzymes differs considerably (in a 40 : 1 ratio), the relationship between the percentage of the bound enzyme and its concentration is virtually identical. The effect of the incubation time on the capacity of N6-(6-aminohexyl)-S'-AMPSepharose to bind lactate dehydrogenase in the batchwise arrangement (Lowe ef QZ., 1974a) is illustrated in Fig. 5.10B. The greatest increase in the binding of the enzyme on the immobilized nucleotide occurs in the initial phase of the process, which then continues more slowly up to 100% binding, achieved after 16 h, while the half-time is about 20 min. The course of the sorption of chymotrypsin on NH2-Spheron with attached carbobenzoxyglycyl-D-phenylalanine, shown in Fig. 3.1 ,indicates a much faster attainment of equilibrium. Fig. 10.4 shows how this attainment of equilibrium is affected by dilution.

5.5 EFFECT OF TEMPERATURE Adsorption of a dissolved substance from the mobile phase on to the stationary phase is generally exothermic, and thus according to Le Chatelier's principle, elevated temperatures will move the equilibrium in the direction of heat absorption. Under chromatographic conditions, the increase in temperature shifts the equilibrium to a higher relative concentration in the mobile phase, and a higher temperature usually leads to more rapid

AFFINANT-SORBENT BONDING

72

migration through the chromatographic bed. In general, the more exothermic the adsorption of a certain enzyme, the more sensitive it will be to temperature (Harvey el d., I 974b). The dependence of the distribution constant, R , on the absolute temperature, T , is given by the equation

where AS" is the standard entropy and AHo the standard enthalpy of adsorption, and R is the gas constant. When enzymes with different enthalpies of adsorption (-AHo) are separated, the dependence on temperature can be utilized for separation. The effect of temperature on the capacity of N6-(6-aminohexyl)-5'-AMP-Sepharose to bind alcohoi dehydrogenase and glycerokinase at two concentrations of the immobilized nucleotide is shown in Fig. 5.1 1 (Harvey ef al., 1974b). The increasing temperature affects substantially the amount of enzyme bound to the affinity sorbent with a lower content of affinant (1.5 pmole/ml of AMP). However, if an adsorbent with a higher concentration of the bound nucleotide (4.0 pmole/ml of AMP) is used, the increase in temperature will have no practical consequences. The difference in the concentration of the immobilized -----

I

- -7-

TEMPERATURE.'c

Fig. 5.1 1. Effect of temperature on the capacity of N6-(6-aminohexyl)-5'-AMP-Sepharose.The enzyme sample (5 U) plus 1.5 mg of bovine serum albumin (100pl) was applied to a column (50 X 5 mm) containing 0.5 g of N6-(6-aminohexy1)J'-AMP-Sepharoseaccording to the brocedure reported in Fig. 4.6. The capacity was determined as the percentage of the enzyme activity that was retained by the adsorbent. Bovine serum albumin was located in the void volume. The immobilized ligand concentration was 1.5 rmole/ml of AMP for yeast alcohol dehydrogenase (0) and glycerokinase ( 0 ) and 4.0 fimole/ml of AMP for glycerokinase (e). Reproduced with permission from M.J.Harvey el al., Eur. J. Biochem., 41 (1974)353-357.

13

TEMPERATURE

AMP did not, however, have any influence on the effect of temperature on the binding

(0) of alcohol dehydrogenase and glycerokinase to N6-(6-aminohexyl)-5‘-AMP-Sepharose, in all examples investigated a decrease in the strength of interaction (0) taking place with the increase in temperature (Harvey et al., 1974b). The Arrhenius plot for the binding of glycerokinase on immobilized AMP gave a linear dependence in the temperature range 0.5-35°C. For an affinity adsorbent with a Sepharose containing 4.0 pmole/ml of AMP the energy of adsorption is 20.2 kJ/mole (4.8 kcal/mole), and for a sorbent with a Sepharose content 1.5 pmole/mi of AMP it is 24.6 kJ/mole (5.9 kcal/mole). An analogous binding of alcohol dehydrogenase displays a biphasic Arrhenius plot with adsorption energies of 24.6 kJ/mole (5.9 kcal/mole) and 58.8 kJ/mole (14.1 kcal/mole) above and below the transition temperature of 15°C: respectively. The reason for this behaviour may lie in the conformational change of the enzymes induced by temperature.

I-

4

o\

I

0

O

I

I

I

0

I

20

10

0

30

TEMPERATURE ,‘C

I

I

I

I

I

I

I

I

I

I

I

1

32

33

3.4

3.5

3.6

3.7

1.8

10’1 T,’K-’

Fig. 5.12. Binding of pig heart muscle lactate dehydrogenase to N6-(6-aminohexyl)-5’-AMP-Sepharose in response to temperature. The enzyme sample (5 U) containing 1.5 mg of bovine serum albumin (100pl) was applied to a column (50 X 5 mm) containing 0.5 g of N6-(6-aminohexyl)-5’-AMP-Sepharose (1.5 pmolelml of AMP). (A) Binding was determined by the procedure reported in Fig. 4.6 tising a linear gradient of NADH (0 to 5 mM, 20 ml total volume). (B) Arrhenius plot of the above data. Reproduced with permission from M J . Harvey etal., Eur. J. Biochem., 41 (1974)353-357.

74

AFFINANT-SORBENT BONDING

T E M P E R A T U R E ,"C

Fig. 5.13. Effect of varying temperature on binding ( P ) (defined in Fig. 5.5) of alcohol dehydrogenase (0) and phosphofructokinase ( 0 ) to N6-(6-aminohexy1)-5'-AMP-Sepharose.The enzyme extract (0.5 ml, 81 U of phosphofructokinase, 20 U or 18.7 mg of alcohol dehydrogenase per millilitre) was dialysed against 10 rnM potassium phosphate buffer, pH 6.8, and adsorbed on to a column of substituted AMP-Sepharose. Elution was carried out with a linear salt gradient (0 to 1 M KC1,40 ml) in 10 rnM potassium phosphate buffer, pH 6.8, at 0.4 ml/min. Reproduced with permission from M.J. Comer et al., Eur. J. Biochem., 55 (1975) 201-209.

The same adsorption energy of glycerokinase, determined in the range 35-O.S°C, was obtained if the determination was carried out in the opposite direction of temperature change. From this result, it follows that even prolonged exposure of these columns to increased temperature does not change their subsequent chromatographic behaviour at a lower temperature. The mentioned apparent energies of adsorption were calculated without regard to the effect of temperature on the ionic strength of the eluent. If this is taken into consideration, the corrected value of the energy of adsorption for glycerokinase is 8.3 kJ/mole (2.0 kcal/mole). The binding of lactate dehydrogenase on to N6-(6-arninohexy1)-5'-AMP-Sepharoseis so strong that the enzyme cannot be eluted even with 1 M potassium chloride solution at 40°C. However, a linear gradient of NADH can be used for elution. Fig. 5.12 shows a plot of the concentration of NADH necessary for the elution of lactate dehydrogenase against temperature. With increasing temperature, the required concentration of the specific eluting agent decreases, represented by a linear Arrhenius plot. The corresponding adsorption energy is 54.6 kJ/mole (13.0 kcal/mole) (Harvey et al., 1974b). Fig. 5.13 shows the effect of different temperatures on the binding (0) of the thermophilic enzymes alcohol dehydrogenase and phosphofructokinase from Bacillus stearothemophilus to N6-(6-aminohexyl)-5'-AMP-Sepharose(Comer et al.). Hence, with the

pH AND IONIC STRENGTH

15

thermophilic enzymes mentioned, on the contrary, the binding (0) increases first with increasing temperature. Above 50°C destruction of gel already takes place, which is evident from the presence of the nucleotide in the eluate. Figs. 5.14A and 5.14B show that the pH dependence of the binding of both enzymes on to the immobilized AMP is while Fig. 5.14C shows the dependence of the also temperature dependent (Comer et d.), inflexion points of the binding on temperature.

C

B

TEMP.%

3 TEMP. %

4

,

I

5

6

1

8

9

1 0 1 1

6

7

8

9

1

0

P"

8

9

10

PK

Fig. 5.14. Interdependence of binding (p) (defined in Fig. 5.5) to N6-(6-aminohexyl)-5'-AMP-Sepharose on both pH and temperature for (A) alcohol dehydrogenase and (B) phosphofructokinase. Conditions as in Fig. 5.13. The buffer used was 10 mM Tris-phosphate which was adjusted to the required pH at the relevant temperature. (C) Relationship between the point of inflection (apparent pK) and temperature for the binding of alcohol dehydrogenase (0)and phosphofructokinase (0).Reproduced with permission from M.J. Comer et al., Eur. J. Biochem., 55 (1975) 201-209.

The effect of temperature on the sorption and elution of the isolated substances in affinity chromatography is discussed further in Chapter 10. In concluding this section, it should be stressed that the effect of temperature should always be borne in mind, because reproducible results can be obtained only if the temperature is carefully controlled, especially in analytical applications.

5.6 EFFECT OF pH AND IONIC STRENGTH The catalytic effect of enzymes is usually limited to a narrow range of the so-called optimal pH, which reflects the ionization of both the enzyme and its substrate. A shift from the optimal pH range results in a decrease in both the rate of the enzymatic reaction and the affinity of the system for the substrate, Ionic and hydrophobic interactions operative in the binding site of the enzyme with its substrate or inhibitor are further considerably influenced by ionic strength. From this it follows that both pH and ionic strength

76

AFFINANT-SORBENT BONDING

are important factors in both sorption and desorption. The effect of pH and of ionic strength on the sorption of chymotrypsin on immobilized benzyloxycarbonylglycy1-Dphenylalanine has already been mentioned in Chapter 3 (Fig. 3.1). Kasai and Ishii determined an increase in the dissociation constant of trypsin with immobilized glycylglycylL-arginine from 0.23 t o 0.33 mM on addition of 0.3 M sodium chloride, and to 0.75 mM on addition of 1.OM sodium chloride. The effect of the dependence of mutual relationships of the equilibrium constants during sorption and desorption on the course of affinity chromatography was also discussed in detail in Chapter 3 and it was clearly shown by Graves and Wu (Fig. 3.5). The effect of pH on the binding of lactate dehydrogenase on affinity sorbents such as N6-(6-aminohexyl)-5'-AMP-Sepharoseand 6-aminohexanoyl-NAD+-Sepharoseis shown in Fig. 5.15 (Lowe ef al., 1974b). Up to pH 8 the interaction of the enzyme with the immobilized ligand is independent of pH. Depending on the nature of the immobilized affinant, above pH 8 the amount of the bound enzyme decreases with pH. With N6-(6-

100

ao

CI

% a z

60

3 I-

z

3

40

0

$ 20

0

9.0

10.0

11.0

PH

Fig. 5.15. Effect of pH on the binding of pig heart muscle lactate dehydrogenase to (A) €-aminohexanoyl-NAD+-Sepharose and (B) N6-(6-aminohexyl)-AhfP-Sepharose. The results are expressed as percentages based on determinations at pH 6.5:(A) fl/flma at pH 6.5 determined from linear KCl gradient (0 to 1 M);(B) percentage of enzyme activity eluted by NADH pulse (200 pl, 5 mM). The equilibration buffers used were adjusted to a constant conductivity (3.3 m a - ' ) by the addition of 1M KCl, (0)10 mhfKH,PO,-KOH; (e) 10 mM tricine-KOH; (0)10 mhfglycine-KOH and (m) 10 mhf K,HPO,. The enzyme sample (100 rl) plus bovine serum albumin (1.5 mg) was applied to a column containing 0.5 g of N6-(6-aminohexyl)-5'-AMP-Sepharose (1.5 pmole/ml of AMP) according to the procedure reported in Fig. 4.6.Reproduced with permission from C.R. Lowe et al., Eur. J. Biochern.,

41 (1974)347-351.

COMPETITIVE LIGANDS

I1

aminohexy1)-5'-AMP-Sepharose this decrease is characterized by an apparent pK value of about 9.7, and with 6-aminohexanoyl-NAD+-Sepharoseby a pK of 8.5. This difference in pK values may be caused by the effect of different preparations of the affinity adsorbents. While in the former support an already spaced nucleotide, N6-(6-aminohexyl)-5'AMP, is attached to Sepharose, in the latter the attachment of NAD' to 6-aminohexanoylSepharose may leave residual charged groups on the carrier. Winer and Schwert showed that the binding of NAD' to lactate dehydrogenase in free solution is affected by the group on the surface of enzymes with pK = 9.7, which is in good agreement with the value determined for N6-(6-aminohexyl)-S'-AMP-Sepharose. In order to check this assumption, Lowe et al. (1974b) determined titration curves for 6-aminohexanoyl-NAD+-Sepharose, unmodified Sepharose, 6-aminohexanoyl-Sepharose and, for comparison, also for corresponding derivatives of cellulose, and on the basis of the curves obtained they demonstrated a distinct effect of the carrier (see Fig. 8.1). If the affinity adsorbent is prepared from a charged affinant, then a decrease in its affinity for the complementary macromolecule takes place when the ionic strength increases. For sorption a low ionic strength should be used, which then has the undesirable result of an increased amount of contaminating proteins; these are also increasingly sorbed owing to the ionic bond with the ligand. An example is the affinity chromatography of acetylcholinesterase (Kalderon et al.), discussed in Section 5.3. The application of a pH gradient and a salt gradient for the elution of specifically sorbed substances and the effect of the ionic strength on non-specific sorption are discussed further in Chapter 10. 5.7 ELUTION WITH COMPETITIVE AFFINITY LIGANDS A theoretical deduction of elution of enzymes by means of a competitive inhibitor has already been discussed in Chapter 3 (Fig. 3.6). The scheme for the formation of complexes in a system containing an isolated enzyme (E), specifically sorbed on an immobilized ligand (L), and a soluble inhibitor (I) is illustrated in.Fig. 5.16 (Akanuma et al.). The presence of the inhibitor in the mobile phase can affect the migration of the enzyme through the column in three different ways: (1) Competitive effect. If the ternary complex (ELI) is less stable than the binary complex (EI), the increase in the concentration of the inhibitor (I) increases the proportion of the enzyme in the mobile phase, thus reducing the retardation of the enzymes through the column. (2) Non-competitive effect. If the stability of the ternary complex (ELI) is approximately the same as that of the binary complex (EI), the binding of the inhibitor (I) will not affect the affinity of the enzyme for the immobilized ligand. In this instance the presence of an inhibitor will have no effect on the retardation of the enzyme. (3) Uncompetitive effect. If the stability of the ternary complex (ELI) is greater than that of the binary complex (EI), then the presence of the free inhibitor (I) will decrease the proportion of the enzyme in the mobile phase and thus increase the binding of the enzyme. If the ternary complex is less stable than the binary complex, an increase in the

AFFINANT-SORBENT BONDING

I8

I

MATRIX

C OMPETITIVE NONCOMP: NONC OMP UNCOMP:

H I

1

Fig. 5.16. Possible molecular species of a given enzyme within the affinity matrix. L, Covalently fixed affinity &and to matrix; I, unfixed inhibitor; E, enzyme. Reproduced with permission from H. Akanuma et al., Biochem. Biophys. Res. Commun., 45 (1971) 21-33.

concentration of the free affinity ligand will lead to the elution of the enzyme from the column. Many examples of elution with competitive ligands can be found in the literature. In Chapter 10 (Fig. 10.5),the isolation of trypsin and chymotrypsin from pancreatic extracts, carried out on Sepharose with bonded trypsin inhibitor, is described. Chymotrypsin was liberated using a solution of the competitive inhibitor of tryptamine, while trypsin was eluted with a benzamidine solution. A discussion was presented in Chapter 3, indicating that for the elution of enzymes from the affinity matrix with a solution of competitive inhibitor it is important that the latter should be present in a higher concentration than that of the affinant bound in the matrix. This is true under the assumption that both the free and the bound inhibitor have approximately equal affinities towards the isolated enzyme. Also, it is possible to elute the enzyme with a solution of affinant that actually has a higher affinity. In many instances of the elution of specifically sorbed enzymes with buffers that contain high concentrations of competitive inhibitor, the enzyme is obtained more diluted than if the elution is produced by changes in pH or ionic strength. This phenomenon is especially striking in interactions that involve hgh affinities, when elution from the affinity sorbent may represent a timedependent process even when high concentrations of the competitive affinant in the buffer are employed (Lowe and Dean, 1974). The rate of dissociation of the enzyme from the stationary phase is a first-order process, depending on the concentration of the complex alone, and independent of the concentration of the free competing inhibitor. The free substrate or the inhibitor reduces the tendency of the enzyme t o re-associate with the immobilized ligand by preferential formation of a soluble complex, assuming that they occur in a sufficiently high concentration.

79

COMPETITIVE LIGANDS

If the affinity of the complex is very high (KL < lo-' M), the time necessary for complete dissociation of the complex may be considerable. In order to decrease the amount of the enzyme bound t o the immobilized affinity ligand to half of the original value, the so-called half-life, f d 2 ,should be known, which is given by the expression tl/2 =

0.693 lnEo/[E] = -In2 -- k-1 k-1 k-1

where [El is the concentration of the free enzyme, Eo is the initial concentration of the bound enzyme and k-1 is the rate constant of dissociation of the complex: E

-t

L

k+i L E k-1

L

The dependence of the elution on time can be circumvented by temporarily interrupting the flow through the column after the soluble inhibitor has been soaked into it. The necessary time is given by the nature of k-1; as it is difficult to determine k-1, in practice this is done more or less empirically. An alternative means of enhancing specific elution consists in effecting simultaneous changes in pH, ionic strength and temperature. As the affinity of adsorption decreases with increasing temperature, the temperature increase may substantially affect the elution with the competitive inhibitor. The effect of various elution systems on lactate dehydrogenase bound to AMP-Sepharose (Ohlsson el al.) is evident from Fig. 5.17. While a quantitative elution of lactate dehydrogenase with .'no,

y;

../O

>

k

2

I-

U

a

J

a

E

x

T1ME.h

Fig. 5.17. Efficiency of different eluent systems on ox heart lactate dehydrogenase (LDH) bound to an AMP-Sepharose column (40 X 15 mm containing 1.0 g of wet gel). LDH (0.1 mg) in 0.5 ml of 0.1 M phosphate buffer, pH 7.5, was applied. The following systems in the same buffer were used: 0.5 mM NAD+ + 0.5 mM L-lactate ( 0 ) ;0.5 mM NAD++ 0.5 mM pyruvate (A); 0.5 mM oxidized NADpyruvate adduct (0);and 0.5 mM NADH (A). The arrows indicate a pulse of 2.0 ml of 10 mM NADH to permit elution of the remaining bound enzyme. Corrections were made for the inhibition effects in enzyme assays; 5.5-ml fractions were collected at a rate of 6 ml/h. Reproduced with permission from R. Ohlsson et al., FEBS Lett., 25 (1972) 234-238.

80

AFFINANT-SORBENT BONDING

0.5 mM NADH takes 1 h, only 3% of it is eluted after 20 h with 0.5 mM NAD’ + 0.5 mM L-lactate.

5.8 NONSPECIFIC EFFECTS

The efficiency of affinity chromatography is decreased mainly by non-specific sorption of inert substances. Non-specific effects are generally relatively weak and are therefore manifested mainly in interacting systems with low affinities. 5.8.1 Effect of ionic strength on non-specific sorption

Until recently, ion-exchange effects were considered to represent the main cause of non-specific sorption. It has been generally believed that by eliminating the ionic groups in the matrix material and the spacers used, the interference effects could be overcome. Of course, in many instances the affinity ligands themselves are ionic and can bind on the basis of ionic exchange. The studies of O’Carra et d. (1974a) have demonstrated, however, that a much more frequent cause of “mock affinity systems” is the non-ionic spacers which bind non-specifically, or rather non-biospecifically, the proteins on the basis of hydrophobic interactions. On closer scrutiny it became evident that these hydrophobic interactions can be made use of for the separation of a number of substances, and the term hydrophobic chromatography was introduced for this type of chromatography. It will be discussed in greater detail in Section 7.1. Morrow et d. developed a semi-quantitative theory to explain the non-specific binding of proteins on to substituted chromatographic affinity supports, caused by electrostatic and hydrophobic interactions. In Section 5.1 the isolation of Pgalactosidase from Escherichia coli, carried out on Sepharose with attached p-aminophenyl-P-D-thiogalactopyranoside inhibitor (Steers ef al.), was discussed in detail. The active sorbent was obtained when the inhibitor was bonded via a spacer prepared from bis(3-aminopropy1)amine and succinic anhydride. However, its activity was considerably decreased when a hydrophilic spacer was employed (O’Carra et al., 1974a). Robinson et ~ lmade . use of an affinity sorbent used for the first time by Steers et al. for the study of the effect of the ionic strength on the purification of @-galactosidase. The enzyme was bound to the adsorbent reversibly at an ionic strength of about 0.05. At a lower ionic strength (0.01), irreversible binding of the enzyme took place. On the basis of these results, Morrow et al. studied the effect of ionic strength on the equilibrium constant for adsorption and the rate constant for desorption by using 0-galactosidase as the enzyme and Sepharose with bonded bis(3-aminopropy1)amine as the sorbent. Fig. 5.18 shows the chromatography of Pgalactosidase on this sorbent in 0.05 M Tris-hydrochloric acid buffer containing 0.1 M sodium chloride and 0.01 M mag nesium chloride (pH 7.5). About 90% of the enzyme passed through the column unretained. Only 1% of the total amount of enzyme remained bound in the column by electrostatic bonds, and this could be eluted by applying a higher ionic strength, for example 1 M sodium chloride solution. From this chromatography, it can be concluded that under the given conditions the amine part of the ligand is not very operative in the adsorption of the enzyme. In an analogous chromatography of Pgalactosidase, the only

NON-SPECIFIC EFFECTS

81

z

Q

t

K I-

z

w

U

z

s w

s >

N

z

w

FRACTION

NUMBER

Fig. 5.18. Chromatography of Escherichiu coli pgalactosidase on Sepharose 4B substituted with 3,3’diaminodipropylamine.A sample (5 ml,containing 0.5 mg of enzyme) was applied to a column (109 X 16 mm) of substituted gel in pH 7.5,0.05 MTris-HC1 buffer containing 0.10 MNaCl and 0.01 M MgCI,. The flow-rate was 20 ml/h and fractions were collected at 3.5-min intervals. Reproduced with permission from R.M. Morrow et al., Biotechnol. Bioeng., 17 (1975) 895-914.

difference being that salts were not added to the 0.05 M Tris-hydrochloric acid buffer, almost all of the applied enzyme was bound in the column non-specifically; 85% of the enzyme was obtained on increasing the ionic strength of the buffer by addition of 1 M sodium chloride. The authors achieved very good results when the binding of 0-galactosidase on to the substituted gel was carried out at high ionic strength (1.8 M phosphate buffer), and when they used for the subsequent elution a decreasing linear gradient of ionic strength. 5.8.2 Extended Debye-Hiickel theory applied to the study of the dependence of the

ionic strength on the adsorption equilibrium constant and the rate of desorption of the enzyme from the substituted gels* When an enzyme (E) interacts with a ligand (L) bound on a gel surface with the formation of an enzyme-ligand complex, we have (eqn. 3.1):

E

+

L-EL

Further analysis is based on a model similar to the Debye-Huckel theory of the

*

See for list of symbols p. 86.

82

AFFINANT-SORBENT BONDING

solubility of proteins or the interaction of an inhibitor with active sites (Morrow era).). A prerequisite for further deductions is that the enzyme should not display any attraction toward the gel surface. This is not fulfilled with lysozyme and Sepharose. For the above reaction, the equilibrium dissociation constant, K, can be defined in terms of the activities, a ~aL, and aEL, of the enzyme, ligand and enzyme-ligand complex, respectively:

where YE, y~ and YEL are activity coefficients and cE, cL and cEL are the concentrations of single species. Because, in contrast to activity, the concentration (c) can be measured easily, Morrow e l al. defined the concentration equilibrium constant, K c , as CE cL

Kc = -

(5.6)

CEL

Eqn. 5.5 can then be re-written as Kc -

-- - ?EL YEYL

The interactions between the enzyme and the ligand are of both a electrostatic and a hydrophobic nature. The activity coefficient, ri,of a species i that has both electrostatic and hydrophobic properties is given as a modification of the extended Debye-Huckel theory:

where Is is the ionic strength of the solvent, Z i is the charge on the ionic species i and Ki is a constant representing hydrophobic effects. Constants A and B are functions of the dielectric constant, D, of solution temperature, T("C), and ionic radius, a (A):

-

A = 1A246 106/(DT)3/z

B = 50.29 a/(DT)llZ

(5 -9) (5.10)

It can be assumed that during the interaction of the enzyme with the ligand the charge of the enzyme-ligand complex is equal t o the sum of the charges on the enzyme and the ligand: (5.1 1)

and that A and B are constant for all species. Then eqn. 5.8 can be re-written for activity coefficients of all kinds as (5.12)

83

NON-SPECIFIC EFFECTS

where KO = KEL - KE - K L

(5.13)

can be positive, negative or zero. Thus the logarithm of the measured equilibrium constant [log(Kc/K)] will increase with increasing ionic strength for low values of the ionic strength of the solvent, I,, and will decrease with increasing I, if KO < 0. If KO> 0 for large values ofl, log(K,/K) will continue to increase with increasing I,, while for KO= 0, log(K,/K) will remain constant with increasing I,: (5.14)

The measured values of the equilibrium constants as a function of ionic strength for the desorption of 0-galactosidase are shown in Fig. 5.19, and are in good agreement with

6o

40

IONIC STRENGTH, I,

Fig. 5.19. Equilibrium constant ratio versus ionic strength for the desorption of pgalactosidase in potassium phosphate buffer, pH 7.5. Reproduced with permission from R.M. Morrow e? al., Biotechnol. Bioeng., 17 (1975)895-914.

84

AFFINANT-SORBENT BONDING

the prediction based on eqn. 5.12. Constants A and B , and also K O ,are usually considered as semi-empirical parameters. From Fig. 5.19, it is evident that the linear region predicted by eqn. 5.14 begins at an ionic strength higher than 0.8. For lower ionic strengths the binding is primarily electrostatic. This is true when the term dominating K , is the first Debye-Huckel term on the right-hand side of eqn. 5.12. The slope of the straight-line portion of the curve is the hydrophobic binding constant, K O ,in eqn. 5.14. The intercept of this straight line is the term -~AZEZL/B. A similar experiment carried out with serum albumin and lysozyme showed a continuous increase in the binding constant of serum albumin with increasing ionic strength. Even at high ionic strength it indicates K O > 0, while with lysozyme K O it is evidently very close to zero, as shown by the constant value of K , even for high values of I,. In order to determine whether equal values of K , will be obtained when the enzyme is bound first at very low ionic strength and then the ionic strength is gradually increased, and whether re-equilibration will take place, Morrow et al. carried out the experiment illustrated by Fig. 5.20. Curve A refers t o the enzyme bound originally at I, = 1.8 and curve B t o the enzyme bound at very low ionic strength. The effect of hysteresis is clearly evident and hence the difference exists depending on the direction in which the experiment was carried out. Nevertheless, the results of both experiments agree with the prediction of eqn. 5.12. From a knowledge of the total effective concentration of the ligand capable of binding the enzyme (8 pmole per millilitre of gel), the distance between the arms of the ligand bound to the gel, which is about 29 A, can be determined. As the diameter of the P-galactosidase molecule is approximately 140 A it can be deduced that approximately 19 spacer arms can interact with each protein molecule. This explains the high possibility of non-specific electrostatic and hydrophobic reactions taking place in these substituted gels. If an inhibitor is bound to these spacers, a more specific interaction can be assumed at lower substitution of the gel with the ligand. Using a continuously stirred tank and an extended Debye-Huckel theory, MOKOWef al. proved that the adsorption of the enzyme is more reversible at lower ionic strengths, while at higher ionic strengths it is substantially irreversible. With a decreasing linear gradient of the ionic strength, the validity of the derivation of the desorption of the enzyme in a continuously stirred tank has been deduced as a function of time. It was further shown that the presence of other proteins, for example haemoglobin, does not affect the above results. OCarra et al. (1974a) studied glyceraldehyde-3-phosphatedehydrogenase sorbed on the polymer:

and observed that the amount of the elutable enzyme is dependent on the time during which the enzyme is in contact with the substituted support. The yield of the enzyme elutable with NAD+ becomes progressively lower the longer the enzyme remains adsorbed on the gel before elution. The adsorption of the enzyme is not appreciably affected by high concentrations of potassium chloride, the enzyme is purely eluted with 2 mM NAD+

NON-SPECIFIC EFFECTS

16

0

04

85

a8

L2

1.6

J

IONIC STRENGTH, I,

Fig. 5.20. Hysteresis effect of K, versus ionic strength for g-galactosidase. (A) Initial binding at high ionic strength; (B) initial binding at low ionic strength. Reproduced with permission from R.M. Morrow e f al., Biofechnol. Bioeng., 17 (1975) 895-914.

if the elution buffer contains a 0.2 M concentration of salt. Under such conditions, the enzyme is not sorbed either on the support with attached spacer or after replacement of NAD+ with NADP+. From this result, it can be concluded that by its nature the sorption is biospecific. The decrease in the amount of elutable enzyme with the time of its contact with the specific sorbent leads to the hypothesis that this decrease is due to nonbiospecific adsorption on to the hydrophobic spacer arm after it has been previously bound biospecifically on immobilized NAD+.The correctness of this hypothesis is supported by the results obtained with an affinity sorbent in which the hydrophobic spacer was replaced with a hydrophilic spacer. The sorbent prepared in this manner retained its

86

AFFINANT-SORBENT BONDING

strong affinity for glyceraldehyde-3-phosphatedehydrogenase, while the yield of the enzyme eluted with NAD’ solution was greater than 90% and did not decrease significantly even when the enzyme was allowed t o stand in the column for 1% h before elution. After the same time, the yield of NAD’-elutable enzyme which was sorbed on a sorbent with hydrophobic spacer decreased t o almost 10%. Although the non-biospecific adsorption (O’Carra et al., 1974a) generally represents an undesirable complication in affinity chromatography (in the sense of bioaffinity sorption), many examples have been described in which it contributed usefully to an increase in the affinity of weak bioaffinity systems. In such instances a suitable choice and a careful control of the conditions may preserve the predominance of the biospecific nature of the interaction suitably supported by the non-biospecific interaction, so that the latter does not become dominant. Such enhanced bioaffinities behave very similarly to strong bioaffinities and the enzyme can be purely eluted by a biospecific counter ligand. For this effect, OCarra e t al. (1974a) introduced the term “compound affinity”. From the above discussion, it follows clearly that many factors affect the interaction of the immobilized ligand with the complementary molecule. Both biospecific and nonbiospecific sorptions are based, in principle, on the same nature and combination of electrostatic and hydrophobic interactions. The contribution of non-biospecific interactions can best be determined from a comparison of the dissociation constants of the complex of the isolated macromolecule with the immobilized affinity ligand, and with the same ligand in the solution used for elution, as shown in Chapter 4. This characterization of the affinity system is very necessary, especially if affinity chromatography is used not only for isolation purposes but also for the study of the specific interactions that take place in biochemical processes. 5.8.2.1 List of symbols used

Constant in the Debye-Hiickel theory; Ionic radius (A) Activities of enzyme, ligand and enzyme-ligand complex, respectively; Constant in the deb ye-Hiickel theory; Concentrations of enzyme, ligand and enzyme-ligand complex, respectively (molell); Total effective ligand concentration (molell); Dielectric constant of the solution; Enzyme; Enzyme-ligand complex; Ionic strength of the solvent; Hydrophobic constant for the ith species (e.g., K E , K L , K E L ,K x ) ; Binding constant when I, = 0; eqn. 5.14; Binding constant; eqn. 5.6; KEL - K L - K E , eqn. 5.13; Ligand; Time (min); Charge on the ith species (e.g., ZE,ZL,ZEL,2x1; Activity coefficient of the ith species (e.g., TE, TL, TEL, 7 ~ ) .

REFERENCES

87

REFERENCES Akanuma, H., Kasuga, A., Akanuma, T . and Yamasaki, M., Biochem Biophys. Res. Commun., 45

(1971)27-33. Comer, M.J., Craven, D.B., Harvey, M.J., Atkinson, A. and Dean, P.D.G., Eur. J. Biochem., 55 (1975)

201-209. Craven, G.R., Steers, Jr., E. and Anfiisen, C.B.,J. Biol. Chem., 240 (1965)2468-2477. Cuatrecasas, P., J. Biol. Chem., 245 (1970)3059-3065. Cuatrecasas, P., Wilchek, M. and Anfiisen, C.B., Proc. Nut. Acad. Sci. US.,61 (1968)636-643. Dean, P.D.G., Craven, D.B., Harvey, M.J. and Lowe, C.R., Advan. Exp. Med. Biol., 42 (1974)99-121. Graves, D.J. and Wu, Y.-T., Methods Enzyrnol., 34 (1974) 140-163. Harvey, M.J., Lowe, C.R., Craven, D.B. and Dean, P.D.G., Eur. J. Biochem., 41 (1974a)335-340. Harvey, M.J., Lowe, C.R. and Dean, P.D.G., Eur. J. Biochem., 41 (1974b) 353-357. Hipwell, M.C., Harvey, M.J. and Dean, P.D.G., FEBSLett., 42 (1974)355-359. Hixson, Jr., H.F. and Nishikawa, A.H., Arch. Biochem. Biophys., 154 (1973)501-509. Hofstee, B.H.J., Biochem Biophys. Res. Commun., 50 (1973)75 1-757. Holguin, J. and Cardinaud, R., Eur. J. Biochem., 54 (1975)505-5 14. Holroyde, M.J. and Trayer, I.P., Biochem. SOC.Trans., 2 (1974)1310-1311. Holroyde, M.J., Chesher, J.M.E., Trayer, I.P. and Walker, D.G., Biochem J., 153 (1976) 351-361. Kalderon, N., Silman, I., Blumberg, S. and Dudai, Y., Biochim. Biophys. Acta, 207 (1970)560-562. Kasai, K. and Ishii, S., J. Biochem. (Tokyo), 77 (1975)261-264. Lowe, C.R. and Dean, P.D.G.,FEBS Lett., 18 (1971)31-34. Lowe, C.R. and Dean, P.D.G., Affinity Chromatography, Wiley, New York, London, 1974,pp. 272. Lowe, C.R., Harvey, M.J., Craven, D.B. and Dean, P.D.G., Biochem J., 133 (1973)499-506. Lowe, C.R., Harvey, M J . and Dean, P.D.G., Eur. J. Biochem., 41 (1974a) 341-345. Lowe, C.R., Harvey, M.J. and Dean, P.D.G., Eur. J. Biochem., 41 (1974b) 347-351. Morrow, R.M., Carbonell, R.G. and McCoy, B.J., Biotechnol. Bioeng., 17 (1975)895-914. Nishikawa, A.H., Bailon, P. and Ramel, A.H.,Advan. Exp. Med. Biol., 42 (1974)33-42. O’Carra, P., Barry, S . and Griffin, T., Biochem. SOC. Trans., 1 (1973)284-290. O’Carra, P., Barry, S . and Griffii, T.,Methods Enzymol., 34 (1974a) 108-126. O’Carra, P., Barry, S. and Griffin, T., FEBS Lett., 43 (1974b) 169-175. Ohlsson, R., Brodelius, P. and Mosbach, K., FEBS Lett., 25 (1972)234-238. Robinson, P.J., Dunnill, P. and Lilly, M.D.,Biochim Biophys. Acta, 285 (1972)28-35. Schmidt, J. and Raftery, M.A., Biochemistry, 12 (1973)852-856. Steers, E., Cuatrecasas, P. and Pollard, B.,J. Biol. Chem., 246 (1971) 196-200. Winer, A.D. and Schwert, G.W.,J. Biol. Chem., 231 (1958) 1065-1083.

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89

Chapter 6

Choice of affinity ligands for attachment 6.1 HIGHLY SPECIFIC AND GROUP-SPECIFIC MATRICES A compound is a suitable affinant for the isolation of biologically active products if it will bind these products specifically and reversibly. Hence, depending on the different nature of biologically active products, affinants represent very different types of chemical compounds. Their classification can therefore be based on biochemical function rather than chemical structure. A review of affinants used for the isolation of enzymes, inhibitors, cofactors, antibodies, antigens, agglutinins, glycoproteins and glycopolysaccharides, nucleic acids, nucleotides, transport and receptor proteins, hormones and their receptors, lipids, cells, viruses and other substances is given in Chapter 11 (Table 11.1). Affinity ligands with very narrow specificities are also included in that review. For example, when an inhibitor specific for a single enzyme is attached to the support, a sorbent is formed that is specific just for that enzyme. However, the use of specific ligands requires a different and often very tedious synthesis of the sorbent for each separation. Not all affinants that are suitable for a complementary binding of macromolecules also have suitable functional groups for their attachment to a solid support. These groups must first be introduced into the affinant, as well as suitably long spacing arms, indispensable mainly with low-molecular-weightaffinity ligands, necessary to permit bonding interactions. The practical utilization of specific sorbents increases if, instead of the narrowly specific ligands, a so-called “general ligand” (Mosbach) is used for their preparation. As is implied by the name, a group-specific matrix prepared in this manner displays affinity for a larger group of biological macromolecules. For example, the enzymes related to the metabolism of aspartic acid show group-specific adsorption affinity to N-(a-aminohexy1)-L-asparticacid-Sepharose. On this immobilized affinant, asparaginase, aspartase, aspartate-0-decarboxylase and asparaginase modified with tetranitromethane (Tosa et aZ.) could be sorbed. In group-specific affinants, each individual enzyme does not necessarily distinguish the same part of the immobilized ligand in the same manner. Thus, for example, if the ligand is common to several enzymes and if it can be immobilized in various ways, affinity chromatography may give an idea of the nature of the interaction of each individual enzyme with the attached affinity ligand. Table 4.2 which shows the difference in the binding of various dehydrogenases and kinases on 5‘-AMP bound to Sepharose also shows that for the interaction with the enzyme either the free phosphate group or the free adenosine part of the affinant was accessible. The phosphate part of the nucleotide is essential for the binding of, for example, alcohol dehydrogenase and glycerokinase, and it has a completely different role in the interaction of the nucleotide with myokinase or glyceraldehyde-3-phosphatedehydrogenase, where, on the contrary, the adenosine part of the affinant is essential for the interaction. A serious limitation of the use of general ligands in affinity chromatography is their

AFFINITY LIGANDS

90

low selectivity. Therefore, further means are necessary for the differentiation of a complex mixture of enzymes which can be adsorbed. If the immobilized affinity ligand shows affinity t o more than one complementary molecule, then the specific shape of the adsorption isotherm has important consequences. Fig. 6.1 gives as an example adsorption isotherms for four enzymes, each of which displays different affinities for the immobilized affinant (Lowe and Dean). Enzyme 1 possesses a very high affinity for the specific sorbent with a dissociation constant of 10-7-10-8 M. Enzymes 2 and 3 have affinity for sorbents with dissociation constants of about lo-’ M, and enzyme 4 shows a very weak affinity with a dissociation constant of > lo-’ M . For the generalized Langmuir adsorption isotherm

where qj is the specific amount of the adsorbed substance i, Ci is concentration and kl and k2 are constants. For low concentrations of Ci, eqn. 6.1 reduces t o qj = klkzCj, and for high concentrations ofCi to ~i = k2. In general we can write

where n = 0-1. It then follows that when the concentration of the ligand is sufficiently high, so that the adsorbent capacity is not a limiting factor, the specific amount of the adsorbed substance i, 4i,is dependent on its concentration in the mobile phase, Ci, and

ENZY.ME C O N C E N T R A T I O N

(0

Fig. 6.1. Adsorption isoaerms for four enzymes interacting with a single imnobilized affinity :igand. Reproduced with permission from C.R.Lowe and P.D.G.Dean, Affinity Chromatography, Wiley, New York, London, 1974, p. 91.

SPECIFIC MATRICES

91

not on its affinity towards the attached affinant. For a sample containing equimolar amounts of four enzymes, the amount of each of them adsorbed will be q l , q2,q3 and 44. In displacement elution, using a concentration D of the displacer, enzymes 1 , 2 and 3 with concentrations C1,C, and C3will be eluted. Enzyme 4 will appear before the displacing solution because its adsorption isotherm is not intersected by the displacer line. An enzyme with a high affinity does not displace a less strongly bound enzyme even when, after the initial adsorption, a further amount of enzyme of high affinity is added. If the capacity of the adsorbent is exceeded, enzymes will appear in the retention volume of the eluate with both a high and a low affinity, i.e., not only those which are weakly adsorbed. This consequence is important in view of the differentiation of enzymes that display affinity towards general ligands. Sometimes it becomes necessary to eliminate the contaminating proteins before adsorption on a specific adsorbent by inserting the preceding fractionation step. If the conditions of adsorption, such as pH, ionic strength, temperature, flow-rate and dielectric constant, are changed some enzymes can be specifically excluded. Further, an inhibitor or other ligands can be added in order to prevent the adsorption of some enzymes. The use of a solid support with small pores can exclude proteins with a high molecular weight. Increased selectivity can be further achieved by using specific methods of elution. A knowledge of inhibitors or substrates of various enzymes can be utilized for the selective elution of individual enzymes. In Chapter 10, examples are given of the separation of mixtures of enzymes bound to group-specific sorbents utilizing pH, ionic strength or temperature gradient. The selectivity of affinity ligands can also be affected by the nature of the solid support (Fritz et al.). Proteolytic enzymes bound to a negatively charged copolymer of maleic acid with ethylene sorbed only inhibitors, the isoelectric points of which were below 4-5. If the strongly negative charges of the copolymer chain were neutralized by attachment of, for example, hexamethylenediamine and dimethylethylenediamine, the polyamphoteric derivative formed became suitable even for the isolation of inhibitors with lower isoelectric points. As is discussed in detail in Section 6.3, antibodies show a high affinity for corresponding antigens and vice versa. Difficulties with their liberation from complexes ensue from the strength of this interaction. The use of strongly chaotropic eluents in immunoaffinity can be circumvented by chemical modification of the immobilized affinity ligand (Murphy et al.). For example, the elution of anti-glucagon antibodies from a column of immobilized glucagon can be achieved under milder conditions if the steric complementarity to the binding site of the antibody is partly perturbed by selective modification of the hormone, for example by reaction with 2-hydroxy-hitrobenzyl bromide, tetranitromethane or hydrogen peroxide. O'Carra recommends differentiating affinity systems with small ligands and with macroligands. Low-molecular-weight synthetic affinants are advantageous mainly owing to their stability and better accessibility. The specific sorbents prepared from them are usually better characterized, because they are attached via a pre-defined functional group. In order to increase their steric accessibility, a spacer is inserted, in most instances between them and the surface of the solid support. High-molecular-weight affinants are predominantly proteins or nucleic acids. They often undergo denaturation leading to an

92

AFFINITY LIGANDS

irreversible loss of activity, and for them the method of attachment is usually not unambiguously defined.

6.2 ISOLATION OF ENZYMES, INHIBITORS AND COFACTORS Affinity ligands for the isolation of enzymes can be competitive inhibitors, substrates and their analogues, products, cofactors and alosteric effectors, and also antibodies or compounds that contain metal ions or SH groups, as is evident from Table 11.l. As an example of the use of a competitive inhibitor, the isolation of chymotrypsin from a crude pancreatic extract is shown in Fig. 6.2, where the specific sorbent was prepared by attachment of N-benzyloxycarbonylglycyl-D-phenylalanine t o Spheron via 1i3,

1

1

I

I

II

I B

6

PH 4

E 0

8. W

2

u z

a m a

sm a

nc

'I 1A

8

I,

6

PH

4

I 120

.

ci'. +

.

-n

Lzliy-u-rne-Nn,-apneron. A iuu-mg sample of the active pancreatic extract was applied to the column (60 X 15 mm) which was eluted with an aqueous solution of ammonium formate (0.05 M formic acid solution was treated with 25% aqueous ammonia t o a final pH of 8.0). Fractions (6 ml) were collected at 20-min intervals. -, Absorbance at 280 nm; .. pH. (a) Contaminants and trypsin; (b) chymotrypsin; (c) complex of chymotrypsin with lung trypsin inhibitor. In A the arrow ihdicates the change in pH from 8.0 to 3.5 (0.1 M solution of formic acid whose pH had been adjusted t o 3.5 with ammonia), and in B the addition of 20 mg of trypsin inhibitor in 5 ml of aqueous solution of ammonium formate. Data from J. TurkovL ef al., Biochim. Biophys. Acta, 427 (1976) 586-593. uuwairarul;laplly

- - a # ,

UL

sruus: pan~~uanr: exrracr on

2

"

l

93

ENZYMES, INHIBITORS AND COFACTORS

hexamethylenediamine (Turkovii et al., 1976). Elution of the enzymes is carried out both by a change in pH (Fig. 6.2A) or specifically by elution with a solution of competitive trypsin inhibitor (Fig. 6.2B). In order to obtain chymotrypsin from its complex with soluble trypsin inhibitor, an additional gel chromatographic step in acidic medium was necessary. The amount and the activity of chymotrypsin obtained in this way virtually coincided with the amount and the activity of chymotrypsin obtained after simple freezedrying of the material from peak b (Fig. 6.2A). For many DNA-specific enzymes, single-stranded DNA serves as a substrate that can be used as an affinant for a series of enzymes, as is evident from Table 11. l . A very efficient DNA-agarose was prepared, for example, by Schaller et al. It was characterized by a high concentration of DNA, and its non-specific adsorption could be minimized by a suitable choice of conditions. An example of the use of DNA-agarose for the isolation of RNA polymerase is given in Fig. 10.9. Further examples of the use of substrates for the isolation of enzymes are the affinity chromatography of cytochrome oxidase on Sepharose with attached cytochrome C (Ozawa el al.) and protocollagen proline hydroxylase on Bio-Gel A with attached reduced and carboxymethylated collagen (Berg and Prockop). For the broad area of reactions catalysed by enzymes, coenzymes have the function of co-substrates. Such enzymes will contain at least two specific binding sites, one of them for the coenzyme, which will be common to all of them, and one or more for the substrate. The latter site will then be dependent on the nature of the substrate and the catalysed reaction. The immobilized coenzymes will then sorb selectively those groups of enzymes which are utilized in bi- or multi-substrate reactions. Bi-substrate enzymic reactions can be of two types (Lowe and Dean):

(1) Bi-substrate compulsory order:

(2) Bi-substrate random order:

A

B

P

I

4

t

E

E

EA

EAB

AIB)

B(A)

i

4 EA(B)

E A B G==

Q

t

EPQ

EQ

E

P(Q)

t

EPQ

alp)

t EQIP)

E

where E represents the enzyme and A, B, P and Q are reactants or products. (1) In an ordered mechanism, ligand A is bound compulsorily earlier than ligand B can react with the binary complex formed. (2) In the random mechanism, ligands A and B can be bound independently of each other in any order. In this instance, the choice of the ligand will depend on the strength of the affinity for the enzyme and the relative ease of immobilization. For a directed mechanism, the immobilization of ligand A will be subject to the usual limitations of affinity chromatography. In this instance the immobilization of ligand B will not create a competing adsorbent for the complementary molecule, except when ligand A is present in the irrigating buffer. That is, the presence of ligand A is necessary for the binding of enzymes to the adsorbent, because ligand B binds the binary ligand A-enzyme complex. By this process a preliminary selection can be introduced in chromatography, and the subsequent elution can be achieved by elimination of ligand A.

AFFINITY LIGANDS

94

The ordered kinetic sequence of most pyridine nucleotide-dependent dehydrogenases takes place according t o the scheme NAOH

P

i

i

E

ENADH

s t ENAD!+

P

ENAD* S

NAO'

t ENAD+

E

where E is dehydrogenase, NAD' and NADH are the oxidized and the reduced nicotinamide adenine dinucleotide, P is the product and S the substrate. Fig. 6.3 shows the affinity chromatography of glucosophosphate dehydrogenase from human red cells (Yoshida). The enzyme associated with NADPH was sorbed effectively on agarose on which NADP was attached via adipic acid dihydrazide. The enzyme was eluted specifically from the column with NADP in the elution buffer. The coenzyme mentioned is one of the most commonly used in affinity chromatography. Other frequently employed affinants are nucleotides of adenine, uridine, guanine and flavine, coenzymes of pyridoxal, folate and its analogues, biotin, lipoic acid, cobalamines

7

14

W

f

:

3-

W

-

L

2

E L U T I O N V O L U M E . ml

Fig. 6.3. Chromatography of NADPH-bound glucose 6-phosphate dehydrogenase on an agarose-NADP column. Partially purified NADPH-bound enzyme (10 ml, 41 units) was placed on an agarose-NADP column (50 X 5 mm, bed volume 1 ml) equilibrated with 0.01 MTris-HC1, pH 8.0, that was 10 mM in MgCl,. After the enzyme solution had been applied (at the single arrow), the column was washed continuously with buffer. Then (at the double arrow) the column was eluted with buffer that was 1 mM in NADP. The flow-rate was 0.4 ml/min and the chromatography was carried out at 25°C. Reproduced with permission from A. Yoshida,J. Chromatogr., 114 (1975) 321-327.

IMMUNOAFFINITY CHROMATOGRAPHY

95

and porphine derivatives. Examples of their use are given in Table 1 1.l, and they are reviewed and discussed in detail in the book by Lowe and Dean. As an example, the affinity chromatography of alcohol dehydrogenase and phosphofructokinase from a partly purified extract of Bacillus stearothermophilus on N6(6-aminohexyl)-5’-AMP--Sepharoseis illustrated in Fig. 6.4 (Comer et al.). The enzymes are eluted with a pulse of 5 mM NADH (alcohol dehydrogenase) and a pulse of 5 mM ATP-Mg2+ (fructokinase) (Fig. 6.4A), and further with a gradient of KC1 (Fig. 6.4B) and a pH gradient (Fig. 6.4C). An example of the use of an allosteric effector for the preparation of a specific adsorbent is the use of the p-aminophenyl ester of dATP bound to Sepharose for the isolation of T4 ribonucleotide reductase (Berglund and Eckstein). For the isolation of ADP glucosopyrophosphorylase from Escherichia coli, Haugen et al. used affinity chromatography on Sepharose with attached P’(6-phospho-1 -hexyl)-P2(6-amino- 1-hexyl)pyrophosphate, resembling the allosteric activator. An example of the isolation of an enzyme using attached antibody is the affinity chromatography of alkaline phosphatase on Sepharose with covalently bound anti-alkali phosphatase antibody (Pitarra et al.). The isolation of enzymes by means of free SH groups is discussed in Section 6.6. For the isolation of inhibitors and cofactors, bound enzymes are most commonly used as affinants. Fig. 6.5 shows the isolation of chymotryptic inhibitor from the crude extract from potatoes on Spheron with attached chymotrypsin (Fig. 6.5B) in comparison with analogous chromatography on unmodified Spheron (Fig. 6.5A). The latter chromatography was carried out in order to check whether, under the given experimental conditions, elution of the material non-specifically sorbed to a solid support does not take place (Turkovri et al., 1973). If the inhibitor contains glycosyl terminal groups, concanavalin A can be used for its isolation. An example is the isolation of al-antitrypsin from human serum on Sepharose with attached concanavalin A (Murthy and Hercz). Myerowitz et al. (1972a,b) obtained considerably enriched al-antitrypsin from human and mouse serum by eliminating the contaminating albumin by immunoadsorption on Sepharose with attached anti-albumin antibody.

6.3 IMMUNOAFFIMTY CHROMATOGRAPHY The interaction of antibodies with their antigens is comparable, in its specificity, to the binding of substrates with enzymes. The dissociation constants are most commonly within the range 10-5-10-8 M (Murphy et al.). For the isolation of antibodies, antigens or haptens (chemically modified groups which are used as immunoagents after their attachment on proteins or synthetic polypeptides, for example) against which they were induced are used. In Table 1 1.1 a series of examples is given. Antibodies induced by a certain antigen are characterized by considerable heterogeneity. When simple chemically defined haptens are used, the reasons for this heterogeneity may be the following (Lowe and Dean): (1) The haptens may be attached to various parts of the carrier molecule, and consequently they are surrounded by a different micro-environment. This can be partly circum-

AFFINITY LIGANDS

96

3 !O 2 10 1

10

40

C

-20

-11 -10

-9

I,

-10

-8

1

-7

ELUTION VOLUME.ml

Fig. 6.4. Specific elution of alcohol dehydrogenase and phosphofructokinase from N6.(6-aminohexyl)5’-AMP-Sepharose. (A) The enzyme extract (0.5 ml) (40.5 U of phosphofructokinase, 10 U or 9.9 mg of alcohol dehydrogenase per millilitre) was dialysed exhaustively against 10 mM phosphate, pH 6.8, containing 0.2 M KCl (this buffer was also used for equilibration) and adsorbed on a column of the AMP-Sepharose. The matrix was then washed sequentially with (a) equilibration buffer (24 ml); (b) 5 mhf NADH in the same buffer (5 ml); (c) buffer (5 ml); (d) 5 mM ATP, 5 mM Mg’+ in buffer (5 ml), flow-rate 0.4 ml/min. (B) Using a KCl gradient with conditions essentially as in A. Following adsorption, the column was washed with 10 mM phosphate, pH 6.8, (20 ml) containing 0.2 M KC1. The linear gradient (0.2 to 0.8 MKCl) (40 ml) was applied in the same buffer. (C) Using a pH gradient with conditions essentially as in A except that the sample was equilibrated against 10 mM N-2-hydroxyethylpiperazine-N’-2ethanesulphonate (pH 6.8). Following adsorption, the column was washed with the same buffer (20 ml). A pH gradient (40 ml) was then applied (10 mM hydroxyethylpiperazine-ethane sulphate, 5 mMglycylglycine, pH 10.4). (a) Protein; (0)glyceraldehyde-3-phosphate dehydrogenase; (A)alcohol dehydrogenase; (m) phosphofructokinase. Reproduced with permission from M.J. Comer et al., Eur. J. Biochem.,55 (1975) 201-209.

97

IMMUNOAFFINITY CHROMATOGRAPHY I

PH 10 8 6

4

2

F R A C T I O N NUMBER

Fig. 6.5. Chromatography of a crude extract of potatoes on (A)-Spheron 300 and (B) Spheron 300 chymotrypsin columns (20 X 18 mm). A 3-g amount of cmde extract of potatoes was applied to the column and 10-ml fractions were collected at 1-h intervals. -, Absorbance at 280 nm; - -,inhibitor activity; ...-.,pH value. Vertical arrow, elution buffer changes from pH 8.0 (0.2 M Tris-HC1 buffer) to pH 2.0 (0.2 M KCl-HCl). Data from J. TurkovP et al., Biochim Biophys. Acta, 322 (1973) 1-9.

-

vented by using carrier proteins containing a single amino acid capable of a binding reaction, or by employing a synthetic polypeptide containing a single type of amino acid. ( 2 ) The hapten can be oriented in various ways with respect to the surface of the antigen molecule. (3) The antibodies may be directed against various parts of the hapten molecule. With antibodies against proteins, the situation is much more complex because the protein contains various antigenic groups that are far less defined than in the case of simple haptens. Further, serum contains several classes of proteins with antibody activity, such as IgG, IgM and IgA immunoglobulins. The heterogeneity of the antibody binding sites results in a spectrum of dissociation constants for the antigen-antibody combinations. On binding of antigen to a solid support, a specific immunoadsorbent is formed, which should possess the following properties: (1) It showed be able to adsorb the complementary antibody from a mixture of componen ts. (2) The liberation of the adsorbed antibody from the specific adsorbent should be quantitative and carried out under conditions harmless for the specific antibody activity.

98

AFFINITY LIGANDS

(3) It should possess a high capacity for the adsorption of the specific antibody. (4) It should retain its biological activity after repeated use and storage. ( 5 ) It should possess adequate mechanical properties, permitting centrifugation, filtration and use in a column. The fulfilment of these requirements is not dependent on the quality and the amount of the bound antigen only, but also on the nature of the solid support and the nature of the bond. If, during the isolation of anti-glucagon antibodies on Sepharose with attached glucagon (Murphy et ~ l . )a, serum with a low titration value (200-300) was applied on to the column, the antibody activity could be eluted with 0.15 M sodium chloride solution adjusted to pH 11 with aqueous ammonia both in one step and in a gradient. A total separation from non-specifically sorbed proteins was achieved in the latter instance only. If the titration value of the antiserum was about 700, release of the antibodies from the column occurred only when 30 column volumes of 0.15 Msodium chloride solution of pH 11 had passed through it. If the titration value of the applied antiserum was about 1000, antibodies could not be eluted even with a 100-fold column volume of 0.15 M sodium chloride solution of pH 11, and they began t o leave the column only with the solvent front of 0.1 M acetic acid the pH of which was adjusted to 2.2 with formic acid. However, if the titration value of the applied antiserum was in the range 1400-9000, not even a 100-fold column volume of 0.1 M acetic acid of pH 2.2 could release the antibodies from the column. The antibodies were eluted only when 4 M guanidine hydrochloride was applied. These results were achieved with antibodies obtained from the same animal at various periods during the immunization programme. Similar behaviour was also observed with antibodies of various rabbits. When a modified glucagon was used for the preparation of the specific adsorbent, prepared by oxidation with hydrogen peroxide, alkylation with 2-hydroxy-5-nitrobenzyl bromide or nitration with tetranitromethane, the application of much milder conditions sufficed for the elution of the antibodies from the complex with the immobilized affinant. Whereas 4M guanidine hydrochloride was necessary for an efficient elution (peaks appeared in 2 column volumes) of columns of immobilized, unmodified glucagon, an analogous elution of columns of immobilized glucagon modified by oxidation with peroxide, alkylation with 2-hydroxy-5-nitrobenzyl bromide, or nitration with tetranitromethane, could be effected by 0.15 M NaCl at pH 1 1. Immobilized antibodies are used, on the contrary, for the isolation of antigens. Antigens may belong to the most varied types of substances and therefore Table 11.1 presents antibodies as affinants for the isolation of many different compounds. In Fig. 6.6, the isolation of human leucocyte interferons on Sepharose with attached corresponding antibodies is given as an example (Anfinsen et d.).Interferons are glycoproteins with a molecular weight of about 25,000 which possess a genus- or family-specific antiviral activity against a broad spectrum of viruses. The use of immobilized antibodies is discussed further in Section 6.7 from the point of view of their use for the isolation of specific peptides. The use of immobilized antidinitrophenyl antibodies for the isolation of trypsin in a complex with dinitrophenylated soybean trypsin inhibitor (Wilchek and Corecki) is also interesting. An important role is also played by immobilized antibodies in the isolation of cells, and this problem is discussed in detail in Section 6.10. However,

LECTINS, GLYCOPROTEINS AND SACCHARIDES

FRACTION

99

NUMBER

Fig. 6.6. Purification of human leukocyte interferon (2.3.10' units) on an anti-leukocyte interferon column (110 X 20 mm). After loading the sample, the column was washed with phosphate-buffered saline and then with Mc Ilvaine phosphate-citrate buffer pH 3.8, containing 500 rg/ml of cytochrome C Interferon was eluted with a pH gradient, starting as indicated by the arrow. About 80% of the interferon units was recovered in the two peaks of interferon activity. Reproduced with permission from C.B. Anfinsen et al., Proc. Nat. Acad. Sci. US.,71 (1974)3139-3142.

the use of immunoadsorbents is not limited to isolations only, but can also be useful in the detection of structural differences, for example between nuclear and mitochondrial dehydrogenases (Di Prisco and Casola).

6.4 ISOLATION OF LECTINS, GLYCOPROTEINS AND SACCHARIDES

Lectins are proteins or glycoproteins of vegetable (phytohaemagglutinins) or animal origin displaying a higher or lower selective affinity for carbohydrates or groups of carbohydrates. These proteins, which resemble antibodies, react with cell membrane components and agglutinate erythrocytes, tumour and embryonal cells (Lowe and Dean). Agglutinins with a defined specificity for sugars may thus be a useful means for the study of surface structures of cells transformed by malignant tumours or by viruses. The isolation of a series of lectins by affinity chromatography is mentioned in Table 11.I. For the isolation of a number of lectins commercially available carbohydrate polymers have been used, such as Sephadex and Sepharose. In Fig. 6.7, the biospecific affinity chromatography of phytoagglutinins from the crude extract of sun hemp (Crotolaria juncea) seeds on ECD-Sepharose after 3-h treatment with 0.2 M hydrochloric acid at 50°C is represented. Galactan chains were hydrolysed without complete degradation of

AFFINITY LIGANDS

100

c C

0 ul P

w

0

z

a m

L

$ a

8m a

I

3

CC

8 m =x

4.. TITER

3.. 1048

1024

532

2-

256 118 6.

10

20 FRACTION

30

40

NUMBER

Fig. 6.7. Chromatography of clarified dialyzed crude extract on (A) untreated ECD-Sepharose 6B (prepared from Sepharose 6B by treatment with epichlorohydrin in alkaline medium, followed by alkaline hydrolysis), and (B) ECD-Sepharose 6B, treated with acid for 3 h (0.2 M HC1,SO"C). Columns, 100 X 19 mm; flow-rate, 15 ml/h; fractions, 2.5 ml. -,A2aonm; ---, haemagglutination titre; ---,A 540nm(sugar concentration as determined by the orcinol method). Arrow indicates start of elution with buffer containing lactose. Reproduced with permission from B. Ersson et nl., Biochim. Biophys. Acta, 310 (1973) 446-452.

the gel matrix. By this the number of end galactosyl groups necessary for specific adsorption of hemagglutinins increased. The importance of this hydrolysis is best evident from a comparison with the chromatography of the same extract, under analogous conditions, on ECD-Sepharose which was not treated with acid (Fig. 6.7A). The isolation of wheat germ agglutinin (Lis et a/.) on Sepharose with covalently attached ~-acetamido-N-(e-aminocaproyl)-2-deoxy-~-D-glucopyranosylamine is an example of the use of a specific adsorbent prepared by covalent attachment of a monosaccharide on an insoluble carrier. HoiejSi and Kocourek prepared a series of specific sorbents for the isolation of phytohemagglutinins from various sources by copolymerization of alkenyl-0glycosides with acrylamide and N',N'-methylenebisacrylamide.The hydrophlic gels thus

LECTINS, GLYCOPROTEINS AND SACCHARIDES

101

obtained contain sugars bound by 0-glycosidic bonds to the alkyl-side chains of the matrix. As an example, the partial tentative structure of 0-a-L-fucopyranosyl derivatives is given (when ally1 a-L-fucopyranosyl is used for copolymerization): HO NH,-CO-CH

/

\

HC-(CH21n-CH2-

H24 CH-CO-NH2

p

2

HCt-CO-NH2

c H3

0.C H2-

HOHO

NH~-CO-C'H

- \ NH2-CO-CH

H2C\ C H , H24 NH2-CO-CH

HC(C ;/Ch2 H2

HO

,,) - C H 2 - o o o H

-CO-NH2 /CH2 NH~-CO-CH \ CH3

For the affinity chromatography of glycoproteins, antibodies or lectins are used as affinants. The use of antibodies as affinity ligands was discussed in Section 6.3. The isolation of glycoproteins by means of immobilized lectins makes use of their differing affinities for terminal carbohydrate residues characteristic of single glycoproteins. For the elaboration of a suitable procedure for the purification of the given glycoproteins or glycopeptides by means of lectins, Kristiansen recommended the following stages: (1) identification of the terminal sugar or sugars in the carbohydrate part of the substance under consideration; (2) selection of a lectin with a corresponding specificity; (3) preparation of the selected lectin; (4) immobilization of the lectin by a covalent bond to a solid support; ( 5 ) choice of optimal conditions for the adsorption of the isolated substance on the immobilized lectin; ( 6 ) choice of conditions for desorption. It is possible either to choose non-specific elution, consisting mainly in a change in pH or salt concentration, or a specific method can be used, i.e., the displacement of the adsorbed glycoprotein by competing carbohydrates. Assuming that the terminal sugar or sugars of glycoproteins have been determined, the choice of a suitable lectin can follow. In most instances lectins are not specific for one sugar only, although great differences exist in the degree of specificity. For example, lectin from the seeds of Lotus tetragonolobus has a narrow specificity for L-fucose, while concanavalin A from Canavalia ensiformis has a broad specificity and binds most glycoproteins from human serum. In Table 6.1 the classification of lectins is given

AFFINITY LIGANDS

102

TABLE 6.1 CLASSIFICATlON OF LECTINS ACCORDING TO THEIR MAIN AFFINITIES ______-

Group

Specificity

Lectins

I

L-Fucose

Lotus tetragonolobus, weakly inhibited by L-galactose Ulex europaeus (gorse); contains another lectin belonging to group VII Ulex pawiflorus; weakly inhibited by other sugars

11

N-Acetyl-D-glucosamine

Group inhibited by N-acetylated chitodextrins Triricurn uulgare (wheat germ); also inhibited by N-acetylneuraminic acid (NANA) Solanurn tuberosum (potato tuber); also inhibited by muramic acid

111

N-Acety I-D-galactosamine

Dolichos biflorus (horse gram) Phaseolus Iunatus (lima bean, also called P. Iimensis) Phaseolus vulgaris (red kidney bean, black kidney bean, yellow wax bean; bean meal is source) Vicia cracca; also contains a non-specific lectin in group VIII Euonymus europaeus Helix pornatia (snail)

IV

DGalactose

Group also inhibited by L-arabinose, D-fucose, lactose, raffinose, and melibiose Crotalaria juncea (sun hemp), @specific Ricinus cornmunis (castor bean) Abrus precatorius Griffonia simplicifolia

V

N-Acet yl-D-galact own ine and D-galactose

These lectins are inhibited almost equally by both sugars Sophora japonica (japanese pagoda tree) Glycine max (soybean), a-specific Caragana arborescens Bandaeirea simplicifolia, a-specific Bauhinia variegata, var. candida Momordia charantia Erythrina subrosa Coronilla varia, a-specific Crotalaria zanzibarica Arachis hypogea, p-specific

VI

DGlucose

Sesamurn indicurn Pisum sativum (garden pea); inhibited about four times better by D-mannose

VII

pGlycosides and @N-acetylglucosaminides

Group inhibited most strongly by N,N'diacetylchitobiose, but also by salicin [24hydroxymethyl)phenyl-p-D-glucopyranoside], phenyl-p-D-glucopyranoside and cellobiose Ulex europaeus (gorse); contains another lectin belonging to group I Ulex galli

___

- _ _ _ ~ _ . _ _ . _ _

-

RECEPTORS, BINDING AND TRANSPORT PROTEINS

103

TABLE 6.1 (continued) Group

Specificity

Lectins

Ulex nanus Cytisus sessilifolius; inhibited also by lactose Laburnum alpinum, inhibited also by lactose Clerodendrum viscosum; pulp is source

VIII

Methyl*-D-mannoside, D-mannose, Sugars listed in decreasing order of inhibition D-glucose, N-acety1-D-glucosamie, Pisum sativum (garden pea); also inhibited by L-sorbose D-glucose, but only about one quarter as efficiently Lens culinaris (common lentil) Canavalia ensiformis (jack bean; gives con A); bean meal is source. Yicia crassa; also contains a lectin in group 111 Lathyrus sativus L.

IX

N-acetylneuraminic acid (NANA)

Limulus polyphemus (haemolymph of horseshoe crab) Triticum vulgaris (wheat germ); also in group I1

according to their main affinities. If not stated otherwise, plant seeds served as the source of lectin (Kristiansen). Examples of the use of lectins for the isolation of glycoproteins and glycopeptides are given in Table 11.l. Using two immobilized lectins, viz., the lectin from Crotalariajuncea, specific for galactose configuration (it reacts with carbohydrates and glycoproteins containing galactose), and the broadly specific concanavalin A, Ersson and Porath elaborated the fractionation of serum proteins. It is known that concanavalin A interacts with polysaccharides with unsubstituted a-D -glucopyranosyl, a-D -mannopyranosyl, or &D-fructofuranosyl residues. Many biological membranes contain glucoproteins, mostly with unknown structure and function. In an effort to elucidate the role of these glycoproteins in the structure of microsomai membranes, Winquist et al. isolated the glycoproteins from liver microsomal membranes with the aid of Sepharose with attached concanavalin A. In Fig. 6.8 the use of the same specific sorbent (concanavalin A-Sepharose) is illustrated for the isolation of teichoic acid from the autolysate of the .cell membranes of BaciZlus subtilis (Doyle eta/.). The column mentioned had a higher capacity for polysaccharides than for teichoic acid. Under the same conditions, when about 60-70 mg of teichoic acid were sorbed, 450 mg of glycogen from rabbit liver could be retained. The use of immobilized concanavalin A for the isolation of cells is discussed in Section 6.10.

6.5 ISOLATION OF RECEPTORS, BINDING AND TRANSPORT PROTEINS The primary effect of some hormones is aimed at the plasma membrane of the target cells. Under the term “receptor” the components of plasma membranes are usually under-

AFFINITY LIGANDS

104

20

60

100 140 180 ELUTION VOLUME, ml

220

Fig. 6.8. Affinity chromatography of a cell wall autolysate from Bacillus subtilis 168 on concanavalin A-Sepharose 4B.The autolysate (45 mg) was dissolved in 5.0 ml of 0.03 M Tris-HCI buffer, pH 7.3, and added to an affinity column (200 x 25 mm). The column was eluted with 0.03 M Tris-HC1, pH 7.3, at a flow-rate of 160 ml, and 10-ml fractions were collected. The glycan, peptide and peptidoglycan fragments emerged as shown by peak I. The teichoic acid was retained on the column. At the arrow, a 0.05 M solution of a-methyl-D-glucose in 0.03 M Tris-HC1, pH 7.3, was added and the teichoic acid emerged as shown by peak 11. Reproduced with permission from R.J. Doyle et al., Prep. Biochem., 3 (1973) 13-18.

stood which are involved in the effect of the particular hormone. It seems that they are localized exclusively on the surface of the membrane cells. In order to elucidate the effect of hormones on a molecular basis, it is necessary to purify and identify these specific membrane receptor structures. The amount of these structures in the tissues is very small in comparison with other material present. For example, the concentration of glucagon receptor on liver cells membranes is very low, 2.6 pmolelmg of protein (Krug et d.).The interaction of such a small amount with the immobilized hormones must be very effective in order to permit a strong binding of large membrane fragments. The interaction of hormones with their complementary receptors is specific and of high affinity. The dissociation constants for glucagon are 10-9-10-'0M,for insulin 5*10-"M and for norepinephrine 10-6-10-7M(Lowe and Dean). It is very difficult to isolate such small amounts by conventional isolation methods. The use of biospecific chromatography on hghly effective immobilized receptors permits such amounts to be concentrated selectively and to be isolated in a relatively high yield. Although technical difficulties in the development of suitable methods of determination of receptor activities and difficulties with solubilization prevented the development of the

105

RECEPTORS, BINDING AND TRANSPORT PROTEINS

use of affinity chromatography for the purification of receptors for a long time, a series of membrane fragments with specialized receptor activity has been isolated. In Table 1 1 . I the receptors isolated and the affinity ligands used are given. In Section 5.3 the isolation of a-bungarotoxin-binding membrane components from the electric organ of Torpedo califomica (Fig. 5.9) was discussed. Affinity chromatography of an insulin receptor solubilized with a detergent by use of diaminodipropylaminosuccinyl-N-phenylalanylinsulin-agarose is shown in Fig. 6.9 (Cuatrecasas). The restricted supply of vitamins and hormones in animals led to the development of mechanisms for adsorption, transport and conservation of these trace substances. In such processes, specific transport or binding proteins play an important role, preventing rapid urinary loss which would occur if the vitamins or the hormones were not bound in plasma

\

-3

n

s

-2

t 0 *

20 -1

44

ELUTION V O L U M E , m l

Fig. 6.9. Affinity chromatography of detergent-solubilized insulin receptors of liver-cell membranes on Liver-cell affinity columns containing diaminodipropylaminosuccinyl-N-phenyl~anyl-insu~-ag~ose. membranes were homogenized, extracted with 2%(v/v) Triton X-100 by shaking at 24°C for 40 min, and centrifuged. The supematant was dialyzed for 16 h at 4°C against Krebs-Ringer hydrogen carbonate buffer, ptf 7.4, containing 0.1% (v/v) of Triton X-100. Then 12 ml of the supernatant were slowly chromatographed at 24°C on an affinity column ( V t = 1.3 ml, in a Pasteur pipette) that had been washed for 20 h with 0.1 M NaHCO, buffer, pH 8.4, followed by equilibration (2 h) with Krebs-Ringer hydrogen carbonate buffer containing 0.1% (v/v) of Triton X-100. The column was washed thoroughly (note break in abscissa) before elution (arrow) with 0.05 M sodium acetate buffer, pH 6.0, containing 4.5 Murea and 0.1%(v/v) of Triton X-100.After application of this buffer to the column, the flow was stopped for 15 min before resumption of chromatography. Fractions of 1 ml were collected for determinations of protein, and the specific binding of ['zsI]insulin was determined with the polyethylene glycol assay. Reproduced with permission from P. Cuatrecasas, Proc. Nut. Acud. Sci. US., 69 (1972) 1277-1281.

106

AFFINITY LIGANDS

in corresponding complexes. Binding proteins are present in very low concentrations. Proteins firmly binding vitamin B12 transcobalamin I and I1 are present, for example, in concentrations of 80 and 20 mg per 1000 1 of human plasma, respectively. However, they ?rsually have a high affinity for complementary vitamins or hormones. The dissociation M(Lowe and Dean). In view of constants of these complexes range from lo-' to their low concentration, they could not be obtained by classical purification procedures, and large volumes of the starting material combined with a specific interaction of high affinity led to the use of affinity chromatography. As in the antibody-antigen interaction, the subsequent dissociation of the protein from the affinity adsorbent becomes the crux of the isolation. For example, in order to set avidin free from biocytin-Sepharose, 6 M guanidine-hydrochloric acid solution of pH 1.5 had to be used (Cuatrecasas and Wilchek). In Table 11.1 further examples are given of the isolations of binding and transport proteins with the affinity ligands used.

6.6

ISOLATION OF -SH PROTEINS AND PEPTIDES

For the isolation of proteins and peptides containing free SH groups, it is advantageous to make use of the high affinity of mercapto compounds for heavy metal ions, mainly mercury. In Table 11.1 a number of examples are given of the isolations of both proteins and peptides on the basis of the formation of the mentioned complex. In Fig. 6.10, the isolation of SH-protease from a crude extract from beans is given, carried out on a column of hydroxyalkyl methacrylate gel containing a mercury derivative of methacrylanilide (Turkovi et al., 1975). SH-protease with an optimal proteolytic activity at pH 8 could be isolated in this way by a single chromatographic run from a mixture of proteolytic enzymes. After the elimination of inactive material by gel filtration on Sephadex G-75, homogeneous protease could be obtained directly (Vavreinovi and Turkovi). For the isolation of papain, bromelain, chymopapain, ficin, propapain, creatine phosphokinase and phosphofructokinase, Brocklehurst et al. employed a polymer with 2,2'-dipyridyl disulphide. The isolation of protein (ESH) containing a thiol group takes place according to the following scheme:

(Py-2-SH 1

t th'Oldted Sepharosel

(D)F S - S Q

(c! V S - S - E

+

ESH (thiocontaining protein)

-

excess of R S H

/

H

St4

+

ESH

+

R-5-5-R

-SH PROTEINS AND PEF‘TIDES

S A M PLE EDTA+ N a 3 0 ,

- -_

107

STANDARD BUFFER

--

_-

HgCI,

2 .c

E 0 OD

N

1.:

W

0

z

U m K

0 m m

1.0

U

0.5

FRACTION

NUMBER

Fig. 6.10. Isolation of SH-protease from a raw extract of beans on a column of hydroxyalkyl methacrylate gel with 15% methacrylanilide (100 X 10 mm). Fractions of 5 ml each were taken at 15-min intervals. Standard buffer solution: 0.5%butanol, 10%dimethyl sulphoxide, 0.1 M KC1,0.05 MCH,COONa. Elution buffer solution: standard buffer solution adjusted to 0.5 mM HgCl,. Data from J. Turkovl er al., Biochim. Biophys. Acfa, 386 (1975) 503-508.

A covalent bond is formed between the isolated protein and the solid support, which is then split with excess of low-molecular-weight thiol (RSH) after the unretained material has been washed out. In view of the formation of the covalent bond, this type of chromatography is called “covalent chromatography” and is discussed in Section 7.2. Egorov et al. made use of the thiol-disulphide exchange on agarose-(glutathione-2pyridyl disulphide) conjugate as a rapid and specific method for the isolation of thiolcontaining peptides from large proteins, They attached parvalbumin (a protein with 109 amino acid residues and one cysteine residue), mercaptalbumin (565 amino acid residues and one cystein residue), and ceruloplasmin (1065 amino acid residues and three cystein residues) on a modified carrier by means of disulphide bridges. The immobilized proteins were degraded proteolytically. After washing, the elution of peptides containing a thiol group was carried out with a reducing agent, then a single preparative paper electrophoresis step sufficed to obtain pure peptides in a good yield. The advantage of this method is that it gives homologous peptides from related proteins by a very simple method. Among other heavy metal ions that can be used for the formation of complexes with the thiol groups of proteins or peptides, Zn2+and Cu2+should be mentioned. For the

AFFINITY LIGANDS

108

utilization of chelate gels containing these ions, Porath et al. introduced the term “metal chelate affinity chromatography”. This method is discussed in greater detail in Section 7.6.

6.7 ISOLATION OF SPECIFIC PEPTIDES When determining amino acids present at the active site or on the surface of the protein molecule, chemical modification of proteins with subsequent isolation of the labelled peptides is used successfully. The isolation of a peptide containing a modified residue is not easy, however, mainly because the modifying reagent often reacts with different residues to give products in various yields, For this reason the protein hydrolysate contains several modified peptides, each of which is present in amounts less than 1 M. The conventional methods for the isolation of peptides require tedious and time-consuming procedures in which each step usually decreases the final yield of the peptide considerably. The use of affinity chromatography on a sorbent specific for a modifying reagent permits a one-step isolation of the modified peptide. Wilchek (1 974) distinguished three categories of modifications of proteins: (1) site-directed modification or affinity labelling of the residues at the active site of the protein; (2) selective modification of one or several residues due to their hyper-reactivity or the localization on the protein surface; (3) general modification of all side-chains of certain amino acids with a group-specific reagent, aimed at sequencing. To the first group belong reactions of analogues of substrates with the amino acids of the enzyme active site or the reaction of the hapten in the active sites of antibodies. In these instances the specific and reversible binding of analogues at the binding site of the

0-&I UNFOLDING

PROTEIN

ENZYMIC

-

DIGESTION

A F F.CHROMAT.

Fig. 6.1 1. General scheme for the isolation of labelled peptides from affinity-labelled protein. Reproduced with permission from M.Wilchek, Advun. Exp. Med Biol., 42 (1974) 15-31.

109

SPECIFIC PEF‘TIDES

protein is followed by the formation of a covalent bond at or near to the site of the labelling. In Fig. 6.1 1 a general scheme for the isolation of labelled peptides from an affinity-labelled protein is shown. The native protein (enzyme or antibody) bound covalently to a solid support sorbs from the hydrolysate of the same affinity-labelled protein only the affinity-labelled peptide. After washing the column, the specifically sorbed labelled peptide can be eluted under conditions such that dissociation takes place. Wilchek (1970) made use of this method for the isolation of affinity-labelled peptides from staphylococcal nuclease after reaction with the bromoacetyl derivative of deoxythymidine3’-paminophenylphosphate 5‘-phosphate and with bromoacetyl-p-aminophenylphosphate. Fig. 6.12 shows the affinity chromatography of labelled peptides from tryptic hydrolysate of modified nuclease on nuclease-Sepharose. Table 11.1 gives further examples of isolations of affmity-labelled peptides.

2.10’

4

8

12

ELUTION VOLUME ml

Fig. 6.12. Affinity chromatography on a nuclease-Sepharose column (20 X 5 mm) of affinity-labelled peptides with reagents I and 11. The columns were equilibrated with 0.05 M borate buffer, pH 8.0, containing 10 mM CaC1,. Tryptic digests of modified nuclease (1.7 mg) were applied in 0.5 ml of the same buffer. After 10 ml of buffer had passed through, the bound peptides were eluted with ammonia solution, pH 11.0 (arrow). Reproduced with permission from M.Wilchek, FEBS Lett., 7 (1970) 161163.

AFFINITY LIGANDS

110

TABLE 6.2

USE OF ANTI-DNP ANTIBODIES FOR THE ISOLATION OF PEPTIDES Amino acid

Cysteine

Reagent RX

Condition

Products

pH 1.5, 3 h, 25°C

-NH-CH-CO-NH I

y

-

2

S R

PH 5 , l h, 25°C

Me thionine

24 h, 25"C, 8 M urea,

-NH-CH-CO-NHI

CH2

pH 3.5

:H2

@S-R I C H3

Tryptophan

50% acetic acid, 1 h, 25°C

-NH-$H-CO-NH-

Histidine

pH 5 , 2 4 h, 25°C

-NH-CH-CO-NHI

H N, ,N R

Lysine

pH 5 , 2 4 h,

25°C

s2

-NH-cH-CO-NHI

(CH,!, I NH

R

Specific antibodies with a high affinity can be induced against almost all small molecules. The specific antibodies induced in this manner, when bound covalently to a solid support, represent suitable specific sorbents for peptides that contain the corresponding small molecules. An example is the isolation of nitrotyrosyi peptides from a tryptic digest of nitrotyrosyl lysozyme by Helman and Givol, using anti-nitrotyrosyl antibodies attached to Sepharose as the affinant. If an enzymatic digest of the nitrated protein is passed through a column containing the attached nitrotyrosine antibody, all peptides (except those which contain nitrotyrosine) emerge as the first peak. The nitrotyrosine peptides are then eluted with 1 M ammonia. The procedure described can be used in

NUCLEIC ACIDS AND NUCLEOTIDES

111

topographical studies aimed at the determination of tyrosine residues located on the surface of the molecule. After the sequenator has been introduced in the determination of amino acid sequences of proteins, the purification of peptides became the rate-determining step. The development of sensitive and efficient methods for the selective isolation of modified peptides makes it possible to obtain even very small amounts of peptides from proteins that are not accessible in larger amounts (receptors, transport proteins, etc.). In Table 6.2 the use of anti-dinitrophenyl antibodies for the isolation of peptides containing cysteine, methionine, tryptophan, histidin? and lysine residues is given as an example (Wilchek, 1974). The affinity chromatography of S-peptide and S-protein.formed by proteolytic cleavage of bovine pancreatic ribonuclease has been discussed in Section 4.4. Chromatography can even be used for the purification of synthetic analogues of S-peptides (see Table 11.1). The isolation of peptides containing a free SH group was discussed in Section 6.6.

6.8 ISOLATION OF NUCLEIC ACIDS AND NUCLEOTIDES

Mononucleotides, oligonucleotides and nucleic acids bound covalently to inert matrices represent suitable materials both for isolation purposes and for the study of the physicochemical properties of nucleic acids and enzymes participating in their synthesis and degradation. Immobilized nucleic acid bases, nucleosides or oligonucleotides may be used for separation, fractionation and structure determination of various nucleic acids. As an example can serve sequence study of nucleic acids carried out by Gilham and Robinson. They separated for example heptanucleotides on a column of thymidine-polynucleotidecellulose. DNA-cellulose chromatography has been reviewed by Alberts and Herrick (1971). In Fig. 6.13 separation of nucleic acid bases and nucleosides by high-performance affinity chromatography employing a column packed with porous spherical resins of diameter 12-15 pm, coupled with thymine, is shown (Kato e l al., 1977). Many animal and viral messenger RNAs are rich in polyadenylic acid. This was made use of in a number of instances (see Table 11.1) for isolations carried out by chromatography on supports containing oligothymidylic acid. In Fig. 6.14 the chromatography of RNA from duck reticulocytes on oligothymidylic acid-cellulose (Pemberton et al.) is illustrated as an example. About 94-96% of the RNA was eluted in peak 1 and contained ribosomal and transfer RNA. Peak 2 contained, in addition to ribosomal RNA, 10s globin messenger RNA. On the basis of the analysis of single peaks by centrifugation in sugar gradients, a very good effect of the separation was proved. De Larco and Guroff tested a series of celluloses in comparison with their derivatives for their ability to bind homonucleotide oligomers. From Table 6.3 it is evident that celluloses display considerably sorption, the degree of which varies with various celluloses. As the treatment of cellulose with sodium hydrogen sulphite caused a reduction in the amount of sorbed polyadenylic acid, De Larco and Guroff concluded that the binding was caused by lignin-like contaminants. The specificity of commercially available oligodeoxythymidylic acid-cellulose T3 for molecules containing polyadenylic acid is given in Section 6.1 1 (Table 6.6). Lindberg and Persson (1974) and Wetekam et al. used Sepharose with covalently bound

112

AFFINITY LIGANDS

polyuridylic acid for the isolation of RNA from animal cell polysomes. For the purification of DNA, Edelman also used an affinity sorbent prepared from Sepharose. However, he did not use a material with affinity for DNA in this instance, but immobilized lectins for the elimination of polysaccharidic contaminants which can be eliminated from DNA with great difficulty. Most often concanavalin A-Separose is used because the most commonly occurring contaminating polysaccharides are glycogen fractions or starch-like substances. If polysaccharides are present, which in addition to glucose, fructose or mannose also contain other terminal groups, still further immobilized lectins must be used. In Section 4.4 template chromatography (Schott et al., 1975), employed for the investigation of the interactions between peptides and oligodeoxythymidylic acid, was discussed. Schott et at. (1974) also made use of immobilized defined oligonucleotides for the selective separation of free nucleotides on the basis of a base-pairing mechanism.

25

40

20

30

15

20

10

I0

RETENTION TIME ,min

Fig. 6.13. (A) Chromatogram of a mixture of five nucleic acid bases obtained by using resins coupled with 8.0% thymine (the porous spherical resins were synthesized by suspension polymerization of glycidyl methacrylate in the presence of diluent. The attachment of thymine to the glycidyl metacrylate in the resin so formed was conducted in dimethylformamide at 65°C in the presence of potassium carbonate). The chromatographic measurement was carried out in a stainless-steel column (610 X 7.6 mm) at 25°C on a high-performance liquid chromatograph (HLC-802U, Toyo Soda Manufacturing Co.) using distilled water as solvent. The eluate from the column was monitored with a UV detector at 254 nm. The flow-rate was 3.5 ml/min and the pressure drop was 45 kg/cm'. (B) Chromatogram of a mixture of five nucleosides obtained by using resins coupled with 16.4% thymine. Resin particle size, 15-20 Mm;flow-rate, 3.1 ml/min; temperature, 30°C. The course of the chromatography was analogous to that described previously. Reproduced with permission from Y. Kato et al.,J. Chromatogr., 134 (1977) 204-206.

NUCLEIC ACIDS AND NUCLEOTIDES

113

E

t E

E

0

a N W

u z

3U

1

4

sm a

2

c

0.4

0 m m

a

- 0.2

B,

10

20

F R A C T I O N NUMBER

Fig. 6.14. Chromatography of duck reticulocyte RNA on oligodeoxythymidylic acid-cellulose. The column (40 X 5 mm) was equilibrated with 0.5 MNaCl-0.5% sodium dodecyl sulphate-0.01 M Tris buffer, pH 7.5 (starting buffer). The samples applied t o the column were dissolved in water and then diluted with an equal volume of twice-concentrated starting buffer. The column was washed with approximately 30 ml of starting buffer and then eluted with 0.5% sodium dodecyl sulphate-0.01 M Tris buffer, pH 7.5. All of the operations, after the addition of starting buffer to the samples, were carried out at room temperature. RNA was precipitated with two volumes of ethanol at 20°C after adjusting the NaCl concentration to 0.1 M. Reproduced with permission from R.E. Pemberton et al., Anal. Biochem., 66 (1975) 18-28.

Complementary oligonucleotides in the mobile phase are selectively adsorbed on the immobilized template if chromatography takes place under the conditions necessary for base pairing. Desorption is then carried out with a temperature gradient. Template chromatography permits the study of the specificity of oligonucleotides for the formation of bases, and the interaction of oligonucleotides with peptides. For the chromatography of mononucleotides, oligonucleotides and transfer FWA molecules, Schott et a/. (1973) used sorbents containing a dihydroxyboryl derivative:

114

AFFINITY LIGANDS

Use is made here of the complex formation between unsubstituted 2',3'-diol groups, localized at the 3'-terminus of polynucleotides and RNA, and the borate anion. 3-Aminobenzene-boric acid attached to succinylated aminododecylcellulose was used by Sprinzl et al. for the isolation of tRNAPhe.

TABLE 6.3 BINDING CAPACITIES FOR THE HOMOPOLYMERSTO DIFFERENT CELLULOSES AND THEIR DERIVATIVES Column

Polyadenylic acid* (A,,, units bound to 100 mg cellulose)

Polyinosinic acid** (A,,, units bound to 100 mg cellulose)

Polyuridilic acid*** (A,,, units bound to 100 mg cellulose)

Oligodeoxythymidylic acid-cellulose (CF-11)

3.56

0.90

0.33

Oligodeoxycytidylic acid-celluiose (CF-11)

0.60

1.32

0.36

Treated cellulose (CF-I 1)

0.48

1.06

0.21

Untreated cellulose (CF-11)

0.54

1.05

0.22

Untreated cellulose (CC-41)

1.26

1.33

0.37

Untreated cellulose (Sigmacell-38)

1.98

1.83

0.28

*Added 4.52 A,,, units to each column. **Added 2.40 A,,, units to each column. ***Added 4.12 A,,, units to each column.

6.9 ISOLATION OF LIPIDS, HORMONES AND OTHER SUBSTANCES Dodecylamine is a useful affinity ligand for the isolation of lipids (Deutsch et al.). For the isolation of hormones, corresponding antibodies, transport proteins or lectins serve as affinity sorbents (see Table 11.1). An example is the affinity chromatography of sheep luteinizing hormone on Sepharose with covalently bound anti-luteinizing hormone immunoglobulin fraction (Gospodarowicz). Cholera toxin, exotoxin from Vibrio cholerue, is the protein responsible for gastrointestinal manifestations of clinical cholera. In the medium of a Vibrio cholerue culture, the toxin occurs in a very low concentration of up to about 1 mg per litre of medium. The toxin is bound very strongly and specifically on certain brain gangliosides. It seems that monosialogangliosides(GM~)localized in the membrane are natural receptors that interact specifically with cholera toxin, which results in a stimulation of the activity of adenylate cyclase in the tissue. Ganghosides bound to solid supports are affinity sorbents for cholera

LIPIDS, HORMONES AND OTHER SUBSTANCES

115

toxin. This toxin also binds certain glycoproteins, such as fetuin and thyroglobulin. The affinity chromatography of '251-labelledtoxin from Vibrio cholerue on agarose with attached fetuin or on ganglioside-diaminodipropylaminoagarose (Parikh and Cuatrecasas) is shown in Fig. 6.15. Agarose with attached ganghosides can also be used for the purification of other toxic proteins, such as botulin or tetanus toxin, which are also known to form firm complexes with free gangliosides. Occurrence in low concentrations and the absence of practically utilizable detection methods are typical of genetic regulator proteins. A regulator protein, uru C protein from L-arabinose operon, was isolated by Wilcox and Clementson on Sepharose with attached 4-[4-(4-aminophenyl)butanamido]phenyl-0-D-fucopyranoside. Further examples of affinity ligands for other compounds are given in Table 11.l.

:

A

CnlOO-

s

a u

rn

-

0

z

5 100-

B

z

a

u

-

I-

a

E

%-

m -

p!

1

2

3

4

FRACTION NUMBER

Fig. 6.15.Affinity chromatography of 'z51-labelled toxin from Vibrio cholerae on fetuin-agarose (A) and on ganglioside-diaminodipropylamino-agarose (B). Pasteur pipettes containing 0.5 ml of the indicated gel were washed for 4 h at 24°C with 50% (v/v) methanol followed by 40 ml of 7 M guanidine hydrochloride and 40 ml of Krebs-Ringer hydrogen carbonate buffer containing 0.1% of albumin. The samples (0.5 ml) applied on the columns contained 5.1 * lo5 cpm of 1z51-labelledcholera toxin (6.7 pCi-pg") in Krebs-Ringer hydrogen carbonate buffer containing 0.1% of albumin. The columns were washed at 24°C with 20 ml of the same buffer (note break in axis)before elution with 7 M guanidine hydrochloride (arrows). The radioactivity in each fraction (0.5-1 ml) was determined. Reproduced with permission from I. Parikh and P. Cuatrecasas, Methods Enzymol., 34 (1974)610-619.

AFFINITY LIGANDS

116

6.10 ISOLATION OF CELLS AND VIRUSES

The separation of cells on the basis of their affinity to specific supports, mainly beads coated with a biospecific reagent, is used in various systems. Plastics, glass, polyacrylamide and agarose are commonly used as solid supports. Substances that have the necessary specificity, such as antigens, antibodies, lectins and hormones, are either adsorbed on to these supports or covalently bound. In Table 1 1.1 examples are given of the isolation of cells and of the affinity ligands used. Affinity cell fractionation has most often been applied in the analysis of lymphoid cell populations. The fractionation of lymphoid cells from rat spleen and thymus and mouse spleen by means of Sepharose with attached aggregated rat immunoglobulin was elaborated by Matthews et ~ l . Fibre fractionation of cells (Edelman and Rutishauser) separates the cells on the basis of their ability t o be bound specifically to strung fibres derivatized with molecules such as antigens, antibodies and lectins. A Petri dish containing a polyethylene frame with strung nylon fibres (the length of the longest fibre being 2.5 cm) makes a very simple, efficient and cheap separation device. This device circumvents many difficulties that arise

/

SPECIFIC CELL BINDING

+ 0

0

SPECIFIC INHlBlTlON OF BlNDlNG

MECHANICAL CLEAVAGE

Fig. 6.16. General scheme for fibre fractionation. Reproduced with permission from G.M. Edelman and U. Rutishauser, Methods Enzyrnol,, 34 (1974) 195-225.

CELLS AND VIRUSES

117

in the use of columns for the separation of cells. Fractionation can be achieved on the basis of specific bonding to a single component on the cell surface, or on the basis of the differences in binding affinity, or on the number and distribution of cell surface receptors of equal specificity. The basic principle of fractionation by means of fibres is shown in Fig. 6.16. Suitable molecules or macromolecules are coupled in a suitable chemical form with nylon fibres strung on a frame. The dissociated cells are then shaken with the fibre in a suitable medium and the non-sorbed cells are washed out. The coupled cells can then be transferred into another medium and there further characterized, or they can be set free into the medium by plucking them from the taut fibre with a needle, which serves to split off the cells from their points of attachment. Affinity ligands can also be coupled with the fibres through special linkers, permitting the liberation of the cells by a specific chemical or enzymatic cleavage. Fig. 6.17 shows the binding of erythrocytes and thymocytes on various fibres as a function of the number of concanavalin A molecules bound per centimetre of the nylon fibre. In Table 1 1.I, several examples of the isolations of viruses are also presented. One of the most frequently employed affinants is bound antibodies. An example is the use of Sepharose with coupled IgG immunoglobulin from chronically infected mink for the isolation of Alleutian disease virus (Yoon et d).

CON A / FIBRE, MOLECULES~cm-’x10”

Fig. 6.17. Binding of erythrocytes (0)and thymocytes ( 0 )to different fibres as a function of the number of concanavalin A (con A) molecules per centimetre of nylon fibre; 2-10’ cells in 4 ml of phosphate-buffered saline were incubated with different fibres for 120 min with standard mixing conditions. The number of cells bound was determined by microscopic examination of a 1-mm fibre segment. Reproduced with permission from G.M. Edelman and U. Rutishauser, Methods Enzymol., 34 (1974) 195-225.

118

AFFINITY LIGANDS

6.1 1 COMMERCIALLY AVAILABLE INSOLUBLE AFFlNANTS In parallel with the development of affinity chromatography, the number of commercially available immobilized affinity ligands is also increasing. In order to obtain an idea of how and which products are mostly used in practice, commercial names of the supports and immobilized affinants used are listed in Table 11.l. From the references included in the table, an idea can be easily formed of how the application of commercial immobilized affinants is increasing at present, for example concanavalin A bound to Sepharose (Con ASepharose). It can be assumed that the development will be the same as for other chromatographic materials. Thus, in the same way as DEAE- and CM-derivatives of cellulose are prepared in very few laboratories today, it can be expected that the use of commercial biospecific sorbents will steadily increase. This is true, of course, for groupspecific adsorbents or specific sorbents for substances currently prepared in laboratories. This is so because in affinity chromatography its specific nature should always be borne in mind. From the diversity of biologically active substances, a very wide range of necessary specific sorbents follows, and therefore a considerable number of research workers will have to prepare special highly effective sorbents themselves. Firms* which supply immobilized affinity ligands are mainly the following: Miles Labs., (Slough. Great Britain), Miles-Yeda (Elkhart, Ind., U.S.A.), Pharmacia (Uppsala, Sweden), PL Biochemicals (Milwaukee, Wisc., U.S.A.), Koch-Light Labs. (Colnbrook, Great Britain), Merck (Darmstadt, G.F.R.) and Collaborative Research (Waltham, Mass., U.S.A.). In Table 6.4 immobilized enzymes are listed which were first introduced before 1970 by Miles-Seravac and Miles-Yeda. This firm produces a number of affinity ligands for the isolation of a series of biological macromolecules. For the isolation of chymotrypsin it is agarose-e-aminocaproyl-D-tryptophanmethyl ester (capacity 2.5 mglml), for the isolation of papain it is agarose-gly-gly-tyr(0BZ)-arg (capacity 3.7 mg/ml), for proteinkinase it is agarose-casein (content of casein 15-20 mdml), for ribonuclease it is agarose-5’(4aminopheny1)phosphoryluridine 2’(3‘)-phosphate (capacity 4.7 mglml), for the isolation of trypsin and chymotrypsin it is agarose-trypsin inhibitor (capacity 10-14 pmole/ml) and for the isolation of mercaptopapain and other mercapto compounds it is agarosepaminophenylrnercury(I1) acetate (capacity 1-2 mdml). For the isolation of specifically binding proteins the following materials are supplied: agarose-e-aminocaproylfucosamine, agarose-L-phenylalanine (containing 2.4 m d m l of phenylalanine), agarose-thyroxine (1-2 m d m l of thyroxine), agarose-triiodothyronine (content 1.3 mglml), agarose-Ltryptophan (content 10-14 pmole/ml), agarose-D-tryptophan (content 3-4 pmolelml) and agarose-L-tyrosine (content 3-8 pmole/ml). For the isolation of serum proteins the following are produced: agarose-L-lysine of 6.8 O.D. unitslml capacity for the isolation of plasminogen, agarose-anti-BSA of 0.6 m d m l capacity for the isolation of bovine albumin and agarose-anti-human IgG of 5 m d m l capacity for the isolation of human immunoglobulin G. For the purification of immunoglobulin, Miles supplies Agarosedinitrophenol of 2 mdml capacity and Agarose-arsanilic acid of 5 mg/ml capacity. For the purification of carbohydrates and glycoprotein, Agarose-SBA is available, which is

*

Only firms known to the author are mentioned in the text; therefore the list is necessarily incomplete and it should in no case be considered as implying a recommendation of any particular firm or product.

119

COMMERCIALLY AVAILABLE AFFINANTS

TABLE 6.4 REVIEW OF INSOLUBILIZED ENZYMES PRODUCED BY MILES-SERAVAC AND MILES-YEDA Enzyme

Enzymes bound Percentage to (I) CM-cellulose of protein or (11) DEAEcellulose

Enzymes bound to copolymer of ethylene with maleic anhydride (EMA)

Percentage of protein

Enzymes bound to agarose

Trypsin

Enzitetrypsin (1)

5-10

Enzite-EMAtrypsin

65-70

Enziteagarosetrypsin

Chymotrypsin

Enzitechymotrypsin (I)

5 -10

Enzite-EMAchymotIypsin

65 -70

Enziteagarosechymotrypsin

Papain

Enzitepapain (1)

5-10

Enzite-EMApapain

60-65

Enziteagarosepapain

Protease (Streptomyces griseus)

Enziteprotease (I)

1-10

Subtilopep tidase

Enziteagaroseprotease Enzite-EMAsubtilopeptidase A Enzite-EMAsubtilopep tidase B

Leucine aminopep tidase

Enziteleucine aminopeptidase (11)

5-10

Bromelain

Enzitebromelain (I)

5-10

Ficin

Enzite-fich (I)

5-10

Alcohol dehydrogenase

EnziteYADH* (11)

1-5

Glucose oxidase

Enzite-glucose oxidase (11)

5-10

Peroxidase

Enziteperoxidase (I)

1-10

Ribonuclease

Enzite-RNAse (I) 5-10

Urease

Enzite-urease(I1)

Amylase

Enzite-amylase (I) 1-5

Cytochrome c

Enzitecytochrome (I)

YADH = yeast alcohol dehydrogenase.

5-10

5-10

50-55

50-55

120

AFFINITY LIGANDS

agarose-bound soybean aglutinin. Immobilized fucose-binding protein is supplied under the name Fucosylex, immobilized wheat germ aglutinin under the name Glycaminosylex and immobilized concanavalin A under the name Glycosylex. All of the sorbents mentioned are also prepared by binding on agarose. Agarose-bound concanavalin A is also produced by Pharmacia under the name Con ASepharose. Concanavalin A is bound to Sepharose 4B using the cyanogen bromide method, and its content is about 8 mg per millilitre of swollen gel. Con A-Sepharose is supplied as a suspension in 0.1 M acetate buffer of pH 6, containing 1 M sodium chloride, 1 mM calcium, magnesium and manganese chlorides and 0.02% of merthiolate added as a protecting reagent. The gel should be stored in a refrigerator at 3-8°C. Another immobilized lectin, supplied by Pharmacia, is wheat germ lectin-Sepharose 6MB, which is used for the fractionation of cells, subcellular particles and soluble carbohydrate-containing molecules. Lectin is bound on macrobeads of Sepharose 6 MB (200300 gm in diameter) by the cyanogen bromide method. The swollen gel contains about 5 mg of bound lectin per millilitre and its binding capacity for ovomucoid (molecular weight 28,000) is approximately 1 mg per millilitre of bed volume. It is supplied in suspension, 10 ml of sedimented gel in 0.9% sodium chloride solution containing 0.01% merthiolate as protecting agent. The producer recommends storing the gel in a refrigerator at 3-8OC. In order to prevent the dissociation of lectin into sub-units, the gel should not be in a medium the pH of which is lower than 3.5. For the affinity chromatography of immunoglobulin G-type antibodies, immunoglobulin molecules of subclasses 1 , 2 and 4 and their fragments containing the F, region, protein A-Sepharose CL-4B is supplied. It is a protein from the cell walls of Staphylococcus aureus, called A, composed of a single polypeptide chain of molecular weight 42,000. The characteristic biological property of protein A is its ability to interact with various immunoglobulin G molecules from various species. The protein is covalently bound to Sepharose CL4B by the cyanogen bromide method. The content of protein is 2 mg/ml and the binding capacity for human immunoglobulin G is approximately 25 mg of immunoglobulin per millilitre of gel. As Sepharose CL-4B (i.e.,agarose crosslinked with 2,3-dibromopropanol and desulphonated by alkaline hydrolysis under reductive conditions) is used as a carrier, protein A-Sepharose CL4B is characterized by high chemical and mechanical stability. The sorbent is stable in the pH range 2-1 1 and it can withstand relatively high concentrations of denaturating agents, such as urea and guanidine hydrochloride, or chaotropic salts, such as 3 M potassium isothiocyanate solution, which is generally used for the elution of bound molecules from the immunosorbent. The gel is supplied freeze-dried in the presence of dextran and lactose. One gram of freeze-dried powder is approximately equivalent to 3.5 ml of swelled gel. If stored in a refrigerator at temperatures under 8’C, this immunosorbent retains its immunoglobulin-binding capacity for at least 2 years. Among other immobilized affinity ligands produced by Pharmacia, 5’-AMP-Sepharose 4B should be mentioned, which is prepared by binding N6(6-aminohexyl)-5’-AMP on Sepharose 4B by means of the cyanogen bromide method. This sorbent permits a groupspecific separation of enzymes that require cofactors which have the adenylic acid moiety of 5’-AMP in common. These enzymes include dehydrogenases with NADH as cofactor, as well as some kinases with ATP as cofactor. The concentration of bound 5’-AMP is approximately 2 pmole per millilitre of swollen gel. When crystalline lactate dehydrogenase is

COMMERCIALLY AVAILABLE AFFINANTS

121

used, the binding capacity is approximately 10 mg of enzyme per millilitre of swollen gel in 0.1 M phosphate buffer of pH 7, at 20°C. The gel is supplied as a freeze-dried powder in 5-g packets that are equivalent t o 20 ml of swollen gel. The gel contains additives to presene the swelling characteristics of the gel. For chromatography of ribosomal RNA and for the isolation of plasminogen, lysineSepharose 4B is produced. It is made by binding L-lysine on Sepharose 4B after cyanogen bromide activation. The concentration of the bound lysine is 4-5 pmole per millilitre of swollen gel. Lysine-Sepharose 4B is supplied as a freeze-dried powder in 15-g packets that are equivalent to 60 ml of swollen gel. Additives are added to preserve the swelling characteristics of the gel. If the gel is stored dry at temperatures below 8°C no detectable loss of the bound ligand can be observed, even after 1 year. Another immobilized affinant is poly(U)-Sepharose 4B, which is formed by a stable covalent bonding of polyuridylic acid [poly(U)] with Sepharose 4B, after cyanogen bromide activation. This binding method gives multi-point covalent bonding via tautomeric enolate ions of the nucleotide, and a no less favourable one-point esterification via the terminal free phosphate group. A chain of 30-40 nucleotides permits simultaneous multipoint binding to the matrix and binding to the polyadenylic parts of the messenger RNA. Owing to its length, this chain acts simultaneously as a spacer and thus permits good steric accessibility for the formation of the complex. The amount of the bound uridylic acid is approximately 0.5 mg per millilitre of swollen gel. The binding capacity is about 150 pg of mRNA per millilitre of swollen gel, which is approximately equivalent to 5 absorbance units, when polysomal preparations from KB cells (heteroploid cell line of human origin) are used. Recoveries of mRNA are about 90%. Poly(U)-Sepharose 4B is supplied in the form of a freeze-dried powder in 5-g packets, corresponding to 25 ml of swollen gel. For the preservation of the swelling characteristics lactose and dextran are added to the gel. If the freeze-dried powder is stored at temperatures not exceeding 8°C about 2% of the nucleotide material is lost after 1 year. However, this is caused rather by the splitting off of the monomer from the polymer than by the splitting off of the affinant from the matrix. For the isolation of transfer RNA and synthetically protected oligonucleotides, BDSephadex is used. This is a lipophilic derivative of DEAE-Sephadex A-25 prepared by its benzoylation. The degree of benzoylation corresponds approximately to 5 mequiv. of benzoyl groups per gram of dry gel. The capacity for tRNA is approximately 20 mg per millilitre of bed volume. In view of the possibility of hydrolysis of the ester bonds between the aromatic groups and the matrix at extreme pH values, the producer recommends washing the material immediately before use. For hydrophobic interaction chromatography, octyl8epharose C U B and phenylSepharose CL4B are produced. Octyl or phenyl groups are introduced into the matrix by reaction of Sepharose CL4B with the corresponding glycidyl ether, ie., by uncharged, chemically stable ether bonds. The concentrations of the bound ligands are approximately equal for both gels, viz., 40 pmole per millilitre of gel bed, which corresponds to a degree of substitution of about 0.2 mole of hydrophobic substituent per mole of galactose. The binding capacities of the gels are dependent on the experimental conditions and the nature of the proteins isolated. In 0.01 M phosphate buffer of pH 6.8 containing 1 M ammonium sulphate, both gels bind approximately 15-20 mg of human serum albumin (molecular

c-.

w

TABLE 6.5

h)

Ligand

P-L names

Content

Mode of attachment

Stored

Coenzyme

Agarose-hexane-coenzyme (Type 1)

A

0.5-4.0 pmol/ml

Reaction of CoA with agarose-hexanoic acid using carbodiimide (DCC)

In 50% glycerol at -25°C

Agarose-hexane-coenzyme (Type 5)

A

2.0-6.0 pmol/ml

Reaction of CoA with N-hydroxysuccinimide ester of agarose-hexanoic acid

At 4°C as a suspension containing 0.02% of sodium azide

Agarose-hexane-nicotinamide adenine dinucleotide (Type 1)

At least 3.0 pmol/ml

Reaction of NAD with agarose-hexanoic acid using carbodiimide (DCC)

In 50% glycerol at -25°C

Agarose-hexane-nicotinamide adenine dinucleotide (Type 3)

1.0-4.0 gmol/ml

Hexamethylenediamine attached to NAD at the adenine C-8 position before attachment to agarose

In 50% glycerol at -25°C

Agarose-hexane-nicotinamide adenine dinucleotide (Type 4)

1.0-4.0 pmol/ml

Periodate oxidation of ribose followed by hydrazide coupling using adipic acid dihydrazide as spacer

In 50%glycerol at -25°C

Agarose-hexane-nicotinamide adenine dinucleotide phosphate (Type 4)

1.0-4.0 pmol/ml

Periodate oxidation of ribose followed by hydrazide coupling using adipic acid

In 50%glycerol at -25°C

Lectin

Agarose-concavalin A

6-8 mg/ml

Cyanogen bromide activation

At 4°C as a suspension containing 0.02% of sodium azide

Nucleic acid

DNA-agarose

1.0-3.0 mg/ml

Denatured calf thymus DNA trapped in 4% agarose

At 4°C as a suspension containing 0.0 1 M TrisHCI, pH 7.5 + 1.0M NaCl + 1 mM EDTA + 0.02% of sodium azide

DNA-cellulose (denatured DNA)

0.5-1.5 mg/ml

Denatured calf thymus DNA is adsorbed to cellulose

As a frozen slurry containing 0.01 M Tris-HC1, pH 7.5 + 0.001 M EDTA + 0.15 MNaCl

DNA-cellulose (native DNA)

0.5-1.5 mg/ml

Native calf thymus DNA is adsorbed to cellulose

As a frozen slurry containing 0.01 M Tris-HC1, pH 7.5 + 0.001 M EDTA +0.15MNaCl

Nucleoside

Agarose-hexane-adenosine (Type 3)

2.0-5.0 pmol/ml

Hexamethylenediamine attached to adenosine at the adenine C-8 position before attachment to agarose

At 4°C as a suspension containing 0.02%of sodium azide

Nucleotide

Agarose-hexane-adenosine 2',5'-diphosphate (Type 2)

2.0-5.0 pmol/ml

Hexamethylenediamine is attached to 2',5'-ADP at the purine 6-position before attachment to agarose

At 4°C as a suspension containing 0.02%of sodium azide

Agarose-hexane-adenosine 3', 5'-diphosphate (Type 2)

2.0-5.0 pmol/ml

Hexamethylenediamine is attached to 3',5'-ADP at the purine 6-position before attachment to agarose

At 4" C as a suspension containing 0.02% of sodium azide

Agarose-hexane-adenosine 5'-diphosphate (Type 4)

2.0-8.0 pmol/ml

Periodate oxidation of ribose followed by hydrazide coupling using adipic acid hydrazide as spacer

In 50% glycerol at -25°C

Agarose-hexane-adenosine 3',5'-cyclic phosphate (Type 3)

3.0-8.0 pmol/ml

Hexamethylenediamine is attached to CAMPat the adenine C-8 position before attachment to agarose

At 4" C as a suspension containing 0.02% of sodium azide

Agarose- hexane-adenosine 5'-phosphate (Type 2)

3.0-8.0 pmol/ml

Hexamethylenediamine is attached to AMP at the purine 6-position before attachment to agarose

At 4°C as a suspension containing 0.02%of sodium azide

Agarose- hexane-adenosine 5'-phosphate (Type 3)

3.0-8.0 pmol/ml

Hexamethylenediamine is attached to AMP at the adenine C-8 position before attachment to agarose

At 4°C as a suspension containing 0.02%of sodium azide

Agarose- hexane-adenosine 5'-phosphate (Type 4)

2.0-5.0 pmol/ml

Periodate oxidation of ribose followed by hydrazide coupling using adipic acid dihydrazide as spacer

In 50% glycerol at -25°C

Agarose- hexane-adenosine 5'-triphosphate (Type 4)

2.0-8.0 pmol/ml

Periodate oxidation of ribose followed by hydrazide coupling using adipic acid dihydrazide as spacer

In 50% glycerol at -25°C

-

(Continued on p . 124)

TABLE 6.5 (continued) Ligand

Oligonucleotide

Polynucleo tide

c

P-L names

Content

Mode of attachment

Stored

Agarose- hexane-uridine S'diphosphate (Type 4)

2.0-8.0 Mmol/ml

Periodate oxidation of ribose followed by hydrazide coupling using adipic acid dihydrazide as spacer

In 50% glycerol at -25°C

Agarose- hexane-uridine S'-triphosphate (Type 4)

2.0--8.0 pmol/ml

Periodate oxidation of ribose followed by hydrazide coupling using adipic acid dihydrazide

In 50% glycerol at -25°C

5'-AMP-cellulose (Type 7)

1.5-3.0 pmol/ml

Attached by phosphodiester linkage between cellulose OH and S'-phosphate of the nucleotide (has no spacer)

5'GMP-cellulose (Type 7)

1.5-3.0 pmollml

Attached by phosphodiester linkage between cellulose OH and S'-phosphate of the nucleotide (has no spacer)

5'-UMP-cellulose (Type 7)

1.5-3.0 Mmol/ml

Attached by phosphodiester linkage between cellulose OH and 5'-phosphate of the nucleotide (has no spacer)

Oligo(dT)-cellulose (Type 7)

Binding capacity for poly(A) 61 A,,, units per gram

Polymerization of thymidine-5'-monophosphate on cellulose using N,N-dicyclohexylcarbodiimide reaction

As a dried powder (2.5-3.5 ml/g)

Oligo(dT)-cellulose (Type 7B)

Binding capacity for poly(A) 250 nmol/ml of poly(A)

Polymerization of thymidine-5'-monophosphate on cellulose using N,N'-dicyclohexylcarbodiimide reaction

Contains 0.02% of sodium azide

Agarose- polyriboadenylic acid (Type 6 )

0.25-1.0 mg/ml

Cyanogen bromide activation

At 4°C as a suspension containing 0.02% of sodium azide

Agarose-polyribocytidylic acid (Type 6 )

0.25-1.0 mg/ml

Agarose-polyriboguanylic acid (Type 6 )

0.25-1.0 mg/ml

h)

P

9

Cyanogen bromide activation

Cyanogen bromide activation

At 4°C as a suspension containing 0.02% of sodium azide At 4°C as a suspension containing 0.02% of sodium azide

-0

3 z 'c(

el

4

5* z

z

Protease substrate or Inhibitor

Thiol

Agarose-polyriboinosinic acid (Type 6)

3.0-8.0 mg/ml

Cyanogen bromide activation

At 4°C as a suspension containing 0.02% of sodium azide

Agarose-polyriboinosinic-polyribocytidylic acid (Type 6)

6-16 mg/ml

Agarose polyriboinosinic acid treated with polyribocytidylic acid

At 4°C as a suspension containing 0.1 M NaCl + 0.02 M Na,FQ,, pH 7.5 t 0.02% of sodium azide

Agarose-polyribouridylic acid (Type 6 )

3.0-8.0 m g / d

Cyanogen bromide activation

At 4°C as a slurry containing 0.02% of sodium azide

Agarose-polyribouridylic acid (Type 6A)

0.5 - 1.O mg/ml

Cyanogen bromide activation

At 4°C as a slurry containing 0.02% of sodium azide

Agarose-haemoglobin

2.0-6.0 mg/ml

Cyanogen bromide activation

In 50% glycerol at -20°C

Agarose-lima bean trypsin inhibitor

2.0-6.0 mg/ml

Cyanogen bromide activation

In 5 0 % glycerol at -20°C

Agarose-soybean trypsin inhibitor

2.0-6.0 mg/ml

Cyanogen bromide activation

In 50% glycerol at -20°C

Agarose-cysteamine

1.0-5.0 pmol/ml of SH

Cyanogen bromide activation

At 4°C as a suspension containing 0.02% of sodium azide

Agarose-hexane-thiol

4.0-10.0 pmol/ml Reaction of N-acetylhomocysteine of SH thiolactone with agarose-aminohexane

At 4°C as a suspension containing 0.02% of sodium azide

126

AFFINITY LIGANDS

weight 68,000) or 3-6 mg of P-lactoglobulin (molecular weight 18,000) per millilitre of gel. Octyl-Sepharose CLAB and phenyl-Sepharose CLAB are supplied in suspension sterilized with diethyl pyrocarbonate, and in admixture with 0.02% of sodium azide. The suspension of the octyl derivative also contains 25% of ethanol. Owing to the presence of Sepharose CLIIB, which is crosslinked with 2,3-dibromopropanol and desulphonated, the gels possess very good properties, mainly high chemical and physical stability. They are resistant to denaturing agents, such as urea and guanidine hydrochloride, and they can be used in organic solvents and ionic and non-ionic detergents. The producer recommends storing them in suspension, in a refrigerator at temperatures not exceeding 8”C, but without freezing. A large number of immobilized affinity ligands are also produced by PL Biochemicals, and a list of products is given in Table 6.5. As it is often possible to bind an affinity ligand on the matrix in several ways, type numbers are included in the table, indicating the method of attachment: Type I . The linkage is formed by reaction of the matrix, which may or may not contain a spacer, directly with the affinity ligand using a coupling reagent, such as carbodiimide. The specific point of linkage is unknown (Mosbach e f al.). Type 2. The linkage is through the N6-aminogroup of the adenosine ring (Guilford et

Ql.). Type 3. The linkage is through the C8-position of the adenine ring (Guilford et al.; Wilchek et al.), Type 4 . The linkage is through the hydroxyl group of ribose (Lamed e f d). Type 5 . The linkage is through the mercapto group of the ligand (Cuatrecasas and Parikh). Type 6. The linkage is formed by reacting the affinity ligand with cyanogen bromideactivated agarose. The linkage is unknown (Wagner et al.; Lindberg and Persson, 1972). Type 7, The linkage is through the terminal phosphate of a nucleotide, generally by a phosphodiester bond (Gilham). The enzymes bound to Enzacryls are supplied by Koch-Light Labs., and include a-amylase, catalase, a-chymotrypsin, dextranase, glucose oxidase, trypsin, urease and uricase . Oligodeoxythymidylic acid (1 0- 12 bases long) bound to cellulose is produced under the name oligo(dT)-cellulose T2 and T3 by Collaborative Research. Three grades of this sorbent were tested by Bantle et QI. for their binding capacity of Escherichia coli RNA. The T2 grade has the lowest binding capacity and the highest background “noise” [poly(A)RNA binding]. The T3 product has a 2-5 times higher binding capacity and a lower nonspecific binding than T2. The lowest non-specific binding is in grade T31, which is oligo(dT)cellulose prepared from Whatman CF 11 cellulose, washed according to the method of Alberts and Herrick and supplied by Collaborative Research also as T3. The specificity of oligo(dT)-cellulose T3 for molecules containing polyadenylic acid is given in Table 6.6 (Bantle ef QL). Cellulose as a carrier for the immobilization of affinity ligands is also supplied by Merck. For the isolation of a number of proteases they attach 4-aminobenzamidine on succinylated aminododecylcellulose(90- 110 pmole per millilitre of bed volume), while the bed volume is 1.2-1.4 ml per gram of wet sorbent. For the isolation of proteases a further immobilized

REFERENCES

127

TABLE 6.6 SPECIFICITY OF OLIGODEOXYTHYMIDYLIC ACID-CELLULOSE (GRADE T3) FOR POLYADENYLIC ACIDCONTAINING MOLECULES* Nucleic acid

Bound (%)

Polyadenylic acid homopolymer Polyurid ylic acid homopolymer Polyuridylic acid-poly(A)triplex Sheared singlestrand DNA Sheared double-strand DNA 4s E. coli RNA Polyadenylic acid mRNA

97 1 1 0.1 0.3 0.2 98.7

* "-Labelled nucleic acids applied to pre-emptied columns prepared from 0.25 g of oligodeoxythymidylic acid-cellulose. In all instances <1.0 Hg (4000-15000 cpm) of nucleic acid was applied.

inhibitor is used, i e . , trypsin inhibitor of the Kunitz type from soybean, attached on succinylated aminohexylcellulose (the binding capacity for trypsin is 15-20 mg per millilitre of bed volume, while 1 g of wet sorbent has a 1.2-1.4-ml volume). For the affinity chromatography of sugars, sugar derivatives and nucleosides, 3-aminobenzylboricacid attached to succinylated aminododecylcellulose is used (90-1 10 I,tmole/ml, while the bed volume is 1.2-1.4 ml per gram of wet sorbent). In view of the fact that 3-aminobenzylboric acid also functions as an inhibitor for serine proteases, the producer recommends the mentioned derivative of cellulose also as a specific sorbent for the isolation of subtilysine and other bacterial serine proteases.

REFERENCES Alberts, B. and Herrick, G., Methods Enzymol., 21D (1971) 198-217. Anfinsen, C.B., Bose, S., Corley, L. and Gurari-Rotman, D.,Proc. Nut. Acad. Sci U S . , 71 (1974)

3 139 -3142. Bantle, J.A., Maxwell, I.H. and Hahn, W.E., Anal. Biochem., 72 (1976)413-427. Berg, R.A. and Prockop, D.J.,J. Biol. Chem., 248 (1973)1175-1182. Berglund, 0.and Eckstein, F., Eur. J. Biochem., 28 (1972)492-496. Brocklehurst, K.,Carlsson, J., Kierstan, M.P.J. and Crook, E.M., Methods Enzymol., 34 (1974)531-544. Comer, M.J., Craven, D.B., Harvey, M.J., Atkinson, A. and Dean, P.D.G., Eur. J. Biochem., 55 (1975)

201 -209. Cuatrecasas, P., Proc. Nat. Acad. Sci. US.,69 (1972) 1277-1281. Cuatrecasas, P. and Parikh, I., Biochemistry, 11 (1972)2291-2299. Cuatrecasas, P. and Wilchek, M., Biochem. Biophys. Res. Commun., 33 (1968)235-239. De Larco, J. and Guroff, G., Biochem. Biophys. Res. Commun., 50 (1973)486-492. Deutsch, D.G., Fogelman, D.J. and von Kaulla, K.N., Biochem. Biophys. Res. Commun., 50 (1973)

758-764. Di Prisco, G. and Casola, L., Biochemistry, 14 (1975)4679-4683. Doyle, R.J., Birdsell, D.C.and Young, F.E., Prep. Biochem., 3 (1973) 13-18. Edelman, M., Methods Enzymol., 34 (1974)499-502. Edelman, G.M. and Rutishauser, U., Methods Enzymol., 34 (1974) 195-225.

128

AFFINITY LIGANDS

Egorov, T.A., Svenson, A., Ryden, L. and Carlsson, J., Proc. Nut. Acad. Sci. U.S., 72 (1975) 3029-3033. Ersson, B., Aspberg, K. and Porath, J., Biochim. Biophys. Actu, 310 (1973) 446-452. Ersson, B. and Porath, J., FEBS Lett., 48 (1974) 126-129. Fritz, H., Gebhardt, B.M., Fink, E., Schramm, W. and Werle, E., Hoppe-Seyler’s Z. Physiol. Chem., 350 (1969) 129-138. Gilham, P.T., Biochemistry, 7 (1968) 2809-2813. Gilham. P.T. and Robinson, W.E., J. Amer. Chem. SOC.,86 (1964) 4985-4989. Gospodarowicz, I)., J. Biol. Chem., 247 (1972) 6491-6498. Guilford, H., Larsson, P.O.and Mosbach, K., Chem. Scr., 2 (1972) 165-170. Haugen, T., Ishaque, A., Chatterjee, A.K. and Preiss, J., FEBS Lett., 42 (1974) 205-208. Helman, M. and Givol, D., Biochem. J . , 125 (1971) 971-974. HokjJi, V. and Kocourek, J., Methods Enzymol., 34 (1974) 361-367. Kato, Y., Seita, T., Hashimoto, T. and Shimizu, A., J. Chromatogr., 134 (1977) 204-206. Kristiansen, T.,Methods Enzymol., 34 (1974) 331-341. Krug, F., Desbuquois, B. and Cuatrecasas, P., Nature New Biol., 234 (1971) 268-270. Lamed, R., Levin, Y.and Wilchek, M., Biochim. Biophys. Acta, 304 (1973) 231-235. Lindberg, U. and Persson, T., Eur. J. Biochem., 31 (1972) 246-254. Lindberg, U. and Person, T., Methods Enzyrnol., 34 (1974) 496-499. Lis, H., Lotan, R. and Sharon, N.,Methods Enzymol., 34 (1974) 341-346. Lowe, C.R. and Dean, P.D.G., AfFnity Chromutography, Wiley, New York, London, 1974, pp. 272. Matthews, N., RoUand, J.M. and Naim, R.C.,J. Immunol. Methods, 9 (1976) 323-335. Mosbach, K., Biochem. SOC.Trans., 2 (1974) 1294-1296. Mosbach, K., Guilford, H., Ohlsson, R. and Scott, M., Biochem. J., 127 (1972) 625-631. Murphy, R.F., Imam, A., Hughes, A.E., McGucken, M.J., Buchanan, K.D., Conlon, J.M. and Elmore, D.T., Biochim. Biophys. Acta, 420 (1976) 87-96. Murthy, R.J. and Hercz, A., FEBS Lett., 32 (1973) 243-246. Myerowitz, R.L., Handzel, Z.T. and Robbins, J.B., Clin Chim. Acta, 39 (1972a) 307-317. Myerowitz, R.L.,Chrambach, A., Rodbard, D. and Robbins, J.B., Anal. Biochem., 48 (1972b) 394409. O’Carra, P., Biochem. Soc. Trans., 2 (1974) 1289-1293. Ozawa, T., Okumura, M. and Yagi, K., Biochem. Biophys. Res. Commun., 65 (1975) 1102-1107. Parikh, I. and Cuatrecasas, P., Merhods Enzymol., 34 (1974) 610-619. Pemberton, R.E., Libexti, P. and Baglioni, C., Anal Biochem., 66 (1975) 18-28. Pitana, G.C., Charm, S.E. and Green, S., Biotechnol. Bioeng., 17 (1975) 607-611. Porath, J., Carlsson, J., Olsson, I. and Belfrage, G., Nature (London), 258 (1975) 598-599. Schaller, H., Nusslein, C., Bonhoeffer, F.J., Kurz, C. and Nietzschmann, I., Eur. J. Biochem., 26 (1972) 474-481. Schott, H., Eckstein, H. and Bayer, E., J. Chromatogr., 99 (1974) 31-34. Schott, H., Eckstein, H., Gatfield, I. and Bayer, E., Biochemistry, 14 (1975) 5541-5548. Schott, H., Rudloff, E., Schmidt, P., Roychoudhury, R. and Kossel, H., Biochemistry, 12 (1973) 932-938. Sprinzl, M., Scheit, K.H., Stembach, H., Von der Haar, F. and Cramer, F.,Biochem. Biophys. Res. Commun.,51 (1973) 881-887. Tow, T., Sato, T., Sano, R., Yamamoto, K., Matuo, Y. and Chibata, I., Biochim. Biophys. Acta, 334 (1974) 1-11. Turkovi, J., Blha, K., Valentovi, O., toupek, J. and Seifertovi, A., Biochim. Biophys. Acta, 427 (1976) 586-593. Turkovi, J., Hubdkovi, O., Kfivikovi, M. and coupek, J., Biochim. Biophys. Acta, 322 (1973) 1-9. Turkovi, J., Vavreinovi, S., Kfivikovi, M. and Coupek, J., Biochim Biophys. Acta, 386 (1975) 503508. Vavreinovi, S. and Turkovi, J., Biochim Biophys. Actu, 403 (1975) 506-513. Wagner, A.F., Bugianesi, R.L. and Shen, T.Y., Biochem Biophys. Res. Commun., 45 (1971) 184-189. Wetekam, W., Mullinix, K.P., Deeley, R.G., Kronenberg, H.M.,Eldridge, J.D., Meyers, M. and Goldberger, R.F.,Proc. Nat. Acad. Sci. US.,72 (1975) 3364-3368.

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Wilchek, M., FEBS Lett., 7 (1970) 161-163. Wilchek, M., Advan. Exp. Med. Biol., 42 (1974) 15-31. Wilchek, M. and Gorecki, M., FEBS Lett., 31 (1973) 149-152. Wilchek, M., Salornon, Y., Lowe, M. and Selinger, Z., Biochem. Bfophys. Res. Commun., 45 (1971) 1174-1184. Wilcox, G . and Clementson, J.,Methods Enzyrnol., 34 (1974) 368-373. Winquist, L., Eriksson, L.C. and Dallner, G., FEBS Lett., 42 (1974) 27-31. Yoon, J.W., Kenyon, A.J. and Good, R.A., Nature New Biol., 245 (1973) 205-207. Yoshida, A, J. Chromatogr., 114 (1975) 321-327.

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131

Chapter 7

Hydrophobic chromatography,covalent affinity chromatography, affinity elution and related methods In addition to biospecific affinity chromatography, hydrophobic chromatography, covalent chromatography, affinity elution, affinity density perturbation and affinity electrophoresis, metal chelate affinity chromatography is also included among the affinity methods (Jakoby and Wilchek; Lowe and Dean). All of these methods have many common features; and this is also true of typical biospecific affinity chromatography. In general, they involve the formation of specific complexes of biologically active substances, one of the components of the complex being immobilized on a solid matrix. The methods for the preparation of specific sorbents are virtually identical, and similar problems, such as the effects of the solid support and steric accessibility of the immobilized ligand are also common. Often it is difficult to determine whether a typical biospecific interaction is involved or whether a non-specific hydrophobic interaction already prevails, for example with a hydrophobic spacer. Nevertheless, it seems that at least some of the procedures mentioned will develop into independent procedures, especially hydrophobic chromatography, which utilizes non-covalent interactions of the hydrophobic areas present on the surface of proteins with the hydrophobic ligands bound to solid supports. However, it is possible for sorbents prepared in this manner to be used also for the separation of low-molecularweight substances, such as peptides. The use of various concentrations of a suitable bound ligand and the establishment of optimal conditions for the use of various concentrations of salts during sorption and desorption may make hydrophobic chromatography, in addition to gel and ion-exchange chromatography, a standard procedure for the purification of various types of compounds.

7.1 HYDROPHOBIC CHROMATOGRAPHY

Hydrophobic chromatography was first used as a method for the purification of proteins and other biological molecules by Shaltiel. For separation purposes one can utilize homologous series of, for example, hydrocarbon-coated agaroses (Sepharose-NH(CH-3, X, where X = H, NH2, COOH, OH, C6H5,etc.). Each member in the series offers hydrophobic “arms” or “yard sticks” of various sizes and distributions, which interact with the available hydrophobic pockets or regions in various proteins. Retardation or retention is achieved by means of lipophilic interactions between these pockets and suitably large hydrocarbon sidechains on a solid support. Shaltiel based this suggestion on the following observations (1) A series of alkylagaroses with the structure S ~ ~ ~ ~ ~ O S ~ - N H ((where C H ~ n) =~ C H ~ 0-7) and o-aminoalkylagaroseswith the structure Sepharose-NH(CH&NH2 (where n = 2-8) had a similar content of alkyl side-chains per gel bead (Er-el et al.; Shaltiel and Er-el). Under identical conditions of pH, ionic strength, buffer composition and temperature, the ability of Sepharose-NH(CHz), CH3 to retain phosphorylase b depended on the length of the hydrocarbon chains. On Sepharose derivatives where n = 0-1, phosphorylase b was

132

AFFINITY CHROMATOGRAPHIC METHODS

excluded; at n = 2 retardation of the enzyme took place and at n = 3 the enzyme was adsorbed. The elution of phosphorylase b from the derivative of Sepharose with n = 3 was possible when “deforming” buffers were used, which were shown to cause reversible structural changes on the enzyme. In a derivative with n = 5 the binding of phosphorylase was very strong, so that the enzyme could not be eluted from the column even when the pH of the deforming buffer’was decreased to 5.8, although at this pH an increase in the deforming power took place. The recovery of this derivative was possible only after washing the column with 0.2 M acetic acid, when phosphorylase b could be obtained in an inactive form. Agarose itself contains negative charges and the binding of alkyl- or arylamines on to cyanogen bromide-activated agarose introduces positive charges into the gel (see Section 8.2.4). In connection with this fact Jost et al. stressed that the binding of proteins on Sepharose, on t o which alkylamines were bound after previous activation with cyanogen bromide, takes place mostly at pH values higher than the isoelectric point of the proteins isolated. Hence, they considered it probable that in these instances electrostatic interactions with a positively charged N-substituted isourea are more operative than the hydrophobic interactions with the hydrophobic side-chain. Nonetheless, the agarose beads did not bind phosphorylase b unless alkyl side-chains of a certain minimum length were bound to them. In addition, the above-mentioned charges were equally present in all of the members of the homologous series and therefore they could not affect the differing degree of retardation, which was dependent mainly on the length of the hydrophobic chain. (2) As no similarity between the 4carbon-atom afkyl group in the alkylagarose chain and the known substrate or the phosphorylase b effector is known, and because under the same conditions other proteins can also be bound on to this type of column, it seems that this interaction is not operative in the catalytic or regulation site of the enzyme exclusively. The fact that some enzymes retain their catalytic activity even after sorption on a hydrophobic adsorbent is also in agreement with this suggestion. ( 3 ) Proteins with a similar charge and molecular weight require various arm lengths for retention on alkylagarose columns. (4) The strength of bond of a given protein increases up to a certain limit with prolongation of the hydrophobic arm, as can be shown on the basis of the drastic procedures that have to be used for the desorption of the protein from the column. (5) The proteins bound on the alkylagarose column can be desorbed by increasing the hydrophobicity of the eluent. The elution patterns on alkylagaroses can be considerably affected by ionic interactions. The content of ionic interactions does not, however, necessarily lead to an increase in column affinity: rather the opposite is true. For example, glycogen phosphorylase b is retained, as already mentioned, on alkylagarose that contains four carbon atoms in the chain. In the w-aminoalkyl series, the enzyme is retained on a six-carbon-atom side-chain only. Similarly, glycogen synthetase is bound on Sepharose-NHCH3, while in the w aminoalkyl series the S e p h a r ~ s e - N H ( c H ~ ) ~ NisHessential. ~ The introduction of a charged group at the end of the hydrocarbon chain not only leads to the creation of ionic interactions but also reduces the hydrophobic character of the column. In some instances, the retained proteins may be liberated from alkyl agaroses by increasing the ionic strength or by changing the pH of the eluent. In these instances, Shaltiel considered the possibility that the ionic interactions are directly present in the

133

HYDROPHOBIC CHROMATOGRAPHY

bond of the macromolecules on modified agarose. With proteins, the conformation and the aggregation state of which are strongly dependent on intramolecular ionic interactions, it is possible that the ionic strength and the pH affect the structure of the protein molecules and thus change the size and the accessibility of their hydrophobic pockets. The assumption that hydrophobic interactions play a key role in the resolution achieved by the w-aminoalkyl series of agaroses was confirmed by several observations. Firstly, minimization of ionic interactions by using eluents with a high salt concentration does not suffice for the liberation of some proteins retained in these columns. For example, protein J from Salmonella typhimurium, which is negatively charged at pH 7.0, is virtually unretained at a low ionic strength on all w-aminoalkylagaroseseven though they are charged positively at this pH. The elution courses observed during the separation of proteins on w-aminoalkylagaroses differ from those on DEAE-cellulose, where a separation of proteins on the basis of ionic interactions is generally assumed. Nonetheless, it is DEAE-cellulose,containing diethylaminoethyl groups, that may in fact represent a special case of a mixed ionic and hydrophobic adsorbent, which might explain some abnormalities in the binding of proteins on ion exchangers on the “wrong” side of their isoelectric point. The series of w-aminoalkyl-Sepharoses further display a gradation in the retention of specific proteins with increasing length of the w-aminoalkyl chains. This gradation is similar to that observed in the series of alkylagaroses. Hofstee has shown that proteins differ significantly in their hydrophobicity, which can be measured on the basis of their adsorption on hydrophobic gels. Nishikawa and Bailon confirmed the observation of Hofstee that the order of increasing hydrophobicity for the three proteins tested is ovalbumin < 0-lactoglobulin < bovine serum albumin, by using a non-ionic hydrophobic gel (Sepharose 4B with bonded caprylic hydrazide). From Fig. 7.1, it is evident that a 4%agarose gel containing 5.5 microequivalents of caprylate per millilitre binds bovine serum albumin very strongly and requires 3 M guanidine hydrochloride solution for desorption. 0-Lactoglobulin is merely retarded in its passage through the sorbent, while ovalbumin is eluted with no retardation. From Table 7.1 (Nishikawa and Bailon), it is evident how the adsorption of bovine serum albumin and 0-lactoglobulin depends on the concentration of caprylic hydrazide bound on 1 ml of Sepharose 4B. As the proteins tested differ in their hydrodynamic sizes, the comparison of their chromatographic behaviour is carried out by referring the observed elution volume, V,, to Vs,i.e., the elution volume of the protein if the column is pre-equilibrated with 2 M guanidine hydrochloride solution. Using this solution, all proTABLE 7.1 PROTEIN ADSORPTION AS A FUNCTION OF AFFINITY LIGAND CONCENTRATION Protein*

Bovine serum albumin P-Lactoglobulii p-Lactoglobulin+ 3 M NaCl

Ligand concentration (pequivlml) 5.5

2.1

1.5

bound retarded bound

retarded not bound slightly retarded

not bound not bound not bound

*Dissolved in 0.05 M sodium acetate buffer at pH 6.0.

AFFINITY CHROMATOGRAPHIC METHODS

134

z

0

z

-

0 a

1 1

!

I

I

L.’ , :

I

ELUTION VOLUME

mi

Fig. 7.1. Chromatography of tipophilic proteins on caprylic hydrazide-agarose. Chromatographic studies were carried out with 4% agarose gels containing 5.5 wquiv./ml of caprylate packed into 90 X 16 mm columns and 0.05 M sodium acetate buffer at pH 6.0 was used. Proteins (5-10 rng) were introduced at the point indicated. The flow-rate was futed at 43 ml/h and column effluents were monitored at 280 nm. Three separate runs are arranged to make comparisons convenient. Abbreviations: ovalbumin, OVAL; p-lactoglobulin, BLG; bovine serum albumin, BSA; guanidine hydrochloride, GuCl. Reproduced with permission from A.H. Nishikawa and P. Bailon, Anal. Biochem., 68 (1975) 274-280.

teins migrate unretained by the sorbent. In terms of the measured elution volumes, “bound” in Table 7.1 means that Ve >> V s , “retarded” means V, > V s and “not bound” means Ve = V s . The sensitivity of the adsorption of bovine serum albumin towards small changes in ligand concentration may be caused by simultaneous interactions of the sorbent with a multiplicity of sites on the protein molecule. With 3-iactoglobulin the effect of various concentrations of the ligand was more pronounced if sodium chloride was present in the buffer at a concentration of 3 M.In agreement with the known saltingout effects of sodium chloride, the presence of this salt increased the adsorption of 0-lactoglobulin on the hydrophobic sorbent. In connection with the effect of salts the fractionation of proteins should be mentioned, based on hydrophobic salting-out adsorption on non-ionic amphiphilic gels, for which Porath ef al. (1973) used the term “hydrophobic saltingout chromatography”.

HYDROPHOBIC CHROMATOGRAPHY

135

Amphiphilic gels are prepared by introducing a limited number of hydrophobic groups into hydrophilic gel-forming substances, yielding crosslinked polymers that are highly permeable and capable of swelling in both water and many organic solvents. In aqueous solutions they have an affinity for dissolved hydrophobic substances or substances with hydrophobic groups. They do not contain ionic groups, in contrast to adsorbents prepared by coupling aliphatic amines, tryptophans and their esters, etc., on to cyanogen bromideactivated agarose. Hence, the non-ionic amphiphilic gels display a “pure” hydrophobic interaction with the hydrophobic regions on the surface of protein molecules or other dissolved substances instead of a mixed ionic-hydrophobic adsorption of the agarose derivatives. Under certain conditions, their adsorption capacity for proteins may be very high. As an example, Porath et al. (1973) mentioned the chromatography of kidney bean extract on Sepharose 6B benzyl ether. In the presence of 3 M sodium chloride the sorption of the extract and the elution of single fractions is carried out by increasing the pH, decreasing the ionic strength and decreasing the polarity of the solvent. After using this column more than 20 times and with a 50-fold amount of proteins (with respect to the dry weight of the gel), the hydrophobic sorbent did not change its chromatographic properties. The principle of salting-out adsorption is not completely clear. Evidently, the prevailing force is the increase in entropy due to the changes in the structure of the water surrounding the interacting hydrophobic groups. Hydrophobic chromatography can be used with advantage at high ionic strength for the isolation of unstable enzymes that require a high salt concentration for stabilization, for example in the isolation of aisopropyl malate isomerase (Bigelis and Umbarger). From the above discussion, it follows that in hydrophobic chromatography a distinction must be made between sorbents that contain hydrophobic groups only and materials that additionally contain ionic groups. Among the supports of the latter type Hjerten included benzoylated DEAE-cellulose, introduced by Gillam et al. (1967) for the fractionation of transfer RNA, polyacrylic acid with bonded aliphatic amines (Weiss and Bucher), used for chromatography of mitochondria1 membrane proteins, and succinoylated aminodecylagarose, used by Yon for the purification of transcarbamoylase. These and other which also includes the use of Sepharose 4B, on similar examples are listed in Table 11.l, which n-propylamine was bound after activation with cyanogen bromide, and which was used for the isolation of 0-galactosidase (Raibaud et al.). In Fig. 7.2, the effect of the concentration of various anions on the adsorption of 0-galactosidaseon to the mentioned adsorbent is shown. This effect is strongly dependent on the nature of the anions, which can be classified in the following series according to decreasing desorption: thiocyanate = iodide > chloride > acetate > citrate. This order is identical with Hofmeister’s lyotropic series of neutral salts (Green). The effect of anions on the adsorption of proteins on alkylSepharose is very similar to the lyotropic, rather than the purely electrostatic, effect. The effect of salt concentration on the stepwise elution of enzymes of glycogen metabolism from methylamine-Sepharose of increasing hydrophobicity is shown in Table 7.2 (Jennissen and Heilmeyer).

136

AFFINITY CHROMATOGRAPHIC METHODS

ANION CONCENTRATIONM

Fig. 7.2. Influence of neutral salts on the adsorption. A lOO-pI volume of a suspension of Sepharose 4B with coupled trimethylenediamine, having adsorbed 0.24 unit/ml of p-galactosidase, was added to 0.5 ml of standard buffer (0.01 M Tris-acetate, pH 7.1, containing 0.1 M NacI, 0.01 M MgCI, and 0.01 M kmercaptoethanol) supplemented with 0.1 M NaCl and different concentrations of neutral salts (pH adjusted to 7.1, if necessary). All of the neutral salts employed were potassium salts; only the anion is indicated in the figure. After 15 min of incubation at room temperature, the supernatant obtained by filtration was assayed for enzyme activity. The activities are indicated as a percentage of the initial adsorbed activity. Reproduced with permission from 0. Raibaud et al., FEBS Lett., 50 (1975) 130-134 TABLE 7.2 AMOUNT OF ACTIVITY OF ENZYMES O F GLYCOGEN METABOLISM THAT CAN BE ELUTED WITH DIFFERENT SALT CONCENTRATIONS FROM METHYLAMINE-SUBSTITUTED SEPHAROSES OF INCREASING HYDROPHOBICITY Content of NaCl in buffers used for stepwise elution 50 mM

120 mM

1M

pmoles of methylamine per ml of packed sepharose

Units per ml of packed sepharose* Phosphorylase b

Phosphorylase kinase

5.1 12.1 20.0 32.5

0.8 0.65 32.0

6.5 301 6554 353

5.1 12.1 20.0 32.5

16.0

5.1 12.1 20.0 32.5 ______

-

14.0 -

-

1.3 2839 1304 -

3.6 210 6666

Phosphorylase phosphatase

Protein kinase

Glycogen synthetase

0.12 2.0 0.3

0.147 1.4 0.014

21.1 146 32.0

1.6 1.8

1.2 0.04

58.0 35.0

-

-

-

-

-

-

-

1.2 16.6

0.09 4.2

*Dashes indicate that enzyme activity was not detectable in the eluate.

12.0 161

COVALENT AFFINITY CHROMATOGRAPHY

137

7.2 COVALENT AFFINITY CHROMATOGRAPHY

The selective isolation of biologically active macromolecules by affinity chromatography is based on reversible interactions between the immobilized affinant and the free macromolecule. The principle of affinity chromatography can also be used, however, even with an adsorbent that contains an “irreversible” inhibitor, when, after the sorption of the complementary macromolecule, a covalent bond is formed in the specific complex. A suitable chemical reaction must be used for the release of the isolated substance from the affinity adsorbent. An example of covalent affinity chromatography is the isolation of acetylcholinesterase by means of immobilized organophosphates (Ashani and Wilson; Voss et al.). Acetylcholinesterase belongs to the serine esterases, of which the inhibition by binding with organophosphates or organophosphonate esters containing a good leaving group is typical. An example is the reaction with 0,O-diethyl phosphofluoridate:

EtO, 0 it ,P-F EtO‘

+

E

k, = A105

EtO,?

k,=

EtO’

P-E

~

10

+

F-

The reaction is reversible, but the equilibrium is shifted far to the right, as is evident from the values of the second-order rate constants, given in litres/moles/min for acetylcholinesterase from the electric eel. Therefore, the reaction is usually one-directional. The hydroxy group of the active serine is the enzyme nucleophile, and this group is normally acetylated during the hydrolysis of acetylcholine. The phosphorylated enzyme is relatively stable and is hydrolysed only very slowly in aqueous solution. However, if a potent nucleo(2-PAM), the latter phile is added to the solution, such as N-methylpyridinium-2-aldoxime reacts rzpidly with the inhibited enzyme with liberation of free enzyme:

It 0

+‘

‘

CH3 H

+ E ‘OE t

The scheme for the isolation of acetylcholinesterase on a covalent affinity carrier is

138

AFFINITY CHROMATOGRAPHIC METHODS

i

"1.

0

0

Sepharose-/ h j H ! ( 3 i - r 2 ~ ~ ~ ~ ~ ~ -NcHH cH Z~ccH H ~~- c o-P-o

i

AChE

+

CH3 (fl

proteins

=1-3)

+ other unretarded substances

0

I1 Sepharose-arm -NHCH2CH2-0-P-0-AChE

+

HO

CH3

I

/ \

' 0 /

NO2

+

proteins

+

other unretarded substances

2-PAM

0

II Sepharose-arm- NHCH2CHZ-O- P-OH \ CH3

+

AChE

In the first step, the column would be treated with a mixture of acetylcholinesterase (AChE) and other proteins. The AChE would be covalently bound, together with any other serine esterases, and unreacted proteins could be washed from the column. In the second step, the column would be treated with a solution of 2-PAM, selectively releasing the AChE. The affinity carrier binds acetylcholinesterase, chymotrypsin and probably even other serine esterases if they react rapidly with the ligand. For the release of the enzyme from the covalent bond with the inhibitor, fluorides could be used, but for practical reasons more active nucleophiles are used, for example the already mentioned Nmethylpyridinium-2-aldoxime. At a flow-rate that permits the enzyme to remain in contact with the immobilized "irreversible" inhibitor of about 20 /.IM concentration for at least 3 min, all of the enzyme is retained. This reaction rate corresponds to a second-order rate constant higher than 105M-'*min-'. If the enzyme was inhibited completely by organophosphate before its application on the affinity sorbent, no acetylcholinesterase is retained in the column. The capacity of the covalent affinity column is remarkably high. If the gel contained 0.2 mM of bound affinant, 0.1 pmole of chymotrypsin, i.e., 2.5 mg of protein, was bound on 1 ml of gel. With acetylcholinesterase, 1 mg of enzyme per millilitre of gel could be obtained on a column with a 0.5 mM concentration of bound inhibitor. Another example of covalent chromatography, i.e., thiol-disulphide interchange, used for the isolation of proteins and peptides with free-SH groups, has already been discussed in Section 6.6. Blumberg and Strominger (1972, 1974) applied covalent affinity chromatography to the isolation of penicillin-binding components from solubilized membranes of Bacillus subtilis. The membranes were solubilized with 2% Nonidet P-40and the extract was sorbed on 6-aminopenicillanic-substituted Sepharose in a batchwise arrangement at room temperature. The penicillin-binding components form a covalent bond with penicillin, which is evidently a thioester bond between the cysteine residue of the protein and the carbonyl group of the P-lactam ring of penicillin. The penicillin-enzyme bond csn then be split by mild treatment with neutral hydroxylamine. The enzymes obtained in this manner, primarily D-alaninecarboxypeptidasefrom Bacillus subtilis, retain their full enzymatic activity. Examples of the isolation of substances by covalent affinity chromatography are also given in Table 11 .l.

AFFINITY ELUTION

139

7.3 AFFINITY ELUTION

An alternative method of affinity chromatography, utilizing the formation of a specific complex of the macromolecule under isolation with an affinity ligand, is biospecific elution from non-specific adsorbents; this method is known as “affinity elution” and in some instances as “specific elution by the substrate” (Von der Haar, 1974a). In this instance, for example, the mixture of enzymes is non-specifically bound to a polymer which carries the functional groups interacting with the enzymes. The required enzyme is then eluted specifically with a solution of substrate, inhibitor or some other affinity ligand. The most commonly used polymers for affinity elution are ion exchangers. If the binding of the sorbed molecule takes place by means of groups located at the binding site, then any ion exchanger can be considered as an affinity polymer containing “general ligands”. When the complex of a protein with a free affinity ligand is formed, the charged groups at their binding sites become protected against interaction with the ion exchanger. This protection may be caused by steric factors or, in the most favourable situations, by neutralization with opposite charges on the affinity ligand. If the differences in the accessibilities of the charged groups between the enzyme and the enzyme-affinant complex are significant, then the enzyme can be bound under such conditions of pH and ionic strength that the enzyme-affinity ligand complex is not bound. It is difficult to determine in advance the extent to which the enzyme charge should be changed in order to prevent its binding on to the sorbent. The maximum number of charges on an affinity ligand, capable of overcoming the binding of the enzyme on an ion exchanger under the given conditions, is four in the purification of fructose-l,6-diphosphataseusing fructose-l,6-diphosphate, three in the isolation of isocitrate dehydrogenase by elution with sodium isocitrate or sodium citrate, and two in the elution of glucose-6-phosphatedehydrogenase with glucose6-phosphate. As the most effective affinants for elution are those which contain charged groups that are identical with the charged groups on the ion exchanger (positively charged groups in the case of anion exchangers and negative in the case of cation exchangers), the assumption was made that desorption is a result of the differences between the net charge on the free enzyme and the enzyme-affinant complex. Theoretically, however, even a neutral ligand can lead to enzyme desorption if the charged groups responsible for adsorption are located on the binding site for the affinity ligand and if they are sterically protected, after the formation of the complex, against interaction with the ion exchanger, or if a conformational change takes place that prevents the interaction of the charged groups with the ion exchanger (Von der Haar, 1974a, b). In addition to the changes in charge between the enzyme and the enzyme-substrate complex, the strength of the bond between the enzyme and the ion exchanger and the affinity between the enzyme and the affinity ligand represent further parameters that affect the efficiency of elution. Mutually dependent equilibria exist between the enzyme (E) bound to the ion exchanger (IE) (eqn. 7.1) and the enzyme bound to the ligand (L) in solution (eqn. 7.2):

140

AFFINITY CHROMATOGRAPHIC METHODS

In this instance the ion exchanger is considered as a macromolecule containing a finite number of binding sites. This assumption is valid under the given conditions, i.e., pH, ionic strength and the exact amount of enzyme bound to the polymer. As K 1 decreases continuously with increasing ionic strength of the elution medium, theoretically conditions could be found for the enzyme-affinity ligand complex for any system. However, in practice, a low ionic strength should be maintained in order to minimize the elution of the contaminating proteins. Eqn. (7.3) is derived under the assumption that the concentration of the free enzyme, [El,is equal in eqns. 7.1 and 7.2. In fact, this is a limiting case for effective elution because [El in eqn. 7.2 should not exceed [El in eqn. 7.1, because otherwise the enzyme would remain bound to the polymer. The necessary concentration of the affinity ligand for effective elution can be determined. The purification of a given enzyme in this system will generally be given by the specificity of the enzyme-affinant interaction, as in other affinity systems. If several complexes can be formed, for example (EIL)and (E2L),then it is difficult to determine the order of elution of the enzymes, because it is difficult t o determine K 1 in practice. If we consider, theoretically, that K1' is the constant of association of enzyme Elwith the polymer and K l Zis the constant of association of enzyme E2 with the polymer, then, if both constants are significantly different, the enzyme can easily be separated by simple chromatography with a salt gradient. If the association constants of El with ligand (K2l), and Ez with are approximately equal, both enzymes will be eluted simultaneously, and if ligand (Kz2) K,' >K z 2 ,then El will be eluted first. Thus, for example, it is possible to separate ~ from lactate dehydrogenase glyceraldehyde phosphate dehydrogenase ( K r n= ~5 ~lo-') by means of 0.15 mM NAD' as eluent on an affinity polymer carry( K ~ N A D= 1 ing NAD' as a general ligand (Mosbach et al.). In this instance KZ1is only half of K2'. Similarly, fructose-l,6-diphosphatase can be separated from aldolase (Pogell, 1962) and from pyruvate kinase (Carminatti et aL) when low concentration of fructose-l,6-diphosphate is used for elution and a cation exchanger is employed as a polymer. A typical example of the use of biospecific elution is the isolation of aminoacyl transfer RNA synthetases (Von der Haar, 1973). These enzymes interact with three differently charged substrates, transfer RNA, ATP and amino acids. In Table 7.3, examples are given of the use of both transfer RNA and ATP as specific elution reagents. Amino acids are unsuitable for the elution of synthetases from anion exchangers because at neutral pH they exist as zwitterions and they are adsorbed themselves. Therefore, for example, 2aminoethanol was used for the elution of seryl transfer RNA synthetase, phenylethylamine for phenylalanine transfer RNA synthetase and tyramine for tyrosyl transfer RNA synthetase. These amines are competitive inhibitors in aminoacylation reactions of the corresponding amino acids (Von der Haar, 1974a). Further examples of the isolation of substances by affinity elution are given in Table 7.3 7.4 AFFINITY DENSITY PERTURBATION Membranes of animal cells contain genetically regulated specific systems that contain receptors for hormones, toxins, drugs, etc. Much attention is being paid at present to the

141

AFFINITY DENSITY PERTURBATION

study of the structure and the function of these systems. A method that permits the fractionation of membrane fragments carrying specific receptors has been developed by Wallach and co-workers (Wallach; Wallach and Lin; Wallach et al,), and it has been named “affinity density perturbation”. The fractionation principle of the method is represented schematically in Fig. 7.3. The membranes are first physically sheared into minute vesicles. Particles of higher density are added to the membrane fragments carrying the given receptor, to which a specific affinity ligand for the isolated receptor is bound covalently. The complex formed between the added affinant and the membrane particle is then rapidly separated on an ultracentrifuge on the basis of the increase in density. In order to avoid difficulties stemming from the small amount of the occurring corresponding receptors, the membranes and affinity ligands are radioactively labelled with different isotopes. The formation of complexes between the membrane fragments and the affinity particle can be blocked or cancelled, if necessary, by the addition of reagents with a higher affinity for the affinity ligand than in the case of the given receptor, or by an excess of receptor analogue with a similar affinity.

-

@x

1>

DENSITY PERTURBANT-

-

LIGAND PHAGE

LIGAND RECEPTOR ANALOGUE

0

-MEMBRANE VESICLE BEARING

a @

LIGAND RECEPTOR

= LIGAND = K 29 phage

Fig. 7.3.The principle of affinity density perturbation. A plasma membrane bearing multiple receptors (a) is sheared into membrane fragments carrying different numbers of receptors in varying distributions. These are reacted with the ligand coupled to the density perturbant, i.e., K29 phage, producing a membrane-receptor-ligand-phage complex with a higher density than that of the membrane itself and a lower density than that of the density perturbant. Addition of a low-molecular-weightdissociating agent (a) returns the membrane and density perturbant to their original densities. Reproduced with permission from D.F.H. Wallach et al., FEBS Lett., 21 (1972)29-33.

r

TABLE 7.3

P h)

USE OF AFFINITY ELUTION FOR THE JSOLATION OF ENZYMES Enzymes isolated

Affinity elution agent

Adsorbent

References

Aldolase

Fructosel,6diphosphate

CM-cellulose

Pogell(1962) Pogell ( 1966)

Aminoacyl t RNA synthetases

tRNA

Phosphocellulose

Von der Haar (1973)

Aminoacyl tRNA synthetases (arginyl, histidyl, isoleucyl, leucyl, lysyl, phenylalanyl, seryl, threonyl, tyrosyl and valeryl)

Unfractionated tRNA

Phosphocellulose

Von der Haar (1974a)

Amylase from Pseudomoms saccharophila

Soluble starch

Insoluble starch

Thayer

Diphosphopyridine nucleotide specific isocitrate dehydrogenase (E.C.1.1.1.4 1)

Citrate

Phosphocellulose

Barnet et al.

Fructose 1,6diphosphatase (E.C. 3.1.3.1 1)

Fnrctose-l,6diphosphate

CM-cellulose

Pogell (1962) Pontremoli et al. Pogell ( 1966) Pon tremoli Sarngadharan et al. Pogell and Sarngadharan

From rabbit liver

Phosphocellulose

From rabbit muscle

Rosen et al. (1965) Rosen et al. (1966)

From Candidautilis From Poly sphondyliu m pallidu m Glucose phosphate isomerase (D-glucose-6-phosphate ketol isomerase, E.C. 5.3.1.9) from human leukocyte From human erythrocyte

Fernando et al.

Glucose 6-phosphate

*

?1

9

5

.e

85FI 0

F

CM-Sephadex

Rosen

3 n

Phosphocellulose

Tilley ef al.

%

76

T:

c, Bertrand e f al. Phillips et al.

E

3 z

0 U m

Glucose-6-phosphatedehydrogenase (E.C. 1.1.1.49) from rat liver

Glucose-6-phosphate

Inorganic pyrophosphatase from yeast

Pyrophosphate

CM-cellulose

Matsuda and Yugari Watanabe and Taketa

Alluminium hydroxide gel

Heppel

NADPdependent isocitrate dehydrogenase from pig liver cytoplasm

Sodium isocitrate

CM-cellulose

Illingworth and Tipton

Nuclease from chicken pancreas

Ribonucleic acid

Phosphorylated cellulose

Eley

Orotate phosphoribosyl transferase (E.C. 2.4.2.10)

Orotic acid

DEAErcellulose

Brown et al.

Orotidylate decarboxylase (orotidine-5-phosphate decarboxylase, E.C. 4.1.1.23)

Orotic acid

DEAEcellulose

Brown et al.

Phosphoglycerate kinase from mammalian, fish and chicken muscle, mammalian liver and yeast

Negatively charged substrates

Phosphocellulose

Scopes and Fifis

Pyruvate kinase (ATP pyruvate phosphotransferase, E.C. 2.7.1.40) from rat liver

Fructose 1,6diphosphate

CM-cellulose

Carminatti et al.

SephadexG-200

Blume e l al.

Phosphoenolpyruvate, fructose 1,6-bisphosphate, adenosine S-diphosphate of pyrophosphate

Phosphocellulose

Schulz et al.

Tyrosine tRNA

Phosphocellulose

Yamada

Tyrosyl tRNA synthetase (E.C. 6.1.1.1) from baker’s yeast

-3% I7

4

4 U

m

zj 4

2

144

AFFINITY CHROMATOGRAPHIC METHODS

Density-perturbing particles can be made visible under an electron microscope, which enables the receptor topology to be mapped. A model system is the use of homogeneous concanavalin A, labelled with ‘*’I, as an affinity ligand. Particles with a high density were prepared from it by means of glutaraldehyde by covalent bonding to coliphage K29, a stable icosahedron of diameter 450 A and density 1.495 g/ml. The membrane fragments were prepared from hog lymphocytoplasmatic membranes, and contained a large amount of receptor for concanavalin A. The interaction of membrane fragments carrying the receptor with the perturbant increased reversibly the buyoant density in a caesium chloride gradient from about 1.18 for untreated membranes to a broad zone with maximum density at 1.30-1.40. This relatively broad density distribution of the membrane-concanavalin A-K29 complex reflects the microheterogeneity in the distribution of the receptor sites. An addition of excess of a,atrehalose, which does not possess too great an affinity for concanavalin A (K = 5.38 l/mole), was used for the dissociation of the complex of concanavalin A with its receptor. Instead of concanavalin A, antibodies can be used as an affinity ligand, as well as hybrids of antibodies, peptide hormones, etc., and also other binding methods for the particles can be used. In general, the above method can be applied not only for the isolation of receptor sites for hormones, transmitters, drugs, lectins and specific antigens and antibodies, but also for the mapping of the topology of the membrane and cell.

7.5 AFFINITY ELECTROPHORESIS

Affinity electrophoresis on polyacrylamidc gel is a separation technique that makes use of the advantages of both affinity chromatography and polyacrylamide gel electrophoresis (Holejsi and Kocourek, 1974a, b). On a micro-scale it permits the rapid analysis of protein mixtures with the selective separation of those components which have their binding site complementary to the immobilized specific affinity ligands. The latter are covalently bound to a part of the polyacrylamide gel matrix, and thus form a layer of “affinity gel”. The principle of t h s method is best illustrated by a typical example of the separation of active proteins by affrnity electrophoresis as shown in Fig. 7.4. In glass tubes of equal diameter complete polyacrylamide gel sticks with three layers are prepared: (a) large-pore gel; (b) affinity gel prepared by copolymerization of alkenyl O-glycosides with acrylamide, using N,N ’-methylenebisacrylamideas a crosslinking agent; and (c) small-pore gel. A standard gel, consisting of a layer of (a) and (c) (tube I), is always used as a control. In Fig. 7.4, tube 2 contains an affinity gel consisting of a copolymer of polyacrylamide with 0-a-D-galactopyranose, and tube 3 contains an affrnity gel consisting of a layer of a copolymer of polyacrylamide with 0-a-D-mannose. The protein fraction from pea seeds (Pisurn sativurn L.), possessing haemaglutinous activity, contains five protein components, as is evident from the separation in the control tube 1. None of these components interacts with the a-D-galactosyl structure of the affinity gel in tube 2, which is evident from the agreement of the electrophoretic patterns in the first and second tubes. Two of the components are, however, phytohaemagglutinins that interact with the a-D-mannosyl structure of the affinity gel in the third tube, and they therefore remained retained in the

AFFINITY ELECTROPHORESIS

145

1

Fig. 7.4. Electrophoresis of the protein fraction with haemagglutinating activity from pea seeds. (a) Largepore gel; (b) affinity gel; (c) small-pore gel. (1) Control standard gel. (2) Affinity gel (b),formed by 0-a-Dgalactopyranosyl polyacrylamide. (3) Affinity gel (b), formed by 0-cr-Dmannosylpolyacrylamide. Arrows in (2) and (3) indicate boundary between largepore gel and affinity gel. Reproduced with permission from V. Holejuand J. Kocourek, Methods Enzyrnol., 34 (1974) 178-181.

affinity gel in the form of a narrow band at its start (in Fig. 7.4, they are indicated with arrows). The mobility of the remaining three proteins in the mixture remained unchanged, as is evident from the distribution of the bands of the proteins in the low-porosity gel (c). This method permits both the detection of inactive protein molecules and, in the case of subunits and fragments, a decision as to whether their binding sites are unchanged. In addition, it shows which type of affinity adsorbent should be chosen for preparative affinity chromatography. A similar method has been elaborated independently by BbgHansen (1973) and Bbg-Hansen et al. Affinity gel electrophoresis can be used in a similar manner to affinity chromatography for the determination of the dissociation constants of protein-ligand complexes. The principle of this method consists in the following of the mobility of the given protein as a function of the concentration of the bound ligand in the gel. This ligand may be bound to the gel either covalently, or it may be only entrapped in the gel as a consequence of its high-molecular-weight character. The first paper describing such a use of electrophoresis was that by Takeo and Nakamura, although they did not use the term affinity electrophoresis. The dissociation constants of phosphorylase-polysaccharide complexes have been determined by this method, using electrophoresis on polyacrylamide gel containing various concentrations of covalently bound polysaccharide. Bbg-Hansen (1 976) used electrophoresis on Sepharose with covalently bound concanavalin A for the determination of the dissociation constants of complexes of concanavalin A with serum glycoproteins.

146

AFFINITY CHROMATOGRAPHIC METHODS

The principle of affinity electrophoresis can be used not only for the determination of the dissociation constants of complexes of proteins with bound ligands, but also for the determination of the dissociation constants of complexes with free ligands. The presence of a free, specific ligand increases the mobility of the protein zone in electrophoresis on an affinity carrier according to the concentration of the free ligand, at a constant concentration of the bound ligand. This procedure was used for the determination of the dissociation constants of lectin-sugar complexes (V. Ho’fejli, M.Tichi and J. Kocourek, to be published). As a sugar sorbent , polyacrylamide gel that is formed by copolymerization of acrylamide, N,N ’-methylenebisacrylamide and allyl glycoside can be used. In this instance, the remaining monomeric allyl glycoside must be removed, the gel equilibrated with the buffer used in electrophoresis and introduced again into the tube. The preparation of the soluble copolymer of acrylamide and allyl glycoside, which can be obtained in the form of a freeze-dried product with a known content of sugar and a defined molecular weight, is technically simpler. The necessary amount of this saccharide-containing polymer with any bound sugar is added to the polymerization mixture, as is usual in the preparation of polyacrylamide gel. It? addition, a given amount of free sugar is also added to the polymerization mixture, and the gel is allowed to polymerize. In this manner a set of gels is prepared that contain a constant concentration of the bound sugar and an increasing concentration of the free sugar. The obtained dependence of the mobilities of lectins on gels prepared in this manner (with known concentration of free sugar) is evaluated according to the relationship

where Ki = dissociation constant of the lectin-bound sugar complex; K = dissociation constant of the lectin-free sugar complex; Ci = concentration of the bound sugar; c = concentration of the free sugar; 1 = mobility of lectin at a given Ci and c; lo = mobility of lectin not affected by the interaction with the bound sugar (the mobility 1 approximates to the value of lo found at high values of c; for the determination of l o , electrophoresis on a gel containing an inert polyacrylamide copolymer can also be used). This relationship follows directly from the relationship derived by Dunn and Chaiken for the determination of dissociation constants by affinity chromatography. In this manner, the dissociation constants for the interaction of concanavalin A and lectin from Lens esculenta seeds with methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, D-glucose and methyl 0-D-glucopyranosidehave been obtained, and further, for the interactions of lectins from the seeds of Glycine soja. Ricinus communis and Maclura pomifera with Dgalactose, and lectins from the seeds of Lnex europeus and eel serum with L-fucose. The determined values (of the order of 10-2-10-4)agreed well with those obtained by other workers in various ways (equilibrium dialysis, spectroscopic methods, etc.). It was found that the Ki values are in almost all instances lower (5-10 times) than K

METAL CHELATE AFFINITY CHROMATOGRAPHY

147

for the same free sugar, which, evidently, is a consequence of the fact that the lectins investigated contain more than one binding site. Using affinity electrophoresis, relatively high values of dissociation constants could be determined (for example, the interaction of one of the isolectins from eel serum with L-fucose has been characterized by a dissociation constant of K = 2 Theoretically, interactions at least ten times weaker could still be studied. The advantages of the determination of dissociation constants by means of affinity electrophoresis are as follows: (a) small amount of sample, which can even be considerably inhomogeneous; (b) simultaneous determination of Kj and K for all electrophoretically differing components interacting with the ligand studied, present in the sample (for example, isolectins); (c) at all ci and c the bands are sharp and their positions can be determined well (in affinity chromatography, considerable broadening of the peaks takes place at low c); the sharpest band is observed at c = 0, while in affinity chromatography zone techniques usually cannot be applied under these conditions; (d) speed of performance, simplicity of laboratory equipment, analysis of a large amount of sample simultaneously; (e) wide range of K and Ki;possibility of determining high values of K; the main disadvantage is the impossibility of determining K for charged (electrophoretically mobile) ligands. This technique could even be used for the study of the interactions of other proteins (for example, antibodies and enzymes) with specific ligands.

7.6 METAL CHELATE AFFINITY CHROMATOGRAPHY The affinity of proteins for heavy metal ions can be exploited as a basis for their purification and analysis (Porath et al., 1975). Its basis is the formation of stable complexes of histidine and cysteine with zinc or copper ions in neutral aqueous solutions. Hydrophilic gels which have firmly fixed Zn2+or Cu2+ions can be used as selective adsorbents, mainly for histidine- and cysteine-containing peptides and proteins. In addition to the ions mentioned, cadmium, mercury, cobalt and nickel also form coordination compounds with histidine and cysteine. Porath et al. (1975) prepared chelate-forming adsorbents for proteins and peptides from agarose derivatives. As the metal is a transition element, the affinity is pH dependent. At pH 6-8, adsorption of proteins will take place rather on the basis of the selectivity for histidine and probably also for cysteine. At alkaline pH values, coordination with amino acids will take place, which will cause the adsorption to be more effective but at the same time less selective. In order to achieve easy regeneration of the adsorbent for chromatography, it is necessary for the metal ions to have a much higher affinity for the gel than for the substances which have to be isolated. This principle can be illustrated as follows: @-L-Men* v

+

higher pH

XP

,

lower pH

@-LzMen+---XP

148

AFFINITY CHROMATOGRAPHIC METHODS

where M is the gel matrix, L the chelate-forming ligand, Men+the metal ion and X the metal affinity substituent in the protein or in the peptide XP. As an example of XP,separated serum proteins are shown in Fig. 7.5. As the gel-forming matrix M oxirane-activated agarose with attached biscarboxymethylamino groups such as chelate-forming ligands L was used. ZnZ+and Cuz+were used as the metal ions Me". The components of individual peaks were identified as follows: peak I, albumin; peak 11, atbumin, y-globulins, prealbumin, trace amounts of crl-antitrypsin; peak 111, albumin, transferin, haptoglobins, 0-lipoprotein, trace amounts of y-globulins; peak IV, transferin, a,-antitrypsin, acid glycoprotein, yglobulins, celluloplasmin; peak V, transferin, trace amounts of haemoglobin, trace amounts of y-globulin; peak VI, cwz-macroglobulin, trace amounts of haemoglobin. During chromatography, none of the components were denatured. The metal chelate gel retained its adsorption ability even in 1 M sodium chloride solution, which excludes the possibility of ordinary ionic adsorption having taken place on this gel. On the basis of orienting experiments, it was found that among the amino acids present in protein, histidine and cysteine are adsorbed most strongly. At pH 8-9 other amino acids are also adsorbed, but their desorption takes place much earlier than that of histidine on decreasing the pH. Hence, it can be assumed that the chromatographic behaviour of proteins is governed mainly by the number and the density of exposed imidazole and thiol groups on the surface of the molecules. Indole groups also could play a role.

6 1

3

B

C

14

4

ELUTION VOLUME

k

ml

Fig. 7.5. Composite chromatogram obtained by elution of the coupled Zn'+ and Cu2+columns (A). The two columns were washed with 0.05 M Tris-HC1 buffer, pH 8,0.15 M in NaCl(60 ml each). The adsorbed material was removed from each column separately, according to the following scheme. CuZ+ column (B): 1,O.l M sodium phosphate buffer, pH 6.5, 18 ml; 2, 0.1 M sodium phosphate buffer, 0.8Min NaCI, pH 6.5, 20 ml; 3,O.l Msodium acetate buffer, 0 . 8 M i n NaCI, pH 4.5, 17 m1;4,0.05 M EDTA, 0.5 M in NaCI, pH 7.0, 25 ml. Znz+column (C): 5,0.1 M sodium phosphate buffer, pH 6.5,47 ml; 6,O.l M sodium phosphate buffer, 0.8 M in NaCI, pH 6.5, 38 ml;7, 0.1 M sodium acetate buffer, 0.8 M in NaCl, pH 4.5, 12 ml; 8, 0.5 M EDTA, 0.5 M in NaCI, pH 7.0, 10 ml. Reproduced with permission from J . Porath et al., Nature (London), 258 (1975) 598-599.

REFERENCES

149

Among the metal chelates tested, the adsorption capacity for serum proteins decreased in the order Cu > Zn > Ni > Mn. The copper chelate is very effective, while the manganese chelate gel has an almost negligible adsorption capacity. Their considerable adsorption capacity and power of separation make these gels suitable for large-scale fractionations.

REFERENCES Ashani, Y. and Wilson, I.B., Biochim. Biophys. Acta, 276 (1972) 317-322. Barnes, L.D., Kuehn, G.D. and Atkinson, D.E., Biochemistry, 10 (1971) 3939-3944. Bertrand, O., Kahn, A., Cottreau, D. and Boivin, P., Biochimie, 58 (1976) 261-267. Bigelis, R. and Umbarger, H.E., J. Biol. Chem., 250 (1975) 4315-4321. Blumberg, P.M. and Strominger, J.L.,Proc. Nut. Acad. Sci. US.,69 (1972) 3751-3755. Blumberg, P.M. and Strominger, J.L., Methods Enzymol., 34 (1974) 401-405. Blume, K.G., Hoffbauer, R.W., Busch, D., Arnold, H. and Ldhr, G.W., Biochim. Biophys. Acta, 227 (1971) 364-372. BQg-Hansen,T.C., Anal. Biochem., 56 (1973) 480-488. BQg-Hansen,T.C., Abstr., 10th Int. Congr. Biochem., Hamburg, (1976) 188. BQg-Hansen, T.C., Bjermm, O.J. and Ramlau, J., Scand. J. Immunol., 4, Suppl. 2 (1975) 141-147. Brown, G.K., Fox, R.M. and O'Sullivan, W.J.,J. Biol. Chem., 250 (1975) 7352-7358. Carminatti, H., Rozengurt, E. and Jimdnez de Asua, L., FEBSLett., 4 (1969) 307-310. Dunn, B.M. and Chaiken, I.M., Biochemistry, 14 (1975) 2343-2349. Eley, J., Biochemistry, 8 (1969) 1502-1506. Er-el, Z., Zaidenzaig, Y. and Shaltiel, S., Biochem. Biophys. Res. Commun., 49 (1972) 383-390. Fernando, J., Enser, M., Pontremoli, S. and Horecker, B.L., Arch. Biochem. Biophys., 126 (1968) 599-606. Gillam, I., Blew, D., Warrington, R.C., Von Tiegerstrom, M. and Tener, G.M., Biochemistry, 7 (1968) 3459-3468. Gillam, I., Millward, S., Blew, D., Von Tiegerstrom, M., Wimmer, E. and Tener, G.M., Biochemistry, 6 (1967) 3043-3056. Green, A.A., J. Biol. Chem., 93 (1931) 495-516. Heppel, L.H., Methods Enzymol., 2 (1955) 570-576. Hjertdn, S., J. Chromatogr., 87 (1973) 325-331. Hofstee, B.H.J., Anal. Biochem., 52 (1973) 430-448. Hoiejg;, V. and Kocourek, J., Methods Enzymol., 34 (1974a) 178-181. Hofejxi, V. and Kocourek, J., Biochim. Biophys. Acta, 336 (1974b) 338-343. Illingworth, J.A. and Tipton, K.F., Biochem. J., 118 (1970) 253-258. Jakoby, W.B. and Wilchek, M.,Methods Enzymol., 34 (1974) 1-810. Jennissen, H.P. and Heilmeyer, L.M.G., Jr., Biochemistry, 14 (1975) 754-760. Jost, R., Miron, T. and Wilchek, M., Biochim Biophys. Acta, 362 (1974) 75-82. Lowe, C.R. and Dean, P.D.G., Affinity Chromatography, Wiley, New York, London, 1974, pp. 272. Matsuda, T. and Yugari, Y., J. Biochem., 61 (1967) 535-540. Mosbach, K., Guilford, H., Ohlsson, R. and Scott, M.,Biochem. J., 127 (1972) 12P-13P. Nishikawa, A.H. and Bailon, P., Anal. Biochem., 68 (1975) 274-280. Phillips, T.L., Talent, J.M. and Gracy, R.W., Biochim. Biophys. Acta, 429 (1976) 624-628. Pogell, B.M.,Biochem. Biophys. Res. Commun., 7 (1962) 225-230. Pogell, B.M., Methods Enzymol., 9 (1966) 9-15. Pogell, B.M. and Sarngadharan, M.G., Methods Enzymol., 22 (1971) 379-385. Pontremoli, S., Methods Enzymol., 9 (1966) 625-631. Pontremoli, S., Traniello, S., Luppis, B. and Wood, W.A., J. Biol. Chem., 240 (1965) 3459-3463. Porath, J., Carlsson, J., Olsson, I. and Belfrage, G., Nature (London), 258 (1975) 598-599.

150

AFFINITY CHROMATOGRAPHIC METHODS

Porath, J., Sundberg, L., Fornstedt, N. and Olsson, I., Nature (London), 245 (1973)465-466. Raibaud, O.,Hugberg-Raibaud, A. and Coldberg, M.E.,FEBS Lett., 50 (1975)130-134. iiosen, O.M.,Arch. Biochern Biophys., 114 (1966)31-37. Rosen, O.M.,Rosen, S.M.and Horecker, B.L., Arch. Biochem. Biophys., 112 (1965)411-420. Rosen, O.M.,Rosen, S.M. and Horecker, B.L., Methods Enzymol., 9 (1966)632-636. Sarngadharan,M.C.,Watanabe, A. and PogeU, B.M.,J. Biol. Chem., 245 (1970) 1926-1929. Schulz, J., Wilhelm, G., Lorenz, G. and Hofmann, E., Acta Biol. Med. Cer., 34 (1975) 1321-1332. Scopes, R.K. and Fifis, T., R o c . Aust. Biochem Soc., 8 (1975) 17. Shaltiel, S.,Merhods Enzymol., 34 (1974) 126-140. Shaltiel, S. and Erel, Z., Proc. Nut. Acad. Sci. US.,70 (1973)778-781. Takeo, K. and Nakamura, S., Arch. Biochem. Biophys., 153 (1972) 1-7. Thayer, P.S., J. Bacteriol., 66 (1953)656-663. Tilley, B.E., Gracy, R.W. and Welch, S.G., J. Biol. Own., 249 (1974)4571-4579. Von der Haar, F., Eur. J. Biochem., 34 (1973)84-90. Von der Haar, F., Methods Enzymol., 34 (1974a)163-171. Von der Haar, F., Biochem Soc. Trans., 2 (1974b)1297-1298. Voss, H.F., Ashani, Y. and Wilson, I.B.,Merhods Enzymol., 34 (1974)581-591. Wallach, D.F.H.,Methods Enzymol., 34 (1974) 171-177. Wallach, D.F.H., Kranz, B., Ferber, E. and Fischer, H., FEBSLert., 21 (1972)29-33. Wallach, D.F.H. and Lin, P.S., Biochim. Biophys. Acta, 300 (1973)211-254. Watanabe, A. and Taketa, K., J. Biochem., 72 (1972) 1277-1280. Weiss, H.and Bucher, T., Eur. J. Biochem., 17 (1970) 561-567. Yamada, A.,J. Biochem. (Tokyo), 74 (1973) 187-190. Yon, R.J., Biochem J., 126 (1972)765-767.

151

Chapter 8

Solid matrix supports and the most used methods of binding One of the most important factors in the development of affinity chromatography and immobilized enzymes is the development of solid supports. This development is the controlling factor of whether and when it will be possible to introduce into practice procedures that have mainly been used in laboratories so far. As a carrier that fulfills all possible requirements does not yet exist, it is necessary to evaluate all aspects and factors during the selection of a support and the method of attachment, including the possibility of practical use, and economic aspects must also be taken into consideration. The key position of solid supports is presently parallelled by their rapid development. Increasing numbers of different types of supports are becoming commercially available, so that Tables 8.3 and 8.4, listing gels presently supplied by Pharmacia and Bio-Rad Labs.*, will be outof-date by the time this book is published. The selection of solid supports and the methods of attachment will always depend on which gels will respond best at a certain time to the requirements put on them, including their price. For a considerable number of research workers, only basic supports, which must be activated before attachment, will be available for a long time. Therefore, after the introductory section, which sets out the requirements for ideal supports, a section is given that deals with the most common solid supports and methods for their activation and attachment. As agarose is at present undoubtedly the most commonly used material (see Table 1l.l), Section 8.2.4 is devoted to it and is much more detailed than the sections describing cellulose, dialdehydestarch-methylenedianiline, dextran gels, ethylene-maleic anhydride copolymer, polyacrylamide and hydroxyalkylmethacrylate gels and glass. Separate sections are devoted to the question of spacers (8.3), blocking of unreacted groups (8.4) and the splitting off of attached affinity ligands (8.5). The last section (8.6) summarizes the properties of individual supports and methods.

8.1 REQUIRED CHARACTERISTICS An ideal matrix for successful application in affinity chromatography and for the immobilization of enzymes should possess the following properties (Porath): (1) insolubility; ( 2 ) sufficient permeability and a large specific area; (3) high rigidity and a suitable form of particles; (4) zero adsorption capacity; (5) chemical reactivity permitting the introduction of affinity ligands or enzymes. (6) chemical stability under the conditions required for the attachment, adsorption, desorption and regeneration; (7) resistance to microbial and enzymatic attack; *Only fiims known to the author are mentioned in the text. Therefore, the list is necessarily incomplete and it should in no case be considered as implying the recommendation of any particular firm or product.

SOLID MATRIX SUPPORTS AND BINDING METHODS

152

(8) hydrophilic character. Complete insolubility is essential, not only for prevention of losses of affinity adsorbent, but mainly for prevention of contamination of the substance being isolated by dissolved carrier. The requirement of sufficient permeability of the solid support, permitting sufficient freedom for the formation of complexes of macromolecules with complementary affinity ligands, has already been discussed in detail in Section 5.1. A high porosity of the solid carrier is further essential for the isolation of substances with a relatively weak affinity for the bound affnant (dissociation constant 2 lo-’). The concentration of the attached affinant, freely accessible to the isolated substance, should be very high in this instance, in order to achieve a strong interaction which retains physically the isolated substances migrating down the column. Table 8.1 shows the amount of chymotrypsin and glycine bound to 1 ml of hydroxyalkyl methacrylate gels of various pore sizes, depending on the exclusion molecular weights and the different specific surface areas (Turkovi er al., 1973). It is obvious that the amount of bound chymotrypsin depends directly on the specific surface area, which is largest with Spheron 3 0 0 and 500. The amount of bound glycine indicates that there are relatively small differences in the number of reactive groups. The relative proteolytic activity is also given in Table 8.1. The requirement of rigidity and a suitable shape of the particles is connected with the problem of flow-through rate. For a smooth course of affinity chromatography, good flow properties are important, i e . , the eluent should penetrate the support column at a sufficient rate even when the affinant is bound on to it. The particle size of the gel should not exceed 200 pm and should not be less than 5 pm. The sorbent should have minimal non-specific adsorption. When an insoluble affinant is prepared, it is important that it should be attached to the carrier in the form of covalently bound molecules only, and the molecules of the affinant that are not attached covalently must be washed out. This is difficult with supports that strongly adsorb the affinant molecules. Similarly, when substances that form a specific and reversible complex with the bound affinant are isolated, it is important that, as far as possible, only their retention TABLE 8.1 AMOUNTS OF CHYMOTRYPSIN AND GLYCIN BOUND TO HYDROXYALKYL METHACRYLATE GELS (SPHERONS) AS A FUNCTION OF THEIR SPECIFIC SURFACE AREAS Cel

Exclusion mol.wt.

Specific surface area (m’ /ml) ~~

Spheron los Spheron lo’ Spheron 700 Spheron 500 Spheron 300 Spheron 200 Spheron 100

1O8 lo6 700,000 500,000

300,000 200,000 100.000

0.96 5.9

3.6 23 19.5

0.6 0.2

Amount of bound glycine (mdml) ___-

Amount of bound chymotrypsin (mdml)

0.5 3.1 2.8 2.6 3.15 2.3 2.6

0.73 7.8 6.7 17.1 17.7 6.9

2.6

Relative proteolytic activity

(%I -

44 49

31 44

53 38

SOLID SUPPORTS AND COUPLING PROCEDURE

15 3

should take place on the column of insoluble affinant and only in the form of a specific complex with the bound affinant. This is one of the main reasons why carriers that contain ionogenic groups, such as the copolymer of ethylene with maleic anhydride, which sets carboxyl groups free after the affinant has been attached, have never been as widely applied as neutral agarose in affmity chromatography. The support must possess a sufficient number of chemical groups that can be activated or modified in such a manner that they become able to bind affinants. The capacity of a specific adsorbent prepared by the attachment of the affinant to the solid support is dependent on the number of these groups present. The activation or modification should take place under conditions that do not change the structure of the support. No less important are the chemical and mechanical stabilities of the carrier under the conditions of attachment of the affinant, and also at various pH values, temperatures and ionic strengths, in the possible presence of denaturating agents, etc., which may be necessary for good sorption and elution of the isolated substance. The possibility of the repeated use of a specific adsorbent depends on these stabilities. A further requirement is connected with the above, viz., that the specific sorbents should not be attacked by microorganisms and enzymes. This requirement is best fulfilled by inorganic supports, such as glass, or by synthetic polymers, such as polyacrylamide or hydroxyalkyl methacrylate gels. A hydrophilic character of a solid support is desirable not only because of the necessity of minimizing non-specific sorption and inactivation, but also because a hydrophobic character of the support can decrease the stability of some bound enzymes, on the basis of denaturation analogous to that produced by organic solvents.

8.2 SURVEY OF THE MOST COMMON SOLID SUPPORTS AND COUPLING PROCEDURES 8.2.1 Cellulose and its derivatives

Cellulose forms linear polymers of /3-1,4-linked D-glucose units with an occasional 1,6bond:

Commercially available celluloses are generally crosslinked with bifunctional reagents, such as 1-chloro-2,3-epoxypropane, and they are very stable to chemical attack. Glycosidic bonds are sensitive to acid hydrolysis, and under extreme conditions an almost quantitative decomposition to pure crystalline D-glucose may take place. On interaction with oxidative reagents, such as sodium periodate, aldehyde and carboxyl groups are formed. Cellulose can be attacked, for example, by microbial cellulases. Cellulose and its derivatives are produced by a number of firms. In addition to What-

SOLID MATRIX SUPPORTS AND BINDING METHODS

154

man (Maidstone, Great Britain) and Schleicher & Schull (Zurich, Switzerland), Serva (Heidelberg, G.F.R.) lists both cellulose derivatives and bromoacetylcellulose (BA-cellulose) p-aminobenzoylcellulose (PAB-cellulose), benzoyl-DEAE-cellulose (BD-cellulose) and benzoylnaphthoyl-DEAE-cellulose (BND-cellulose). Bio-Rad Labs. (kchmond ,Calif., U.S.A.) supply p-aminobenzoylcellulose under the trade-name Cellex PAB and aminoethylcellulose under the name Cellex AE. Miles Labs (Slough, Great Britain) produce a hydrazide derivative of CM-cellulose (Enzite-CMC-hydrazide), bromoacetylcellulose (BAC) and maminobenzyloxymethylcellulose(ABMC). In addition to the supports for the binding of affinants, they also supply insoluble affinants, as already mentioned in Section 6.1 1. The binding of affinants of a predominantly proteinic nature on to cellulose and its derivatives was discussed in a review by Silman and Katchalski, the binding of enzymes by Crook et al. and the binding of nucleotides, polynucleotides and nucleic acids by Gilham. These reviews mention many varied methods of bindings. In view of the present limited use of cellulose in affinity chromatography, we shall briefly mention some of them here. The most commonly used method of binding substances with a free amino group on to cellulose (CEL) is the Curtius azide method, used for the first time by Micheel and Ewers and today applied mostly in the modification of Hornby et al.: CEL--OH + Cl-CH,-COOH+

CEL-0-CH,-CO-NH-Protein

NaOH

CEL-0-CH,-COOH

-

Protein-NH,

CEL-0-CH,-CO-N,

PH 8

CH ,OH CEL-0-CH,-COOCH,

-

NaNO,

1

H,N-NH,

CEL-0-CH,-CO-NH-NH

HCI

After the preparation of carboxymethylcellulose a i d e by Curtius rearrangement, an isocyanate is formed on t o which the amino group of the affinant is bound. Affinants with basic amino groups can be further coupled to the carboxyl groups of carboxymethylcellulose in the presence of carbodiimide (Weliky et al.): CEL-0-CH,-COON

+ R-NH,

W CEL-0-CH,-CO-NH-R

+ H,O

N,N ‘-Dicyclohex ylcarbodiimide

Kay and Lilly developed the triazine method of protein binding. 2-Amino-4,6-dichloros-triazine is bound to the hydroxyl group of cellulose and reacts further with the amino group of the protein: ci

F’

N A N

NH-protein

2-Amino-4,6-dichloro-s-triazine was prepared from cyanuric acid and then allowed to react with various polysaccharide carriers, such as cellulose, DEAE-cellulose, CM-cellulose and also Sephadex and Sepharose. Solution A was prepared by dissolution of 10 g of 2amin0-4~6-dichloro-s-triazine in 250 ml of acetone at 50°C with addition of 250 ml of water at the same temperature. Solution B was a 15% (w/v) aqueous solution of sodium carbonate to which a 0.6-fold volume of 1 M hydrochloric acid was added. Cellulose (20 g) or CM-cellulose was then added to 100 ml of solution A and the mixture stirred at 50°C

155

SOLID SUPPORTS AND COUPLING PROCEDURE

for 5 min. After addition of 40 ml of solution B, the stirring was continued at 5OoC for a further 5 min. The pH of the suspension was rapidly decreased to below 7 by addition of concentrated hydrochloric acid. After filtration, the modified cellulose was washed with a mixture of acetone and water, pure water and finally 0.1 M phosphate buffer of pH 6.7, in which it was also stored. The binding of approximately 1.5% chymotrypsin solution in 0.5 M borate buffer of pH 8.75 gave, at 23°C and after 4.5 h, a product containing 19 mg of enzyme per gram, with an enzyme specific activity retention of 70%. Recently, dichloro-s-triazinyl resin was used by Morikawa et ul. as a carrier of immobilized enzyme. Jagendorf et ul. developed a method of protein binding based on the acylation of the hydroxyl group in cellulose with bromoacetyl bromide and subsequent alkylation of the amino group of the protein: CEL-OH

+ Br-CO-CH,-Br

+ CEL-0-CO-CH,-Br

Protein-NH , -CEL-0-CO-CH,-NH-protein

The preparation and properties of aminoacylase covalently attached to halogenoacetyl celluloses have been described by Sat0 et al. Coupling of 0.6% aminoacylase in 0.2M phosphate buffer of pH 8.5 for 24 h at 7OC yielded a product that contained 56 mg of enzyme protein per gram with a specific activity retention of 30%. The first attachment of an affinant on to cellulose was carried out by means of diazonium groups (Campbell et al.):

The affinants are bound by their aromatic residues (in the case of proteins mainly by tyrosine and histidine), but also non-specifically and more slowly by their amino groups (Gundlach et al.; Tabachnik and Sobotka, 1960). Nucleic acids are bound to aminoethylcellulose mostly by using periodate oxidation (Gilham; Lowe and Dean): C EL -'O -CH2-

C HZ- NH2 Nal04 H '

Alkaline solution

w

OH OH Nucleatides

sugars

CEL-0-CH,-CH,-N=CH-@

I

NaBH4

CEL-O-CH~-CH,-NH-CH,-@

(Schtff's base)

SOLID MATRIX SUPPORTS AND BINDING METHODS

156

Although cellulose was used as a carrier mainly during the initial period of the develop ment of affinity chromatography, it is still used, as is evident from Table 1 1 . l . Lowe et ai. compared the properties of sorbents prepared from both cellulose and Sepharose. They determined titration graphs for 6-aminohexyl-NAD+-Sepharose,unmodified Sepharose, 6-aminohexanoyl-Sepharose in comparison with corresponding cellulose derivatives (Fig. 8.1). From the curves given the formation of ionizing groups after activation with cyanogen bromide is evident; this was also demonstrated on cellulose when dicyclohexylcarbodiirnidewas used for the coupling reaction (Larsson and Mosbach). The deviation observed for Sepharose is substantially smaller than that for cellulose, and this is usually given as a reason for the unsuitability of cellulose for the preparation of affinity sorbents. Non-specific preferential sorption of nucleotides on cellulose with an increased content of lignin contaminants was mentioned in Section 6.8 (Table 6.3) (De Larco and Guroff).

I

6

I

,

t

I

I

I

1

I

I

1

2

3

4

5

VOLUME O F 0.01N HCI. ml

Fig. 8.1. Titration curves of 6-aminohexaonyl-NAD+ polymers. The solid lines represent Sepharose 4-B alone; 0 , coupled to 6-aminohexanoic acid; a, as 6aminohexanoyl-NAD '-Sepharose. The broken lines represent cellulose alone; 0 and as above. Reproduced with permission from C.R.Lowe et al., Eur. J. Biockern., 41 (1974) 347-351.

157

SOLID SUPPORTS AND COUPLING PROCEDURE

8.2.2 Dialdehyde starch-methylenedianiline (S-MDA) S-MDA resin is prepared from the dialdehyde of starch (a product of the periodate oxidation of starch) by condensation with 4,4'-diaminodiphenylmethane(methylenedianiline) and subsequent reduction of the Schiff s bases formed (Goldstein): CH20H

x

...0

o

CH I1 0

L

HC II 0

o

y

L

CH II 0

o---

HC

ti

-

-

NH2-@H2eN~2

+

4,4'-Diarn1nodiphenylrnethane

0

Dialdehyde starch

CH2OH

CH20H

FOL CH

HC

0

66 66 II

NH2

II

$,

NH2

N

I1

-

-

CH,

QQ N II CH

II

CH

HC

-

o

CH2

~

o

N

I1

HC

~

CH20H

o

~

o

~

o

-

-

CH20H

NaBH4

CH20H

...o ~

CH20H

o CH2 H2C I I NH NH

00 CH2

CH2

QO NH2

NH2

CH H C 1 2

OH

2

~O-.-

o NH2

AH

~

o

NH2

00

0

6 66 NH

---oxor

I

OH

CH, H2C

CH,OH

NH

o

~

I

NH

;

CH,OH

I

~

o

-

-

-

158

SOLID MATRIX SUPPORTS AND BINDING METHODS

For binding with proteins, the support is first converted into polydiazonium salts:

The diazotization capacity of S-MDA resins is 0.24-0.26 mequiv./g, and the maximum capacity for the binding of proteins is 80-100 mg of protein per gram of resin. The suppor mentioned is used rather for the binding of enzymes. S-MDA-mercuripapain (Wolodko and Kay) is an example. Dialdehyde starch is produced by Miles Chemical Co. (Elkhart, Ind., U.S.A.) under the trade-name Sumstar 190. 8.2.3 Dextran gels Dextran is a branched-chain glucose polysaccharide produced in solutions containing sugar by various strains of Leuconostoc mesenteroides. Soluble dextran, prepared by fractional precipitation with ethanol of partially hydrolysed crude dextran, contains more than 90%of a-l,6-glucosidic linkages, and it is branched by 1,2-, 1,3- and 1,Cglucoside bonds. When crosslinked with 1-chloro5,3-epoxypropane in alkaline solution, dextran affords a three-dimensional gel with the partial structure -0-Ch2

0 CHOH I

OH

$"2

0

-S-CH2

The most important producer of dextran gels, supplied under the trade-name Sephadex, is Pharmacia (Uppsala, Sweden). The gels are very stable to chemical attack; for example, exposure for 2 months to 0.25 M sodium hydroxide solution at 60°C has no influence. The glucosidic bond is sensitive to hydrolysis at low pH, although it is stable for 6 months in 0.02 M hydrochloric acid, or for 1-2 h in 0.1 M hydrochloric acid or 88%formic acid (Lowe and Dean). Under the effect of oxidizing agents, aldehyde or carboxyl groups are formed. As regards thermal stability, dextran gels can withstand heating in an autoclave at 1 10°C (in solution) for 40 min, or at 120°C when dry. Drying and swelling is reversible. The gels swell to a certain extent even in ethanol, ethylene glycol, formamide, N,N-dimethy formamide and dimethyl sulphoxide.

SOLID SUPPORTS AND COUPLING PROCEDURE

159

For the attachment of affinity ligands to dextran gels a number of coupling methods that have been described for cellulose can be employed, and will be described for agarose, such as binding after activation with cyanogen bromide, the triazine method, binding using epoxides or difunctional derivatives or binding via acylazido intermediates. A simple method for the binding of proteins to insoluble polysaccharides has been described by Sanderson and Wilson. The reaction sequence for the binding of proteins to polysaccharides is CHzOH

-0@o-

Oxidation, NaI04

-0

f y o -

-

CHzOH

-o$lo-

OHC CHO

OH

NaBH4 /BH4

/

reduction

k

Pio- -0T7-

-HOCH,

CH,OH

NI

R

The polysaccharide is “activated” by oxidation with 0.01-0.5 M sodium periodate for 1 h. The aldehyde formed reacts with the protein. For example, the oxidized polysaccharides were washed with water by centrifugation and 10 mg (dry weight) suspended in 1 ml of phosphate-buffered saline (pH 8) containing 10 mg of bovine serum albumin and agitated continuously for 20 h. Subsequent reduction with sodium borohydride (a freshly prepared 1%solution) led to the stabilization of the bonds between the protein and the polysaccharide, and to the reduction of the residual aldehyde groups. The use of dextran gels is partly restricted by their rather low porosity (see Section 5.1). Examples of their use in affinity chromatography are given in Table 11.l. They are widely used without any modification as specific sorbents for the isolation of a series of lectins. 8.2.4 Agarose and its derivatives

Agarose is a linear polysaccharide composed of alternating D-galactose residues and 3,6-anhydro-~-galactoseunits:

-0

D-Galactose

3.6-Anhydro-L-galactose

and is the carrier most commonly used in affinity chromatography at present. As demonstrated by Cuatrecasas and Anfinsen (1971a), agarose fulfills almost all of the requirements of an ideal carrier. The main producers of agarose are Pharmacia (Uppsala, Sweden), under the trade-name

SOLID MATRIX SUPPORTS AND BINDING METHODS

160

Sepharose, and Bio-Rad Labs. (Richmond, Calif., U.S.A.), under the trade-name Bio-Gel A. Sepharose is an agarose gel with spherical particles, and is sold in the swollen state, suspended in water containing 0.02% of sodium azide as a bacteriostatic agent. The following three types first appeared commercially: (1) Sepharose 6B with a cu. 6% concentration of agarose of swollen particles of size 40-210 pm for the fractionation of substances of molecular weight 10’-lo6; (2) Sepharose 4B with a cu. 4% concentration of agarose, the swollen particles of which are of 40-190 pm in size and suitable for the fractionation of molecules of molecular weight 3 105-3* lo6;and (3) Sepharose 2B with a 2% concentration of swollen agarose of 60-250 pm particle size, suitable for the fractionation of substances of molecular weight 2 - 106-25 * lo6. From Table 1 1.1, it is evident that Sepharose 4B is the most commonly used gel; a comparison of some properties of Sepharose 2B, 4B and 6B are given in Table 8.4. In 1975, Sepharose CL (2B, 4B, 6B) was introduced, prepared from appropriate types of Sepharoses by crosslinking with 2,3-dibromopropanol in strongly alkaline medium, and further desulphurization by alkaline hydrolysis under reductive conditions. Bio-Gels A are produced in a variety of types, as follows:

-

Bio-Gel A-0.5 m Bio-Gel A-1.5 m Bio-Gel A-5 m Bio-Gel A-15 rn Bio-Gel A-50 m Bio-Gel A- 150 m

Agarose content

Molecular weight exclusion limit

10% 8% 6% 4% 2% 1%

0.5 * lo6 1.5- lo6 5.106 15~10~ 50. lo6 150- lo6

All of these gels are produced in three particle sizes, viz., 149-290,74-149 and 38-74 pm. They are delivered fully hydrated, in suspension, containing 0.02%of sodium azide, 0.001 M Tris and 0.001 M EDTA. Agarose gels under the name SAG(Ago-Gel)-10, -8, -6, -4 and -2 with molecular weight exclusion limit of 25 104-15 lo’ are also supplied by Seravac Labs. (Maidenhead, Great Britain) and Mann Labs. (New York, N.Y., U.S.A.). Recommended solvents are dilute aqueous solutions of salts. Derivatives of Sepharose adjusted directly for the attachment of affinity ligands are listed in Table 8.2. The newly introduced cyanogen bromide-activated Sepharose 6MB (macro-beads of Sepharose 6B; 200-300 pm) is intended for the affinity chromatography of cells. Activated thiol-Sepharose 4B (mixed disulphide formed between 2,2 ’-dipyridyl disulphide and glutathione coupled to cyanogen bromide-activated Sepharose 4B) is intended for covalent chromatography (see Section 7.2). Derivatives of agarose (Bio-Gel A15 m, 74-149 pm) modified for use in affinity chromatography are produced by Bio-Rad Labs. under the trade-name Affi-Gel, and are listed in Table 8.3. As regards the stability of agarose, it is stable in the pH range 4-9, and temperatures below 0°C or above 4OoC are not recommended. Sepharose is resistant to high salt concentrations, urea (7 M) and guanidine hydrochloride (6 M) (Cuatrecasas). It is stable even when exposed at room temperature to 0.1 M sodium hydroxide solution and 1 M hydro-

161

SOLID SUPPORTS AND COUPLING PROCEDURE

TABLE 8.2 SELECTIVE COUPLING FOR AFFINITY CHROMATOGRAPHY Type of ligand

Functional WUP

Sepharose derivative

coupling conditions

Comments

Carbohydrates and other hydroxyl compounds

-OH

Epoxy-activated Sepharose 6B

16 h at 2O-4S0C, pH 9-13

Organic solvents (e.g., up to 100% formamide) can also be used

Amino acids and peptides;

-NH,

Activated CHSepharose 4B

1 h at room temp., pH 5-9

Epox y-activated Sepharose 6B

16 h at 20-45"C, pH 9-13

Direct coupling of amino acids and peptides. Other functional groups (e.g., -CO,H) require no protection

CNBr-activated Sepharose 4B and 6MB

2 h at room temp., or 16 h at 4"C, pH 8-10

The method of choice for coupling proteins

Proteins

Amino acids, keto acids and carboxylic acids

-CO,H

AH-Sepharose 4B

carbodiimide, 16 h at room temp., pH 4.5-6.0

Organic solvents (e.g., up to 50%dioxane) can also be used

Thiol compounds

-SH

Activated ThiolSepharose 4B, epoxy-activated Sepharose 6B

Wide range of conditions

Coupling reaction is readily reversible

chloric acid for a short time (2-3 h). For the affinity chromatography of weakly watersoluble substances, 50%dimethylformamide or 50% ethylene glycol can also be used. Freeze-drying can be carried out only after the addition of protective substances, for example dextran, glucose and serum albumin. The stability of an agarose matrix can be considerably increased by crosslinking with epichlorohydrin.(Kristiansen; Porath et al., 197I), 2,3-dibromopropanol (Kristiansen; L%s, 1975) or divinyl sulphone (Kristiansen; Porath et al., 1975) before activation with cyanogen bromide. The stability in aqueous medium increases with crosslinking, in both acidic and alkaline regions (pH 3-14). The possibility of using chaotropic salts, mainly for the elution of antibodies, is discussed in Section 10.2. Through crosslinking, the gels acquired further stability in organic solvents, such as ethanol, dimethylformamide, tetrahydrofuran, acetone, dimethyl sulphoxide, chloroform, dichloromethane, dichloroethane and dichloroethane-pyridine (1 :1).

Coupling of affinants on cyanogen brornide-activated agarose The most commonly utilized method of affnant bonding on Sepharose activated with cyanogen bromide was developed by Porath et al. (1967) and Axin et al. The affnants are bound by means of primary aliphatic or aromatic amino groups in their non-protonated form. A x h and Ernback assumed the formation of three different structures during the

c

TABLE 8.3

o\ N

PROPERTIES OF AFFI-GEL SUPPORTS

Affi-Gel

Arm length

Formula*

Terminal reactive group

Ligand bonding Coupling group

Type of bond

Activated carboxyl: N-hydroxysuccinimide ester

Primary aliphatic or aromatic amino

Peptide

Amino

Free carboxyl

Peptide

Amino

Alkyl halides

C-N covalent

(A) 0

~ f f i - ~ eo l l

1o**

$-OCH,C

HC ,

H~N: H

c H,C

H,CON

0

cn

h

U

Affi-Gel401

IS***

&O!;H(CH~

,NHCOC H3 ) 3 ~ ~ ( ~ ~ 2 (C \ H2~).,SH~ C ~ C ~ ,

Carboxyl

Free primary aliphatic Peptide or aromatic amino

Carboxyl

Sulphydryl hydroxyl

Sulphydryl

Sulphydryl free carboxyl

Disulphide ester

E

U

z

2;

E

X cn

Disulphide thioester !a3

-I m

NH

Affi-Gel 501

15***

$ - O ~ NH (CH,$NH

(CH,),NHCO

~

60 = Agarose gel matrix (Bio-Gel A-15 m, 100-200 mesh). *Arm linked to agarose gel by ether linkage. -Arm linked to agarose gel by cyanogen bromide coupling.

H

~

C

I

Organomercury(I1) chloride

Active sulphydryl

Mercaptide

s

2:

U

m

SOLID SUPPORTS AND COUPLING PROCEDURE

163

binding, i. e., N-substituted carbamates, N-substituted imidocarbonates and N-substituted isoureas. N-Substituted isourea was assumed by Svensson as the predominant form on the basis of isoelectric focusing studies. Wilchek et al. presented various chemical evidence to prove that N-substituted isoureas are indeed the main reaction products in the reaction between amines and cyanogen bromide-activated Sepharose, represented by the following scheme: Sepharose Sepharose OH

Sepharose

Sepharose

CNBr

0-CIN

[OH

[OH

Activation

coupling

NH*R

B

NH

T;1H20 0-C-NHR

foH

C

+

It

A-NH-C-NHR’

The attachment of amines on cyanogen bromide-activated Sepharose thus produces Nsubstituted isoureas. So, in the neutral and alkaline region a group capable of protonation is formed, which functions as an ion exchanger. Further addition of amines on N-substituted isoureas brings about the formation of N1,N2-disubstitutedguanidines. In combination with the hydrophobic chains of alkylamines, the positive charges formed act as detergents. By this means non-specific sorption is increased, which can be considered a reversible denaturation of proteins by the “detergent-like” agarose derivatives. As a consequence, in some instances, such as with penicillinase eluted from ethylSepharose with 1 M sodium chloride solution, the enzyme is inactivated (Wilchek, 1974). The formation of cationic groups on the carrier at stage B can be prevented by using hydrazides instead of aliphatic amines. The resulting derivatives have a pK of 4.2 and therefore they do not carry a charge at neutral pH (Lamed et al.; Nishikawa and Bailon, 1975b; Wilchek and Miron, 1974a, b). The advantages of the use of hydrazides as spacers is discussed in detail in Section 8.3. The reaction of N-substituted isourea conjugates of Sepharose with amines and bovine serum albumin (Wilchek et al., 1975) helped to elucidate the discrepancies between various published data on the stability of conjugates. Compounds containing nucleophiles, for example amines or proteins, cleave the isourea bonds as shown in the preceding scheme. If a cleavage of the bond was observed in the absence of the compounds reacting with isourea bonds, then it is due rather to the liberation of the adsorbed molecules of the affinity ligand, which were not well washed out. The amount of material released, formed by the very slow hydrolysis of isourea, is usually very small and does not interfere during the normal course of affinity chromatography. As will be discussed in Section 8.5, the release of the affinity ligand from the specific adsorbent is critical mainly in systems with a high affinity, or when a small amount of substance is isolated. Schnapp and Shalitin circumvented the leakage of the affinity ligands after coupling with cyanogen bromideactivated Sepharose in the presence of nucleophiles by replacing the isourea bond with the more stable guanidine bond; this they performed by using an amine group-containing carrier for the activation with cyanogen bromide. The degree of agarose activation, measured on the basis of the capacity for binding of small peptides (Axe’n and Ernbach), is directly proportional to the pH. Activation takes place at pH 11. The whole procedure for the activation of Sepharose with cyanogen bromide (Axe’n et al.; Cuatrecasas; Prath et al., 1967) is described in detail below.

164

SOLID MATRIX SUPPORTS AND BINDING METHODS

Cyanogen bromide activation of agarose by use of a p H meter

Washed and decanted Sepharose is suspended in distilled water (1 :1). The suspension is placed in a well ventilated hood, a pH meter electrode pair is immersed in the suspension and finely divided cyanogen bromide (50-300 mg per millilitre of packed Sepharose) is added gradually, with constant stirring. The suspension is maintained at pH 11 by addition of sodium hydroxide solution. The concentration of the sodium hydroxide solution used depends on the amounts of Sepharose and cyanogen bromide present; for 5-10 ml of packed Sepharose and 1-3 g of added cyanogen bromide, Cuatrecasas recommended the use of 2 M sodium hydroxide solution, and for 100-200 ml of packed Sepharose and 20-30 g of cyanogen bromide an 8 M solution is suitable. The temperature should not exceed 20°C; if cooling is necessary, ice can be added. The reaction is completed in 8-12 min. After rapid transfer of the suspension on a Buchner funnel under constant suction, the activated Sepharose is washed with cooled buffer solution of the same composition as is to be used for the subsequent coupling of the affinant. The buffer volume should be 10-1 5 times that of the Sepharose to be activated. The washed Sepharose is suspended as rapidly as possible in an equal volume of the affinant solution. According to Cuatrecasas, the washing, addition of affinant solution and mixing should not take more than 90 sec. Even at a low temperature, the activated Sepharose is unstable. Cyanogen bromide-activated Sepharose 4B is produced commercially by freeze-drying with the addition of dextran and lactose, which must be washed out before use. The manufacturer (Pharmacia) gives the following procedure for coupling the affinant with cyanoger bromide-activated Sepharose. The required amount of gel is allowed to swell in low3M hydrochloric acid and the solution is then used for washing the gel for 15 min. The volume of 1 g of the freeze-dried gel when swollen is approximately 3.5 ml. It is recommended that 200 ml of the solution per gram of dry gel should be used for the washing, in several batches. Immediately after washing, the solution of the affinant to be coupled is added. The optimal conditions for coupling the affinant, i.e., pH, buffer composition and temperature, are dependent to an appreciable extent on its character. In general, the coupling reaction takes place most effectively at pH 8-10, but if the nature of the coupled affinant requires it, lower pH values can also be used. The binding of a sufficient amount of affinant can usually be carried out successfully even at that pH if the amount of cyanogen bromide is increased during activation and the amount of affinant is increased during the binding. The affinant, especially if it is of a proteinic character, is dissolved in a buffer of high ionic strength (about 0.5) in order to prevent non-specific adsorption. The higher ionic strength then facilitates subsequent washing. Carbonate or borate buffers with sodium chloride added can be used. The amount of the affinant coupled depends on the proportion of the affinant in the reaction mixture and the volume of gel, the pH of the reaction, the nature of the coupled affinant (number of reactive groups, etc.) and also on the reaction time and temperature. For example, when chymotrypsin is coupled with 2 ml of cyanogen bromide-activated Sepharose at pH 8, only 5 mg were coupled when 10 mg of protein were present, with 20 mg of protein ca. 8 mg were coupled, and with 30 mg of protein the amount coupled was ca. 10 mg. At room temperature (20-25OC), the coupling is usually completed after 2 h, while at lower temperatures it is recommended that the mixture is allowed to stand over-

SOLID SUPPORTS AND COUPLING PROCEDURE

165

night. During the coupling, the reaction mixture must be stirred. Stirring with a magnetic stirrer is not recommended, as it may cause mechanical destruction of the gel. When the coupling is completed, the gel with the coupled affinant is transferred on to a sintered-glass filter and washed with the buffer used during the coupling. In order to eliminate the remaining active groups, the manufacturer (Pharmacia) recommends blocking them with 1 M 2-aminoethanol at pH 8 for 2 h. The final product should then be washed four or five times alternately with buffer solutions or high and low pH. For example, acetate buffer (0.1 M, pH 4) and borate buffer (0.1 M, pH 8.5), each being 1 M in sodium chloride, can be used. As already stated, all non-covalently bound substances should be eliminated during the washing.

Qanogen bromide activation of agarose in concentrated phosphate buffer (Porath et al., 19 73) For the activation of agarose on a large scale and for the activation of agarose membranes or thin layers of agarose coating glass beads, Porath et al. (1973) developed a very simple and reproducible method of activation in alkaline phosphate solution of very high buffering capacity, during which control of pH and intensive stirring are not necessary. Conditions for high, medium and low degrees of activation, which must be selected with respect to the nature of the bound affinant, were established. For highly activated gels the conditions of activation are further modified both for gels with a low content of agarose (p = 1-4%) and for a high content of agarose 01 = 4-8%); in all of these procedures the percentage of agarose in the swollen gel (indicated by p ) is considered.

Procedure A : Highly activated agarose gels ( p = 1-4%) The swollen gel (10 g, with p% of agarose) is suspended in 2 . 5 ml ~ of cold (5-1OoC) 5 M potassium phosphate buffer (3.33 mole of K3P04 t 1.67 mole of KzHP04are dissolved per litre of solution) and the suspension is diluted with distilled water to a total volume of 20 ml. A 0.1~-mlaqueous solution of cyanogen bromide (100 mg/ml) is then added in small portions over 2 min. The solution is gently stirred at 5-10°C for 10 min (including the addition of cyanogen bromide). The product of activation is washed on a glass filter with cold water until neutral. Procedure B: Highly activated agarose gel ( p = 4-8%) The agarose gel is washed with 2 M phosphate buffer and filtered off under suction. A 10-g amount of a p% agarose gel is suspended in 2 . 5 ~ml of cold phosphate buffer and activated as under A with 1.Op ml of an aqueous solution of cyanogen bromide. Procedure C: Medium activated agarose gels ( p = 1-8%) The procedure is identical with that described under A, but the volume of the buffer is 0 . 5 ml ~ and the volume of the cyanogen bromide solution is 0 . 2 ml. ~ Procedure D: Weakly activated agarose gels { p = 1 4 % ) The procedure is analogous to that described under A, but the volume of buffer is 0 . 1 2 ~ml and the volume of the cyanogen bromide solution is 0.05 ml.

166

SOLID MATRIX SUPPORTS AND BINDING METHODS

The activated products were rapidly washed with a 0.25 M solution of sodium hydrogen carbonate (pH 9) in wluch the binding of chymotrypsin was carried out, applying a proceh r e similar to that used for commercially activated Sepharose.

Simplified method of cyanogen bromide activation of agarose according to March et al. (19 74a, b )

Cyanogen bromide is dissolved in acetonitrile (2 g/ml = 19 M). This solution is stable at -20°C and can be stored at this temperature. Equal volumes of a 50%(v/v) suspension of agarose in water and 2 M sodium carbonate solution are mixed and cooled to ice temperature. A corresponding volume of the cyanogen bromide-acetonitrile solution is then added (0.2 g of cyanogen bromide per packed millilitre of agarose) and the mixture is stirred for 1-2 min. The activated gel is then washed and added to the solution of the substance that is to be bound (for example, in 0.2 M sodium hydrogen carbonate solution, pH 9.5). The advantage of this method is its simplicity and a substantial shortening of the unpleasant and dangerous manipulation with cyanogen bromide during weighing. The high solubility of cyanogen bromide in N-methyl-2-pyrrolidone was used by Nishlkawa and Bailon (1975a) for the study of the effect of the amount of cyanogen bromide on the activation of agarose. Fig. 8.2 shows the effect of the amount of cyanogen bromide used for activation on the amount of 6-aminohexanoic acid bound on 6%, 4% and 2% agarose. Cyanogen bromide was added in a 16% (v/v) aqueous solution of Nmethyl-2-pyrrolidone. By varying the amount of cyanogen bromide during activation, the final amount of the bound affinant can be regulated. Cyanogen bromide should be colourless, with minimal “sweating” on the crystals. Yellowish preparations are poorly reactive. On the basis of the study of the effect of the concentration of 6-aminohexanoic acid on the binding on cyanogen bromide-activated Sepharose, it can be inferred that a high concentration of affinant during the short incubation time with the activated gel gives better results than a low concentration and long incubation times. Table 8.4 gives the amounts of affinity ligand bound to 2%, 4% and 6% agarose, and also includes the calculated values for the expected hydroxyl density in the different agarose gels. The yield of the chemical coupling for all types of gels appears to be about 1-2% of the possible hydroxyl groups. Table 8.4 also gives the data obtained by Sundberg and Kristiansen for the binding of elastase t o cyanogen bromide-activated Sepharose 2B, 4B and 6B,and the maximum flow-rates determined by Robinson et al. with individual types of Sepharose before and after the attachment of p-aminophenyl4-D-thiogalactopyranoside. Another important result from the work of Nishikawa and Bailon (1975a), concerning this method, is the necessity to keep the temperature between 4 and 10°C during the activation reaction with cyanogen bromide, while the binding reaction of the affinity ligand can be carried out at room temperature. In a number of gels, E-aminocaproic acid was bound under various conditions, and its amount was determined both by titration and on the basis of nitrogen determination. In all instances more nitrogen was incorporated during the action of cyanogen bromide than would be expected from the content of bound e-aminocaproic acid, determined by titration, the ratio of these values ranging between 3: 1

SOLID SUPPORTS AND COUPLING PROCEDURE

167

4(

-al -m E

\

.-5 J

D

m

-

a

60 2c

x

Y

IBrCNI. g/100ml gel

Fig. 8.2. Effect of cyanogen bromide on e-aminocaproic acid incorporation. 6% Agarose (Sepharose 6B, o), 4% agarose (Sepharose 4B, A) and 2% agarose (Bio-Gel A-SO m, 0)were treated with cyanogen bromide at 4-10°C. Usually comparable weights of e-aminocaproic acid (0.018 mole per 100 ml of activated gel) were used in the coupling step. The carboxylic groups in the gel were determined by titration. Reproduced with permission from A.H. Nishikawa and P. Bailon, Anal. Biochem., 64 (1975) 268-215.

and 9: 1. The increased content of the incorporated nitrogen increased with an increase in the temperature used during the activation step (at 2°C a 3.3 times higher amount, at 4°C a 4.9 times higher amount and at 20°C an 8.3 times higher amount). As activation is very slow at 2"C, this temperature cannot be used in practice. A temperature of 10°C seems to be a practical compromise between the activation rate and the minimization of side-reactions. These side-reactions, characterized by an increased nitrogen content in immobilized preparations, constitute one of the sources of the cationic groups that cause non-specific sorption. These are discussed in detail in Section 10.3. Lowe and Dean stressed the small but significant content of bromine in activated gels, which might be caused by the presence of tribromotriazine in cyanogen bromide preparations, and which also can paricipate in the binding reaction.

'TABLE 8.4 SUMMARY OF PROPERTIES OF AGAROSE GELS Polymer

Sepharose 28 Bio-Gel A50m Sepharose4B Sepharose6B

Gel (%)

2 2 4 6

Calculated values for the expected hydroxyl density (MI*

Exclusion Limits of' chemical molecular incorporation* weight )(lo6* (coupling of 1 6 diaminohexane) beqiv./ml)

-

50 15

0.29 0.58 0.88

5

5 20 60

*From A.H. Nishikawa and P. Bailon, Anal. Biockem., 64 (1975) 268-275. **From L. Sundberg and T.Kristiansen, FEBS Lett., 22 (1972) 175-177. ***From P.J. Robinson et al., Biochim. Biophys. Acta, 285 (1972) 28-35.

Amount of elastase in the reaction mixture per 1500 mg of dry gel (mg)**

50 50 50

Elastase bound per gram of dry conjugate (mg)**

Coupling yield** based on amount of enzyme added

Max. flow-rate (ml/min/cm2) before and after cnupling of p-aminophenyl-p-Dthiogalactopyranoside***

(%)

Before coupling

After coupling

37.5

0.95

1.1 -

12.5 -

-

25 33

80 99

-

1.8 3.6

2.2 3.8

m

2

SOLID SUPPORTS AND COUPLING PROCEDURE

169

A further source of non-specific sorption in agarose is the content of sulphur (Gas, 1972), which can be almost completely eliminated by alkaline desulphurization in the presence of sodium borohydride followed by reduction with lithium aluminium hydride in dioxane. The effect of a decreased content of sulphur in various agarose preparations on the adsorption capacity for cytochrome C was described by Porath et al. (1971). Attachment of affinity ligands to agarose by means of bifunctional oxiranes (Sundberg and Porath) Bisoxiranes (e.g., 1,Cbutanediol diglycidyl ether) were used for the introduction of reactive oxirane groups into agarose. These groups can further react with compounds that contain amino, hydroxyl or thiol groups:

OH

+

C p 7 C H - C H2-0

- ( C H 2)4-0-C

0

-#-#-

O-CH,-CH-CH,-O-1CH2)4-O-CH2-CH-CH2 dH

O-CH2-CH AH

H2- C H - C H 2 \ / 0

+

H2N-R

'0'

-CH2-O-(CH2),-O-CH2-CH-CH2-NH I OH

-

-R

The method is suitable for the binding of sugars that form ether linkages through their hydroxyl groups. With proteins and peptides the method forms alkylamine linkages through their primary amino groups. With substances that contain thiol groups, thioether linkages are formed. When 1,4-bis(2,3-epoxypropoxy)butane is used, a spacer corresponding to a 12-carbon chain is introduced between the ligand and the agarose chain. Through the effect of bisoxirane, crosslinking of carbohydrate chains of the gel matrix takes place, which increases its stability. This permits more drastic conditions for the binding and elution to be applied. These gels are available commercially under the trade-name epoxyactivated Sepharose.

Activation, crosslinking and binding One gram of suction-dried agarose (Sepharose 6B) is washed with water on a glass filterfunnel and then mixed with 1 ml of diglycidyl ether and 1 ml of 0.6 M sodium hydroxide solution containing 2 mg of sodium borohydride per millilitre. The suspension is stirred (by rotation) at 25°C for 8 h, then the reaction is stopped by washing the gel with large volumes of water (500 ml) on a glass filter-funnel. The binding is carried out by mixing 1 g of the suction-dried oxirane-agarose with 2 ml of a solution of substance to be bound in a buffer of adequate pH. The proteins are bound at pH 8 5 - 1 0 at 25"C,the reaction time being 15-48 h. For amino acids, amines, carbohydrates and other more stable compounds, a pH of 9-1 1 can be applied at 25-75°C with a reaction time of 4-15 h. An increased binding yield can be achieved at higher pH and temperatures. A decrease in the yield at lower pH and the temperature used for the binding of proteins can be compen-

SOLID MATRIX SUPPORTS AND BINDING METHODS

170

sated for by prolongation of the reaction time. The remaining oxirane groups capable of further bindings are blocked, for example, with 2 M glycine or 2-aminoethanol, preferably at a pH above 8.5, at 23°C in 24 h. In addition to aqueous solutions, organic solvents can also be used, such as dimethylformamide or dioxane (50% of the final mixture). The ether and alkylamine linkages are very stable, as is evident from Fig. 8.3, in which the stability of glycylleucine coupled to epoxy-activated Sepharose 6B is shown at 70°C and pH 3 , 7 , 11 and 13.

I

0'

I

1

2

4

6

TIME OF HYDROLYSIS .weeks

Fig. 8.3. Stability at 70°C of glycylleucine coupled to epoxy-activated Sepharose 6B. Reproduced by permission of Pharmacia Fine Chemicals AB, Affinity Chromafogruphy,principles and methods, Pharmacia Fine Chemicals, Uppsala, Sweden.

Reversible covalent immobilization of enzymes by thiol-disulphide interchange (Brocklehurst et al.)

Carlsson et d.employed epoxide-activated agarose as the basis for the preparation of the mercaptohydroxypropyl ether of agarose gel, which they used for covalent immobilization of &-amylase and chymotrypsin by thiol-disulphide interchange. This technique consists of two steps: (a) thiolation of enzymes with methyl 3-mercaptopropioimidate; (b) binding of thiolated enzymes to a mixed disulphide derivative of agarose obtained by reaction of the mercaptohydroxypropyl ether of agarose with 2,2 '-dipyridyl disulphide. The immobilized preparations formed possessed high activity. Immobilized &-amylase was used for continuous hydrolysis of starch. When the preparation had lost its enzymatic activity, the inactive protein was reduced off and the gel used for the binding of a new active thiolated a-amylase. The thiol enrichment of enzyme was carried out in the following manner: 30 mg of

SOLID SUPPORTS AND COUPLING PROCEDURE

171

enzyme were dissolved in 5 ml of 0.1 M sodium hydrogen carbonate solution, pH 8.2. The solution was deaerated in a nitrogen atmosphere for 15 min and 0.1-2 mg of methyl 3-mercaptopropioimidate was added. The thiolation was carried out at room temperature under nitrogen for 60 min. Excess of imidate was eliminated by gel filtration on Sephadex G-25, using 0.1 M sodium hydrogen carbonate solution as eluting reagent. In order to prevent the oxidation of thiolated enzymes, dithiothreitol(1 mM final concentration) was added to the solution just before the gel filtration. Activated thiolSepharose was prepared according to Brocklehurst et al. Epoxideactivated agarose (50 g) was washed on a glass filter with 0.5 Mphosphate buffer (4.1 g of NaH,PO, H20 t 2.8 g of Na2HP04 2H20 dissolved in 100 ml of distilled water, pH 6.3). The gel was sucked free from interstitial buffer and suspended in the same buffer to a final volume of 100 ml. A 2 M solution of sodium thiosulphate (50 ml) was added and the reaction mixture was shaken for 6 h at room temperature. The gel was washed free from sodium thiosulphate with distilled water. The thiosulphate ester gel (50 g) was suspended in 0.1 M sodium hydrogen carbonate solution (1 mM EDTA) to a total volume of 100 ml. Dithiothreitol(60 mg) dissolved in 4 ml 1 mMEDTA solution was added to the suspension. The reaction time was 30 min at room temperature. The gel was washed on a glass filter with 0.1 M sodium hydrogen carbonate solution (1 Min sodium chloride and 1 mM in EDTA) and finally with 1 mM EDTA solution. The thiol-agarose (50 g) was washed on a glass filter with 1 mM EDTA solution. The washed gel was rapidly added to 200 ml of 2,2'-dipyridyl disulphide solution (1.5 mM in 0.1 M sodium hydrogen carbonate solution). The mixture was stirred during the reaction, which was allowed to proceed for 30 min at room temperature. The product was washed with 0.1 M sodium hydrogen carbonate solution, 1 M sodium chloride solution and finally with 1 mM EDTA solution. The degree of substitution was determined by nitrogen determination according to Kjeldahl. The product, called activated thiol-agarose, is stable to storage. The binding of the thiolated enzyme was carried out in the following manner: 1-20 mg of thiolated enzyme in 10 ml of 0.1 M sodium hydrogen carbonate solution were mixed with 3.0 ml of sedimented activated thiol-agarose (pre-washed with 0.1 M sodium hydrogen carbonate solution) and allowed to react at 23OC for 24 h under constant stirring. The conjugate was washed on a sintered-glass funnel with 100 ml of 0.1 Msodium hydrogen carbonate solution, transferred into a column and washed at the rate of 10 mllh with the following solutions: (1) 0.1 M sodium hydrogen carbonate solution containing 0.2 M sodium chloride (24 h); (2) 0.1 M sodium acetate solution of pH 5.4,containing 0.2 M sodium chloride (24 h); (3) 0.2 M sodium chloride solution (24 h). The inactivated enzyme was eliminated from the carrier in the column (0.5 ml) by washing with 50 ml of a 20 mM solution of dithiothreitol in 0.1 M sodium hydrogen carbonate solution at a flow-rate of 20 ml/h. The reduced carrier was washed with 150 ml of 1 M sodium chloride solution and activated by passing 100 ml of a 1.5 mM solution of 2,2 '-dipyridyl disulphide in 0.1 M sodium hydrogen carbonate solution. The activated thiol gel was washed with 100 ml of 1 M sodium chloride solution and 100 ml of 0.1 M sodium hydrogen carbonate solution and re-used for the immobilization of a new thiol system. In addition to the binding of affinant on to agarose, described above, the triazine method of binding affinants on agarose is also used. It was originally developed by Kay

-

172

SOLID MATRIX SUPPORTS AND BINDING METHODS

and Lilly for binding affinants to cellulose (see Section 8.2 .l), using 2-amino-4,6-dichloros-triazine.

The attachment of affinim ligands by means of N-hydroxysuccinimide esters of agarose Cuatrecasas and Parikh described the preparation of N-hydroxysuccinimide esters of succinylated aminoalkyi agarose derivatives. These active ester derivatives of agarose, when stored in dioxane, are stable for several months. These derivatives very rapidly form stable amide bonds (at 4OC) with non-protonated forms of primary aliphatic or aromatic amino groups at pH 6-9. Among the functional groups of amino acids tested, only sulphydryl groups compete effectively with the amino grups during the binding reaction. The reaction takes place according to the following scheme:

Diaminodipropylaminoagaroseis treated with succinic anhydride in saturated sodium borate buffer to obtain the corresponding succinylated derivative (A). The latter is made to react with N,N‘-dicyclohexylcarbodiimide and N-hydroxysuccinimide in dioxane to yield the active agarose ester (B). After removing dicyclohexylurea and the unreacted reagents (dioxane and methanol washes), the active ester of agarose is subjected to reaction in aqueous medium with ligands or proteins to yield stable amide-linked derivatives (C) Using the esterification of the carboxyl groups of CH-Sepharose 4B (i.e.,Sepharose to which Eaminocaproic acid is bound after activation with cyanogen bromide) with the application of N-hydroxysuccinimide, Pharmacia (Uppsala, Sweden) produce “activated CH-Sepharose 4B”.The pH range suitable for binding on this derivative is indicated by Pharmacia to be 5-10, with an optimum of pH 8. The advantage of lower pH values consists in the decreased hydrolysis of esters, but on the other hand the reaction is slower. For binding, buffers that contain amino acids cannot be used (Tris or glycine buffers). An agarose derivative also containing N-hydroxysuccinimide ester has been introduced by Bio-Rad Labs. (Richmond, Calif., U.S.A.) under the name Affi-Gel 10.

173

SOLID SUPPORTS AND COUPLING PROCEDURE

Covalent attachment of proteins to polysaccharide carriers, including agarose, by means of benzoquinone (Brandt et al.) The mechanism of activation and binding by this method probably has the following course:

f O - 00

+

fko6 /

R - N H 2 d

OH

N-R H I

The binding of proteins on activated gel takes place at alkaline pH. When binding serum albumin, most of the protein was bound at pH 8; during the binding of chymotrypsin, the amount of attached enzyme increased with pH up to pH 10, when the maximum amount was bound. Affinants that contain free carboxyl groups can be bound to aminoethylagarose by using water-soluble carbodiimides. This method is discussed in detail in Section 8.3. 8.2.5 Copolymer of ethylene and maleic anhydride

The linkage of affinants to a copolymer of ethylene and maleic anhydride (EMA) was discussed in a review by Goldstein. The method of binding enzymes to this support was developed by Levin et al. The protein is bound to anhydro groups of the polymer by its amino groups: '

-CH2-CH

I

o=c, -CH2-CH I

o=c

- CH -CH2-CH2-CH I 1

0'

c=o

o=c,

'0

- C H -CH2-CH,-CH I I

coo-

- CH -CH, - CH21 c=o

- CI H -CH2-CH2-

coo- coo-

NH I Drotein /

o=c I

AH

coo-

-CH2-CH-CH

I

coo- coo-

-CH2-CH2-CH

I

1 - CH-CH2-CH2-

+

N H2- protein - N H2

-

SOLID MATRIX SUPPORTS AND BINDING METHODS

174

When the affinant is bound (for example, in 0.2 Mpotassium phosphate buffer, pH 7.51, carboxyl groups are set free (either after the binding with proteins or hydrolysis in aqueous medium), which give the support a polyanionic character. The copolymer of ethylene and maleic anhydride is produced by Monsanto (St. Louis, Mo., U.S.A.). The firm Miles-Yeda binds the enzyme trypsin, chymotrypsin, papain and subtilopeptidases A and B on to this polymer and supplies them under the trade-names Enzite-EMAXenzyme name). These preparations are characterized by a high content (about 60%) of the bound enzyme. The properties of enzymes bound to EMA carriers have been intensively investigated (Goldstein and Katchalski; Silman and Katchalski). This support, with bound proteases, was utilized mainly by Fritz, Werle and co-workers for the preparation of a series of inhibitors of proteolytic enzymes (Fritz et al., 1967, 1968, 1972; Hochstrasser et al.) and with bound inhibitors for the isolation of proteases (Fritz et ~ l .1969). , 8.2.6 Polyacrylamidesupports and their derivatives

Polyacrylamide gels are composed of a hydrocarbon skeleton on to which carboxamide groups are bound: -CH2-

F

H-CHI-

CO-NHZ

F

H-CH2-CH-

I

CO-NH2 CO-NH2

The main producer of polyacrylamide geis is Bio-Rad Labs., under the trade-name Bio-Gel P, prepared by copolymerization of acrylamide and N,N'-methylenebisacrylamide. Bio-Gel P is produced with various pore sizes range, from Bio-Gel P-2 with a molecular-weight exclusion limit of 1800 up to Bio-Gel P-300 with a molecular-weight exclusion limit of 400,000. All brands are available with 50-100, 100-200,200-400 and 400 mesh size. In addition to these gels, Bio-Rad Labs. produce ion-exchanging derivatives of the gels, for example the weakly acidic cation exchanger Bio-Gel CM, and also intermediates for affinity chromatography, such as the aminoethyl and hydrazide derivatives of Bio-Gel P-2 and P-60. For the linking of affinants, mainly enzymes, Koch-Light (Colnbrook, Great Britain) produces Enzacryls. Enzacryl AH is a hydrazide derivative of polyacrylamide gels, and Enzacryl AA is a polyacrylamide gel containing aromatic acid residues. Enzacryl Polyacetal is a copolymer of N-acryloylaminoacetaldehyde dimethyl acetal with N,N '-methylenediacrylamide, which binds proteins through their NH2 groups. Enzacryl Polythiol is a crosslinked copolymer of acrylamide and N-acryloylcysteine. In the presence of oxidants it binds proteins through their -SH groups. For the introduction of the -SH groups into the enzyme, the reaction of the enzyme with N-acetylhomocysteine thiolactone is recommended in this instance [ l o mg of this reagent are dissolved in 0.5 ml of carbonate buffer of pH 10.6 and added to a solution of 100 mg of enzyme in 10 ml of the same buffer. After 60-min reaction at 4OC the mixture is submitted to gel filtration on Sephadex G-25 (50 X 2 em), again in the carbonate buffer of pH 10.61. Enzacryl Polythiolactone contains a thiolactone ring and binds proteins directly through the aliphatic amino groups and the aliphatic and phenolic hydroxyl groups. Polyacrylamide gels are stable in the pH range 1-10 and they support well all common

SOLID SUPPORTS AND COUPLING PROCEDURE

175

eluents. They do not contain charged groups, and so ion exchange with the chromatographed substances is minimal. They are biologically inert and, as they are synthetic polymers, they are not attacked by microorganisms. As the gel particles adhere strongly to clean glass surfaces, Inman and Dintzis recommend the use of siliconized glass or polyethylene laboratory vessels. On reaction with a suitable compound, they can be converted into solid carriers suitable for the binding of a series of affinants (Inman and Dintzis). Their aminoethyl derivatives can be prepared by using a large excess of ethylenediamine at 90"C, and hydrazide derivatives by using an excess of hydrazine at 50°C. Aminoethyl derivatives of polyacrylamide gels can be converted into their p-aminobenzamidoethyl derivatives by reaction with pnitrobenzoylazide in the presence of N,N-dimethylformamide,triethylamine and sodium thiosulphate. After activation with nitrous acid, the hydrazide derivative can bind affinants with its amino groups: -CH-CH;

I

CO-NH-NH2

-CH-CH2HNo2 Protein-NH2 CO-N3 ------+

-I

TH2CO-NH-protein

Polyacrylamide gels containing residues of aromatic amines, when diazotized with nitrous acid, bind affinants mainly through their aromatic residues:

\CH~- protein The same gels, when activated with thiophosgene, bind affinants by means of their free amino groups:

-CH

-CH2-

S

-

LO-NHeNH-!-NH-protein

The procedures for the binding of proteins on to all three derivatives of polyacrylamide gels are given below. Coupling of proteins with commercially produced polyacrylamide derivatives (Enzacryls) Coupling of affinants on polyacrylamide gels containing aromatic amino acid residues (Enzacryl AA) after activation with nitrous acid To a suspension of 100 mg of Enzacryl AA in 5 ml of 2 M hydrochloric acid, cooled to O"C, 4 ml of an ice-cold 2%solution of sodium nitrite are added and the mixture is stirred

176

SOLID MATRIX SUPPORTS AND BINDING METHODS

magnetically for 15 min. The diazo-Enzacryl formed is then washed four times with the buffer in which the affinant will undergo coupling (for example, a phosphate buffer of pH 7.5). After centrifugation and decantation, the affinant is added, for example an enzyme (2.5 mg) in a suitable buffer (0.5 ml). The coupling is allowed to proceed with magnetic stirring for 48 h. The reaction is terminated by addition of an ice-cold solution of phenol (0.01%) in sodium acetate (10%). After a further 15 min, the Enzacryl with the coupled affinant is first washed with a dilute buffer, then with the same buffer made 0.5 M in sodium chloride. This washing should be carried out very carefully. The manufacturer (Koch-Light) recommends carrying out the whole experiment first with non-diazotized Enzacryl, in order to determine the best conditions for washing out all of the adsorbed material. Affinity ligands bound in this manner can be set free under mild conditions, i.e., with 0.1 M sodium dithionite in 0.2 M sodium borate at pH 9.

Coupling of affinantson polyacrylamide gels containing aromatic amino acid residues (Enzacryl A A ) after activation with rhiophosgene To a suspension of 100 mg of Enzacryl AA in 1 ml of phosphate buffer (3.5 M , pH 6.8-7.0), well stirred with a magnetic stirrer, 0.2 ml of a 10%thiophosgene solution in chloroform is added. After vigorous stirring for 20 min, a further 0.2 ml of the thiophosgene solution is added and, after additional stirring for 20 min, the NCS-Enzacryl is washed once with acetone, twice with 0.5 M sodium hydrogen carbonate solution and twice with a buffer suitable for coupling (for example, a borate buffer of pH > 8.5). After centrifugation and decantation, 0.5 ml of an affinant solution (for example, 2.5 mg of enzyme) is added and the coupling is carried out as described in the preceding section. Activation of the hydrazine derivative of polyacrylamide gel (Enzacryl AH) with nitrous acid and subsequent coupling are carried out in the same manner as described for Enzacryl AA. Coupling of proteins on polyacrylamide gels by using glutaraldehyde Weston and Avrameas developed a method for the direct binding of affinants on to polyacrylamide gels using glutaraldehyde, which, if present in excess, reacts via one of its two aldehyde groups with the free amide group present in the polyacrylamide gel. The remaining free active group then reacts with the amino group of the affinant added during the subsequent binding reaction. Thus a firm bond is formed between the support and the affinant . Bio-Gel P-300 is allowed to swell in water and is washed twice with a four-fold volume of 0.1 M phosphate buffer of pH 6.9. Then 19.4 ml of gel (1 g of dry beads per 45 ml) are mixed with glutaraldehyde solution (4.8 ml; 25%, vlv) and incubated at 37°C for 17 h. The gel is washed and centrifuged four times with a four-fold volume of 0.1 M phosphate buffer of pH 6.9, then three times with 0.1 M phosphate buffer of pH 7.7. The coupling of the protein is carried out after mixing of 3 ml of gel in 13.5 ml of a buffer of pH 7.7 with 0.3 ml of enzyme solution (20 mg/ml) at 4°C for 18 h on a shaker. After the reaction, the gel is centrifuged and washed. Using this method, 70 mg of acid phosphatase could be coupled per gram of dry gel. A number of other bifunctional derivatives, mentioned mainly by Lowe and Dean, are listed in Table 8.5.

177

SOLID SUPPORTS AND COUPLING PROCEDURE TABLE 8.5 BIFUNCTIONAL REAGENTS Reagent

Formula

Principal reaction

Bisdiazobenzidine

Phenolic HOHS-

Bisoxiranes

-OH to NH,

Diethyl malonimidate

H,N-

p,p '-Difluoro-m,m '-dinitrodiphenyl sulphone

H2NPhenolic HO-

Dimethyl adipimidate

NH, *C OCHf

Dimethyl suberimidate

+NH, \)C-(CH2)6-C, OCH3

+

-(C H

NH2 L- Ct, OCH,

p

2 OCH3

H2N-

H,N-

0 II

Divinyl sulphone

CH,=CH-S-CH II 0

=CH2

HS-

N,N '-Ethylenebisiodoacetamide Glutaraldehyde

HO-

0*C-(CH2)3-C
H'

H

H,N-

Hexamethylene diisocyanate

H,N-

N,N'-( 1,3-Phenylene)bismaleimide

HS-

S0,Cl

Phenol-2,4-disulphonyl chloride

Woodward's K

H2NHo&

S0,CI

@Go'

-COOH to NH, k - C H2- CH,

SOLID MATRIX SUPPORTS AND BINDING METHODS

178

A review of the use of acrylamide gels in affinity chromatography is included in Table 11.1. 8.2.7 Hydroxyalkyl methacrylate gels

Hydrophilic hydroxyalkyl methacrylate gels, introduced by Wichterle and Lim, were prepared by Coupek et al. by polymerization of a suspension of hydroxyalkyl esters of methacrylic acid and alkylene dimethacrylate by varying the ratio of the concentrations of monomer and inert components. The number of reactive groups, the porosity and the specific surface area of the gel may be changed within broad limits. The gel has the following structure:

CKJ

I

-C

c H3

-CCH2----C-CH2-C-CH2-CI I CO

cc I

0

I OCH2CH20il

I

CH3 I I

7

H3

I

co

co

OCH2CHZOH

0

I

I I

fH2

CH* I Ch,

;

0

0 I

,

C 0

i -CH2-

-C

I CH-1

H2

c

H3 C -CH2-

I

7%

C -CH2--CI

cI o

cI o

OCHzCH20H

OCHzCH20H

co I I CH3

The gels are produced under the trade-name Spheron both by Lachema (Brno, Czechoslovakia) and Realco Chemical Co. (New Brunswick, N.J., U S A . ) . The binding of chymotrypsin and glycine on seven types of Spherons has already been shown in Table 8.1. The gels form regular beads with excellent chemical and physical stabilities. They withstand chromatography under pressure well and do not change their structures alter heating for 8 h in 1 M sodium glycolate solution at 150°C or after boiling in 20%hydrochloric acid for 24 h. They are biologically inert and, like acrylamide gels, are not attacked by microorganisms. They can be employed in organic solvents, which was taken advantage of during the binding of peptides on to these gels (Turkova' et al., 1976). The hydroxyl groups of the gel possess analogous properties to those of agarose. After cyanogen bromide activation they bind the affinants in the same manner as Sepharose by their amino groups (Turkova; Turkovh et al., 1973). The gel activation and the affinant binding are virtually identical with those described in the procedures for binding on agarose. Binding of proteins and peptides to hydroxyalkyl methacrylate gels by using active esters (Turkova; eoupek et al., 1977) An amino acid or protein is bonded to the p-nitrophenol ester derivative of the hydroxy. alkyl methacrylate gel (NPAC) according to the scheme

SOLID SUPPORTS AND COUPLING PROCEDURE

c

H?

-C I

7H3

-CI CONH(CH2)5CONHR

179

-

+

NH2-R

+

H O O N 0 2

-

The rate of liberation of p-nitrophenol can be followed by measuring the yellow coloration of the solution at 400 nm. Simultaneously with the binding of amino acids or proteins on to NPAC gel, hydrolysis of p-nitrophenol esters takes place in aqueous medium with an excess of hydroxyl ions. In Fig. 8.4 the liberation of p-nitrophenol during the binding of phenylalanine on WAC gel is shown as a function of pH. Table 8.6 indicates the amount of phenylalanine and serum albumin bound to NPAC gels as a function of pH. Table 8.6 shows that the largest amount of phenylalanine was bonded at pH 9. This pronounced optimum is connected with the pKvalue of the &-aminogroup of amino acids (7.6-8.4). As NPAC gel binds amino acids only by their amino groups in the non-protonated form, only a very low concentration of these acids is available at pH 7 and 8.At pH > 8.5,the amount of attached phenylalanine has increased considerably. On the other hand, at pH

0

I

I

20

I TIME.min

I

40

I

I

60 I

Fig. 8.4. Release of p-nitrophenolduring binding of phenylalanine on NPAC gel depending on pH (Britton-Robinson buffers). pH: (1) 7; (2)8; (3) 9; (4) 10;(5) 11; (6)12.

180

SOLID MATRIX SUPPORTS AND BINDING METHODS

TABLE 8.6 DEPENDENCE OF THE AMOUNT OF IMMOBILIZED PHENYLALANINE AND HUMAN SERUM ALBUMIN ON NPAC GEL ON THE pH OF THE MEDIUM pH I.(

Phenyk&-he (I.(mole/g)

___ __ ___ 7 Traces 8 1.12 9 16.1 10 3.83 11 1.68 Traces 12 __--_____

Serum albumin b m W d ~

~

0.138 0.156 0.151 0.112 0.125

10 and higher, there is a large decrease in the content of bonded phenylalanine due to an increase in the concentration of competitive hydroxyl ions, which greatly supports hydrolysis. Table 8.6 shows also the dependence of the amount of bonded serum albumin on pH. The largest amount was again bound at pH 9. At higher pH values, in the case of a protein, with increasing concentration of hydroxyl ions the number of NH2 groups capable of bonding also increases (pK of the €-amino groups of lysine = 9.4-10.6). From the amount of phenylalanine bound to W A C gel, determined as a function of the concentration of phenylalanine during the binding, it was ascertained that a relatively high concentration of bound amino acid is necessary for effective binding. For example, at a concentration of 10 pmole of phenylalanine per millilitre of solution, 10 pmole of phenylalanine was bound on to 1 g of gel, while at a concentration of 50 pmole/ml of phenylalanine only about 30 pmole of phenylalanine was bound on to 1 g of gel. Methacrylate gels with epoxide groups have also been employed for the binding of peptides and proteins. Experiments with glycyl-L-leucine and acetyl-L-leucine have shown that the binding of the a-amino group to the epoxide group depends considerably on pH (optimum at pH 9.7), whereas the binding of the carboxyl group is virtually independent of pH. Tlus fact, together with the rate of the reaction of the e-amino group with the epoxide group, which is one order of magnitude lower than that of the a-amino group, are reasons why serum albumin and trypsin were attached t o these gels in a greater extent at acidic than at alkaline pH (TurkovA el al., to be published). A review of the applications of Spherons is included in Table 11.1.

8.2.8 Glass and its derivatives The most important inorganic solid support is undoubtedly glass, which is used more for the preparation of immobilized enzymes than for affinity chromatography. In Table 8.7, some commercially available porous glass packing materials are listed, together with the names of th-eir producers. Glass derivatives have outstanding stability and are not attacked by microorganisms. However, in some instances they cause undesirable non-specific adsorption (Cuatrecasas a d Anfinsen, 1971b). Corning Glass Works have demonstrated that glass, when treated with y-aminopropyltriethoxysilane,becomes a suitable support for a series of affinants

181

SOLID SUPPORTS AND COUPLING PROCEDURE

(Messing; Weetall; Weetall and Filbert). The silanization process was found to be a reaction between the glass surface hydroxyl groups and the amino-functional silane coupling agent: I

0 - CH2- CH3

0

I -0-51-OH

I 0

+

I

f -NH2

CH3-CH2-O-SI-O-(CH

I

2 3

0-CH,-CH,

I

-

I 0

0-CH2-CH3

I

1

-O-SI-O-SI-(CH,),-NH~ I I 0 0-CH2-CH, I

The reaction proceeds in methanol and water at 25°C and the resulting alkylamino-glass can subsequently be used to immobilize affinity ligands and enzymes. Affinants can be bound on to the amino groups of the glass derivatives by their carboxyl group using soluble carbodiimide, and by their amino groups after activation with thiophosgene, and by aromatic residues with the azo bond to arylamine derivatives, as is evident from the fol- ' lowing scheme:

0 I 0 I -O-SI(CH~),NH~ I

+

0

I

P

+

II HO-C-protein

+

-O-Si(CH2)3NH-C-proteln I

+

I C=O I

I -O-SI(CH~)~NCS

ClCCl

0 I

I

-O-SI(CH~)~NCS I

II

I 0

? 0 I

,

NH

0

?

S

-O-SI(CH~)~NH~

c,

0

N II C I1

+

H2N-protein

0

-

I

I 0

S

II - o - s ~ I( C H , ) ~ N H C N H - p r o t e l n

0 I

0 I

I

I

OH

+ 0

@ protein

-

I

P P

0

-O-Si(CH2)3NHe I

protein

The utilization of glass in affinity chromatography is shown in Table 11.1 8.2.9 Other supports

In the lists of carriers used for affinity chromatography (Table 11.l), further carriers are also given, such as copolymers of amino acids, vinyl copolymers, nylon and polystyrene matrices and nickel oxide, and further details can be found in the original litera-

SOLID MATRIX SUPPORTS AND BINDING METHODS

182

TABLE 8.7 POROUS GLASS PACKING MATERlALS .Type Molecular-weight exclusion limit

__

Dextran standards

Bio-Glass 200 Bio-Glass 500 Bio-Glass lo00 Bio-Glass 1500 Bio-Glass 2500 CPG 10-75 CPG 10-125 CPG 10-175 CPG 10-240 CPG 10-370 CPG 10-700 CPG 10-1250 CPG 10-2000

Polystyrene standards 30,000 100,000 500,000 2,000,000 9,000,000

28,000 48,000 68,000 95,000 150,000 300,000 550,000 1,200,000

Producer

120,000 400,000 1,200,000 4,000,000 12,000,000

Bio-Rad Labs., Richmond, Calif., USA.

Waters Assoc., Milford, Mass., U.S.A.; Corning Glass Works, Corning, N.Y., U.S.A.

ture cited. Among the carriers mentioned, polystyrene gels are commercially available, for example under the trade-names Poragel, Styragel and Aquapak from Waters Assoc. (Milford, Mass., U.S.A.) and Bio-Beads from Bio-Rad Labs. (Richmond, Calif., U.S.A.); Merckogel [poly(vinyl acetate)] gels are the products of E. Merck (Darmstadt, G.F.R.).

8.3 SPACERS If a low-molecular-weight substance functions as an affinity ligand, then for the preparation of an effective specific sorbent it is necessary, in most instances, to insert a spacing arm, generally called a spacer, between the affinant and the surface of a solid support. The question of steric accessibility of low-molecular-weight affinity ligands for the complementary sites of biological macromolecules has already been discussed in detail in Section 5.1. When synthesizing an affinity gel, two approaches are possible. The spacer ligand can be synthesized in free solution by orthodox methods of organic chemistry, then purified and bound on a gel matrix i~ the last step. Some workers prefer this approach because it does not introduce into the gel the groups of unsubstituted spacer molecules that contain ionic groups, which can affect the non-specific sorption, as will be discussed in detail in Section 10.3. The second approach consists in the buildingin of the spacer into the gel matrix in a first step, and then the binding of the affinity ligand in a second step. Barry et al. compared the two procedures and demonstrated the advantages of the latter. The binding of spacers on a gel with subsequent attachment of the affinant is usually technically less demanding, especially for those without expertise in synthetic chemistry. The problem of the introduction and the elimination of protecting groups is thus avoided. By the synthesis on a solid phase, the elimination of unreacted compounds is greatly facilitated,

SPACERS

183

because they can be eliminated by a simple wash. However, the first method usually requires a complicated fractionation of spacered affinity ligands. Although the binding of the spacer on agarose, for example, is carried out in aqueous medium, in a number of instances non-aqueous solvents, such as pyridine or methanol, could also be used for the binding of affinants on the spacer. In a series of binding reactions, crosslinking of the gel takes place during the attachment of the spacers, and thus leads to stabilization of the gel. Barry et af. stressed that the origin of non-specific sorption lies not so much in unsubstituted spacer molecules, but rather in the nature of their bond with the carrier (for example, the formation of a positive charge after the binding of alkylamine on to agarose after cyanogen bromide activation; cf., Section 8.2.4), and further in the nature of the carrier itself (hydrophobic interactions in the case of hydrophobic spacers), and also in side-reactionsduring the binding. As example they gave the generally employed method of binding the affinant by means of water-soluble carbodiimides. When NAD' is bound to an immobilized spacer, amide bonds between the amino groups of the adenine residues and the carboxyl group on the spacer arm should be formed when carbodiimide condensation is applied. Barry and O'Carra demonstrated, however, that an ester bond is formed with the hydroxyl groups of the ribosyl residues. Therefore, for the binding of nucleotides on the spacer, they recommended other methods. From Table 11.1, where it is indicated in all instances whether the spacer was bound to the affinity ligand or to the solid support, an idea can be gained of the use of individual procedures. At present, the binding of spacers to a solid support predominates. As is evident from Table 11.l, one of the most frequent combinations of gel and spacer, used for the binding of low-molecular-weightaffinity ligands, is Sepharose with attached hexamethylenediamine (trade-name AH-Sepharose) or e-aminocaproicacid (trade-name CH-Sepharose). For their binding with affinants carrying primary aliphatic or aromatic amine or carboxyl groups, the condensation reaction with a carbodiimide-promoted method is used. The scheme of this reaction is as follows

I

c=o

// I

R3-NH2

I

I

R2

R2

\

R3- S H

+

NH

I

c=o I R

NH I R2

I R2 Ri

I

Pj

NH

I

c=o I

NH I

+

S I

c=o I

R

184

SOLID MATRIX SUPPORTS AND BINDING METHODS

(a) A nucleophilic attack gives an acyl-nucleophile product and the urea of the correspofiiing carbodiimide. (b) Thiol ester bonds are formed. (c) The 0-acylisourea intermediate is converted into N-acylurea by an intramolecular acyl shift. The binding reaction between the carboxyl group and the nucleophile can be almost quantitative in the presence of excess of carbodiimide and the nucleophilic reagent. For the synthesis of adsorbents, water-soluble carbodiimides are predominantly used, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC):

CH3-CH2-N=C=N-CH2

FH3

-CH2-CH2 -N'-HCl

I

CH3 and 1 -cyclohexyl-3-(2-morpholinoethyl)carbodiimidemetho-p-toluenesulphonate (CMC):

Their main advantage is that their corresponding urea derivatives are soluble in water and that they can therefore be easily eliminated from the gel by washing with water. The pH range used for the carbodiimide condensation is 4.7-6.5 according to Lowe and Dean, and the reaction time is 1.5-72 h at a carbodiimide concentration of 2-100 mg/ml. As an example, the preparation of estradiolSepharose (Cuatrecasas) is described below. A 300-mg amount of 3-0-succinyl-[3H]estradiol dissolved in 400 ml of dimethylformamide is added to 40 ml of packed aminoethylSepharose 4B. The dimethylformamidt is needed in order t o solubilize the estradiol, and it is not required for affinants that are soluble in water. The suspension is maintained at pH 4.7 with 1 N hydrochloric acid. Then SO0 mg (2.6 mmole) of 1-ethyl-3-(dimethylaminopropyl)carbodiimide, dissolved in 3 ml of water, are added to the suspension over 5 min and the reaction is allowed to proceed at room temperature for 20 h. Substituted Sepharose, after being transferred into the column is washed with SO% aqueous dimethylformamide until the eluate is no longer radioactive. It is recommended that the derivative should be washed with about 10 1 of the washing liquid over 3-5 days. Using this procedure, about 0.5 pmole of estradiol can be bound per millilitre of packed Sepharose. Of other Sepharose derivatives, Cuatrecasas prepared bromoacetamidoethyl-Sepharose by reaction of 0-bromoacetyl-N-hydroxysuccinimide with aminoethyl-Sepharose, succinylaminoethyl-Sepharose by reaction with succinic and also anhydride, diazonium derivatives from p-aminobenzamidoethyl-Sepharose, tyrosyl-Sepharose and sulphydryl-Sepharose. He bound affinants with free carboxyl groups to sulphydryl-Sepharose by thiol-ester bonds using water-soluble carbodiimides. On the contrary, on a carrier containing carboxyl groups, compounds can be bound using carbodiimide, which contains thiol groups. The thiol-ester bond can be cleaved specificall: with hydroxylamine, which permits a simple separation of intact ligand-protein complex from the matrix. In Section 5.1 the hydrophobic and the hydrophilic nature of the spacer was discussed.

SPACERS

185

O'Carra et al. mentioned three selected affinity ligands immobilized through hydrophobic (A, D and G ) and hydrophilic (B, C, E, F and H) spacer arms (A-C, immobilized oxamate; D-F, 8-linked azo-NAD'; G and H, 6-linked 6-mercaptopurine analogue of NAD'). The hatched portions on the left represent the Sepharose 4B matrix:

FNH

H,-

-cH,-CH,-CH,-c

(A) OH

0

c H,-c

H-,

OH

(8) $--NH-CH~-CH-CH,-NH-C-CH,-NH-CH~-CH-CH~I II I

$-

(C)

(D)

NH a CH,-

OH I

c H-C H-,

~-NH-cH,-cH,-cH,-cH,-cH~-cH~-NH-

OH ~NH-CH,-~H-CH,-NH-

(F)

(GI $-NH-CH~-CH,-CH~-CH~-CH,-CH,-W-

j-

NH-

(H)

-C -CH,-S-deaminoNAD'

OH

c H2-&-

(6-linked)

cH2-N H-

The following scheme of the synthesis of a hydrophilic spacer arm is also taken from the paper by O'Carra et al.: Sepharose 4 0

i

1.3-d iamino-2-propanol

OH

1

(brornoacetylat ion )

OH I

N H-C H2-C

0

- - CI1-C

H - C H2 NH

H, B r

11,3 -diamino-2-propanol

c I c NH -CH,-CH

OH I

-CH,-NH

0 II

- C -CH2-

NH -CH,-C

OH I H -C ti2- NHZ

(brornoacetylatton)

OH I NH -CH2-CH -CH2-NH

0 OH It I -C -CH2-NH -CHZ-CH-CH2-NH

0 II -C-CH,Br

SOLID MATRIX SUPPORTS AND BINDING METHODS

186

In most instances the hydrophobic character of the spacers leads to considerable complications through the introduction of non-specific hydrophobic interactions, and therefore it is preferable to avoid them by using a hydrophilic spacer. Further examples of the incorporation of hydrophilic spacers have already been mentioned in Section 8.2.4 in connection with binding by means of bisoxiranes. As an example of hydrophilic spacers, oligopeptide chains can also be mentioned:

T

N H -C HI-C

NH-

s::

4‘ H - C H I - C

FI

-N H-CH,-COOH

R

c H ~ - C - N H -CH,-C - N H - C H

COOH

’

‘c Ho2@0 NH-cH2--2!

R -NNH-cCH2-c--N~-~~<

H

COOH CHzSH

In Section 8.2.4, the problem of the liberation of alkylamine spacers bound to cyanoge bromide-activated agarose was discussed, and this problem will be treated further in Sectio 8.5. The escape of ligands from specific sorbents can best be prevented by using polyvaleni spacers (Wilchek; Wilchek and Miron, 1974a, b). Stable agarose derivatives with a high capacity can be obtained by attachment of polylysine or multi-poly-DL-alanylpolylysine on cyanogen bromide-activated Sepharose. The disadvantage of the derivative prepared is their ion-exchanging properties, but these properties can be avoided by using hydrazides for the binding on cyanogen bromide-activated Sepharose. In view of the low pK value (4.2) of the attachment of spacers on the carrier, at neutral and alkaline pH a sorbent without ion-exchanging properties is obtained.

Preparation ofpolyacrylic hydrazidoagarose Preparation of poly(methyl acry la te) Sodium Iauryl sulphate (0.25 g), freshly re-distilled methyl acrylate (5 mI), 1 % thioglycolic acid (1 ml) and ammonium persulphate (0.125 g) are added successively to 50 ml of distilled water under magnetic stirring (operated in a hood). The emulsion is refluxed for 2.5 h in a water-bath (80°C) until the odour of methyl acrylate nearly disappears. After polymerization, the emulsion is poured with stirring into 100 ml of ice-cold water, to which 100 ml of cold 2 N hydrochloric acid are added. The product, which coagulates in the form of white flakes, is kept for 30 min in the cold. The product is washed by decantation with large amounts of cold water. Preparation of polyacrylic hydrazide Poly(methy1 acrylate) (5 g) is dispersed into 70 mi of hydrazine hydrate (98%). The reaction mixture is stirred vigorously and heated at 100°C in a boiling water-bath. After 3 h, a clear, viscous solution is obtained. The reaction mixture is cooled to room temperature, the insoluble particles are filtered through gauze and the filtrate is poured with constant stirring into 500 ml of icecold methanol containing 1 ml of glacial acetic acid. The precipitate is filtered off, washed with cold methanol-acetic acid (500: 1) and re-dissolved

BLOCKING OF UNREACTED GROUPS

187

in 150 ml of water. The insoluble particles are removed by filtration. This solution can be used immediately for coupling to Sepharose, or can be freeze-dried, taking care to remove any trace of moisture to prevent crosslinking of the product.

Preparation of polyacrylic hydrazido-Sepharose Sepharose 4B is activated with cyanogen bromide. The activated Sepharose is suspended in three volumes of the above cold polyacrylic hydrazide solution in water or 0.1 N sodium hydrogen carbonate solution, and the reaction is allowed to proceed overnight at 4°C with slow stirring. The conjugate is washed with 0.1 M sodium chloride solution until the washings show no colour on reaction with sodium 2,4,6-trinitrobenzenesulphonate. Columns containing up to 120 pmole of available hydrazide per millilitre of agarose are obtained. Derivatives containing an average capacity of 15.25 pmole of available hydrazide per millilitre of agarose were used for further substitutions by the same method as described for the monovalent coupled hydrazides. Procedures for preparing derivatives of Sepharosehydrazide are shown in Fig. 8.5. Polyacrylic hy drazido-Sepharose derivatives behave as ideal insoluble carriers for affinity chromatography. They retain the properties of Sepharose, mainly a minimum non-specific interaction with proteins, good flow-through properties and high porosity. In addition, they are mechanically and chemically stable. These derivatives also possess the advantages of acrylamide gels. At neutral pH they carry no charge and they contain a large number of modifiable groups.

8.4 BLOCKING OF UNREACTED GROUPS

When the binding of the affinity ligand on to the solid support is terminated, it is recommended that the remaining active groups that are capable of binding should be eliminated. In the methods that make use of the amino groups for the binding of the affinant ,low-molecular-weight primary amines are most often used as inactivating reagents. For example, according to the recommendation of Pharmacia, for the binding to Sepharose by cyanogen-bromide activation the inactivation of the remaining active groups can be effected simply by reaction with 1 M 2-aminoethanol at pH 9 and room temperature for 2 h, while after the binding to epoxide-activated Sepharose 4 h are required under the same conditions. The buffer used is the same as during the binding, for example 0.1 M sodium hydrogen carbonate solution containing 0.5 M sodium chloride. For blocking, other low-molecular-weightprimary amines can also be used, such as glucosamine. Sundberg and HGglund used a saturated solution of glycine (in carbonate buffer of pH 9.5) and demonstrated that it does not contribute to non-specific sorption. Smith also investigated the inactivation of cyanogen bromide-activated groups of Sepharose with glycine. The effect of 1OMglycine at 2°C in Tris-buffered saline (pH 8.5) virtually prevented the subsequent binding of the 311-labelledyG-globulin. Roche et al. blocked cyanogen bromideactivated groups of Sepharose after the binding of submaxillary mucine with 2-amino-2hydroxymethylpropane-1,3-diol,while Ratner, after having bound RNA polymerase, quenched the remaining active groups by washing with a 0.1 M solution of 2-mercaptoethanol. The producer states that it is sufficient for the elimination of cyanogen-bromide-

'

.-I

m m

0 C-NH-NH-C-CH2-CH2-COOH

0-C-NH-NH-C-CH

SH

CR

9-42

C-NH-NHC-CH-COCH,

+!-NHNH!-CH;Br 0

Na2S204

\

# ! - N H N H : e0 NH;

zz 4! z

n

Fig. 8.5. Procedures for preparing derivatives of Sepharose-hydrazide; CD = carbodiimide. Reproduced with permission from M. Wilchek, Advan. Exp. Med. Biol., 4 2 (1974) 15-31.

LEAKAGE OF COUPLED AFFINANT

189

activated groups of Sepharose to suspend the gel in a Tris buffer of pH 8 for 2 h. The effect of the small number of charges, introduced by the use of Tris or glycine, can be overcome by using relatively high concentrations of salts during affinity chromatography. Almost complete elimination of these active groups can be achieved, however, even by merely standing the gel overnight in a mildly alkaline solution. In gels that contain active ester groups for spontaneous covalent bonding (for example, activated CH-Sepharose, Affi-Gel 10 or hydroxyalkyl methacrylate gels with a p-nitrophenyl ester group), the unreacted groups are eliminated by addition of 0.1 MTris buffer of pH 8. After standing for 1 h virtually no groups capable of binding proteins or peptides remain on the gel. In carriers that contain aldehyde groups as active binding groups, reduction with sodium borohydride is applied with advantage after the binding. This leads both to the reduction of the remaining aldehyde groups and to the stabilization of the bond between the protein and the solid matrix. If the affinity ligand is bound on a spacer, it could happen that a part of the spacer molecules most commonly containing at their end amino or carboxyl groups will remain unoccupied. The unreacted carboxyl groups on the extending arms were eliminated by Rafestin et al. by blocking with 0.5 mM Tris[(2-amino3-hydroxymethylpropane-I,3-diol)] and with 1.6 mM N-cyclohexyl-N'-[2(4-morpholinyl)ethyl] carbodiimide metho-p-toluenesulphonate. Whiteley et a2. blocked the carboxyl groups in the following manner: 15 ml of Sepharose on to which 5-fluoro-2'-deoxyuridine-5'-(p-aminophenyl) phosphate was bound via hexamethylenediamine and succinic anhydride were suspended in 50 ml of a 1:1 (v/v) mixture of dimethylformamide and 0.1 M cacodylate buffer (pH 4.5) containing 1.1 g of glycinamide. A 2-g amount of 1-ethyl-3-(3'-dimethylaminopropy1)carbodiimidehydrochloride dissolved in 10 ml of the same mixture was added to the suspension, which was then stirred at room temperature for 12 h. When the affinity ligand has to be bound to CH- or AH-Sepharose by means of the carbodiimide binding reaction, the producer recommends continuing, after the binding of the affinant, with the binding reaction by carrying out further carbodiimide reactions with glucosamine (Fransson) or 2-aminoethanol in the case of CH-Sepharose, or with acetic acid as blocking agent for the amino groups of AHSepharose. The unsubstituted amino groups can also be eliminated with acetic anhydride (Kanfer et al.). A suspension of 60 ml of Sepharose, on to which D-galactono-cw-lactone had been bound through benzidine, in 100 ml of water was sonicated with 2 ml of acetic anhydride for 10 min in a bath-type sonicator. The blocking of unreacted amino groups with acetic anhydride was also used by Hierowski and Brodersen, but it led to the liberation of bilirubin bound by the carbodiimide method on to AH-Sepharose. Another step in the preparation of specific sorbents before the affinity chromatography proper consists in the thorough washing out of all substances that are not covalently bound to the surface of the solid matrix. Most commonly the best results were obtained if the washing was carried out alternately with alkaline and acidic buffers of high ionic strength. A series of examples was mentioned in Section 8.2.

8.5 LEAKAGE OF THE COUPLED AFFINANT

A serious limitation to the use of affinity chromatography in systems with a high

SOLID MATRIX SUPPORTS AND BINDING METHODS

190

affinity (for example, hormone-receptor interactions) and in the isolation of picomole and nanomole amounts of proteins is due to the relatively easy release of affinity ligands into the solution. These are primarily affinity ligands bound monovalently through a spacer on to cyanogen bromide-activated agarose. The mechanism of leakage of affinants bound on cyanogen bromide-activated Sepharose in the presence of compounds that contain nucleophiles was discussed in Section 8.2.4.The setting free of ligands from agarose 3 ,5 ‘-cyclophosphate as a with bound 8-(~-aminocaproyl-3-aminoethylthio)adenosine function of time and pH is shown in Fig. 8.6 (Tesser el al., 1972). In order to elucidate the leakage of affinity ligands from specific sorbents more closely, Tesser et al. (1 974)prepared sorbents by binding adenosine 3 ,5 ‘-cyclic monophosphate and other substances through variously long spacers to agarose, cellulose and crosslinked

0

30

60

90

120

TIME .min

Fig. 8.6. Liberation of ligand from CAMP-spacer-agarose as a function of time and pH. ( 0 ) Sodium acetate, pH 5; (A) imidazole-HC1, pH 6; (=) Tris-Ha, pH 7; (0)Tris-HC1, pH 8; Room temperature. The incubation medium contained 0.005 M of the buffers plus 0.015 M NaC1. Reproduced with permission from G.I. Tesser etal., FEBS Lett., 23 (1972) 56-58.

LEAKAGE OF COUPLED AFFINANT

19 1

dextran by means of cyanogen bromide-activation, and to polyacrylamide by an amide bond formed with the carboxyl groups of the carrier, according to Inman and Dintzis. From the dependence of the amount of individual detached affinants on pH and time, it followed that the leakage of affinants from the matrices takes place predominantly at the site of the fixation on the surface of the solid carrier, and that it can be enhanced by anchimeric assistance of neighbouring carboxyl and carboxamide groups in polyacrylamide gels and hydroxyl groups in agarose, cellulose and crosslinked dextrans. The releasing reaction is general and independent of the structure of the bound affinity ligand. The affinants bound to polyacrylamide gels by the R-NH-CO-acrylamide gel bonds are detached more slowly. The leakage of deoxycorticosteroids -and estriols from agarose conjugates has been described by Ludens et al., estradiol by Sica er ul., and of e-DNPlysine by Wilchek (1973). Yong observed a steady leakage of catecholamines and propanol from the chemical bond on both Sepharose and glass, and Vauquelin et al. observed the leakage of isoproterenol from the binding to agarose. The leakage of bound ligands can be supressed if binding through polyvalent spacers is used, as already discussed in Section 8.3. A further improvement can be achieved by using bonding reactions that afford a stronger bonding of the affinant to the carrier, such as the periodate oxidation method or the attachment to epoxide-activated agarose. Parikh et al. discussed the variation of the degree of leakage as a function of the carrier used. In the isolation of estrogen and insulin receptors, it is impossible to use, for example, glass and polyacrylamide carriers, owing to the excessively rapid leakage of affinity ligands. Mathematical approach to ligand leakage in affinity chromatography (Gribnau and Tesser) The hydrolytic detachment of a multiply bound ligand molecule from a matrix can be described as a consecutive reaction. With the assumptions of pseudo-first-orderkinetics, which is compatible with the proposed detachment mechanism, and of reaction constants of the same magnitude in all steps, an expression for the concentration of free ligands (CN) can be derived. If the concentration of totally fmed ligand is given in micromoles per millilitre of wet gel, then a=CA+CB+..’.+CN The rupture of the first bond (A + B) is described by the equation

Solution of this differential equation, and of analogous equations for the reactions C + D,

192

SOLID MATRIX SUPPORTS AND BINDING METHODS

D -+ E, etc., by the method of Teorell leads to

Combination of these equations with eqn. 8.1, leads to the expression

For each value of n, a curve can be constructed for CN/a as a function of k t . Results for n = 1,. ..,6 are given in Fig. 8.7. It is clear that a time lag exists between the start of the detachment reactions and the appearance of the first free ligand molecules, in the case of n > 1. This interval increases with increasing values of n and with decreasing values of k . From eqn. 8.2, a leakage half-time (7)can be calculated by setting CN equal to a / 2 :

Because this equation is transcendental, no exact analytical solution can be given. Graphical solution, however, leads to the results presented in Table 8.8. A less time-consuming and more generally useful solution can be attained by application of the Newton-Raphson procedure. Apparently the following generalization is allowed: Tn

%

[(n - 1) f In 2 ] / k

(8.4)

It is also possible t o calculate a value for the time lag, mentioned above. By substituting chosen values of CN/a and n, the same computer program provides the accessory values of kt (Table 8.9). In accordance with expectations, there is a decrease in kt at decreasing values of CN/a and constant n, and an increase in kt at increasing values of n and constant CN/U. A remarkable result is that for increasing values of n the difference between the values of kt at the extremes of CN/a diminishes. Replacement of a monovalently by a bivalently coupled ligand has a much greater effect than, for example, the change from n = 5 t o n = 10. From the experimental results, a value can be computed for the detachment rate constant. At pH 8 and room temperature, this constant appears to be 0.25 * 10-4/min. This value, combined with data from Table 8.9, leads t o the result that after 2-3 sec the concentration of free ligand molecules will reach a value of 2 pmole per millilitre of wet gel, when originally 2 Mmole per millilitre of wet gel were coupled, monovalently. This order

193

LEAKAGE OF COUPLED AFFINANT

i

kt

Fig. 8.7. Graphical representation of CN/Uas a function of kt, for n = 1, ..., 6 (cf., q n . 8.2). Reproduced with permission from T.C.J. Gribnau and G.I. Tesser, Experienfia, 30 (1974) 1228-1230.

TABLE 8.8 NUMERICAL RESULTS OF THE SOLUTION OF EQN. 8.3 FOR n = 1,

n

k7,

1 2 3 4

0.69 1.68 2.67 3.67 4.67 5.67

5

6

...,6

TABLE 8.9

1 2 5 10

lo-*

10-~

o.i0.10-~ 0.15-10° 0.13.10' 0.41 * 10'

o.i0.10-~ 0.14*10-' 0.44*10° 0.22 * 10'

o.io.10-~ 0.14*10-2 0.17.10° 0.13 * 10'

10-8

10-1°

lo-"

o.i0.10-~ 0.14*10-3 0.66*10-' 0.77 10'

o.i0.10-~ 0.14*10-4 0.26*10-' 0.47 * 10'

o.i0.10-~~ 0.14.10-5 0.10.10' 0.29 10'

-

194

SOLID MATRIX SUPPORTS AND BINDING METHODS

of magnitude is in agreement with ligand leakage rates found before. At the same value of k, the 2-3 sec mentioned above become 1 h (n = 2), 5 days (n = 5) and 5-6 weeks (n = 10). This trend corresponds with the findings of Wilchek (1973). Thoni pointed out that the half-lives and the time course of ligand release can be calculated conveniently from tabulated x2 values using the well known relationship between the cumulative probability function of the Poisson distribution and the x2 distribution. The derivation of the leakage function of Gribnau and Tesser rests on four basic assumptions: (a) at time t = 0 all ligand molecules are attached to the matrix by the maximal number of bonds, n ;(b) the cleavage of the ligand-matrix bonds is pseudo-first order (approximately constant hydroxyl concentration in a buffered solution); (c) all bonds are similar and split with the same rate constant, k ; and (d) the bonds are split in a consecutive order, i.e., given a bond numbering which is not further specified, bond 2 will be attacked by OH- only if bond 1 is cleaved, and so forth. Retaining assumptions (a) to (c), it is possible to assume a random nucleophilic attack of the hydroxyl ions. This is equivalent to the statement that the cleavage of the ligandmatrix bonds does not depend on numbering (Lasch). If a is again ihe total ligand concentration (micromoles per millilitre of wet gel) and s the number of bonds hydrolysed, then at any time u = c ~ + C ~ t-c,, _,+...+

C,

_st...+ co

where co is the concentration of free ligand. The number of different forms of the ligand species with s bonds split is = n!/s!(n- s)!. These forms are kinetically degenerated because of assumption (c), that is, any one of the remaining (n - s) bonds will be attacked with the same probability in the next step. In writing the differential equations, care must be taken with statistical factors. If k is the pseudo-first-order rate constant, which is proportional to the probability of cleavage of a given bond during a fixed time interval, then the probability that any one bond of the ligand species with n points of attachment will be cleaved is proportional to nk. The statistical factor for ligands with n - 1 points of attachment is n - 1, and so on. It is possible to write

(t)

dcnldt

(t)

= -nkc,

dc,_,ldt=nkC,-(n-l)k~,_

1

dcn - 2/dt = (n -- 1)kcn - 1 - (n - 2 ) k ~ n- 2 etc. The solutions to these differential equations are easily found by the procedures of Bernoulli or Lagrange: C,

= aexp(-nkt)

Cn -

= M {exp[+n - I)kt] - exp(-nkt)}

cn - = n(n - 1)a/2 {exp [-(n - 2)kt] - 2 exp [-(n - I)kt]

+ exp(-nkt) }

SORBENTS, SPACERS AND COUPLING AND BLOCKING PROCEDURES

195

It then follows that the leakage function is

This equation can be rearranged to (8.7)

k is a measurable quantity, so that half-lives, T ~ can , be computed from:

(S") (-

1/ 2 =

1)s - 1 exp(-sk.r,)

s= 1

The time course of ligand release is computed from eqn. 8.7. The shapes of the curves obtained are qualitatively the same as in the Gribnau and Tesser model, although the analytical expression of the leakage function is different (cf., eqn. 8.7). kTn values were computed from eqn. 8.8 using the iterative procedure of Newton and Raphson, and are compared with the values calculated by Gribnau and Tesser (in Table 8.10). It is especially noteworthy that the increase in the kTn values with increasing n is not so steep as in the Gribnau and Tesser model. The interpretation of this surprising result is that the stability gained by an additional point of attachment is partially offset by an increased probability of cleavage of a ligand-matrix bond. For n = 1, both models must yield the same hl. This condition is fulfilled by the random model. TABLE 8.10 CALCULATION OF HALF-LIVES,

7n, USING

EQN. 8.8 AND k = 2.5 * lO-'/min

Determined experimentally by Gribnau and Tesser. n

1 2 3 4 5 6

7n (days)

Consecutive order model

Random order model

19.16 46.67 14.67 101.94 129.72 157.50

19.16 34.17 43.89 51.11 56.67 61.67

8.6 GENERAL CONSIDERATIONS IN THE CHOICE OF SORBENTS, SPACERS AND COUPLING AND BLOCKING PROCEDURES

As already mentioned in the introductory part of this chapter, during the choice of

TABLE 8.1 I

GUIDE To CHOICE OF MATRIX ~

~

Matrix

Flow properties

Leakage at pH 7, 25°C

Permeability for high molecular weight solutes

Non-specific adsorption capacity

Chemical stability pH>12

pH<3

Swelling properties in organic solvents

Density of potentially reactive groups

-

Low

?

-

High Moderate

Porous glass

Excellent

Negligible

Very high

Very high

Very low

Polyacr ylamide Hydroxyalkyl methacrylate gels Cellulose

Good Excellent

Low Negligible

High High

Low Low

Low Very high

Very high (except in HF) Moderate Very high

Fair

Moderate low

Moderate

Poor

High

Good Good Good

Low Moderate Negligible

Moderatehigh Low Low Very low

Very high

Sephadex Agarose Glycer yl-bridged, de-sulphated agarose Glyceryl-bridged, hydroxylated agarose Divinyl sulphons crosslinked agarose

Moderate (variable) Moderate Very high Very high

Very high Very high Very high

Moderate Low Moderate

Poor Good Very good*

High Moderate Moderate

Good

Negligible

Very high

Very low

LOW**

Moderate

Very good

High

Excellent

Negligible

Very high

Very low

Low

Moderate

Very good

Moderate

~~

~

*This is valid on the assumption that the primary swelling has taken place in aqueous solution. **Hydroxylation with polyphenols yields products that are easily oxidized at high pH.

SORBENTS, SPACERS AND COUPLING AND BLOCKING PROCEDURES

197

the carrier not only the properties following from the nature of the affinity chromatography or of the immobilized enzymes should be taken into consideration, but also their field of use. A further important factor is the rapid development of solid supports. If at present some carrier is neglected owing to some undesirable quality, it may become better after suitable modification. Table 8.1 1 completes the “guide to matrix choice” published by Porath and Kristiansen in 1975. From this, it is clearly evident how, for example, divinyl sulphone crosslinking considerably improved the flow-through properties of agarose. Specific sorbents supplied in 1976 by Pharmacia are already being prepared from Sepharose CL, i.e., from agarose crosslinked with 2,3-dibromopropanol and desulphated by alkaline hydrolysis under reducing conditions. In addition, for example, in the case of octyl- or phenyl-Sepharose C U B ,octyl and phenyl groups are bound to monosaccharide units of the agarose matrix via uncharged, chemically stable ether linkages. These are undoubtedly sorbents with good flow-through properties, without the risk that they will be destroyed or that the affinity ligand will be split off, with excellent permeability and with minimum non-specific adsorption. They are chemically stable in the pH range 3-14, can be autoclaved at 120°C at pH 7 and can be used in organic solvents. However, the question remains of whether their practical application on a larger scale is feasible from the economic point of view. On the other hand, the development of carriers such as glass and ceramic materials is more suitable for practical use. Their disadvantage consists in the relatively large non-specific sorption and also sensitivity to alkalis. However, a suitable modification or treatment of the surface overcomes these disadvantages to a considerable extent. In view of the fact that cellulose is one of the cheapest carriers, it is mainly employed by experimenters interested in practical applications. Its disadvantage is that it often shows considerable adsorption and permeability that is not easy to control (Porath and Kristiansen). Biospecific adsorbents based on cellulose very often deviate more from the behaviour expected on the basis of the degree of substitution than do other adsorbents. Hence, the substitution obviously takes place in regions that are sterically unfavourable for the formation of highly polymeric complexes. Relatively poor flow-through properties can be improved by suitable adjustment of the matrix or by mixing with an inert solid support, such as glass beads. Although cellulose can be attacked by bacteria or fungi, if certain conditions are observed it displays considerable stability. In Table 8.1 1 the copolymer of ethylene with maleic anhydride is not mentioned because, owing to its high content of carboxyl groups, it is gradually becoming less frequently used in affinity chromatography and in the preparation of immobilized enzymes, as is evident from Table 1 1.1. From Table 11.1 it is also evident that the polyacrylamide gels are also relatively little used. It is true that they have a number of good properties, but their stability in alkaline medium is poor. By alkaline hydrolysis and in strongly acidic media, free carboxyl groups are formed. Hydroxyalkyl methacrylate gels seem to have very good prospects; their production is not yet fully developed but useful experience has been obtained with them. The use of dextran gels has already been decreased by the use of agarose gels (except for some special cases); at present agarose gels are among the most widely used gels, as can be seen from Table 11.l. Therefore, greater attention has been devoted to them in this chapter. The high permeability of the solid supports, which is a prerequisite for adequate free-

198

SOLID MATRIX SUPPORTS AND BINDING METHODS

dom for the formation of specific complexes of high-molecular-weight substances, is not a sufficient condition for the steric accessibility of the binding sites. Mainly with lowmolecular-weight affinity ligands, spacers must be inserted between them and the matrix surface. Much attention has already been devoted to the nature of these spacers. The hydrophobic character of the first spacers employed gave rise to a new branch of chromatography that is already becoming independent, namely hydrophobic chromatography. In this technique the hydrophobic spacer plays an important role. However, if endeavours are made in affinity chromatography to utilize predominantly the specific interactions of the binding sites, for example of the enzyme and the inhibitor, it is better to give preference to spacers of hydrophilic character. Owing to the increasing use of affinity chromatography in studies of specific interactions and the mechanism of the action of biologically active substances, it became imperative that the interaction of the substance studied with the immobilized affinity ligand should correspond with their interaction in solution. In these instances, it is desirable if both the effect of the carrier and that of the attachment are excluded. On the other hand, it is the immobilization that permits a study of the effect of micro-environment. Very often a new environment affects the properties of the immobilized substance favourably. Among the many properties involved, the increased stability of many immobilized enzymes should be mentioned first. The dependence of this property on the type of binding and the support was shown by Weetall and Mason. They prepared a series of immobilized papains bound in different ways on to glass and determined the time during which the activity of the preparation decreased by half. While papain bound t o an amino derivative of glass by means of carbodiimide had an operational half-life of 1.9 days, and that bound by means of glutaraldehyde 2.2 days, the enzyme bound to a carboxyl derivative of glass via the amide linkage had a half-life of 4.1 days, that bound to arylamine derivative of glass 7.2 days, and that bound to the zirconium dioxide-coated glass via an amide linkage had a half-life as high as 35 days. From the above, it follows that in addition to the nature of the solid support, the method of linkage also plays a considerable role. In Table 8.12 the methods included in Sections 8.2 and 8.3 are listed. For the selection of the method of linkage, the primary consideration is which groups of the affinity ligand can be used for the linkage to a solid matrix without affecting its binding site. If several groups are available, it is advisable to choose the most selective method, because a linkage specifically through one particular functional group is desirable. The attachment should not introduce non-specifically sorbing groups into the specific sorbent. From this point of view, it is better to link the spacer to an affinity ligand, and only when it has been modified in this manner to attach it to a solid support. The linkage between the surface of a solid support and an affinity ligand should not introduce non-specifically sorbing groups either, and it should be stable during adsorption, desorption and regeneration (see Section 8.5). In the choice of a method, the dependence of the stability of the affinity ligand on pH should also be borne in mind. Acid proteases, for example, cannot be bound at alkaline pH values because they would be inactivated. Therefore, in Table 8.12 the pH value that is used for the binding is also given. However, in many methods good results can be obtained even at lower pH values (binding to agarose after cyanogen bromide activation in neutral media or by means of glutaraldehyde in acidic media). In these instances, however, it is necessary to modify the currently used method. When the coupling reaction is terminated, it is suitable to block the unreacted groups, which is discussed in detail in Section 8.4.

199

SORBENTS, SPACERS AND COUPLING AND BLOCKING PROCEDURES

TABLE 8.12 REVIEW OF BINDING PROCEDURES INCLUDED IN CHAPTER 8 Group on the affinity ligand

pH of the coupling reaction

Group on the solid matrix

Activation reagent

Section where mentioned

-NH, -NH, -NH,

1.5-8 4.5-6.0 8.7

-CO-NH-NH, -COOH -OH

8.2.1, 8.2.6 8.2.1,8.3 8.2.1

-NH, -NH, -NH,

8.5 8 8-10 (down to 6)

-OH -OH

NaNO, + HC1 Carbodiimide 2-Amino-4,6-dichloros-triazine Bromoacetyl Oxidation with NaIO, BrCN

-NH,

8.5-13

C -, -’ C,

-OH

8.2.1 8.2.3 8.2.4 8.2.4

H2

0

5-10

-NH,

,CO-CH2

I

-C-0-N,

1:

-

8.2.4

Benzoquinone

8.2.4

-

8.2.5

Cl,C=S

8.2.6, 8.2.8

Glutaraldehyde

8.2.6

CO-CHI

-NH,

8-10

-OH

-NH,

1-5

-CH-CHI I

o=c,

,c=o

0

N H e -N H 2

-NH,

8-5

-CO-

-NH,

1.7

-NH,

-NH,

9-12

-COOH -COOH -OH

4.5 -6 4.5-6.0 Alkaline solution

-NH, -SH -NH,

Carbodiimide Carbodiimide Oxidation with NaIO.

8.2.8, 8.3 8.2.4,8.3 8.2.1

-OH

9-13

-CH--CH*

-

8.2.4

8.2.1

‘0’

O

O

H

1.5-8.5

-SH

9-13

-SH

Wide range of conditions

-SH

4.5-6.0

Alkaline solution

-c,

/o

-

c

H

~

~

N

H

~NaNO,

+ HCl

8.2.1, 8.2.2, 8.2.6 8.2.4

-CH--CH2 ‘0’

-

8.2.4

-COOH

Carbodiimide

8.2.4, 8.3

-NH,

-

8.2.3

4-S-Q

N-

200

SOLID MATRIX SUPPORTS AND BINDING METHODS

When bifunctional compounds are used for the coupling, complications arising from the crosslinking of both the carrier and the proteins with one another should be borne in mind, and therefore suitable reaction conditions, such as pH, temperature and time, should be maintained carefully during the binding. For the binding of proteins, the most commonly used groups are the N-terminal aamino group and e-amino group of lysine, and also the C-terminal carboxyl group and the carboxyl groups of glutamic and aspartic acids. Phenolic hydroxyl groups of tyrosine or the -SH groups of cysteine residues may also participate in the binding. With carbohydrates and their derivatives, the groups that often take part in the binding are the hydroxyl and the amino groups, in nucleic acids they are the phosphate groups, the sugar hydroxyl groups, and amino or enolate groups in the bases. If high-molecular-weight compounds, which possess more groups for the binding, are bound, they are attached at several places. As a consequence, the risk arising from the detachment of the bound molecules decreases considerably but, on the other hand, there is a certain risk that the multiple binding will bring about a deformation of the native structure of the immobilized molecule and thus also a change in its properties. In contrast, more labile methods of binding are sometimes applied, for example through the thiol-ester bond between the thiol derivatives of the solid supports and the molecules containing carboxyl groups, and vice versa. These bonds can be cleaved specifically with hydroxylamine, which causes the separation of the intact complex of the affinity ligand and the substance to be isolated from the matrix. From the point of view of ease of detachment, the binding of proteins via S-S bridges seems advantageous mainly for the preparation of immobilized enzymes. Inactivated enzyme molecules are eliminated by reduction and the carrier can be used, after activation, for the binding of a new active enzyme. As relatively few enzymes contain free -SH groups, these are introduced into the molecule by thiolation (see Section 8.2.4). On the contrary, with sorbents prepared from stable low-molecular-weight molecules, it is advantageous to use a strong bond (for example, binding to epoxide-activated gels), because their activity can be regenerated by washing with denaturing reagents, such as 6 M guanidine hydrochloride (see Section 10.3). A strong bond is also formed between aldehydes and amino groups if the S c h f f s base formed is stabilized by reduction with sodium borohydride. The attachment of affinants on to carriers that contain active ester groups also leads t o a strong bond. However, some carboxyl groups are also formed during the process, the effect of which should be eliminated by using an increased ionic strength during the affinity chromatography proper. The specific sorbents prepared by diazo coupling fall short of the high stability required for immobilized proteins (Porath and Kristiansen). The reason is probably that the bond is not formed at the phenol and imidazole nuclei of tyrosine and histidine residues only, but also at a-terminal and e-amino groups. These side-reactions lead to the formation of triazene linkages (-N=N-NH-), which are less stable and can be easily hydrolysed. The affiiity ligands bound by an azo linkage can be detached with sodium dithionite.

REFERENCES

20 1

REFERENCES Ax&, R. and Emback, S., Eur. J. Biochem., 18 (1971) 351-360. Ax&, R., Porath, J. and Emback, S., Nature (London), 214 (1967) 1302-1304. Barry, S., Griffin, T. and O’Carra, P., Biochem. SOC.Trans., 2 (1974) 1319-1322. Barry, S. and O’Carra, P., Biochem. J., 135 (1973) 595-607. Brandt, J., Anderson, L.O. and Porath, J., Biochim. Biophys. Actu, 386 (1975) 196-202. Brocklehurst, K., Carlsson, J., Kierstan, M.P.J. and Crook, E.M., Biochem. J., 133 (1973) 573-584. Campbell, D.H., Luescher, E.L. and Lerman, L.S.,Proc. Nut. Acad. Sci. US.,37 (1951) 575-578. carlsson, J., Axeh, R. and Unge, T., Eur. J. Biochem., 59 (1975) 567-572. coupek, J., KKvdkovd, M. and Pokornp, S., J. Polym. Sci. Symp., 42 (1973) 185-190. Coupek, J., Labskp, J., Kdlal, J., Turkovd, J. and Valentovd, O., Biochim. Biophys. Actu, 481 (1977) 289-296. Crook, E.M., Brocklehurst, K. and Wharton, C.W., Methods Enzymol., 19 (1970) 963-978. Cuatrecasas, P., J. Biol. Chem., 245 (1970) 3059-3065. Cuatrecasas, P. and Anfinsen, C.B., Methods Enzymol., 22 (1971a) 345-378. Cuatrecasas, P. and Anfinsen, C.B., Annu. Rev. Biochem., 40 (1971b) 259-275. Cuatrecasas, P. and Parikh, I., Biochemistry, 11 (1972) 2291-2299. De Larco, J. p d Guroff, G., Biochem Biophys. Res. Commun., 50 (1973) 486-492. Fransson, L.A., Biochim. Biophys. Actu, 437 (1976) 106-115. Fritz, H., Brey, B. and Bdress, L., Hoppdeyler’s Z. Physiol. Chem., 353 (1972) 19-30. Fritz, H., Brey, B., Schmal, A. and Werle, E., Hoppe-Seyler’s Z. Physiol. Chem., 350 (1969) 617-625. Fritz, H., Hochstrasser, K., Werle, E., Brey, E. and Gebhardt, B.M., Z. Anal. Chem., 243 (1968) 452463. Fritz, H., Schult, H., Hutzel, M., Wiedemann, M. and Werle, E., Hoppe-Seyler’s Z. Physiol. Chem., 348 (1967) 308-312. Gilham, P.T., Methods Enzymol., 21 (1971) 191-197. Goldstein, L., Methods Enzymol., 19 (1970) 935-962. Goldstein, L. and Katchalski, E., Z. Anal. Chem., 243 (1968) 375-396. Gribnau, T.C.J. and Tesser, G.I., Experientia, 30 (1974) 1228-1230. Gundlach, G., Kohne, C. and Turba, F., Biochem Z., 336 (1962) 215-228. Hierowski, M. and Brodersen, R., Biochim. Biophys. Actu, 354 (1974) 121-129. Hochstrasser, K., Reichert, R., Schwarz, S. and Werle, E., Hoppe-Seyler’s Z. Physiol. Chem., 353 (1972) 221-226. Hornby, W.E., Lilly, M.D. and Crook, E.M., Biochem. J., 98 (1966) 420-425. Inman, J.K. and Dmtzis, H.M., Biochemistry, 8 (1969) 4074-4082. Jagendorf, A.T., Patchornik, A. and Sela, M.P., Biochim. Biophys. Acta, 78 (1963) 516-528. Kanfer, J.N., Petrovich, G. and Mumford, R.A., Anal. Biochem., 55 (1973) 301-305. Kay, G . and Lilly, M.D., Biochim. Biophys. Actu, 198 (1970) 276-285. Kristiansen, T., Biochim. Biophys. Actu, 362 (1974) 567-574. L%s, T., J. Chromutogr., 66 (1972) 347-355. L%s, T.,J. Chromatogr., 111 (1975) 373-387. Lamed, R., Levin, Y. and Oplatka, A., Biochim. Biophys. Actu, 305 (1973) 163-171. Larsson, P.O. and Mosbach, K., Biotechnol. Bioeng., 13 (1971) 393-398. Lasch, J., Experientia, 31 (1975) 1125-1126. Levin, Y., Pecht, M., Goldstein, L. and Katchalski, E., Biochemistry, 3 (1964) 1905-1913. Lowe, C.R. and Dean, P.D.G., Affinity Chromatography, Wiley, New York, London, 1974, pp. 272. Lowe, C.R., Harvey, M.J. and Dean, P.D.G., Eur. J. Biochem., 41 (1974) 347-351. Ludens, J.H., De Vries, J.R. and Fanestil, D.D.,J. Biol. Chem., 247 (1972) 7533-7538. March, S.C., Parikh, I. and Cuatrecasas, P., Advan. Exp. Med. Biol., 42 (1974a) 3-14. March, S.C., Parikh, I. and Cuatrecasas, P., Anal. Biochem., 60 (1974b) 149-152. Messing, R.A. (editor), Immobilized Enzymes for Industrial Reactors, Academic Press, New York, 1975, pp. 232. Micheel, F. and Ewers, J.,Mukromol. Chem., 3 (1949) 200-209.

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Morikawa, Y., Tezuka, T., Teranishi, M., Kimura, K., Fujimoto, Y.and Samejima, H., Agr. Biol. Chem., 40 (1976) 1137-1174.

Nshikawa, A.H. and Bailon, P., Anal. Biochem., 64 (1975a) 268-275. Nishikawa, A.H. and Bailon, P., Arch. Biochem. Biophys., 168 (1975b) 576-584. O’Carra, P., Barry, S. and Griffin, T., FEBS Left., 43 (1974) 169-175. Parikh, I., Sica, V., Nola, E., Puca, G.A. and Cuatrecasas, P., Methods Enzymol., 34 (1974) 670-688. Porath, J., Methods Enzymol., 34 (1974) 13-30. Porath, J,, Aspberg, K., Drevin, H. and Axdn, R.. J. Chromtogr., 86 (1973) 53-56. Porath, J., Axdn, R. and Emback, S., Nature (London), 215 (1967) 1491-1492. Porath, J., Janson, J.C. and L g s , T., J. Chromatogr., 6 0 (1971) 167-177. Porath, J. and Kristiansen, T., in H. Neurath and R.L. Hill (Editors), The Proteins, Academic Press, New York, 3rd edn., 1975, pp. 95-178. Porath, J., L%s, T. and Janson, J.C.,J. Chromtogr., 103 (1975) 49-62. Rafestin, M.E., Obrenovitch, A., Oblin, A. and Monsigny, M., FEBS Lett., 40 (1974) 62-66. Ratner, D., J. Mol. Biol., 88 (1974) 373-383. Robinson, P.J., Dunnill, P. and L a y , M.D., Biochim Biophys. Acta, 285 (1972) 28-35. Roche, A.C., Schauer, R. and Monsigny, M., FEBS Lett., 57 (1975) 245-249. Sanderson, C.J. and Wilson, D.V., Immunology, 20 (1971) 1061-1065. Sato, T., Mori, T., Tosa, T. and Chibata, I., Arch. Biochem. Biophys., 147 (1971) 788-796. Schnapp, J. and Shalitin, Y.,Biochem Biophys Res. Commun., 70 (1976) 8-14. Sica, V., Parikh, I., Nola, E., Puca, G.A. and Cuatrecasas, P., J. Biol. Chem., 248 (1973) 6543-6558. Silman, 1.H. and Katchalski, E.. Annu. Rev. Biochem., 35 (1966) 873-908. Smith, B.R., J. Endocrinol., 52 (1972) 229-237. Sundberg, L. and Hoglund, S., FEBS Lett., 37 (1973) 70-73. Sundberg, L. and K&iansen, T., FEBS Lett., 22 (1972) 175-177. Sundberg, L. and Porath, J., J. Chromtogr., 90 (1974) 87-98. Svensson, B., FEBS Lett., 29 (1973) 167-169. Tabachnick, M. and Sobotka, H.,J. Biol. Chem., 235 (1960) 1051-1054. Tesser, G.I., Fisch, H.-U. and Schwyzer, R., FEBS Lett., 23 (1972) 56-58. Tesser, G.I., Fisch, H . 4 . and Schwyzer, R., Helv. a i m . Acta, 57 (1974) 1718-1730. Thoni, H., Experientia, 3 1 (1975) 251. Turkovil, J., Methods Enzymol., 44 (1977) 66-83. Turkovi, J., Bliha, K., Malanikovi, M., Vanckrovi, D., Svec, F. and KBlal, J., Biochim Biophys. Acra, in press. Turkovli, J., Bldha, K., Valentovil, O., coupek, J. and Seifertovil, A., Biochim. Biophys. Acta, 427 (1976) 586- 593.

Turkovil, J., Hubdlkovli.,O., KGvAkovA, M. and Coupek, J., Biochim. Biophys. Acta, 322 (1973) 1-9. Vauquelin, G., Lacombe, M.L.,Hanoune, J. and Strosberg, A.D., Biochem Biophys. Res. Commun., 64 (1975) 1076- 1082.

Weetall, H.H., Sep. Punt Methods, 2 (1973) 199-229. Weetall, H.H. and Filbert, A.M., Methods Enzymol., 34 (1974) 59-72. Weetall, H.H. and Mason, R.D., Biotechnol. Bioeng., 15 (1973) 455-466. Wetiky, N., Weetall, H.H., Gilden, R.V. and Campbell, D.H.,Immunochernistry, 1 (1964) 219-229. Weston, P.D. and Avrameas, S., Biochem Biophys. Res. Commun.,45 (1971) 1574-1580. Whiteley, J.M., Jerkunica, 1. and Deits, T., Biochemistry, 13 (1974) 2044-2050. Wichterle, 0. and Lim, D., Nature (London), 185 (1960) 117-118. Wilchek, M., FEBS Left., 33 (1973) 70-72. Wilchek, M., Advan. Exp. Med. Biol., 42 (1974) 15-31. Wilchek, M. and Muon, T., Methods Enzymol., 34 (1974a) 72-76. Wichek, M. and Muon, T.,Mol. Cell. Biochem., 4 (1974b) 181-187. Wilchek, M., Oka, T. and Topper, Y.J.,Proc. Nor. Acad. Sci. US.,72 (1975) 1055-1058. Wolodko, W.T. and Kay, C.M., Can. J. Biochem., 53 (1975) 175-188. Yong, M.S., Science, 182 (1973) 157-158.

203

Chapter 9

Characterization of supports and immobilized affinity ligands To master the affinity chromatography method successfully, it is important to know the characteristics of both the solid supports employed and the immobilized affinity ligands. One of the main characteristics that determines the suitability of solid supports for affinity chromatography is their sorption properties. In Section 9.1, some examples of procedures for the determination of non-specific sorption are given. For the linkage of affinants proper, the amount of activatable or active groups capable of binding the molecules of the affinity ligand is important. In Section 9.2, methods are given for the determination of carboxyl, amino and hydrazide groups, including the colour test with sodium 2,4,6-trinitrobenzenesulphonate.Further methods for the determination of oxirane groups, p-nitrophenol esters and vinyl and sulphydryl groups are also given. The number of groups that can be utilized for immobilization is often determined on the basis of the attachment of low-molecular substances, for example glycine or glycylleucine for epoxy-activated gels. Its content can be then determined by some of the methods described in Section 9.3. From Table 8.1,giving the amounts of glycine and chymotrypsin attached on various types of Spheron, it is evident how important it is for coupling that the size of the immobilized molecules and the surface of the solid support are taken into consideration. The most commonly used methods for the determination of the content of immobilized affinity ligands are listed in Section 9.3. For example, spectroscopic methods, acid-base titrations, determination of affinants after their release by acid, alkaline or enzymatic hydrolysis, determination on the basis of elemental analysis, and also by the measurement of radioactivity. With immobilized enzymes, in addition to the content of the bound protein and the total or relative activity, the titration of the active site may also provide further valuable information. Examples of such determinations are given in Section 9.4. Examples of procedures used for the observation of conformational changes are given in Section 9.5 and the determination of the distribution of the proteins bound on the surface of the solid support in Section 9.6. The thorough characterization of an immobilized affinity ligand is necessary mainly when affinity chromatography and the immobilized enzymes are used for the study of questions such as the interaction of enzymes with inhibitors and the effect of various micro-environmentson the stability and the catalytic properties of enzymes. 9.1 METHODS FOR THE DETERMINATION OF NON-SPECIFIC SORPTION

Non-specific sorption is a complicating factor in affinity chromatography. This has been stressed briefly several times in the text, and it is discussed in detail in Section 10.3. In agar, one of the most commonly used supports, Porath et al. (1971) mentioned, for example, two types of groups that cause sorption, viz., sulphate monoester and carboxyl groups.

204

CHARACTERIZATION OF SUPPORTS AND LIGANDS

9.1.1 Determination of adsorption capacity

A solution of cytochrome C (0.1%) in 0.01 M ammonium acetate solution is applied at pH 4.1 on to a gel column (3 X 0.5 cm) equilibrated with the same buffer until saturated, then the column is washed until no protein appears in the eluate. The adsorbed cytochrome is then displaced with 0.15 M ammonium acetate solution of pH 4.1, and the amount of cytochrome desorbed is determined on the basis of the absorbance at 280 nm. After each determination the gel should be washed with 0.5 M sodium hydroxide solution and then with distilled water until neutral. The washed gel is then freeze-dried and weighed. In synthetic polymers, for example hydroxyalkyl methacrylate gels of the Spheron type, the reason for non-specificity may lie in the residues of uneluted organic solvents that were used during the preparation. The adsorption capacity can also be controlled by dividing a solution of serum albumin in 0.05 M sodium acetate solution into two halves, one half is applied on to the column prepared from the tested gel equilibrated with the same buffer. After passage through the column, the fractions containing the protein are combined and made up to a certain volume with 0.05 M sodium acetate solution. The second half, serving as a control, is made up to the same volume. Agreement between the absorbances at 280 nm of the two solutions shows that no non-specific sorption took place on the gel. With Spheron, if the two solutions do not show the same absorbance, the gel must be washed with organic solvents, dilute acid and then thoroughly with distilled water. Gels that display non-specific sorption should not be used for affinity chromatog raphy. 9.1.2 Determination of residual negatively charged groups (according to Porath et al., 1975)

On a glass filter, about 5 g of wet gel are washed first with water and then with 25 ml of 1 M hydrochloric acid. The gel is then washed with distilled water until neutral and until the test for chlorides (with silver nitrate) is negative. The weighed gel is then suspended in 5 ml of 2 M potassium chloride solution and titrated with 0.01 M sodium hydroxide solution.

9.2 DETERMINATION OF ACTIVATABLE AND ACTIVE GROUPS 9.2.1 Determination of carboxyl, hydrazide and amino groups on the basis of acid-base titration (Inman)

9.2.1.1 Dry weight determination

For a better reproducibility, it is best to refer the amount of functional groups to the dry weight. Inman recommended transferring the titrated sample into a weighed glass filter, washing it first under suction with 0.2 M hydrochloric acid (for carboxyl groups) or with 0.2 M sodium carbonate solution (for amino groups) to render the groups uncharged. Thorough washing with distilled water eliminates all of the electrolyte, while water is

ACTIVATABLE AND ACTIVE GROUPS

205

eliminated by washing with methanol. The material is further dried in a vacuum over anhydrous calcium chloride. The sample should be weighed immediately after taking it out of the desiccator. For example, vacuum-dried polyacrylamide absorbs moisture from the air and increases its weight by about 2%per hour.

9.2.1.2Determination of carboxyl groups (according to Inman) The washed sample of gel is suspended in 0.2 M sodium chloride solution, keeping its volume below twice the bed volume. Using dilute sodium hydroxide solution, the pH of the suspension is adjusted to 6.5-7.5. A standard solution of hydrochloric acid is then added while stirring with a magnetic stirrer until the pH is 2.6. The content of the carboxyl groups of the samples is determined on the basis of the milliequivalents of HCl added, after subtraction of the milliequivalents of free H+ (0.0025v). The amount of the carboxyl groups is finally referred to the dry weight.

9.2.1.3 Determination of hydrazide groups The acylhydrazide groups have pK, values close to 2.6 and direct titration is therefore very difficult. The groups are best converted quantitatively into the succinylhydrazide form by using excess of succinic hydrazide (0.6 g per 25 ml of suspension) at pH 4.0. The derivatized gel is then washed with 0.2 M sodium chloride solution and the content of carboxyl groups is determined as in Section 9.2.1.2.The hydrazide group density, D , obtained from the subsequently determined dry weight, relates to the succinylhydrazide derivative with its incremental mass of succinyl groups. The group density in terms of millimoles of hydrazide per gram of original polyacrylamide is D' = D / ( l - 0.1 150).

9.2.1.4 Determination of aliphatic amino groups The titrated gel is washed and suspended in 0.2 M potassium chloride solution, taking care that the volume of the solution does not exceed twice the bed volume. The pH of the suspension is adjusted to pH 11.O with dilute potassium hydroxide solution. Using a standard solution of hydrochloric acid (v d)of molarity M,the suspension is titrated, bringing the pH to 7 (or pH 6 for a-amino groups). Then the volume of the suspension (V ml) is measured. The content of amino groups is calculated from the milliequivalents of HCl consumed (vM) after subtraction of the milliequivalents of free OH- titrated [0.001 ( V - v)]. The group density in terms of the mass of original poIyacrylamide,D', is obtained from the density with respect to the dry weight of derivative by means of the expression D' = D/(1 - DAr). The quantity Ar must be calculated for the particular derivative and is equal to 0.001 times the molecular weight of all atoms in a residue of the derivative that occur in addition to those in the original carbamyl side-chain. Thus, for the aminoethyl derivative, Ar = 0.043. Amino groups cannot be titrated by this method if diacylhydrazide has been used as the spacer: -CO * NHNH-COR' is ionized to -CO-NHN(-) COR'+ H(+),with pK, = 1 1 .O. Kornbluth et al. also used titration for the determination of the amount of hexamethylenediamine bound to Sepharose. However, in this instance they titrated the gel to a thymolphthalein end-point with 0.1 M sodium hydroxide solution.

CHARACTERIZATION OF SUPPORTS AND LIGANDS

206

9.2.2 Determination of the content of free carboxyl groups (according to Goldstein) Glycine ethyl ester hydrochloride (325 mg) is dissolved in 2.5 ml of dimethyl sulphoxide and an equivalent amount of triethylamine is added to liberate the ester in the free-base form. After stirring the reaction mixture for 1 h over ice, it is filtered to eliminate the triethylammonium chloride precipitate. The solution is added to a test-tube containing the carboxyl derivative of the gel (50 mg). A solution of 325 mg of dicyclohexylcarbodiimide in 3 ml of dimethyl sulphoxide is then added to the magnetically stirred suspension of the gel and the reaction is allowed to proceed at room temperature for 16 h. The gel is then separated by centrifugation, re-suspended in 5 ml of dimethyl sulphoxide and again centrifuged. The washing with dimethyl sulphoxide is repeated three times and is followed by a triple wash with acetone. After drying for 24 h over phosphorus pentoxide under vacuum and acid hydrolysis with 6 M hydrochloric acid for 48 h at 110°C, the amount of glycine in the gel is determined with an amino acid analyser.

9.2.3 Determination of free amino groups in polymers on the basis of the condensation (Esko er al.) reaction with 2-hydroxy-1-naphthaldehyde By the condensation of 2-hydroxy-1-naphthaldehyde with the free amino groups in the polymer, a stable aldimine (Schiffs base) is formed. After diplacement of the chromophore from the polymer with an amine, for example benzylamine, the amount of the soluble aldimine thus formed is determined. Gsko er al. recommended the following procedure. A 10-mg amount of polymer is allowed to react in absolute ethanol with a large excess at room temperature for 12 h. After care(50-100 fold) of 2-hydroxy-1-naphthaldehyde ful washing of the polymer with dichloromethane, 2 ml of 0.4M benzylamine in dichloromethane are added and the mixture is allowed to react for 30 min. The amount of 2hydroxy-1 -naphthylidenebenzylamineis determined spectrophotometrically from the absorbance measured at 420 nm. Excess of benzylamine has virtually no effect on the absorbance.

9.2.4Procedure for azide assay (Brenna er al.) Freshly prepared azido derivatives are washed 2-3 times with ice-cold water in order to remove the acid, then 5- 10 ml of the washed gel are suspended in 20 ml of 0.1 M sodium hydroxide solution and stirred at room temperature for 2 h. A 0.1-ml volume of clear supernatant is then added to 4.9 ml of 0.1 M ammonium iron(II1) sulphate solution. The absorbance at 458.1 nm, which is measured against a blank [4.9ml of 0.1 M ammonium iron(II1) sulphate solution plus 0.1 ml of 0.1 M sodium hydroxide solution], should be read within the first 5 min because in acidic medium hydrazoic acid is slowly released. For azide concentration in the 0.01-1.4 mM range, Beer's law applies ( E ~ cm~ = ~ . ~ 1.2* lo6 1. mole-' * cm'). The concentration of azides can be calculated from the relationship

-

A 5 EN3-] (mequiv./l) = - -

1.2 0.1

ACTIVATABLE AND ACTIVE GROUPS

207

where the concentration of azides is referred to a litre of supernatant. The azide concentration in milliequivalents per gram of dry polymer can be obtained from the relationship [N3-] (mequiv./g) = [N3-] (mequiv./l) .b *-

1

C

where b is the total water content of the suspension in litres and c is the dry weight of the tested polymer in grams.

9.2.5 The sodium 2,4,6-trinitrobenzenesulphonatecolour test Inman and Dintzis described a simple qualitative colour test with which the course of the substitution in gels can be followed. The derivatized gel (0.2-0.5 ml in distilled water) is added to approximately 1 ml of saturated sodium borate solution, followed by three drops of 3% aqueous sodium 2,4,6-trinitrobenzenesulphonate(TNBS) solution: NOa

NO2

The colour is allowed to develop for 2 h at room temperature. In Table 9.1 the colours obtained with individual products are indicated. The progress of substitution of the amino derivatives by carboxyiic ligands, or of hydrazide derivatives by affinity ligands that contain amino groups, can be determined on the basis of the relative colour intensity of the gel. The test can be quantitated by washing the gels to remove picric acid and the unreacted TNBS and solubilizing by warming with 50% acetic acid. From the absorbance at 340 nm, the content of amino groups of gel can be determined. TABLE 9.1 COLOURS PRODUCED BY THE SODIUM 2,4,6-TRINITROBENZENESULPHONATE TEST Derivative

Colour

Unsubstituted agarose or polyacryalmide Carboxylic and bromoacetyl Primary aliphatic amines Primary aromatic amines Unsubstituted hydrazides

Pale yellow Yellow Orange Red-orange Deep red

9.2.6 Fluorescamine test for the rapid detection of trace amounts of amino groups (Felix and Jimenez) Fluorescamine is a very powerful fluorogenic reagent that is capable of reacting almost instantaneously with primary amino compounds, giving rise to fluorophors with extinction and emission maxima at 390 and 475 nm, respectively. The reagent is capable of detecting

CHARACTERIZATION OF SUPPORTS AND LIGANDS

208

picomole amounts of primary amines and it reacts with uncoupled amino groups of the solid support under mild conditions to form highly fluorescent derivatives:

J*"

+

H2N-

Et

N

\ /

A 5-10-mg amount of the tested gel is transferred on to a glass filter and washed with dichloromethane, ethanol, dichloromethane and chloroform (three times each wash). The gel is then treated with 10% trimethylamine in chloroform and washing with chloroform, ethanol and chloroform is repeated (four times each wash, 1 ml per treatment). After filtering off under suction, the gel is saturated with several drops of 10%triethylamine in chloroform. Fluorescamine solution (10 mg/ml) is then added. Triethylamine must be present in the gel before the addition of fluorescarnine in order to prevent the conversion of the fluorophor-containing gel into the non-fluorescent y-lactone. After standing for 10 min at room temperature, the gel is filtered off under suction and washed with chloroform, methanol and chloroform. The funnel containing the gel is viewed under Iongwave ultraviolet light (366 nm) and its fluorescence is compared visually with that of a blank. As the coupling approaches completion, the intensity of the fluorescence decreases and approaches that of the blank. A fluorescamine solution, if stored at 4"C, gives reproducible results on storage for up to 1 month. 9.2.7 Determination of oxirane groups (Sundberg and Porath)

For the determination of the amount of oxirane groups in solution and in the agarose gel, the reaction between the oxirane ring and sodium thiosulphate is used: -CH-CH~ \ / 0

+

2ha'

+ s,o,'- +

H ~ O

-

-CH-CH,-S,O; OH

t 2Na+

+

OH-

The release of OH- was controlled by titration with 0.1 M hydrochloric acid using a pHstat. For the determination of oxirane groups in solution, Sundberg and Porath recommended the following procedure. The oxirane-containing solution (50 pl) is added to 1.5 ml of 1.3 M sodium thiosulphate solution and the pH is kept constant by addition of hydrochloric acid until the reaction is complete. The amount of oxirane present in the solution is then calculated from the amount of hydrochloric acid needed in order to maintain neutrality. For the determination of oxirane groups in agarose gel, they recommended the following procedure. Wet agarose gel (0.5 g) is added to 1.5 ml of 1.3 M sodium thiosulphate solution of pH 7.0 and the oxirane content of the gel is determined by titration with hydrochloric acid. The agarose gel is suction-dried under vacuum on a glass filter-funnel for 5 min and weighed.

209

ACTIVATABLE AND ACTIVE GROUPS

9.2.8 Determination of the capacity of p-nitrophenol ester derivatives of hydroxyalkyl

methacrylate (NPAC) gels (Turkovi) The amount of active groups was determined on the basis of the spectrophotometric measurement of the p-nitrophenol released. A 100-mgamount of NPAC gel was suspended in 10 ml of dilute ammonia (1 :1) and the suspension allowed to stand for 1-4 h with occasional stirring. After the addition of 90 ml of distilled water, the absorbance at 400 nm was measured and the amount of p-nitrophenol released was read from a calibration graph. 9.2.9 Determination of the degree of substitution of benzylated dibromopropanol crosslinked Sepharose ( M s )

On the basis of exhaustive sulphation of benzylated and non-benzylated Sepharose, the degree of substitution can be calculated by determining the difference in sulphur contents. Pyridine (75 ml) was added dropwise to 10 ml of sulphur trioxide in a round-bottomed flask fitted with a stirrer and a reflux condenser and chilled with ice. Five grams of gel were thoroughly washed with pyridine and allowed to react with 7.5 ml of the suspension of sulphur trioxide-pyridine complex in stoppered tubes for 24 h at 40°C. The sulphated gels were then washed on a glass filter with about 200 ml each of ethanol, water, 1 M acetic acid containing 2 M sodium chloride, 1 M sodium carbonate containing 2 M sodium chloride and finally water. The gels were lyophilized and analysed for sulphur. 9.2.10 Determination of vinyl groups (Porath et al., 1975)

For this determination, the reaction

+ S203’-+ HzO +-R-S02-CH2-CH2-S203’-

R-SOZ-CH=CHz

+ OH-

is used. A 2-ml volume of 3 M sodium thiosulphate solution is added to 2 ml of crosslinked gel in water. The hydroxyl ions that are formed during the reaction are titrated with 0.1 M hydrochloric acid; the pH of 1.5 M sodium thiosulphate (5.5) is considered to be the end-point of the titration. The reaction rate is very slow, completion of the reaction taking about 10-15 h. 9.2.11 Determination of sulphydryl groups (Lowe and Dean)

Gel-bound sulphydryl groups can be determined with Elman’s reagent [5,5 ‘-dithiobis(2-nitrobenzoic acid)], which liberates for each thiol group 1 mole of strongly coloured 5-sulphido-2-nitrobenzoate anion:, FSU

+

N

0

-0oc

2

~

S

-

S

~

coo-

N pH0 >6.8 ~

$-S-SQNO2

+

t -5QN02

coo-

H*

cooE

= 1.36 -lo4l/mole/cm at pH 8

CHARACTERIZATION OF SUPPORTS AND LIGANDS

210

Its amount is determined on the basis of the absorbance measured at 412 nm. This method was used by Cuatrecasas for the determination of -SH groups in Sepharose derivatives, and was also used for the determination of dihydrolipoic acid covalently attached to Sepharose (Lowe and Dean). 4,4'-Dipyridyl disulphide (Grassetti and Murray) and 2,2 '-dipyridyl disulphide (Svenson and Carlsson) give similar results:

4,4 '-Dipyridyl disulphide

4-Thioxo-l,4-dihydropy;idine hmax = 324 nm E = 1.98 lo4l/mole/cm

Sepharose-bound thiols can also be determined on the basis of their capability to take up ['4C]iodoacetamide on reaction with 0.01 M iodoacetamide in 0.1 M sodium hydrogen carbonate solution (pH 8.0) at room temperature for 15 min (Cuatrecasas). 9.3 METHODS FOR THE DETERMINATION OF IMMOBILIZED AFFINITY LIGANDS

In Section 5.3,the effect of the concentration of immobilized affinity ligands was discussed in detail. From that discussion, it is evident that after the attachment of affinity ligands to an inert carrier it is very useful to determine the proportion of the molecules that are covalently bound to the matrices before the sorption. A gel prepared for sorption should not contain groups that are capable of coupling (see blocking of the residual active groups in Section 8.4) and it should be washed thoroughly. The preparation of gels is discussed from the practical point of view in Section 8.6, as are methods by which it can be checked that the molecules of affinity ligand covalently bound to the solid support are the only ones present in the gel. 9.3.1 Difference analysis

The amount of the affinity ligand coupled to a gel can be determined from the difference between the total amount of the affinity ligand added to the coupling mixture and that recovered after exhaustive washing. Although this method is sometimes used, the results obtained are inaccurate, especially when only a small proportion of the affinity ligand is covalently attached, or when the affinity ligand is sparingly soluble and requires large volumes for unbound material to be washed out. However, the method is suitable for an approximate evaluation of the amount attached. An example is the determination of the amount of bound DNA on the basis of the difference between the absorbances of the input solution and final wash solution at 260 nm (Schabort). 9.3.2 Spectroscopicmethods

For the determination of immobilized affinity ligands that absorb at wavelengths above 260 nm, direct spectrophotometry of derivatized gels can be employed. The gel is suspend-

IMMOBILIZED AFFINITY LIGANDS

211

ed in optically pure polyacrylamide, ethylene glycol or glycerol in cells of 1 mm width, and the values are read against the underivatized gel in a double-beam spectrophotometer. Lowe et ul. used this method for the determination of the concentration of immobilized NAD' on the basis of the absorbance at 206 nm. The amount of immobilized NADH was determined on the basis of the absorbance at 340 nm and of immobilized FAD on the basis of the absorbance at 450 nm (Lowe and Dean). Immobilized NADH, FAD and pyridoxamine were fluorescent when irradiated with light of an appropriate wavelength (Collier and Kohlhaw; Lowe and Dean). When determining the amount of leucine aminopeptidase and trypsin covalently bound to Sepharose, Koelsch et ul. compared five methods for the determination of bound proteins: (1) on the basis of the protein balance before and after binding; (2) from the amino acid analysis after acid hydrolysis; (3) by a modification of Lowry's method; (4) spectrophotometrically; and (5) by fluorimetric analysis. The advantage of the last two methods, which they elaborated in detail, is that they do not cause destruction of the gel. Together with Lawry's method, these determinations of bound proteins are characterized by simplicity, sensitivity and high reproducibility. For the solubilization of derivatized agaroses, several methods have been proposed that permit the quantitative spectrophotometry of immobilized affinity ligands (Lowe and Dean). Agarose gels can be dissolved in hot water, underivatized agarose can be dissolved at 60"or higher. The subsequent cooling of highly concentrated aqueous solutions of agarose leads to turbidity in the solutions, while more dilute solutions become viscous but remain transparent. These effects are independent of pH over the whole range. In contrast, derivatizable gels do not dissolve, even with heating at 75°C with 8 M urea or 6 M guanidine hydrochloride solution. They can be solubilized by heating with 0.1 M hydrochloric acid or sodium hydroxide solution at 75°C or, most commonly, with 50% acetic acid. When solubilized, no visible precipitate is formed even when the pH is adjusted to neutrality. The spectra of solubilized agarose are shown in Fig. 9.1 (Failla and Santi). Agarose when dissolved in water is transparent at wavelengths above 350 nm, but it absorbs slightly between 240 and 350 nm.This absorbance is directly proportional to the concentration of agarose and therefore it can easily be eliminated by using a suitable blank. After treatment with 0.1 Mhydrochloric acid (at 75°C for 2 h) agarose displays a significant absorption peak at 280 nm, probably produced by the acid-catalysed formation of furfuraldehyde. In contrast, the action of 0.1-1 M sodium hydroxide solution, under the same conditions, brings about partial caramelization of the gel, with a broad absorption in the W-visible spectrum and formation of amber-coloured solutions. This effect can be prevented by adding 0.1%of sodium borohydride prior to heating. In this instance the spectra obtained are similar to those obtained in water. In Section 9.2.5, it has already been mentioned that the solubilization technique can be used in connection with the TNBS test for the quantitative determination of gels containing amino groups. Stepanov et al. solubilized Sepharose with covalently bound mono-N-DNP-hexamethylenediamine with 66% trifluoroacetic acid at 100°C. The solution obtained, when diluted with 20%trifluoroacetic acid, had a maximum at 360 nm in its U V spectrum, which could be used for the calculation of the content of DNP-amine ( E g o = 15,000). Borchardt et al. hydrolysed the modified gel with 2 M sodium hydroxide solution at 100°C before the

CHARACTERIZATION OF SUPPORTS AND LIGANDS

21 2

B

W

0

z U

m

5

-

0.4

v)

m

4

0 WAVELENGTH, nm

Fig. 9.1. Spectra of solubilized agarose. A suspension containing the equivalent of 0.2 ml of the agarose in 5.0 ml of the indicated solvent was dissolved by heating at 7S0C for 2 h. (A) H,O (a); 0.1 N NaOH0.1%NaBH, (b); 50% HOAc (c); 0.1 N HCI (d); 0.1-1 N NaOH (diluted to 10.0 ml with 1 N NaOH) (e). (B) 50%HOAc (a); 0.1 N HCI (b); 0.1 -1 N NaOH (diluted to 10 ml with 1 N NaOH prior to recording spectrum) (c). Reproduced with permission from D. Failla and D.V. Santi, Anal. Biochem., 5 2 (1973) 363-368.

spectrophotometric determination of 3,4-dimethoxy-5-hydroxyphenylethylamine. The determination of the degree of substitution in the series of agarose gels substituted with hydrophobic groups, using H-NMR spectroscopy, has been described by Rosengren et al. Agarose derivatives were washed carefully and shrunk with acetone on a glass filter. A 100-g amount of the suction-dried gel was transferred into a 50-ml flask, 20 ml of 85% formic acid were added and the mixture was heated under a reflux condenser on a waterbath for 1 h. If the polysaccharide carrier did not dissolve during this period, the time of hydrolysis was prolonged. After hydrolysis, the mixture was evaporated t o dryness and, after the addition of 2 ml of 'H,O, the mixture was again dried. The addition of heavy water and the evaporation were repeated and the remaining residue was dissolved in 1 ml of hexadeuteriodimethyl sulphoxide and employed for NMR spectroscopy.

9.3.3 Determination by means of acid-base titration Polar affinity ligands can be determined by titration of the derivatized gel with an acid or a base. Hixson and Nishikawa determined titrimetrically the amount of p-aminobenzamidine bound by the water-soluble carbodiimide to succinylated hexamethylenediamineSepharose together with the remaining unsubstituted carboxyl groups. The Sepharose derivative containing carboxylate ions was washed with a 50-fold volume of 0.5 M sodium chloride solution and the volume of the gel was determined by allowing it to settle for 1 h or by gently centrifuging the beads in a calibrated cone. The gel was titrated from pH 7 to pH 3. The whole procedure was repeated after the attachment of p-aminobenzamidine,

IMMOBILIZED AFFINITY LIGANDS

213

and the concentration of attached affinant was derived from the difference between the titres. The concentration of N-6-(6-aminohexyl)-AMPimmobilized on Sepharose was also determined by direct titration (Craven et al.), 9.3.4 Determination of immobilized proteins, peptides, amino acids, nucleotides, carbohydrates and other substances after liberation by acid, alkaline or enzymatic hydrolysis

9.3.4.1 Determination of immobilized amino acids, peptides and proteins The method most often employed for this determination is quantitative amino acid analysis according to Spackman et al., after acid hydrolysis of the immobilized affinant. The procedure described by Axin and Ernback is most often used. For agarose with attached enzymes, the gels were freed from buffers on a glass fdter (G3) by washing with distilled water and then with a mixture of acetone and water, with increasing concentration of acetone. Finally, the conjugates were washed with acetone, which was eliminated under vacuum, and the preparations were dried under vacuum over phosphorus pentoxide for 48 h. The weighed amount of the modified gel was hydrolysed with 6 M hydrochloric acid at 110°C for 24 h in a closed, evacuated vessel. Amino acid analysis of the hydrolysate was carried out on an amino acid analyser and the protein content was calculated from the content of aspartic and glutamic acid, alanine and leucine. A high content of carbohydrates leads to a dark coloration of the hydrolysate, which, however, does not interfere in the analysis. Koelsch et al. pre-purified the dark-coloured hydrolysate, which results from the acid hydrolysis of the polysaccharide carrier on the ion-exchange column. The hydrolysate was prepared using norleucine as an internal reference. After elimination of HCl on a rotatory evaporator, the samples were dissolved in 2 ml of 0.02 N hydrochloric acid and applied either on a 7 X 1 cm column of Amberlite IR 12O-X8 and eluted with 25 ml of 1 M ammonia solution, or on a 7 X 1 cm column of Dowex 50W-X4 and eluted with 25 ml of 10%(vlv) pyridine-water. Holleman and Weis also determined e-aminocaproicacid eluted shortly before lysine on an amino acid analyser after acid hydrolysis. Svensson determined the number of modified e-amino groups from the difference in the amount of free amino groups before and after the modification. Free amino groups were thus determined by reductive methylation with solutions of formaldehyde and borohydride. The N-e-dimethyllysineand N-e-methyllysine thus formed were then determined using an amino acid analyser (Ottesen and Svensson).

9.3.4.2 Determination of nucleotides The determination of the amount of nucleotides bound to Sepharose was carried out by calculation from the amount of the soluble product, formed after alkaline or nuclease hydrolysis (Hayashi). The material absorbing in the ultraviolet region, formed in the supernatant during hydrolysis, was determined on the basis of the absorbance, and the hydrolysis was stopped when the absorbance no longer increased. During alkaline hydrolysis, 1 ml of the derivatized gel was mixed with 1 ml of 0.6 M sodium hydroxide solution and the

214

CHARACTERIZATION OF SUPPORTS AND LIGANDS

mixture was incubated at 37°C. During enzymatic hydrolysis with T2 ribonuclease, 0.5 ml of the derivatized gel was mixed with 0.5 ml of 0.1M sodium acetate buffer of pH 4.6, containing one unit of T2 ribonuclease. Incubation was again carried out at 37°C.Enzymatic hydrolysis with phosphodiesterase was carried out in 0.1 M Tris-hydrochloric acid buffer of pH 8.5, containing 0.01 M magnesium chloride. One milligram of snake venom phosphodiesterase was used per 0.5 ml of the derivatized gel. Phosphodiesterase and alkaline phosphatase were used simultaneously for hydrolysis of Sepharose with attached p aminophenyl-ATP or dATP by Ekrglund and Eckstein, who determined the release of the nucleotide in the hydrolysate. Similarly, after enzymatic hydrolysis with alkaline phosphatase, the amount of the immobilized AMP was determined on the basis of the phosphate analysis of the terminal phosphate split off (Craven et al.). On the basis of the total determination of phosphorus (King), Moudgil and Toft determined the amount of ATP bound to Sepharose. The sample was first oxidized with a sue pension of 7% perchloric acid in 4.5 M sulphuric acid and then incubated at 240°C for 60 min. 9.3.4.3 Determination of carbohydrate

For the determination of the carbohydrate content of copolymers, HofejSi and Kocourek described the following procedure. A 0.1-g amount of dry pulverized gel was hydrolysed under reflux in boiling 2.5 M sulphuric acid for 6 h. After cooling, the content of carbohydrates in the supernatant was determined by the phenol-sulphuric acid method according to Dubois et al. Galactosyl pyrophosphate bound to Sepharose was determined on the basis of the analysis of phosphates after hydrolysis with 0.5 M hydrochloric acid at 100°C for 1 h (Barker et a/.). Similarly, immobilized N-acetylglucosamine was determined on the basis of the liberated glucosamine determined after acid hydrolysis with 6 M hydrochloric acid at I. 10°C (Barker et a/.). After hydrolysis of a lyophilized sample of heparin-Sepharose, the content of heparin was determined, for example, on the basis of the analysis of sulphates (Sternbach et al.).

9.3.5 Determination of the amount of bound affinant on the basis of elemental analysis The amount of immobilized nucleosides is most commonly expressed on the basis of the determination of the total content of phosphorus (Hipwell et al.; Lamed et al.;Trayer e f al.). In determinations of phosphorus, individual workers refer to the studies of Barlett, King or Meun and Smith. The amount of sulphanylamide bound to Sepharose can be determined on the basis of the determination of sulphur (Falkbring et a/.). The determination of immobilized affinants on the basis of the determination of nitrogen sometimes leads to erroneous conclusions. As already discussed in Section 8.2.4, during cyanogen bromide activation nitrogen is introduced into the gel, and its amount is dependent on the temperature used during activation (Nishikawa and Bailon). The amount of immobilized proteins, especially antibodies, is sometimes calculated from the amount of nitrogen determined by the Kjeldahl method (Matheka and Mussgay).

IMMOBILIZED AFFINITY LIGANDS

215

9.3.6 Determination of labelled affinity ligands

Radioactive affinants have been used in many instances, for binding on to solid supports. The amount of the bound affinity ligand can be determined easily on the basis of the radioactivity measured. For example, Green and Toms determined the amount of biotin bound to Sepharose on the basis of the measured radioactivity. Suspension samples (0.1 ml) were taken with a siliconized 0.25-ml tuberculin syringe or an Eppendorf pipette and transferred into the centre of 4-cm2 planchets. A 0.05-ml volume of 1% sodium dodecyl sulphate solution was added to each sample. The suspensions were dried and their radioactivity was counted. Control experiments with a known amount of [14C]biotin bound to avidin-Sepharose have shown that a self-absorption correction as high as 11% is necessary. 9.3.7 Determination of immobilized diaminodipropylamine by ninhydrin colorimetry (Holleman and Weis)

Aminoalkylagarose (0.1 ml) is diluted to 2 ml with water and 5 ml of ninhydrin solution are added (4 mg of ninhydrin are dissolved in 150 ml of methyl Cellosolve and 50 ml of 4 N acetate buffer of pH 5.5 and 80 mg of tin dichloride are added under a constant stream of nitrogen). The mixture is heated on a boiling water-bath for 20 min and, after dilution with 7 volumes of 50% ethanol, agarose is eliminated by centrifugation at 2000 g for 10 min. The absorbance of the supernatant is read at 750 nm against a blank consisting of a solution prepared from non-derivatized agarose treated in an identical manner. When reading the values from the standard graph obtained with the use of 3,3’-diaminodipropylamine, a correction must be made taking into consideration that 3,3-diaminodipropylamine carries two primary amino groups when free but probably a single amino group when coupled to agarose.

9.3.8 Determination of immobilized proteins on the basis of tryptophan content (Eskamani et al.) Eskamani et al. adapted a method for the determination of tryptophan, based on reaction with ninhydrin in an acid (Gaitonde and Dovey), to the determination of proteins bound to solid supports. To 1 ml of water or dilute buffer (0.02 M) containing the free or the immobilized enzyme they added in each instance 1 ml of ninhydrin reagent (250 mg of ninhydrin in a mixture of 6 ml of 90% formic acid and 4 ml of concentrated hydrochloric acid). After mixing, the capped test-tubes were incubated in a water-bath at 100°C for 20 min and, after cooling, 1-ml aliquots were diluted with 9 ml of 95%ethanol. The absorbance of the solution obtained was read at 400 nm against a blank prepared from the same amount of solid support without the enzyme. As collagen does not contain tryptophan, this method could be used for the determination of the content of proteins covalently bound to collagen. Therefore, the authors determined the calibration graph for lactase and glucoamylase in the presence of collagen (Fig. 9.2). Another colorimetric method for the determination of proteins is that according to Lowry et al. Havekes et al. employed this method for the determination of glutamate

216

CHARACTERIZATION OF SUPPORTS AND LIGANDS

mg T R Y P T O P H A N - C O N T A I N I N G PROTEIN ADDED

Fig. 9.2. Determination of tryptophan-containing protein in the presence of collagen. A standard solution of lactase was prepared by dissolving 100 mg of tyophilized 0-galactosidase in 10 ml of 0.02 M phosphate buffer, pH 7.0. A standard soiution of glucoamylase was similarly prepared in 0.02 M acetate buffer, pH 4.0. Aliquots were diluted to 1.0 ml with the same buffer, and 21.0 mg of collagen membrane were added to each. A 1-mlvolume of ninhydrin reagent was added to these tubes and to a blank with collagen, and solutions were incubated 20 min in a 100°C bath. After cooling, a 1-ml aliquot was diluted with 9 ml of 95%ethanol, and the absorbance of the samples was determined at 400 nm, using the blank with collagen as a reference. 0, Lactase; 0,glucoamylase. The lines were determined by linear least-squares analysis of the data points (omitting the two highest levels of glucoamylase). Reproduced with permission from E. Eskamani er al., Anal. Biochern., 57 (1974) 421-428.

dehydrogenase bound to Sepharose, with the modification that the reaction mixture was stirred for 20 rnin and filtered before the measurement of absorbance.

9.4 ACTIVE-SITE TITRATION OF IMMOBILIZED PROTEASES

After the enzymes are bound to solid supports, their properties can be changed either under the effect of the micro-environment or under the effect of the changes caused by the chemical modification due to the new covalent bond. Therefore, it is very useful to characterize the enzyme in addition to the determination of the total enzymatic activity

IMMOBILIZED PROTEASES

217

and the determination of the amount of the bound protein also by the titration of the active site. The titration of the active site of trypsin, elaborated by Chase and Shaw, makes use of the specific stoichiometric reaction of trypsin with the substrate-like reagent p-nitrophenyl p ‘-guanidinobenzoate (NPGB). The reaction affords a relatively stable acylated enzyme with the simultaneous production of a “burst” of p-nitrophenol. The amount of the spectrophotometrically determined p-nitrophenol in the “burst” is proportional to the amount of the active enzyme. For the determination of active trypsin after its covalent binding to a support, this method was used by Fritz et ul. and Knights and Light. Fig. 9.3 shows the scheme of the re-circulation titration system described by Ford et al. for the titration of the active site of trypsin covalently bound to glass. The volume is adjusted to about 5-6 ml by employing a small (cu. 1 ml) reservoir. The reactor, removed from the system, is empty except for a sample of cu. 50 mg of thoroughly washed immobilized enzyme. The remainder of the system is filled with 0.03 mM NPGB solution (freshly prepared by diluting 3 mM IWGB in dimethyl sulphoxide with 50 mMveronal buffer, pH 8.3). The contents are re-circulated at the rate of 10 ml/min. The spectrophotometer is zeroed at 410 nm with a range of 0.1 absorbance unit and the slow rate of pre-“burst” hydrolysis is recorded. The active site titration is initiated by stopping the pump, inserting the reactor containing the enzyme into the system, starting the pump slowly, inverting the reactor for a short period to purge it of air and finally increasing the re-circulation pump speed to a flow-rate of 10 ml/min. In Fig. 9.4 the increased absorption due to the “burst” and the following slow post-“burst” hydrolysis traced on the strip recorder is shown. The solution contains a large excess of IWGB over its stoichiometric requirement; thus, the initial oscillatory

IMMOBILIZED ENZYME

RESERVOIR SPECTROPHOTOMETER FLOW .THROUGH CUVETTE

Fig. 9.3. Re-circulation titration system. Reproduced with permission from J.R. Ford er uf., Biochim. Biophys. Acfa, 309 (1973) 175-180.

218

CHARACTERIZATION OF SUPPORTS AND LIGANDS

T I M E .min Fig. 9.4. Active-site titration recorder trace: 62.1 mg of 35-pm particle diameter glass-bound trypsin titrated with 5.1 ml of 0.03 mMp-nitropheny1p’-guanidinobenzoate in 50 M v e r o n a l buffer, pH 8.3. A, pump off; B. reactor plugged into system; C, pump restarted; “burst”, 0.0265 absorbance unit. Reproduced with permission from J.R. Ford ef al., Biochirn. Biophys. Acta, 309 (1973) 175-180.

behaviour observed is due to the production of a small “plug” ofp-nitrophenol as a result of the rapid reaction of an approximately stoichiometric volume of titrant solution with the enzyme as it passes through the reactor. The oscillations are quickly damped out as this plug disperses and the p-nitrophenol concentration throughout the re-circulation system becomes constant. The magnitude of the “burst” is obtained by simple extrapolation of pre- and post-“burst” hydrolysis rates to the point of the initial enzyme-titrant contact. The fluid is pumped out of the system and its volume is accurately measured. The immobilized enzyme is carefully removed from the reactor, dried at 90°C and weighed. The moles of active trypsin may be calculated from the following equation: pmoles enzyme/g glass = lo6A A v / E1~o nm wb

(9.1)

where AA = “burst” absorbance; v = total fluid volume (ml); €410 nm = extinction coefficient ofp-nitrophenoI(l.66- lo4 I/mole/cm); w = weight of glass (mg); and b = cuvette path length (cm). From Fig. 9.4 the “burst” is 0.0265 absorbance unit, v is 5.1 ml and a value of 0.13 pmole of active trypsin per gram of glass was calculated. Increasing the concentration of NPGB five-fold did not affect the size of the “burst”. Elimination of dimethyl sulphoxide also did not affect the results. The titration of the active site of chymotrypsin covalently bound t o Sephadex G-200 has been described by Gabel, who used 4-methylumbelliferyl p-(N,N,N-trimethylammonium)cinnamate (MUTMAC) as the titration reagent. The use of this fluorigenic, active-

CONFORMATIONAL CHANGES OF IMMOBILIZED PROTEINS

219

site-titrating reagent permits the determination of even 0.02 nmole of enzyme. MUTMAC (100 pl of a 0.2 mM aqueous solution) is added to 2 ml of 0.1 M borate buffer of pH 7.5, placed in a fluorescence cuvette and the baseline is recorded. A suspension (50-200 pl) of well stirred Sephadex with bound chymotrypsin is then added to the cuvette with stirring and the contents are stirred for 30 sec. The fluorescence of the liberated 4-methylumbelliferone is recorded, a constant value being obtdned after 1 min. On the basis of this value, the concentration of the active site is calculated. The calculated concentration of the active enzyme is independent of the amount of the suspension and of the added MUTMAC, and is constant after stirring for 15 sec-1 min. With proteases that contain one titratable -SH group, which is essential for activity, for example Streptucuccus protease, it is possible to use 5,5 '-dithiobis-(2-nitrobenzoicacid) for the titration of the active site, as has already been described in Section 9.2.1 1.

9.5 STUDY OF CONFORMATIONALCHANGES OF IMMOBILIZED PROTEINS

The changes in the behaviour of affinity ligands after their binding to a solid carrier are mostly attributed to the modification by the binding, to the effect of the newly formed micro-environment, limitation of diffusion, etc. Less often a change in conformation is assumed, in spite of the fact that, in comparison with native enzymes, the changes in the enzyme stability towards thermal inactivation or denaturation agents show clear changes in conformation or in the ease of submitting to these conformational changes as a consequence of the binding of the enzyme to the carrier. Among the various physico-chemical methods elaborated for the study of the conformation of proteins in solution, the fluorescence technique seems to be most easily adapted for conformational studies of immobilized proteins. Both the intensity and the spectrum of the emitted fluorescent light depend on the environment of the fluorescent groups, and changes in the environment are reflected in corresponding changes in the intensity and in the spectrum of the emitted light. However, strong light scattering due to the binding matrix may cause difficulties in the measurement of the fluorescence. Fortunately, the concentration of the proteins in insolubilized enzymes, and thus also the absorbance of the protein absorption bands, are generally high. Thus the light absorption competes effectively with the light scattering. The excited light is absorbed by a very thin layer on the face of a bed of protein-matrix conjugate and the fluorescence should therefore be collected from the front face of the bed. Further, as the emitted light has a longer wavelength than that of the exciting light, fluorescence light can be readily separated from the scattered light. Gabel et al. described a cell that permitted them to study the conformational changes of the immobilized trypsin and chymotrypsin caused by urea, heat or the presence of specific ligands, using the fluorescence method. As the use of this cell is not particularly suitable, Bare1 and Roosens constructed a very simple cylindrical fluorescence cell, which is shown schematically in Fig. 9.5. The cell consists of a stainless-steeltop end-piece (A) with Teflon tubing and a fitting (B), a Teflon adapter (C), fitting into a cylindrical micro-cell (D), and a stainless-steelbottom end-piece (E), with Teflon tubing and a stainless-steelscrew adapter (F). The micro-cell (quartz, 5.00 mm O.D. and 3.0 mm I.D.) has an inserted tightly fitting porous Teflon disk (G). An appropriate holder for this cell was made of aluminium and fits into the ordinary

220

CHARACTERIZATION OF SUPPORTS AND LIGANDS

A

B

C

D

G

E

F

Fig. 9.5. Diagram of fluorescence cell. Reproduced with permission from A.O. Barel and H. Roosens, Anal. Biochem., 60 (1974) 505-511.

cell compartment of the fluorimeter. It was necessary to remove the existing screw-button fixing the cell compartment to the apparatus; it was replaced with a bored screw in order for the Teflon outlet tubing to pass through. The advantages of using the cylindrical cell (even though the scattering may be greater than for rectangular cells) are that the ends of a cylindrical cell are easy to seal with O-rings for its use as a chromatographic column and with easy cleaning for re-use, and that the amount of material required for the cylindrical system is half that required by a commercially available rectangular cell (50 pl compared with 100 pl). Barel and Roosens used an Aminco Bowman SPF spectrofluorimeter equipped with an RCA R4465 photo-tube, an off-axis ellipsoidal condensing system and a Houston 2000 X-Y recorder. Correction of the emission spectra was found to be unnecessary as the spectral response of the detection system (ix., monochromator plus photomultiplier) remains reasonably constant (k 10%)from 300 to 500 nm.It was necessary to narrow the exciting beam with slits of 1-mm diameter in order to decrease the scattering off the round cell. With the combination of slits used, the excitation and emission bandwidths were 10 nm. All experiments were carried out at room temperature. In order to obtain reproducible fluorescence spectra on insoluble proteins, it was necessary to start from the completely dismantled cell. The micro-cell (D and G ) (see Fig. 9.5) was filled with a maximum of 100 p1 (usually 50 pl is sufficient) of the protein-agarose gel by means of a Pasteur pipette and the gel was immediately layered with 100 p1 of the appropriate eluting buffer. The Teflon adapter (C) was fitted into the micro-cell and the cell was partly assembled (C, D, G , E and F). Finally, the cell was introduced into the cell holder (H) and secured in place with the top end-piece (A). The eluting buffer was then passed downwards through the gel with the aid of a peristaltic pump. A flow-rate varying from 0.2 to 0.5 ml/min was used. All of the spectra were recorded after the gel had been washed for 10-15 min with the buffer. The protein-Sepharose 4B gels were illuminated only during the time necessary to record the emission spectra, in order to minimize photoinactivation. By following this procedure, the protein-gel aliquot could be used repeatedly and it was shown that photoinactivation of the protein sample was negligible. It was possible to record easily the fluorescence spectra of about 0.2-300 pg of protein covalently linked to Sepharose 4B. It is worth noting that the same cell device can be used for classical fluorimetry on protein samples in solution. In this instance the protein solution was introduced upwards into the flow cell. This procedure allowed one, while maintaining the overall cell geometry constant, to compare the fluorescence spectra of proteins in solution and on an insoluble

PROTEINS BOUND TO SOLID SUPPORTS

221

matrix. a-Lactalbumin solutions were usually studied at concentrations ranging from 0.05 to 0.15 mg/ml in 0.05 M buffer at pH 7.5. This procedure was used by Bare1 and Prieels for the study of conformational changes of various cu-Iactamines bound to agarose carriers. Berliner et al. studied conformational changes of trypsin spin-labelled with 1 -0xyl2,2,6,6-tetramethyl-4-piperidinyl methylphosphonofluoridate by means of ESR spectroscopy. Benko et al. studied the effect of binding of methaemoproteins on to latex particles, with respect to the conformation of bound proteins, using longitudinal magnetic relaxation rates of solvent protons.

9.6 STUDIES OF THE DISTRIBUTION OF PROTEINS BOUND TO SOLID SUPPORTS As many biologically active substances in nature exist bound to a membrane or in the form of other native complex structures, well characterized solid supports with bonded biologically active molecules also represent suitable models for their study. For this theoretical reason, but also for practical reasons, it is advantageous to know the spacial distribution of covalently bound biologically active molecules. The method most frequently employed is fluorescence microscopy after labelling with fluorescein isothiocyanate to render the protein molecules visible (Fey and Jost; Lasch et al., 1972;Stage and Mannik). In order to make visible leucine-aminopeptidase bound to Sepharose or Sephadex, Lasch et al. (1972)used two procedures, one of which consisted in binding the fluorochrome fluorescein isothiocyanate covalently to the enzyme before its binding to a solid support, and the other in the labelling of the antibody corresponding to the enzyme with the fluorescein derivative. The labelling of the enzyme with the fluorochrome fluorescein isothiocyanate was carried out by dialysing 2 ml of 13.8pM enzyme solution with vigorous stirring against 257 pM fluorescein isothiocyanate solution at 4°C for about 8 h. Both solutions were made up in 0.1 M sodium hydrogen carbonate solution. The labelling was terminated by the application of the mixture on to a Sephadex G-25 column. The enzyme labelled with fluorescein passed through the column unretarded and nearly undiluted, while fluorescein isothiocyanate was strongly adsorbed on to the gel. It could be partly eluted with 0.1 M sodium hydrogen carbonate solution, using a five-fold volume of the column. The labelling of the leucine-aminopeptidase antiserum took place in the same manner. The binding of fluorescein-labelled enzyme to cyanogen bromide-activated Sepharose or Sephadex took place in the same manner as with non-labelled enzyme. The fixation of the fluoresceinlabelled antibody on to the matrix-bound enzyme was carried out in 0.1 M sodium hydrogen carbonate solution. Undiluted labelled antiserum was mixed with the same volume of a suspension of the enzyme bound to a solid support and then allowed to stand in a thermostat at 37°C for 1 h and at 4°C for a further 12 h. After transferring the material on to a glass filter, non-specifically sorbed components were eliminated by intensive washing with 0.1 M sodium hydrogen carbonate solution. In order to determine the distribution of the protein within the support beads, thin sections of the carrier with the attached enzyme had to be prepared, taking care that the structure of the matrix was not damaged. This can be achieved by embedding the support beads in 20% (w/v) gelatin. The

CHARACTERIZATION OF SUPPORTS AND LIGANDS

222

gel particles were soaked in the gelatin solution for 5 h at 37°C and then poured into a suitable small container. After standing for several hours in a refrigerator, the gelatin blocks were frozen and cut with a microtome into 10-pm sections. These sections were spread on microscope slides and protected from drying by covering them with thin glass covers. Extinction and emission spectra of fluorescein isothiocyanate, fluorescein-labelled aminopeptidase, both free and bound to an agarose or dextran carrier, were recorded with a fluorescence spectrophotometer. In studies where the fluorescein technique was used for detection, a uniform distribution of the proteins in the gel was demonstrated. On the other hand, David etaf., who followed the distribution on the basis of the radiography of 2SI-labelledperoxidase bound to Sepharose, found that the binding of the enzyme did not take place within the Sepharose beads. Lasch er al. (1979, however, found that the differences are not caused by the detection method used, but by a different method of preparation of the immobilized protein. Using electron microscopy of ferritin bound to Sepharose, they demonstrated that the application of a very effective method of binding, i.e., with a large excess of ferritin during the binding (more than 50 mg/ml) and a binding efficiency higher than 90%, brought about the formation of a shell-like distribution of the covalently bound ferritin, whereas when the efficiency of the binding was lower than 50%, a uniform distribution of the bound ferritin was again obtained.

REFERENCES Axdn, R. and Ernback, S., Eur. J. Biochem., 18 (1971) 351-360. Barel, A.O. and Prieels, J.P., Eur. J. Biochem., 50 (1975) 463-473. Barel, A.O. and Roosens, H., Anal. Biochem., 60 (1974) 505-511. Barker, R., Olsen, K.W.,Shaper, J.H. and Hill, R.L.,J. Biol. Chem., 247 (1972) 7135-7147. Barlett, G.R., J. Biol. Chem., 234 (1959) 466-468. Benko, B., Vuk-Pavlovii?, S., DeZeliE, G. and Mari’ciC, S., J. Colloid Interface Sci., 52 (1975) 444-451. Berglund, 0. and Eckstein, F., Eur. J. Biochem., 28 (1972) 492-496. Berliner, L.J., Miller, S.T., Uy, R. and Royer, G.P., Biochim. Biophys. Acra, 315 (1973) 195-199. Borchardt, R.T., Cheng, C. and Thakker, D.R., Biochem. Biophys. Rex Commun., 63 (1975) 69-77. Brenna, O., Pace, M. and Pietta, P.G., Anal. Chem., 47 (1975) 329-331. Chase, T., Jr., and Shaw, E., Biochem Biophys. Res. CoMmun., 29 (1967) 508-514. Collier, R. and Kohlhaw, G., Anal. Biochem., 42 (1971) 48-53. Craven, D.B., Harvey, M.J., Lowe, C.R. and Dean, P.D.G., Eur. J. Biochem., 41 (1974) 329-333. Cuatrecasas, P., J. Biot. Chem., 245 (1970) 3059-3065. David, G.S., Chino, T.H. and Resifeld, R.A., FEBSLett., 43 (1974) 264-266. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F., Anal. Chem., 28 (1956) 350-356. Eskamani, A., Chase, T., Jr., Freudenberger, J. and Gilbert, S.G., Anal. Biochem., 57 (1974) 421-428. Esko, K., Karlsson, S. and Porath, J., Acta Chem. Scand., 22 (1968) 3342-3344. Failla, D. and Santi, D.V., Anal. Biochem., 52 (1973) 363-368. Falkbring, S.O., Gothe, P.O., Nyman, P.O.,Sundberg, L. and Porath, J., FEBSLett., 24 (1972) 229235.

Felix, A.M. and Jimenez, M.H., Anal. Biochem., 52 (1973) 377-381. Fey, H. and Jost, R., ExperientL, 29 (1973) 1314-1316. Ford, J.R., Chambers, R.P. and Cohen, W., Biochim. Biophys. Acta, 309 (1973) 175-180. Fritz, H., Gebhardt, M., Meister, R., Illchmann, K. and Hochstrasser, K., Hoppe Seyler’s Z . Physiol. CheM., 351 (1970) 571-574. Gabel, D., FEBS Lett., 49 (1974) 280-281.

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Gabel, D., Steinberg, I.Z.and Katchalski, E., Biochemistry, 10 (1971) 4661-4668. Gaitonde, M.K. and Dovey, T.,Biochem. J., 117 (1970) 907-911. Goldstein, L., Biochim. Biophys. Acta, 315 (1973) 1-17. Grassetti, D.R. and Murray, J.F., Jr., Arch. Biochem. Biophys., 119 (1967) 41-49. Green, N.M. and Toms, E.J., Biochem. J., 133 (1973) 687-700. Havekes, L., Buckmann, F. and Visser, J., Biochim. Biophys. Actu, 334 (1974) 272-286. Hayashi, H., J., Biochem., Tokyo, 74 (1973) 203-208. Hipwell, M.C., Harvey, M.J. and Dean, P.D.G., FEBSLett., 42 (1974) 355-359. Hixson, H.F., Jr., and Nishikawa, A.H., Arch. Biochem. Biophys., 154 (1973) 501-509. Holleman, W.H. and Weiss, L.J., J. Biol. Chem.,251 (1976) 1663-1669. HoiejBi, V. and Kocourek, J., Biochim. Biophys. Acta, 297 (1973) 346-351. Inman, J.K., Methods Enzymol., 34 (1974) 30-58. Inman, J.K. and Dintzis, H.M., Biochemistry, 8 (1969) 4074-4082. King, E.G., Biochem. J., 26 (1932) 292-297. Knights, R.J. and Light, A., Arch. Biochem. Biophys., 160 (1974) 377-386. Koelsch, R., Lasch, J., Marquardt, I. and Hanson, H., Anal. Biochem., 66 (1975) 556-567. Kornbluth, R.A., Ostro, M.J., Rittmann, L.S. and Fondy, T.P., FEBSLett., 39 (1974) 190-194. L%s, T.,J. Chromatogr., 111 (1975) 373-387. Lamed, R., Levin, Y. and Wdchek, M., Biochim Biophys. Acta, 304 (1973) 231-235. Lasch, J., Iwig, M. and Hanson, H., Eur. J. Biochem., 27 (1972) 431-435. Lasch, J., Iwig, M., Koelsch, R., David, H. and Marx, I., Eur. J. Biochem., 60 (1975) 163-167. Lowe, C.R. and Dean, P.D.G., Affinity Chromatography, Wiley, New York, London, 1974, pp. 272. Lowe, C.R., Harvey, M.J., Craven, D.B. and Dean, P.D.G., Biochem. J., 133 (1973)499-506. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.,J. Biol. Chem.,193 (1951) 265-275. Matheka, H.D. and Mussgay, M., Arch. Gesamte Virusforsch., 27 (1969) 13-22. Meun, D.H.C. and Smith, K.C., Anal. Biochem., 26 (1968) 364-368. Moudgil, V.K. and Toft, D.O.,Proc. Nat. Acad. Sci. US.,72 (1975) 901-905. Nishikawa, A.H. and Bailon, P., Anal. Biochem., 64 (1975) 268-275. Ottesen, M. and Svensson, B., C.R. Trav. Lab. Corkberg, 38 (1971) 445-456. Porath, J., Janson, J.-C. and L%s, T.,J. Chromatogr., 60 (1971) 167-177. Porath, J., Lgs, T. and Janson, J.-C., J. Chromatogr., 103 (1975) 49-62. Rosengren, J., Palman, S., Glad, M. and Hjertgn, S., Biochim. Biophys. Acta, 412 (1975) 51-61. Schabort, J.C., J. Chromatogr., 73 (1972) 253-256. Spackman, D.H., Stein, W.H. and Moore, S., Anal. Chem., 30 (1958) 1190-1206. Stage, D.E. and Mannik, M., Biochim. Biophys. Acta, 343 (1974) 382-391. Stepanov, V.M., Lavrenova, G.I., Borovikova, V.P. and Balandina, G.N., J. Chromatogr., 104 (1975) 373-377. Sternbach, H., Engelhardt, R. and Lezius, A.G., Eur. J. Biochem., 60 (1975) 51-55. Sundberg, L. and Porath, I., J. Chromatogr., 90 (1974) 87-98. Svenson, A. and Carlsson, J., Biochim. Biophys. Acta, 400 (1975) 433-438. Svensson, B., Biochim. Biophys. Acta, 429 (1976) 954-963. Trayer, I.P., Trayer, H.R., Small, D.A.P. and Bottomley, R.C.,Biochem. J., 139 (1974) 609-623. Turkovd, J., Methods Enzymol., 44 (1977) 66-83.

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Chapter 10

General considerations on sorption, elution and non-specific binding This chapter reviews briefly the factors that determine the practical course of affinity chromatography. As most of these factors have already been discussed in detail in preceding chapters, the main aim here is to review their mutual relationships.

10.1 SORPTION CONDITIONS

Assuming that an affinity ligand has been attached to a solid support of sufficiently high porosity and at a suitable distance from its surface (see Section 5.1), which has retained its high affinity for the substance isolated (see Section 5.2) even after attachment, a study of optimal conditions for sorption can be undertaken. The capacity of the specific sorbent is given by the concentration of accessible affinity ligands and by their constants of dissociation from the complex with the isolated substance (see Fig. 3.3). In Section 5.3, the requirement of optimal concentration of the immobilized affinant was discussed, which would ensure a sufficient capacity but simultaneously permit the liberation of the isolated substance from the specific complex with immobilized affinant under sufficiently mild conditions, without, however, causing non-specific sorption. The quality of the spacer inserted between the affinity ligand and the surface of the solid matrix (Caron et al.) may also have a similar effect as the concentration. The specific sorbent should be prepared in such a way that all of the groups capable of attachment are blocked (see Section 8.4)and that the number of groups capable of participating in non-specific sorption is minimal (see Section 10.3). However, it is most important that all of the molecules of the affinant that are not coupled on the carrier by a covalent bond should be thoroughly washed out (see Section 8.6). As ionic bonds or hydrogen bonds, hydrophobic interactions and Van der WaalsLondon forces may participate to various extents in the binding of the complementary sites of the affinity ligand and of the isolated substances, the optimal conditions for sorption and desorption will vary in particular instances. In general, the starting conditions for sorption should be selected so that they cause maximal sorption of the isolated substance. The choice of the starting buffer is completely dependent on the optimal conditions of complex formation between the affnant and the isolated substances and, in addition to temperature, pH and ionic strength, it also depends on the content of metal ions and other specific factors. 10.1.1 Effect of temperature, pH and salts

The effect of temperature in affinity chromatography was discussed in Section 5.5. Fig. 10.1 gives a practical example, showing how the amount of a mixture of nucleases sorbed on Sepharose 4B with linked 3 '44 '-aminophenylphosphory1)deoxythymidine5 '-phosphate decreases with increasing temperature (Andria et al.),

SORPTION, ELUTlON AND NON-SPECIFIC BINDING

226

The practical utilization of pH during the sorption of neutral protease from Bacillus subfilis on Sepharose with linked glycyl-D-phenylalanine(through a spacer 23 atoms long, formed by combination of triethylenetetramine, succinic acid and ethylenediamine) is shown in Fig. 10.2. Maximal adsorption of neutral protease occurs at pH values corresponding to minimal values of K m . Between pH 5 and 6.5 neutral protease is effectively adsorbed and thus separated both from subtilisin and other proteins present in the culture filtrate. At higher pH values, an effective separation of neutral protease from subtilisin does not take place. Neutral protease is set free from the complex with the immobilized affinant by increasing the pH to values at which the binding of substrate is already weak, but at which the enzyme is still not denatured. The optimal pH for the elution of neutral protease depends not only on its affinity towards the affinity ligand and on the pH dependence of this affinity, but also on the concentration of the immobilized ligand (Walsh ef al.). Neutral protease is a metalloenzyme and is therefore inhibited by 1,lo-phenanthroline and EDTA. The presence of these chelating agents at a concentration of 1-5 mM prevents the adsorption of the enzyme on to the specific sorbent mentioned. The effect of pH on affinity chromatography was discussed in Section 5.6. It should be borne in mind that the optimal pH for the sorption of a particular enzyme

ELUTION

V O L U M E ,ml

Fig. 10.1. Affiity chromatography of a mixture containing nucleaseT-(6-48), nuclease-(49,50-126), and nuclease(99-149) in the presence of Ca” at various temperatures on Sepharose 4B substituted with 3 ‘44 ‘-aminopheny1phosphoryl)deoxythymidine5 ’-phosphate (Sepharose-pdTp). The mixture containing 0.085 pmole of nuclease-T-(6-48), 0.060 pmole of nuclease-(49;50-126), 0.067 pmole of nuclease-(99-149) and 0.01 M calcium chloride in 0.2 ml of 0.1 M ammonium acetate solution, pH 8 , was equilibrated at a given temperature and applied to a jacketed Sepharose-pdTp column (40 X 10 mm). The elution was carried out with 0.1 M ammonium acetate-0.01 M calcium chloride solution, pH 8. The arrows indicate the points where-the elution with 0.1 N acetic acid was started. The relative intensity of tryptophan fluorescence of fractions was determined in order to monitor nuclease(99-149) with a single tryptophan residue. Reproduced with permission from G. Andria et al., J. Biol. Chern., 246 (1971) 7421-7428.

227

SORPTION CONDITIONS I00 0.

pH 1.5

5 0

0;

Ic

3

'8\

'.

pH 1.0

E

3

1 I II

a

-1

U

2

(v

I

W

0

z

a

I W

0

W

m

t

51m

pH 6.5

a

> k

1

La W

1 Ia

-1 W

a

C FRACTION NUMBER

Fig. 10.2. Effect of pH on the adsorption of neutral protease on Sepharose 4B substituted with triethylenetetramine, succinic anhydride, triethylenetetramine and chloroacetyl-D-phenylalanine. Crude enzyme (10 mg) was dissolved in 0.1 ml of equilibrating buffer and applied to an affmity column (220 X 6 mm) equilibrated with 100 mM sodium chloride, 10 mM calcium chloride, containing 5 mM Tris (pH 7.5 or 7.0) or 5 mM 2-(N-morpholino)ethanesulphonicacid (pH 6.5 or 6.0). After elution for 1 h at 25 ml/h, each column was washed with 100 mMsodium chloride, 10 mM calcium chloride, 50 mM Tris (pH 9.0), as indicated by the arrows. Neutral protease and subtilisin are identified by catalytic activities toward 3-(2-furylacryloyl)lycyl-L-leucinamide (FAGLA) and acetal-N-tyrosine ethyl ester (ATEE). Reproduced with permission from K.A. Walsh et al., Methods Enzymol., 34 (1974) 435-440.

SORPTION, ELUTION AND NON-SPECIFIC BINDING

228

need not be identical with the optimal pH for catalysis. Kasai and Ishii found pH 5.6 to be optimal for the sorption of trypsin on to Sepharose with coupled glycylglycylarginine, which is not the same as that for catalysis. Therefore, it is useful to determine optimal conditions for the formation of each individual complex of the isolated substance with the immobilized affinant. These values can be obtained most easily by using a batchwise arrangement, described in connection with Fig. 3.1, in which the sorption of chymotrypsin as a function of pH and ionic on carbobenzoxyglycyl-D-phenylalanine-NH2-Spheron strength is shown (Turkovi et al., 1976). It is evident from Fig. 3.1 that with increasing ionic strength of the buffer the amount of chymotrypsin sorbed decreases. In contrast, active papain is not bound to Sepharose with attached glycylglycyl-0-benzyltyrosylarginine at low concentration of salts (Blumberg et uf.). In Fig. 10.3, the retardation of

O.OSM PHOS,CIT

2

0.8

1

0.4

5

20

9

;

4 0.25 M

PHOS.CIT

4

1.6

5 C .-

3

1.2

E

-al

W

0.8

2

fa

>' I-

0.4

1

-5t-

2w 1.2

3

W

2

0.8

1

0.4

60

& z

0 . 5 0 M PHOS.CIT

20

I

100

ELUTION V O L U M E . rnl

Fig. 10.3. Increased retardation of L-histidinol phosphate aminotransferaseupon increasing the concentration of a phosphate-citrate buffer. Buffers used contained Na,HF'O, (at the indicated concentration) and were adjusted to pH 6.0 with citric acid. Reproduced with permission from S. Shaltiel et al., Biochemistry, 1 3 (1974) 4330-4335.

SORPTION CONDITIONS

229

L-histidinol phosphate aminotransferase on L-histidinol phosphate-coated agarose is shown, which increases with increasing concentration of the phosphate-citrate buffer (Shaltiel et al.). This increase in retardation cannot be attributed in this instance, however, to the increase in the ionic strength, because the position of the enzyme peak does not change if the ionic strength of the 0.05 M phosphate buffer is increased by addition of 0.9 M sodium chloride solution. A retardation of the enzyme then takes place not only in the phosphatecitrate medium, but also in a sulphate or phosphate buffer; however, this retardation takes place, of course, to various extents. In the series of carboxylic acids used for the adjustment of the pH of 0.5 M disodium hydrogen orthophosphate solution to 6.0, it was found that the retardation of the enzyme increases with increasing valency in the order acetate < succinate < citrate. Raibaud et al. mentioned that, in addition to the length of the aliphatic chain, the concentration and the nature of the salts present in the medium are the two most important factors that affecting the adsorption of proteins on alkyl-Sepharose. The important role of salts in hydrophobic chromatography is discussed in detail in Section 7.1. The effect of individual ions on the hydrophobic interaction can be expressed by the following scheme (from Pharmacia promotional literature): -z----

Increasing salting-out effect

-

Anions:

PO,3-, SO4'-, CH, COO-, C1-, Br-, NO;, ClO,, I-, SCN-

Cations:

NH:,

Rb', K ' , Na', Cs', Li', Mg2+,Ca", Ba2+ Increasing chaotropic effect ---+

At constant ionic strength, the effect of ions on adsorption is strongly dependent on the nature of the ions, some of them causing a decrease and others an increase, which is attributed to lyotropic rather than purely electrostatic effects. The effect of anions classified in the order of decreasing adsorption, thiocyanate = iodide > chloride > acetate > citrate, is identical with Hofmeister's series of neutral salts (Green). If the high ionic strength of the starting buffer does not impair the formation of the affinity complex, it is advantageous to use it because it decreases the non-specific adsorption of polyelectrolytes on the possibly occurring charged groups of the coupled affinant. Therefore, it is recommended to add a ca. 0.5 M sodium chloride solution to the sorption buffer. The effect of ionic strength on the binding of @-galactosidaseon Sepharose with is shown in Table 10.1. With increasing attached p-aminophenyl-0-D-thiogalactopyranoside ionic strength, the amount of the sorbed enzyme decreases, but its specific activity, on the contrary, increases (Robinson et al.). The necessity to add metal ions or other specific factors to the sorption medium is illustrated in Table 10.2. The binding of mitochondria1 ATPase (dispersed with Triton X-100)on Sepharose with covalently bound inhibitor depends on the presence of magnesium ions and ATP (Swanljung and Frigeri). To obtain a high degree of purification of Triton-solubilized brain acetylcholinesterase, the affinity chromatography must be carried out in the presence of Triton (Dawson and Crone). Zanetta and Gombos carried out the affinity chromatography of membrane glycoproteins on concanavalin A-Sepharose in the presence of sodium dodecyl sulphate. For the retention of dihydropteroate synthetase on sulphonamide-Sepharose,the presence of dihydropteroate pyrophosphate and dithiothreitol is essential (Suckling et al.).

230

SORPTION, ELUTION AND NON-SPECIFIC BINDING

TABLE 10.1 EFFECT OF IONIC STRENGTH OF BUFFER ON 8-GALACTOSIDASE BINDING ON TO A COLUMN OF AGAROSE SUBSTITUTED WITH p - AMINOPHENY L-fl-D-THIOGALACTOPYRANOSIDE Bed dimension, 114 X 17 mm;flow-rate, 2.0 ml.min-'.cm-*. Ionic strength

Protein bound (mg)

Activity bound (units)

Specific activity (units/mg)

0.010 0.020 0.035 0.050

75 32 14 1.2

6000 5500 3600 350

80 190 320 340

TABLE 10.2 EFFECT OF Mg2+AND ATP ON THE AMOUNT OF ATPase BOUND TO ATPase INHIBITORSEPHAROSE COLUMN Extract containing adenosine 5 '-triphosphatase (ATPase) was applied to ATPase inhibitor-Sepharose column equilibrated with 0.2 M sucrose, 15 mM Tris-N-tris(hydroxymethyl)methyl-2-aminoethane sulphonic acid (Tris-TES) buffer (pH 6.6), 3 mg/ml Triton X-100 and additions as indicated in the table. The amount of enzyme bound was calculated as the amount eluted with 0.2 M sucrose, 45 mM Tris-TES buffer (pH 8.75), 0.5 M KCI, 1 mM EDTA and 0.3 mg/ml Triton X-100. (Swanljung and Frigeri). Additions -~

None 1 mMMgSO, 0.5 mM MgSO, + 0.5 mM ATF'

ATPase applied (nmole/min)

ATPase bound (nmole/min)

Yield (%bound of that applied)

336 192 678

39 35 221

12 18 33

10.1.2 Practice of sorption

For sorption, it is best to dissolve the sample of the substance to be isolated in the starting buffer and, if necessary, to carry out a change in the composition of salts present in the sample by dialysis or gel filtration. If a substance that forms a strong complex with the immobilized affinity ligand under the given conditions is isolated by column chromatography, the volume of the sample introduced on t o the column is irrelevant. However, if substances of low affinity towards the attached affinant are isolated by affinity chromatog raphy, the volume of the sample applied should not exceed 5% of the hold-up volume in order to prevent the elution of the isolated substance with non-adsorbed material. If a substance with a low affinity for the bound affinant is isolated, its elution from the column often occurs even without a change of buffer. In this instance the isolated substance is obtained in dilute form. An example is a comparison of the course of chromatography of chymotrypsin on Sepharose with both coupled e-aminocaproyl-D-tryptophan methyl ester and with directly attached D-tryptophan methyl ester, shown in Fig. 5.4(Cuatrecasas et d.).

SORPTION CONDITIONS

231

For the elution of chymotrypsin from the solid support on which the inhibitor is bound via e-aminocaproic acid as spacer, a change in pH is necessary for displacement, and the chymotrypsin fraction is eluted as a sharp peak. If the inhibitor is coupled directly on a solid support, its steric accessibility is decreased and chymotrypsin is retarded only. The enzyme is eluted in a much larger volume at the same pH, closely after the inactive material. If a small amount of substance is isolated from a crude mixture by means of an immobilized affinity ligand of high affinity, the batchwise arrangement can be employed with advantage, sometimes combined with elution after a transfer of the material into a column. An example is the isolation of thyroxine-binding globulins by means of affinity chromatography on Sepharose with attached thyroxine. In order to obtain a higher yield, Pensky and Marshall carried out the first part of the sorption in a batchwise arrangement by stirring normal human serum with insolubilized affinant overnight. After washing with 0.1 M sodium hydrogen carbonate solution, they transferred the insoluble carrier with the adsorbed isolated substances into a column from which two thyroxine-binding globulin fractions were then eluted with 0.002 M potassium hydroxide solution. This combination of the batchwise and the columnar arrangement is often met in affinity chromatography. The time necessary not only for the contact of substances to be isolated with the attached affinity ligand is thus prolonged, but also the time necessary for a correct orientation of binding sites. In the columnar arrangement, the prolongation of the time of contact of the substance to be isolated with the immobilized affinant is achieved by stopping the flow through the column after sample application for a certain time. Manen and Russell stated that if the flow of solvent through the column during affinity chromatography of Sadenosyl-L-methioninedecarboxylase on Sepharose with attached p-chloromercuribenzoate was not stopped for 2 h, the enzyme was eluted after several fractions. A sufficiently low flow-rate through the column of a specific sorbent is of prime importance in the isolation of high-molecular-weightsubstances, especially if they occur in high concentrations. The flow-rate, the concentration of the applied sample and the equilibration time were discussed in Section 5.4. The concentration of a substance to be isolated in the sorption solution affects the rate of attainment of equilibrium mainly during the batchwise arrangement. Fig. 10.4 shows an orienting experiment carried out by Porath and Sundberg (Porath and Kristiansen, 1974). One-gram portions of soybean trypsin inhibitor-Sepharose were suspended in solutions containing equal amounts of trypsin but with different concentrations, and residual activity of trypsin was then measured at various time intervals. On the basis of the curves obtained, the authors assumed that for a suspension containing 1% of adsorbing gel particles of diameter 50-100 pm in the solution containing the isolated substance in the molecular-weight range 10,000-100,000 daltons a contact time of 20-30 min is necessary if the gel particles are composed of agarose with a matrix density of 6% or less. Proteins with high molecular volumes, or particles larger than lo6 daltons, will require a much longer time for diffusion to the binding sites of the affinity ligand, even when a gel of high permeability is used. For extra-high-molecular-weightsubstances, it is more advantageous to avoid gel permeation and rather to use adsorption on the beads coated with substances that interact specifically with the particles. The batchwise arrangement of affinity chromatography is particularly suitable for large-

SORPTION, ELUTION AND NON-SPECIFIC BINDING

232

MINUTES

Fig. 10.4. Rate of adsorption of trypsin on to soybean trypsin inhibitor-agarose particles in suspensions of different concentrations. Reproduced with permission from J. Porath and T. Kristiansen, in H. Neurath and R.L. Hill (Editors), The Proteins,Academic Press, New York, 3rd ed., 1975, pp. 95178.

scale isolations if the systems involved have a sufficiently high affinity. Often it is appropriate to insert an initial purification step before the specific sorption, in order to dissociate and separate the components of naturally occurring complexes that contain the required substances. This step may consist, for example, in precipitation, extraction or adsorption on an ion exchanger. Often affinity sorption is carried out directly from crude extracts, sera or exsudatLs. Porath and Kristiansen recommended enclosing the gel particles of specific sorbents into containers with semi-permeable walls, permitting the free passage of dissolved substances and the retention of large particles. For example, nylon net bags can be used. By immersing several bags containing various specific sorbents into the medium, several substances can be isolated simultaneously, or else unwanted substances can be eliminated from the solution, such as proteolytic enzymes, which may decrease the yields of the isolated substances.

10.2 CONDITIONS FOR ELUTION

Theoretical guides to the liberation of isolated substances from specific complexes with the immobilized affinant were discussed in Section 3.1. While the substances that have no affinity for the attached affinant are usually eluted with the hold-up volume, the specifically sorbed material mostly requires displacement with a specific elution agent or by a change in temperature or solvent composition.

ELUTION CONDITIONS

233

10.2.1 Practice of desorption

A practical example of biospecific elution with competitive inhibitors is the isolation of chymotrypsin and trypsin from pancreatic extracts by affinity chromatography on agarose with attached soybean trypsin inhibitor (Fig. 1OS), during which chymotrypsin was displaced with a solution of tryptamine and trypsin with a solution of benzamidine (Porath). This elution is an example of stepwise elution. An example of gradient elution with a specific eluent is shown in Fig. 10.6, which illustrates the elution of native lysozyme specifically sorbed on tri-(N-acety1glucosamine)Sepharose by means of variously steep gradients of tri-(N-acetylglucosamine) (Cornelius et d.).The gradient of the specific elution reagent was always applied after washing with the starting buffer. The amount of the native enzyme applied on to the column, the conditions of the columnar arrangement and the composition of the buffer were the same in all three instances. The gradients differed only in the different rate of change of concentration of tri-(N-acetylglucosamine), as is evident from the course of the procedure. The recoveries of the protein were approximately 90%. Lysozyme was eluted with all three gradients at a 5 lo-’ M concentration of tri-(N-acetylglucosamine), which agrees with the association constant for the binding of tri-(N-acety1glucosamine)-lysozyme (Kassoc = 10’ l/mole). From the theoretical point of view, a more advantageous elution with a competitive affinity ligand has, however, numerous practical limitations, mainly owing to its availability and price. For example, lactate dehydrogenases from immobilized N6-(6-aminohexyl)-5 ’AMP-agarose can be eluted with a solution of competitive ligand of NADH of relatively TRVPTAMINE pH 7.8 0.013M

BENZAMIDINE pH 7.8 0.02 M

INACTIVE

1

TRVPSIN

50

60

FRACTION NUMBER

Fig. 10.5. Affinity chromatography of a pancreatic extract on a column of 6%agarose with bound soybean trypsin inhibitor. Stepwise elution was accomplished with specific inhibitors. Reproduced with permission from J. Porath, Biotechnol. Bioeng. Symp., No. 3 (1972) 145-166.

SORPTION, ELUTION AND NON-SPECIFIC BINDING

234

0

200

400

600

ELUTION VOLUME.mi

Fig. 10.6. Effect of varying the tri-(N-acetylglucosamine) [(GlcNAc),] gradient on the elution of native lysozyme from tri-(N-acetylglucosamine>Sepharose.Fifteen milligrams of native protein in 1 ml of starting buffer (0.1 Mammonium acetate, pH 7.0) were applied to a 260 X 17 mm column. The gradients were begun after washing the column with ca. 5 column volumes ( 5 X 35 ml), finishing with 0.1 mM (GlcNAc), in starting buffer. The gradients were formed using (a) 100 ml of starting buffer vs. 100 ml of finishing buffer, (b) 200 ml YS. 200 ml and (c) 300 ml YS. 300 ml. The flow-rate was 23 ml/h. Reproduced with permission from D.A. Cornelius et al., Methods Enzymol., 34 (1974) 639-645.

ELUTION VOLUME, ml

Fig. 10.7. Resolution of an enzyme mixture on N6-(6-aminohexyl)-5‘-AMP-Sepharose by a temperature gradient. The enzyme sample (100 &, containing 5 U of each enzyme and bovine serum albumin (1.5 mg), was applied to a column (50 X 5 mm) containing 0.5 g of N6-(6-arninohexyl)-S’-AMPSepharose (1.5 mnol/ml of AMP) at 4.7”C. The column was equilibrated at each individual temperature for 5 min prior to elution with 1.6 ml of equilibration buffer, 10 mMglycero1-5 mM MgCI,-l mM EDTA-0.02% sodium azide-10 mM tricine-KOH (pH 7.5). A “pulse” (200 gl) of 5 mM NADH in the equilibration buffer was added as indicated by the arrow. Bovine serum albumin, located in the initial column wash (0-4 ml), hexokinase (m), glycerokinase (o), yeast alcohol dehydrogenase (0)and lactate dehydrogenase H, ( 0 ) were assayed in the effluent. A temperature gradient (----)was achieved through the manual adjustment of a Churchill circulating and heating pump. Reproduced with permission from M.J. Harvey et al., Eur. J. Biochem., 41 (1974) 353-357.

ELUTION CONDITIONS

235

high concentration (1.5 mM), or with a potassium chloride solution of medium concentration (0.13 mM). Therefore, the elution of adsorbed substances by a change in temperature, pH or ionic strength is still the most commonly used procedure. The use of a linear temperature gradient for the differentiation of a mixture of yeast alcohol dehydrogenase, glycerokinase, hexokinase and lactate dehydrogenase in affinity chromatography on N6-(6-aminohexyl-5'-AMP-Sepharose (Harvey et al.) is illustrated in Fig. 10.7. It is interesting that glycerokinase and yeast alcohol dehydrogenase were eluted in the order expected on the basis of their apparent energies of adsorption, with a high recovery (70-90%), while lactate dehydrogenase required a pulse of 5 mM NADH for elution, even at 40°C.Hence, weakly bound enzymes can be eluted with advantage by means of temperature gradients. The advantage of this elution is that the eluted enzymes are not contaminated with elution reagents (salts, nucleotide pulse), and they can be utilized directly for kinetic studies. For firmly bound enzymes, it can be used suitably in combination with other techniques, such as a lower concentration of ligands, NADH pulses or a gradient of ionic strength. An example of the use of a pH gradient for the separation of a mixture of nicotinamide nucleotide-dependent dehydrogenases on N6-(6-aminohexyl)-S'-AMP-Sepharose is shown in Fig. 10.8 (Lowe et d.).Elution with a pH gradient again affords the enzyme free of nucleotide, which can then be further used for kinetic studies. An example of the use of a linear gradient of salt for the isolation of RNA polymerase from Escherichia coli on DNA-agarose (Nusslein and Heyden) is shown in Fig. 10.9. From the fraction of RNA polymerase obtained in preceding chromatography on Bio-Gel A, an

ELUTION VOLUME. r n l

Fig. 10.8. Resolution of a mixture of dehydrogenase on N6-(6-aminohexyl)-5'-AMP-Sepharose by a pH gradient. The enzyme mixture (100 PI), containing bovine serum albumin (1.5 mg) plus 5 U of each enzyme, was applied in 10 mM KH,PO,-KOH, pH 6.0 (A = 3.3 m a - ' ) to a column (50 X 5 mm) containing 0.5 g of N6-(6-aminohexyl)-5'-AMP-Sepharose. The column was washed with 10 mM equilibration buffer of pH 6.0,pnor to development with a pH gradient [pH 6-10; 10 ml of equilibration buffer against 10 ml 30 mM K,HPO,-KOH (pH 11.0) of A = 6.6 m a - ' in a linear gradient apparatus]. 0,Bovine serum albumin; 0 , malate dehydrogenase; glucosed-phosphate dehydrogenase; 0,lactate dehydrogenase;A, yeast alcohol dehydrogenase. Reproduced with permission from C.R. Lowe etal., Eur. J. Biochem., 41 (1974) 347-351.

.,

SORPTION, ELUTION AND NON-SPECIFIC BINDING

-1 4

-

f -10' I

-- 2 L

- 06

-02

FRACTION NUMBER

Fig. 10.9. Affinity chromatography of RNA polymerase on DNA-agarose. The column (150 X 10 mm) M was equilibrated and chromatographed with standard buffer solution (0.01 MTris, pH 8.0, EDTA, 10 - 4 M dithioerythriol, 5% glycerol) and 0.25 M potassium chloride solution. Elution of adsorbed protein was carried out with a 600-ml linear gradient of 0.25-1.25 M potassium chloride in standard buffer solution. The fraction of RNA polymerase (95 ml) was applied on to the column. A constant flow-rate during loading and elution was maintained by use of a peristaltic pump. Fractions of 10 ml were collected and assayed for RNA polymerase activity using calf thymus DNA ( 0 ) and T, DNA ( 0 ) as template. The total recovery of the RNA polymerase activity from the column was 80%. Solid line, absorbance; broken line, potassium chloride concentration. Reproduced with permission from C. Niisslein and B. Heyden, Biochem Biophys. Res. Cornmun., 47 (1972) 282-289.

inactive material was separated by affinity chromatography in the first peak, while the linear gradient of 0.25-1.25 M potassium chloride gave two different RNA polymerases. The isolation of papain sorbed on agarose with bonded glycylglycyl-0-benzyl-L-tyrosyl-Darginine is an example of elution with a decreased ionic strength of the elution buffer. Blumberg el al. displaced papain from the specific complex by using water. The specific complex of the isolated substances with the immobilized affinity ligand can also be decomposed after their steric modification, for example with urea, guanidine salts or chaotropic ions. These reagents disrupt the hydrogen bonds or change the structure of water in the proximity of hydrophobic regions. However, when these reagents are employed, it should be borne in mind that the components of the complex might be irreversibly destroyed during liberation. It is known, however, that mainly with immobilized enzymes the attachment of proteins to solid supports mostly brings about an increase in their stability. By choosing a suitable concentration, temperature and exposure time, minimum conformational changes in the adsorption site can be achieved during desorption, and consequently also reversible conformational changes of whole molecules both of the isolated substances and the immobilized affinant. It is practicable to decide the minimal concentration required for elution on the basis of a preliminary experiment in which the concentration and the biological activity are determined. For example, elution with 1.5 and 2 M guanidine hydrochloride solution was used successfully for the isolation of thermolysin and subtilisin after their specific sorption on benzyloxycarbonyl-L-phenyl-

ELUTION CONDITIONS

231

alanyltriethylenetetraminyl-Sepharose. Both enzymes are stable under the given elution conditions, and the application of the mentioned affinity chromatography gave preparations of high specific activity (Fujiwara et d.).Guanidine hydrochloride solution was used successfully as an eluting agent by Gospodarowicz, who even used a 6 M solution of pH 1.5 for the elution of the luteinizing hormone from an agarose column with a bonded fraction of anti-luteinizing hormone immunoglobulin. Immediately after elution, the pH of the hormone solution was adjusted to 7.3 and dialysis was carried out against a 0.2 M ammonium hydrogen carbonate or 0.01 M phosphate buffer of pH 7.3, containing 0.9% of sodium chloride. Hill achieved elution of antibodies from immunoadsorbents by means of dioxan in admixture with weak organic acids. The use of crosslinked agarose for the preparation of immobilized blood group substance A enabled Kristiansen to use chaotropic ions, namely thiocyanate, trichloroacetate and trifluoroacetate, for elution. The efficiency of chaotropic ions for desorption from immunosorbents has also been demonstrated by Bennich and Johansson. In connection with the elution of antibodies, the finding of Murphy et aZ., concerning the increase in affinity of antibodies during immunization, is interesting. For example, antibodies against glucagon could, at the beginning, be eluted effectively from the immunosorbent with 4.25 Methanol in 4 mMhydrochloric acid, but if they were prepared from the serum of the same rabbit 1 year later, 0.1 M acetic acid, adjusted to pH 2.2 with formic acid, had to be used. The use of solutions of salts for the elution of the isolated substances from sorbents used in hydrophobic chromatography was discussed in Section 7.1. Jennissen and Heilmeyer demonstrated that the elution strength of the anions corresponded directly to the order of Hofmeister’s series, and that of the cations corresponded to the reverse order with respect to the salting-out or -in effect. The salts can penetrate directly into the interphase between the protein and the hydrophobic surface of the sorbent, which leads to desorption. If ionic interactions were responsible for adsorption, then the elution of proteins would depend on the ionic strength and not on the nature of the types of salts used. General aspects of hydrophobic chromatography, including the reagents used for elution, were studied by Hjerten. If the affinant is coupled to the matrix by an azo bond or by thiol- or alcohol-ester bonds, the complex of the affinant with the isolated substance can be detached from the solid matrix and then the affinity ligand separated by dialysis or gel filtration. This, however, prevents the repeated use of the affinity matrix. Carriers of this type were discussed in detail in the section on covalent chromatography (Section 7.2). 10.2.2 Effect of the heterogeneity of the immobilized affmnts When the sorbed substance is released from the specific sorbent, the effect of the heterogeneity of the immobilized affinity ligand should be borne in mind (Amneus et d.). Fig. 10.10 shows the separation patterns of chymotrypsins and trypsins from mouse pancreatic homogenates on various preparations of Sepharose with coupled soybean trypsin inhibitors (STI). Assuming that the difference in elution conditions reflects differences in biological activities, the former can be used for the characterization of the molecule. The applicability of the adsorbent then depends on the functional homogeneity of the immobilized affinity ligand.

SORPTION, ELUTION AND NON-SPECIFIC BINDING

238

A

- B

E

8 N

W

0

z

a m

oc

sm a

2.10’ I ELUTION VOLUME .mi

Fig. 10.10. Separation patterns of mouse chymotrypsin on Sepharose 4B substituted with different preparations of soybean trypsin inhibitor [ STl; STI OK) = inhibitor obtained from Worthmgton Biochemical Corp., STI ( S ) = inhibitor obtained from Sigma]. -, Absorbance or pH; 0 , chymotryptic activity; 0,tryptic activity. (A) Unmodified STI 0-Sepharose, coupled at pH 7.2; (B) Chymotrypsinmodified STI 0-Sepharose, coupled at pH 7.2; (C) chymotrypsin-modified STI 0-Sepharose, coupled at pH 8.5; (D)chymotrypsin-modified STI (S)-Sepharose, coupled at pH 7.2. Reproduced with permission from H. Amneus et al., J. Chromrogr., 120 (1976) 391 -397.

The heterogeneity of an adsorbent in terms of association constants can be caused by (1) the heterogeneity of the biospecific ligand used for the preparation of sorbent, (2)

various changes of the ligand under the effect of immobilization and (3) various modifications of the affinity ligand caused by the molecules present in the fractionated mixture. ( 1 ) Heterogeneity of the ligand before immobilization

High-molecular-weightaffinity ligands of biological origin, such as proteins, nucleic

ELUTION CONDITIONS

239

acids and carbohydrates, used for the preparation of specific sorbent, may be genetically heterogeneous and such heterogeneity can be found in both commercial and non-commercia1 ligand preparations. It is evident that the presence of impurities in immobilized affinants with a similar or stronger affinity towards the isolated molecules may impair the use of a specific sorbent in gradient separations.

(2)Alteration of the affinity ligand by immobilization The effective activity of the ligand can be changed in various ways under the effect of immobilization. The micro-environment formed by the matrix (charge density, steric hindrance, etc.) may affect the interaction of the immobilized ligand with the isolated molecules to a considerable extent and in various ways, and it can also influence the structure of the affinity ligand itself. Immobilization also brings about changes in chemical structure, which variously changes the molecular properties of the affinity ligand as a consequence. Here, for example, the number of bonds between the affinity ligand and the solid support plays a considerable role. From a comparison of Figs. 10.10B and IO.lOC, the unfavourable effect of an increased number of bonds between the molecules of STI and Sepharose 4B, caused by the increase in pH from 7.2 to 8.5 during the binding after cyanogen bromide activation, is evident.

(3)Modification of the affinity ligand after immobilization The fractionated material can contain components that modify the activity of the attached affinity ligand. These components may be similar to the compounds being isolated, although they need not be. The enzymes, for example proteases and nucleases, present in crude extracts may cleave the coupled affinants (proteins, nucleic acids) and thus reduce the capacity of the adsorbent for the binding of the specific complementary compound. In addition to this non-specific degradation of the affinity ligand, the enzymes and other chemicals present in the fractionated mixture can modify the properties of the coupled affinant specifically with the formation of forms with retained but changed activities. In Fig. 10.10, all three of the above-mentioned effects can be followed. Soybean trypsin inhibitor (Kunitz) obtained from both Worthington Biochemical Corp. [STI (W)] and Sigma [STI ( S ) ] was bound on Sepharose activated with cyanogen bromide. The attached STI was further modified with a solution of chymotrypsin: the adsorbent column was washed with a solution of chymotrypsin at pH 8 and a flow-rate of 10 ml/h for 24 h and, after incubation, chymotrypsin was eluted with a buffer of pH 2.5. From Fig. 10.10A, it is evident that when the unmodified STI (W)-Sepharose attached at pH 7.2 was used, only poor resolution between the two peaks with chymotryptic activity and the peak with tryptic activity occurred. Two further peaks with tryptic activity were eluted still later, at much lower pH values. After a modification of STI (W)-Sepharose with a solution of chymotrypsin, elution of the tryptic activity simultaneously with the chymotryptic activity no longer took place. At the same time, good resolution of the two peaks with chymotryptic activities was obtained. If STI (W)-Sepharose, prepared by coupling at pH 8.5 and modified with a solution of chymotrypsin, was used for separation, the resolution of the

SORPTION, ELUTION AND NON-SPECIFIC BINDING

240

two peaks with chymotryptic activities deteriorated considerably, in the same manner as when modified STI (S)-Sepharose coupled at pH 7.2 was used (Fig. 1O.lOD). If various amounts of activated pancreatic homogenate were fractionated on modified STI (W)-Sepharose, attached at pH 7.2, then with increasing load of the adsorbent the two peaks with chymotryptic activities were eluted at higher pH values (see Table 10.3) and they were less well resolved. The gel had a capacity of 9 mg of chymotrypsin per millilitre. After the separation of a total of nine aliquots from pancreatic homogenates, this capacity decreased to 0.2 mglml.

TABLE 10.3 ELUTION CONDITIONS FOR MOUSE CHYMOTRYPSIN (CHT-I AND CHT-11) OF CHYMOTRYPSIN-MODIFIED __ STI(W)-SEPHAROSE, COUPLED AT pH 7.2 Number of CHT activity pancreases not retained applied -~_______

pH of solution CHT-I

CHT-I1

0.5 1 2 4~

4.75 4.90 5.05 >5.10

4.30 4.35 4.50 4.80

0 0

3 _

_

57._._

10.2.3 Establishment of optimal conditions and saturation effect

For finding optimal conditions, Porath and Kristiansen recommended preliminary experiments for the determination of the bed capacity. This determination can best be carried out by frontal chromatography on a column bed with a total volume of a few millilitres. The UV absorption of the effluent is measured at a suitable wavelength and small fractions are collected. The activity in the fractions is determined, and the capacity is calculated from the retention of activity. In addition, frontal chromatography will also provide information about the possible presence of several components with identical affinities but different retention volumes. Sometimes two activities may be determined simultaneously, for example that of trypsin and chymotrypsin, during the elution from a column of immobilized soybean trypsin inhibiter. Information obtained in the preliminary experiment is then valuable for planning the main experiment, which should bring about maximal purification in a bed of a given volume of the sorbent used. In some instances it was observed that when freshly prepared sorbents were used, some activity of the isolated substance was lost, evidently by its irreversible sorption. Thus, for example, Goreclu et al. described the variability of penicillin-Sepharose conjugate. p Aminobenzylpenicillin coupled to Sepharose by the cyanogen bromide method is a very stable conjugate, which preserves its ability to adsorb D-danine carboxypeptidase specifically even after storage in water in the presence of 0.02%sodium azide at 4°C for several months. During the use of this sorbent, the authors found, however, that only about 6Wo of all preparations of substituted gels are suitable for immediate use in the affinity chromatography of D-danine carboxypeptidase. That is, some of the conjugates

NON-SPECIFIC SORPTION

241

sorbed the enzyme irreversibly and no enzymatic activity could be eluted even when high concentrations of salts were applied. Exhaustive saturation of this gel with a definite amount of crude enzyme (about a 10-fold amount of enzyme with respect to that commonly used in normal purification procedures) converted this penicillin-Sepharose into a normal reversible adsorbent, suitable for the affinity chromatography of D-alanine carboxypeptidase. The mentioned saturation effect was highly specific, and no other inert protein, for example bovine serum albumin, could replace D-alanine carboxypeptidase in the irreversible sorption. It was further observed that during the storage at 4°C some of these gels, characterized by irreversible sorption, were converted into gels suitable for normal affinity chromatography. This change can be ascribed to the decrease in the number of highly active “adsorbing sites” on the gel surface, so that the enzyme can then be bound reversibly to these modified specific sorbents.

10.3 NONSPECIFIC SORPTION Non-specific sorption has already been discussed on several occasions (see Sections 5.1, 5.8,7.1 and 8.3). On the one hand it interferes with affinity chromatography based on the formation of a biospecific complex and on the other hand it increases the affinity of weak affinity systems (“compound affinity”). It provides the means for a general separation on the basis of the interactions of hydrophobic surface regions of macromolecules, so-called hydrophobic chromatography, which can be compared with general methods, such as ionexchange chromatography or gel filtration. The most commonly used sorbents in hydrophobic chromatography are agaroses on to which alkylamines are bound after cyanogen bromide activation. Jost et al. demonstrated, however, that by attachment of alkyl- or arylamines on to cyanogen bromide-activated agarose a strong ion exchanger is formed, with an apparent pK value of about 10 for the basic amidine nitrogen. By combining hydrophobic ligands with charges, an adsorbent is obtained that functions as a detergent, which strongly sorbs some enzymes that are partly denatured during the isolation. The elucidation of the mode of attachment of amine to cyanogen bromide-activated agarose (resulting in an isourea bond) led to the explanation of the often observed non-specific sorption of a number of substances (Nishikawa and Bailon). Native Sepharose or Sepharose blocked with ethanolamine immediately after cyanogen bromide-activation does not adsorb proteins (Heinzel et al.). However, if Sepharose is not blocked immediately after activation, non-specific sorption of proteins on it is observed, which becomes stronger as the time between the activation with cyanogen bromide and the blocking of the active sites increases. Human serum albumin-Sepharose blocked 12 h after attachment of the human serum albumin on to Sepharose adsorbed 1.5-2.0 mg of proteins per millilitre of packed gel, while human serum albumin-Sepharose blocked immediately after coupling of the serum albumin adsorbed only 0.4 mg of protein per millilitre of packed gel. Heinzel et al. described the suppression of non-specific protein adsorption of human serum albumin-Sepharose by neutralizing the basic urethane groups (which arise from iminocarbonate groups formed mainly during the coupling of serum albumin to Sepharose) by the anionic dye Blue Dextran 2000 (BD). The immunosorbent prepared in this manner adsorbed 0.7-1 .O mg of proteins per millilitre of packed gel,

242

SORPTION, ELUTION AND NON-SPECIFIC BINDING

while the isolated antibody was pure and preserved its native properties. Lornitzo et al. described non-specific (ionic) binding of fatty acid synthetase subunits on to a specific sorbent prepared by binding pantetheine to e-aminocaproyl-Sepharose by means of a !arger amount of carbodiimide (ethyldimethylamino-n-propylcarbodiimide). Hofstee carried out a similar study concerning the non-homogeneity and “irreversibility” of the binding of proteins on to adsorbents such as Sepharoses substituted with n-alkylamines or 4-phenyl-n-butylamine. For example, he applied on a column ( 2 ml) of Sepharose with attached n-butylamine a 0.1%solution of albumin in 0.001 M Tris-HC1 buffer of pH 8 until the column was saturated. The loaded column contained about 30 mg of protein (at 5°C). Approximately one third could be eluted with the buffer used, but almost 20 mg of protein remained bound in spite of intensive washing and could be washed out only when the ionic power of the eluting agent was increased. Part of the protein was washed out with 0.01 M Tris-HC1 buffer, but most of the protein with 0.01 M Tris-HC1 buffer containing 0.1 M sodium chloride, and a very small amount of protein with 0.1 M Tris-HC1 buffer containing 1.OMsodium chloride. These results indicate that at each level of ionic strength part of the protein was released, while the rest remained bound “irreversibly” under the given conditions. A similar separation into fractions was also achieved during gradient elution with sodium chloride solution. The non-homogeneity obtained by the separation of the protein fractions was attributed by Hofstee to the nonhomogeneity of the binding sites of the adsorbent. This assumption is in agreement with the result that he obtained in the chromatography of 2 mg of ovalbumin on the same column under identical conditions. The elution of this small amount of protein when a gradient of ionic strength (sodium chloride) was used for elution took place at a concentration of salt at which the last part of the applied larger amount of ovalbumin was eluted. These results suggest that certain “strong” binding sites are occupied by the protein in the first place, and that several other sites of decreasing affinity become occupied when more protein is applied on to the column. In agreement with the different bond strengths, analogously, only part of the protein can be eluted with a salt solution (sodium chloride) with a concentration as high as 1 M from adsorbents with a strongly hydrophobic group, whereas the remainder is dislodged only when a polarity-reducing agent (such as ethylene glycol) is added to the eluent. Similar results were also obtained with other proteins and the dye Ponceau S . From this, it follows that small amounts of proteins are bound much more homogeneously on columns of specific sorbents than are large amounts. This could mean that reducing the amount of applied protein causes one type of site on the adsorbent to predominate in the binding. If multiple-point attachment is one of the reasons for strong non-specific binding, one mean of decreasing it would be to decrease the degree of substitution to a point where the distance between single molecules of the affinant is larger than the diameter of the molecules of the substance isolated. This, however, should not affect the specific “one-toone” interaction, such as that between the active site of the enzyme and the immobilized substrate analogue. A further mean of decreasing non-specific sorption is to avoid the formation of charges and hydrophobic regions in immobilized affinity ligands, as was discussed in Sections 5.1 and 8.3.

REFERENCES

243

10.4 REGENERATION AND STORAGE OF AFFINITY COLUMNS The commonest method of regeneration of columns, for repeated use, is to wash them with alternatingly alkaline and acidic buffers, in a similar manner to that during their preparation. For example, Walsh et al. washed Sepharose with bonded glycyl-D-phenylalanine after the isolation of neutral protease from Bacillus subtilis with 2 column volumes of a buffer of pH 9 and then a buffer of pH 5 or 7, and finally they again equilibrated the column with the buffer used for affinity chromatography. The columns regenerated in this manner did not change their chromatographic behaviour, even after 50 runs. Benson et al. used washing with 6 M guanidine hydrochloride solution and re-equilibration with the starting buffer for the regeneration of 19-nortestosterone-17-0-succiny1diaminodipropylaminoagarose. Turkovi et al. (1 975, 1976) also used 6 M guanidine hydrochloride solution for the regeneration of specific sorbents. After experiments involving elution with detergents, the firm Pharmacia recommended the following washing procedure for the regeneration of sorbents used in hydrophobic interaction chromatography: (1) wash with 1 bed volume of distilled water, followed by 1 bed volume of ethanol; (2) wash with 2 bed volumes of n-butanol; (3) wash with 1 bed volume of ethanol, followed by 1 bed volume of distilled water; (4) re-equilibrate the gel with starting buffer, ready for the next experiment. The flow-rate during washing may be similar to or higher than that used during chromatography, and 25-50 cm/h has been found to be suitable. The insoluble affinant, especially if it has a protein character, is often more stable when bound to a solid support than when free. In many instances, for the preservation of activity it is best to store the suction-dried specific sorbent at low temperature in the presence of a suitable bacteriostatic agent (for example, 0.02%of sodium azide). The choice of the storage buffer depends on the properties of the bound affinant.

REFERENCES Amneus, H., Gabel, D. and Kasche, V., J. Chromatogr., 120 (1976) 391-397. Andria, G., Taniuchi, H. and Cone, J.L., J. Biol. Chem., 246 (1971) 7421-7428. Bennich, H. and Johansson, S.G.O., Aduan. Immunol., 13 (1971) 1-55. Benson, A.M., Suruda, A.J., Shaw, R. and Talalay, P., Biochim. Biophys. Acta, 348 (1974) 317-320. Blumberg, S., Schechter, I. and Berger, A., Eur. J. Biochem., 15 (1970) 97-102. Caron, M., Faure, A. and Cornillot, P., Anal. Biochem., 70 (1976) 295-301. Cornelius, D.A., Brown, W.H., Shrake, A.F. and Rupley, J.A., Methods Enzymol., 34 (1974) 639-645. Cuatrecasas, P., Wilchek, M. and Anfinsen, C., Froc. Nut. Acad. Sci. US.,61 (1968) 636-643. Dawson, R.M. and Crone, H.D., J. Chromatogr., 92 (1974) 349-354. Edginton, T.B., J. Immunol., 106 (1971) 673-680. Fujiwara, K., Osue, K. and Tsuru, D., J. Biochem., Tokyo, 77 (1975) 739-743. Gorecki, M., Bar-Eli, A. and Patchornik, A.,Methods Enzymol., 34 (1974) 398-401. Gospodarowicz, D., J. Biol. Chem., 247 (1972)’6491-6498. Green, A.A.,J. Biol. Chem., 93 (1931) 495-516. Harvey, M.J.,Lowe, C.R. and Dean, P.D.G., Eur. J. Biochem., 41 (1974) 353-357. Heinzel, W., Rahimi-Laridjani, I. and Grimminger, H., J. Zmmunol. Methods, 9 (1976) 337-344. Hill, R.J., J. Immunol. Methods, 1 (1972) 231-245.

244

SORPTION, ELUTION AND NON-SPECIFIC BINDING

Hjertdn, S., J. Chromarogr., 87 (1973) 325-331. Hofstee, B.H.J.,Advan. Exp. Med. Biol., 42 (1974) 43-59. Jennissen, H.P. and Heilmeyer, L.M.G., Jr., Biochemistry, 14 (1975) 754-760. Jost, R., Miron, T. and Wilchek, M., Biochim. Biophys. Acta, 362 (1974) 75-82. Kasai, K. and Ishii, S., J. Biochem., Tokyo, 77 (1975) 261-264. Kristiansen, T., Biochim. Biophys. Acta, 338 (1974) 246-253. Lornitzo, F.A., Qureshi, A.A. and Porter, J.W.,J. Biol. Chem., 249 (1974) 1654-1656. Lowe, C.R., Harvey, M.J. and Dean, P.D.G.,Eur. J. Biochem., 41 (1974) 347-351. Manen, C.A. and Russell, D.H., Life Sci., 14 (1974) 1907-1915. Murphy, R.F., Imam, A., Hughes, A.E., McGucken, M.J., Buchanan, K.D., Conlon, J.M. and Elmore, D.T., Biochim. Biophys. Acta, 420 (1976) 87-96. Nishikawa, A.H. and Bailon, P., Arch. Biochem. Biophys., 168 (1975) 576-584. Niisslein, C. and Heyden, B., Biochem. Biophys. Res. Commun., 47 (1972) 282-289. Pensky, J. and Marshall, J.S., Arch. Biochem. Biophys., 135 (1969) 304-310. Porath, J., Biotechnol. Bioeng. Symp., No. 3 (1972) 145-166. Porath, J. and Kristiansen, T., in H. Neurath and R.L. Hill (Editors), The Proteins, Academic Press, New York, 3rd ed., 1975, pp. 95-178. Raibaud, O., Hogberg-Raibaud, A. and Goldberg, M.E.,FEBS Lett., 50 (1975) 130-134. Robinson, P.J., Dunnill, P. and Lilly, M.D., Biochim. Biophys. Acta, 285 (1972) 28-35. Shaltiel, S., Henderson, G.B. and Snell, E.E., Biochemistry, 13 (1974) 4330-4335. Suckling, C.J., Sweeney, J.R. and Wood, H.C.S., Chem. Commun., 1975, 173-174. Swanljung, P. and Frigeri, L., Biochim.vBiophys. Acta, 283 (1972) 391-394. Turkovd, J., Bldha, K., Valentovd, O., Coupek, J. and Seifertovd, A., Biochim. Biophys. Acta, 427 (1976) 586-593. TurkovB, J., Vavreinovi, S., Kiivlkovd, M. and eoupek, J., Biochim. Biophys. Acta, 386 (1975) 503508. Walsh, K.A., Burstein, Y. and Pangburn, M.K., Methods Enzymol., 34 (1974) 435-440. Zanetta, J.P. and Gombos, G., FEBS Lett., 46 (1974) 276-278.

245

Chapter I1

Examples of the use of affinity chromatography The applications of affinity chromatography are becoming increasingly more varied,, as this method makes use of specific interactions of biologically active substances. It is used with advantage mainly for the isolations of a wide range of compounds, as is evident from the review in Table 11.1. It is further used in studies of various systems, from separations of low-molecular-weight enantiomeric pairs to the elimination of undesirable substances from living organisms. By affinity chromatography, for example, D,L-tryptophan can be separated. Using the specific isolation of labelled peptides, the peptides of the active site of an enzyme, of the binding site of antibodies or the site of the peptide chains on the molecule surface can be determined. Affinity chromatography can be used to study the possibility of substituting natural peptide chains of enzymes with various modified synthetic peptides. The active sites of enzymes or antibodies, the binding properties of subunits, the specificity of enzymes towards various inhibitors, the complementarity of nucleic acids, the interaction of nucleotides with peptides, the effect of the presence of various substances on the formation of specific complexes, etc., can be studied by affinity chromatography. The problems of the mechanism of enzymatic activity can be studied on the basis of the course of affinity chromatography, or the molecular structures of, for example, fibroblast or leucocyte interferons can be judged. The application of immunosorbents in solid-phase radioassay and in immunofluorescence assay is now becoming an independent branch of immunology. The application of affinity chromatography for the elimination of undesirable substances from the blood of living organisms is also being investigated. This enumeration of the uses of affinity chromatography does not exhaust its possibilities, which are many and varied. In the subsequent sections some examples of the possibilities will be discussed.

11 .I ISOLATION OF BIOLOGICALLY ACTIVE SUBSTANCES The preparation of specific sorbents utilizing the exceptional properties of biologically active substances to form specific and reversible complexes has facilitated enormously the isolation of a number of enzymes, their inhibitors and co-factors, antibodies and antigens, lectins, glycoproteins, glycopolysaccharides, nucleic acids, nucleotides, fats, transport and receptor proteins, hormones and their receptors, cells and many other compounds, as reviewed in Table 11.1. In addition to isolated substances, Table 11.1 also gives the affinity ligand used, the solid support and the spacer, with an indication of whether it was the affinant or the solid matrix that was modified by it. The review includes, in addition to the isolations carried out by typical bioaffinity chromatography, also those in which hydrophobic or covalent chromatography is made use of. Various conditions applied during the isolation are dependent on the nature of the substances to be isolated. Although in some instances a homogeneous compound was isolated from crude material by a single chromatographc step, combinations of affinity chromato-

TABLE 1 1 . I USE OF AFFINITY CHROMATOGRAPHY FOR THE ISOLATION OF BlOLOGICALLY ACTIVE PRODUCTS Substances isolated

Affinity ligands

ANTlBODlES Anti-A antibody

A substance

Solid supports or immobilized affinity ligands

Sepharose 2B cross-linked with 2,3 d ibro mo pro pan01 Blood group substance A Sepharose 2B with lysine or Anti-A serum antibody (human) aminoethylcellulose Purified blood group substances Purified blood substances copolyAnti-A and anti-B serum antibodies merized with N-earboxyanhydride of L-leucine lon-exchange resins Antibodies Antigens Ethylene-maleic anhydride copolymers Bio-Gel P-300 Sepharose 4B with 6-amino- against adenosine 5'-monophosphate Oligoadenylic acids hexanoic acid - - adrenocorticotropic hormone (ACTH) Synthetic (Y (1-24) fragment of ACTH CNBr-activated Sepharose 4B Sepharose - - a myeloma proteins 315 and 460 Protein 315 and 460 Sepharose 4B - - amyloid fibril protein Amyloid fibril protein Sepharose 4B with serum albumin Angiotensin I1 - - angiotensin I1 Sepharose 4B - - angiotensin-II+-amide Angiotensin I1 Sepharose 4B Apoliprotein A-1 - - apoliprotein A-1 Cellulose - - p-azobenzenearsonate p-(p-Aminobenzeneazo)benzenearsonic acid Insoluble polymer of rabbit serum p-Aminobenzenearsonic acid albumin or yglobulin Sepharose Sepharose Antigen - - p-azophenyl-P-galactoside - - p-azophenyl-P-glucuronide - - p-azophenyl-p-lactoside Antigen copolymerized with - - basement membrane acr yhmide

References

Kristiansen ( 1 974c) Kristiansen et al. (1969) Kaplan and Kabat

Idiker Centeno and Sehon Ternynck and Avrameas Drocourt and Leng Mains and Eipper Sirisinha and Eisen Linke et al. Bauknecht er al. Hurwitz et al. Fainaru et al. Lerman (195 3 a) Onoue et al. Wofsy and Burr Wofsy and Burr Wofsy and Burr Wofsy and Burr Martinez-Hernandez et al.

- - bovine yglobulin

Antigen Mercury(i1) acetate

- - bovine Serum albumin

Antigen Bovine serum albumin (BSA)

- - chains K , h, a from immunoglobulins K-, A- or a-chains - - - rG, r A and r M and K and h (human) Antigens - - - b5 light b5 Light chain (from rabbit immunoglobulin) NH,-terminal extensions from - - chick-tendon procollagen procollagen Clonal IgG copolymerized with bovine - clonal immunoglobulin (IgG) serum albumin (using glutaraldehyde) - - dextran Insolubilized dextran 2,4,6-Trinitrobenzenesulphonicacid - - dinitrophenyl group (DNP) Trinitrophenyl serum albumin

-

- - - from rabbit serum

- - - with high affinity - - - with low affinity - - diphteria toxid - - DNA - - pecdysone - - egg albumin - - erythrocyte - - erythropoietin - - ferritin

Antigen Trinitrophenyl-bovine ?globulin Dinitrobenzenesulphonic acid Antigen DNA p-Ecdysone Antigen Erythrocyte Human urinary erythropoietin Ferritin

Sepharose 4B Cellulose with l-allyloxy-2,3epoxypropane CM-cellulose Bromoacetylcellulose p-Aminobenzylcellulose CMcellulose Cellulose Glutaraldehyde-insolubilizedBSA Antigen copolymerized with acrylamide Sepharose Sepharose 4B Sepharose 2B

Schade and Nosonoff Shainoff Weliky et al. Robbins et al. Campbell et 01. Weetall and Weliky Behrens ef al. Kessler Martinez-Hernandezet al. Morrison and Koshland Mannik and Stage Rejnek et al.

Cross-linked by adding glutaraldehyde

Dehm et al.

BioGel P-300

Stanislawski and Coeur-Joly

Sephadex Cellulose Bromoacetylcellulose

Robbins and Schneerson Cheng and Talmage Jaton et al. Robbins et al. Wofsy and Burr Weinstein et al. Fauci et al. Fauci el al. Robbins et al. Terman et al. Sage and O’Connor Moudgall and Porter Tlaskalovi et al. Ichiki et al. Kist et al. Ghetie and Onica

Sepharose Bromoacetylcellulose Sepharose 2B BioGel P-300 with ethylenediamine Bromoacetylcellulose Cellulose Epoxy-activated Sepharose 6B Aminocellulose Spheron 300 Sepharose 4B CNBr-activated Sepharose 4B Antigens insolubilized in agarose with glutaraldehyde

(Continued on p . 248)

TABLE 11.1 (continued) Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

References

- - follicle-stimulating hormone (FSH) - - gibberellic acid

Human chorion gonadotropin (HCG) Gibberellic acid conjugated to bovine serum albumin Globo side ?Globulin

Sepharose 4B Sepharose 4B

Sato and Cargille Fuchs and Gertman

Aminoeth yl-Sepharose Antigens insolubilized in agarose with glutaraldehyde CNBr-activated Sepharose 4B Sepharose 4B CNBr-activated Sepharose

Laine et al. Ghetie and Onica

Sepharose

Parker et al.

Sepharose 4B Amino group-bearing glass beads CNBr-activated Sepharose CM-cellulose Glass Antigen copolymerized with acrylamide Divinylsulphonyl-Sepharose 6B CNBr-activated Sepharose 4B

Fellows et al. Laine ef al. Bustin and Kupfer Weliky et al. Weetall (1970) Martinez-Hernandez et al.

Antigen copolymerized with acrylamide Ultro-gel Sepharose 4B Sepharose 4B Amino-cellulose p-Aminobenzylcellulose

Martinez-Hernandez et a l . Guesdon and Avrameas Hwang ei al. Guyda and Friesen Moudgal and Porter Webb and Lapresle

Bromoacetylcellulose or Sepharose 4B Spheron 300

Hill Tlaskalovi ef al.

- - globoside - - ?-globulin - - glucagon - - glucose oxidase - - glutamate dehydrogenase

Modified glucagon Glucose oxidase Glutamate dehydrogenase (mitochondrial) - - Group A streptococcal polysaccharide p-Aminophenyl-0-N-acety 1glucosaminide Growth hormone (bovine or human) - - growth hormone Haematoside - - hematoside Histone-H1 - - histone-H1 Antigen - - human ?globulin Human 7-globulin - - - (rabbit) Antigen - - human gonadotropin - - human growth hormone - - human haemoglobin A, from goat serum - - human immunoglobulin

human growth hormone Haemoglobin A, or a-or p-chains

- - human immunoglobulin G - - human pituitary prolactin - - human placental lactogen - - human serum albumin

Human immunoglobulin G Human pituitary prolactin Human placental lactogen Antigen Fragment of human serum albumin called “inhibitor” Human serum albumin

Antigen

Murphy et al. (1976) Valiulis ef al. Di Prisco and Casola

Sairam et al. Tan-Wilson et al.

- - insulin

Human serum albumin with blue dextran dye Insulii

Sepharose 4B

Heinzel et al. Onoue et al.

Short and Kaback Lasch et al. Lugowski and Romanowska Deeley et al. Wetekam et al. Loeffler and Hinds

- interferon - - keyhole limpet haemocyanin - - or-lactalbumins from human, cow, goat and sheep milk - - D-lactate dehydrogenase - - leucine aminopeptidase - - lipid A - - lipovitellin

Interferon Antigen or-Lactalbumin

Insoluble polymer of rabbit serum albumin or yglobulin Sepharose Sepharose 4B, Spheron P-300and both derivatives with hexamethylenediamine CNBr-activated Sepharose 4B Sepharose 4B Sepharose 4B

D-Lactate dehydrogenase Leucine aminopeptidase Lipid A Lipovitellii

Sepharose 4B Sepharose 6B Sepharose 4B Sepharose

- - lysergide from sheep antiserum

D-Lysergic acid

- - lysine-vasopressin - - (8-lysine~vampressinfrom rabbit antiserum - - lysozyme

(8-Lysine)-vasopressin (8-Lysine)-vasopressin

Agarose with long-chain hydrazide derivative Sepharose 4B Sepharose 4B or Spheron P-300

-

Lysozyme copolymerized with bovine serum albumin (using glutaraldehyde) Antigens 6-Succinylmorphine Antigens Myoglobulin

BioGel P-300

Bromoacetylcellulose AH Sepharose 4B Sepharose AE-, BA-, CM-cellulose, EMA or Sepharose 4B - - 3-nitro-4-hydroxy-5-iodophenylacetyl3-Nitro-4-hydroxy-5-iodophenylacetyl-Sepharose 4B determinant ethylenediamine Nitro-yglobulii - - nitrotyrosine Sepharose Ovalbumin - - ovalbumin Ovalbumin cross-linked with glutaraldehyde ICSH - - ovine interstitial cell stimulating Divinylsulphonyl-Sepharose 6B hormone (ICSH) - - ovotransferrin Ovotransferrin Ovotransferrin and bovine serum albumin copolymerized with ethyl chloroformate - - morphine - - myeloma proteins - - myoglobin

Cuatrecasas (1969) Sirakov et al. Hajnicki et al. Schade and Nisonoff Prieels et al.

Fr6noy et al. Van&5kovd et al. Stanislawski and Coeur-Joly Robbins et al. (1967) Simon Wofsy and Burr Boegman and Crumpton Hoffman et al. Helman and Givol Palacios et al. Sairam et al. Faust and Tengerdy

(Continued on p. 250)

N

ln

TABLE 11.1 (continued)

0

Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

References

- - papain - - peroxidase

Antigen Antigen LDH V

Broinoacetylcellulose BioCel P-300 with glutaraldehyde Sepharose 4B

Robbins PI ul. Ternynck and Avrameas Hill

S&bovine T-globulin conjugate Antigen Protein hormones

Sepharose 4 8 Bromoacetylcellulose Divinylsulphonyl-Sepharose 4B

Cheng et al. (1973) Robbins et al. Sairam and Porath

Antigen

Antigen copolymerized with acrylamide Bio-Gel P-300 with glutaraldehyde

Martinez-Hernandez et al.

-

pig lactic acid dehydrogenase Type V (LDH V) - - pneumococcal polysaccharides - - polypeptides and their conjugates - - protein hormones (ovine and human interstitial cell stimulating hormone, the p-subunit and rat pituitary prolactin) - - rabbit 7globulin -

- - rabbit and human immunoglobulin (IgG) - - rat liver malic enzyme - - rhesus and human lymphocyte - - ribonuclease

Rat liver malic enzyme Rhesus lymphocyte stroma Ribonuclease

- - ricinus agglutinin - - serum albumin

Ricinus agglutinin Serum albumin (human and bovine)

- - sheep immunoglobulin G - - sickle hemoglobin

- staphylococcal nuclease - - thyrotropin - _ toxine a1 - _ trypsin Antibodies from rabbit anti-hapten sera -

Antigen

Ternynck and Avrameas

m

$

5r

m

cn

Sepharose 4B Sephadex El00 Bromoacetylcellulose Sepharose 4B CNBr-activated Sepharose 4B Polystyrene

Cellulose Sheep immunoglobulin G Ultrogel Synthetic peptide corresponding to the Sepharose 2B first 13 amino acid residues of the &chain of sickle hemoglobin Nuclease from Staphylococcus aureus Sepharose 4B Thyrotropin Sepharose 4B Toxine a , from Naja nigricollis Sepharose 4B Trypsin BioCel P-300with glutaraldehyde Potassium benzylpenicillin or Benzylpenicylloyl or dinitrophenyl dinitrofluorobenzene hide power

Frenkel Wilson et al. Robbins et al. Igarashi et al. Olsnes and Saltvedt Gyenes et al. Gyenes and Sehon Gourvitch et al. G u e g o n and Avrameas

Young et al.

% C

rn

m

s?

%

Z i

3 4 -

0

I :

a

8 Omenn et al. Tate et al. Ddtrait and Boquet Ternynck and Avrameas

5 8a

Levine and Levytska

4

!i

Anti$, -microglobulin immunoglobulin Anti-phenylarsonic antibodies Anti-protein antibodies

Anti-prothrombin immunoglobulin fraction Antisera to a-fetoprotein Anti-Shigella sonnei sera

p2 -Microglobulin Antigen Proteins [transferrin, Bence-Jones protein (L), immunoglobulins G and M] Proteins (immunoglobulins, BenceJones (type K and L) proteins, serum albumin, glucose oxidase, peroxidase] Prothro mbin

BioGel A-50m CM-cellulose Proteins polymerized with ethyl chloroformate Proteins cross-linked with glutaraldehyde

Robb et al. Weliky er al. Avrameas and Ternynck (1967) Avrameas and Ternynck (1969)

Sepharose 4B

Wallin and Prydz

Estradiol-170-monohemisuccinate

Diarninonane-Sepharose4B CNBr-activated Sepharose 4B or epoxy-activated Sepharose 6B CNBr-activated Sepharose 4B Sepharose 4B CNBr-activated Sepharose 4B

Arnon et al. Romanowska et al.

Shigella sonnei lipopolysaccharides

Anti-soybean agglutinin rglobulin Anti-thyroglobulin autoantibodies Antitoxic components (type A or B) immunoglobulins Bacterial specific antibodies

Soybean agglutinin Human thyroglobulin Toxic components (type A or B)

Bovine anti-mouse lymphocyte antibody Carbohydrate-specific immunoglobulins Globulin (major) from French bean (P.vulgaris) "'1-Labelled immunoglobulin G Immunoglobulins from human serum Immunoglobulin A of normal and myeloma serum Immunoglobulin G monospecific for cytochrome C oxidase Immunoglobulin G3 from human sera Immunoglobulin M - from mouse lymphocyte - Waldenstrom

Acetonedried polymerized bacteria

Nachbar and Oppenheim Hearn et al. Sakaguchi et al. Weetall (1967)

Thymocytes Oligosaccharides Glycoprotein fetuin

Polymerized bacteria in mixture with cellulose Sepharose 2B Sepharose 4B Sepharose 4B

G I Protein from P. vulgaris Staphylococcal protein A Protein A from Staphylococcus aureus Human 7-S rglobulin L-Phenylalanine

Sepharose 4B Sepharose 4B or AHSepharose 4B Sepharose 4B Sepharose 4B Sepharose 4B

Stockman et al. Nilsson et al. Hjelm et al. (1972) Palmer Doellgast and Plaut

Cytochrome C oxidase

Sepharose 4B

Hackenbrock and Hammon

Protein A Concanavalin A

Sepharose 4B Hjelm Donnelly and Goldstein Concanavalin A polymerized with glutaraldehyde Formalin-fixed and heat-killed bacteria Kessler

Cowan I strain of Staphylococcus aureus p-Diazoniumphenylphosphorylcholine CNBr-activated Sepharose 4B with glycyltyrosine

Dresser Jefrey et al. Selaet al. (1975a)

Riesen et al. (Continued on p. 252)

TABLE 11.1 (continued) Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

References

"'I-labelled rabbit antihovine albumin Streptococcal group-specific polysaccharide antibodies from rabbit hyperimmune serum

Serum albumin Partial deacetylated streptococcal polysaccharide

BioGel P-300 CNBr-activated Sepharose 4B or Sepharose 4B with lysine

Inman and Dintzis Robbins and Schneerson

Anti-albumin antibodies Antibodies Antibodies (anti-human serum albumin, anti-immunoglobulin G ) Antibodies (anti-immunoglobulins, anti-serum albumin) Au/SH-antigen antiserum Anti-rabbit IgG

Antibodies polymerized with ethyl chloroformate BioCel P-300 Antibodies polymerized with ethyl chloroformate Antibodies cross-linked with glutaraldehyde Sepharose 4B Cellulose

Avrameas and Ternynck (1967) Ternynck and Avrameas Avrameas and Ternynck (1967) Avrameas and Ternynck (1969) Tripatzis and Horst Houwen et al. (1973)

Rabbit yglobulin Antidinitrophenyl antibodies

Cuprophan Sepharose 4B

Terman er al. (1976a) Tarone et al.

Anti-ovalbumin antibodies Antibodies

Gallop et al. Crook et al.

Antibodies to pancreatic glucagon Antibodies against glucagon

Aminocellulose Disulphide-linked antibodies with Nacetyl homocysteine thiolactone Sepharose 4B Sepharose 4B

Antibodies to human llght chains Concanavalin A Anti-HBsAg antibodies Anti-human yglobulin antibodies Anti-@z-microglobulinimmunoglobulin

Sepharose Sepharose Sepharose 4B Glass BioGel A-50m

Morrison and Koshland Neurath er al. Houwen et al. (1975) Weetall (1970) Robb et al.

Antibodies

BioGel P-300

Sapin er a1

ANTIGENS AND HAPTENS Albumin (human) Antigens

Australia-SH-antigen from urine Australia antigen in complex with rabbit IgG fractions Circulating antigen Erythrocyte membrane proteins after selective labeling with trinitrobenzene Fluorescent ovalbumin YGlobulin Glucagon-like immunoreactivity Glucagon pancreatic) and - (porcine glucagon-like immunoreactivity (pig ileum) J chain from polymeric immunoglobulin Hepatit is pant igen Hepatitis B surface antigen (HBsAg) Human yglobulin Human histocompatibility antigens of the HLA-A and HLA-B loci Human immunoglobulin A and M

Murphy er al. (1971) Murphy er al. (1973)

Lymphocyte-surface immunoglobulins Lysergide (D-lysergic acid diethylamide, LSD) Immunoglobulins from serum IgM globulins Immunoglobulin G subclasses from mouse alloantiserum Immunoglobulin E (IgE) Immunoglobulin M from rat and rabbit serum a, -Macroglobulin Mammalian type C RNA virus antigen (~30) Natural 7 s immunoglobulin M (human serum) Ovalbumin

Protective antigen against anthrax Protein A from Staphylococcus aureus Rabbit immunoglobulin G Secretory immunoglobulin A Thioredoxin from Escherichia coli and phage T, Vitellogenin from plasma estrogen-treated roosters

Antibodies Antibodies to lysergic acid

Sepharose 4B Agarose with long chain hydrazide derivative Sepharose 4B Sepharose 4B CNBr-activated Sepharose 4B

Fromel er aJ. Barros and Lebon Jansen ef al.

Sepharose 2B BioGel P-300

Bennich and Johansson Sapin and Druet

Anti-human ar2 -macroglobulin Antibody

Sepharose 4B Sepharose 4 8

Abe and Nagai Oroszlan et al. (1975)

Anti-IgG antibodies

Sepharose 4B

Dolder

Anti-ovalbumin antibody

Polystyrene Thiolated and cross-linked antibody Aminocellulose Disulphide-linked antibodies with N-acetyl homocysteine thiolactone Polystyrene Sepharose 4B BioGel P-300 with glutaraldehyde CNBr-activated Sepharose 2B Sepharose 4B

Kent and Slade Stephen et al. Gallop et al. Crook et al.

Sepharose

Deeley ef al.

Sepharose 4B Nylon fibres Antigen-coated glass beads

Soderman et al. Rutishauer et al. (1972b, 1973) Abdon and Richter

S-N-2,4dinitrophenyl-L-ornithine .HCI Sepharose 2B BioGel P d with histamine Azophenyl-P-lactoside hapten groups

Trump Robbins and Schneerson

Anti-light chain antibody Anti-human IgM globulins Specific antisera against mouse immunoglobulin subclasses Anti-IgE antibodies Anti-human LC antibody

Antibody Immunoglobulins from human serum Antibodies Antiporcine archain specific antibody yGlobulin fractions of rabbit antithioredoxin Anti-lipovitellin antibodies

CELLS AND CELL ORGANELLES Insulin Adipose cells Antigen-binding cells from spleens of mice Antigens Antigen-reactive cells from normal rabbit bone marrow Anti-hapten plaque-forming cells Antihapten specific lymphocytes

Antigens

Haustein and Warr Loeffler and Hinds

Kent and Slade Hjetm et al. (1972) Ternynck and Avrameas De Buysscher and Berman Sjoberg and Holmgren

(Continued on p. 254)

TABLE 11.1 (continued) Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

References

Bacteriophage SKVI

Shigella sonnei lipopolysaccharides

Romanowska et al.

Cells (erythrocytes, thymocytes) - (spleen)

Concanavalin A Dinitrophenylated serum albumin

- population of specific antibodyproducing and specific memory cells - producing antihapten antibodies

Human or bovine serum albumin or hen ovalbumin Azophenyl-pgl ycoside

CNBr-activated Sepharose 4B or epoxy-activated Sepharose 68 Nylon fibre Dinitrophenylated serum albumin or gelatin fibres Glass beads or Degalan, V26

Cultured tumour cells

Isoproterenol, corticotropin (ACTH), triiodothyronine Anti-erythrocyte antibodies

Erythrocytes

Fat cells Flagelae PGalactosidase-specific polysomes Immune cells Immunoglobulin-bearing lymphocytes Immunospecific precursor cells from unimmunized mice Lymphocytes, antihapten specific - specific immunocompetent T - (T and B cells) from mouse spleens

BioCel P-6 with hydrazine and histamine Glass or Sepharose

Reticulated polyester polyurethane foam Sepharose 6B Concanavalin A Insulin Sepharose 4B Sepharose 4B Anti-Hb globulins p-Aminophenyl-0-D-thiogalactoSepharose with 3-aminosuccinyl-l,6pyranoside diaminohexane Antigen (serum albumin or ovalbumin) Glass and plastic beads coated with antigenic protein molecules Anti-immunoglobulin antibodies Plastic beads coated with antiimmunoglobulin antibodies BioCel P-6 with hydrazine and Azophenyl-P-lactoside histamine &Lactoside haptens Acrylamide Phytomitogens Sepharose 4B Degalan beads coated with antibodies Anti-idiotypic antibodies Nylon fibres Antigens (hapten-bovine serum albumin conjugates, Limulus haemocyanine or concanavalin A) Surface of tissue culture grade Human immunoglobulin (HGG) after treatment with anti-HGG antisera plastic-ware

Edelman and Rutishauer Edelman and Rutishauer Robbins and Schneerson Truffa-Bachi and Wofsy Venter et al. Evans et al. Edelman et al. Soderman et al. Fey and Wetzstein Melcher Wigzell and Anderson Campbell and Grey Henry et al. Wofsy et al. Greaves and Bauminger Binz and Wigzell Rutishauer and Edelman (1972 a) Barker et al. (1975)

c3

- T and B cells from rat thoracic duct lymph Lymphocyte membrane vesicles Lymphoid cells - - from rat spleen and thymus and mouse spleen Membranes Ovalbumin-synthesizing polysomes in complex with anti-albumin antibody Plasma membranes from pig lymph node cells Polyribosomes from mouse plasmacytoma producing IgGl immunoglobulin type x - and pure mRNA Polysomes

Proliferative cells Reticulocyte ribosomes Ribosomes from Escherichia coli Ribosomes synthesizing tyrosine aminotransferase from hepatoma tissue culture cells Thymocytes T4 phage Translating ribosomes COFACTORS AND VITAMINS Antithrombin 111-heparin cofactor Biotin Coenzyme A

Enzyme cofactor NAD from yeast extract Flavin-adenine dinucleo tide

Anti-rat F (ab'), antibody

Sephadex G-200

Concanavalin A Purified coliphage K 29 Bovine serum albumin or its derivatives Nylon fibres Aggregated rat immunoglobulin Sepharose 4B Coicanavalin A Ovalbumin Concanavalin A Complex of mouse immunoglobulin with rabbit antibodies Antigen-antibody complexes Antibody to specific protein Pyridoxamine phosphate Concanavalin A Polyuridylic acid Streptomycin or gentamicin Pyridoxamine-P

Crum and Gregor Wallach Edelman et al. Matthews et al.

Purified coliphage K 29 Ovalbumin cross-liked with glutaraldehyde K29 coliphage

Wallach et al.

Aminocellulose

Sidorova et al. (1973)

Aminocellulose Agarose Sepharose 4B with ethylenediamine and succinic anhydride Con A-Sepharose Sepharose 4B Indubiose 4A Sepharose 4B with 3,3'diaminodipropylamine and succinic anhydride

Wallach Palacios et al.

Sidorova ef al. (1974) Faust et al. Thompson (1974) De Jonge Lee and Heintz Le Goffic et al. (1974) Miller e f al. (1971)

Anti-thymocyte globulin Poly DL-lysine Periodateaxidized polyuridylic acid

Sepharose 4B Sepharose 2B CMcellulose with dihydrazide of dithioglycolic acid

Eshhar et al. Sundberg and Hoglund Belitsha el al.

Heparin Avidin

Sepharose 4B Sepharose 4B

Coenzyme A-affinity protein CoA-binding protein p-Acetoxymercurianie Alcohol dehydrogenase (E.C. 1.1.1.l) p-Acetoxymercurianile

Sepharose 6B

Damus and Wallace Bodanszky and Bodanszky Green and Toms Matuo et al. (1974) Chibata et al. (1974 a) Matuo et QI. (1975) Das e f al. Matuo et al. (1975)

CNBr-activated Sepharose 4B Sepharose 6B

cn

(Continued on p. 256)

0

9

r r 4 9

2

s

2 4

9

z

0 M

cn

w

(n (n

h)

w

TABLE 11.1 (continued)

o\

Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

References

Riboflavin 5'-phosphate and its analogues

Apoflavodoxin

Sepharose 4B

Mayhew and Strating

ENZYMES Trimethylfm-aminopheny1)ammonium BioGel A-50m with 3J'diaminodiAcetylcholinesterase (acetylcholine hydrolase, E.C. 3.1.1.7) from erythrocytes chloride hydrochloride propylamine and succinic anhydride, repeated twice - from electric eel Trimethyl(paminopheny1)ammonium BioGel A-50m with 3,3'diaminodichloride hydrochloride propylamine and succinic anhydride, repeated twice - from Bungarus fasciatus Sepharose 4B AF 201 - from the electric eel and erythrocyte 2% agarose (BioGel) with 3,3'diaminomembranes propylamine and succinic anhydride, repeated twice e-Aminocaproylcholine derivatives Sepharose 2B - from bovine erythrocytes d-Tubocurarine Sepharose 4B with ethylenediamine, succinic anhydride, ethylenediamine and paminobenzoyl residue 2-Aminoethyl p-nitrophenyl Sepharose 4B with bound 1,5diaminopentane and succinic anhydride methylphosphonate - from guinea pig brain mCarboxyphenyltrimethy1Sepharose 4B with 1,4diaminobutane ammonium iodide Sepharose 2B [N-(e-Aminocapro y1)-p-aminophenyl) trimethylammonium bromide hydrobromide Sepharose 4B Sepharose 2B with twice-repeated hexamethylenediamine and succinic anhydride Sepharose 2B

-

1-Methyl-9-[ N@-(e-aminocaproyI)-paminopropylamino]acridinium bromide hydrobromide

Sepharose 2B

Berman and Young

Berman and Young

Kumar and Elliot Dawson and Crone Berman and Young Berman

5r

M

Schwyzer and Frank Jung and Belleau

Ashani and Wilson

cn

%

c,m %

Yamamura er al. Kalderon et al.

Rosenberry et al. Hopff et al.

Dudai and Silman Dudaiet al. (1972a, b) Dudai and Silman

w

5.c

CI

2-Aminoethyl-p-nitrophenyl methylphosphonate * HC1

- from human erythrocytes

- from bovine caudate nucleus

- from fresh electroplax tissue of Elecirophorus electricus - from house-fly head tissue Acetyl CoA apocarboxylase N-Acet yl-pglucosaminidase(2-acetamido2deoxy-0-Dglucoside acetamidodeoxyglucohydrolase, E.C. 3.2.1.30)

- from human urine

- from Trichomoms foetus Acid pgalactosidases A and B (E.C. 3.2.1.23) - from human liver Acid glycosidases(g1ucocerebroside pglucosidase, P-N-acetylglucosaminidase, pgalac tosidase)

m-[6-(6-Aminocaproylamino)caproylamino] phenyltrimethylammonium bromide hydrobromide m-[6-(6-Aminohexanoylamino)hexanoylamino I phenyltrimethylammonium bromide hydroiodide m- and p-isomers of N-(6-aminocaproy1)-1-aminophenyltrimethylammonium bromide hydrobromide (1) Concanavalin A +(2) m-trimethylammoniumaniline Avidin p-Aminophenyl-Nacet y1-8-Dglucosaminide p-Aminobenzyl-1-thio-2-acetamido2deoxyQ-Dglucopyranoside p-Aminophenyl-N-acetyl-8-D-thioglucosamine Proteoglycan glycopeptide Concanavalin A

Sepharose 4B with 1,Sdiaminopentane and succinic anhydride, repeated twice Sepharose 4B

Voss et al.

Sepharose 4B

Ruess et QI.

Sepharose 2B cross-linked and reduced

Morrod er aI.

(1) Con A-Sepharose

+ (2) AffiGel

202 Sepharose 4B Sepharose 4B with 3,3'diaminodipropylamine and succinic anhydride Sepharose 4B with hexamethylene diamine and succinic anhydride Succinylated diaminodipropylaminoagarose Sepharose 4B Sepharose 4B 2-Acetamido-N-(e-aminohexanoyl)-2- Sepharose 4B deoxy-8-Dglucopyranosylamme N-Hydroxysuccinimideester of p-Aminopheny1-Nacetyl-p-D-thioglucosaminide succinylated aminoalkyl agarose 2-Acetamido-2deoxy-D-mannono-l,4-Sepharose 4B with benzidine lactone N-(e-Aminohexanoyl)-2-acetamido-2- CNBr-activated Sepharose 4B deoxy-p-D-glucopyranosyhmine Con A-Sepharose Concanavalm A (1) Concanavalin A; (2) 6-aminohexyl- (1) Con A-Sepharose; (2) Sepharose 4B 1-thio-p-D-galactopyranoside CNBr-activated Sepharose 4B Glucocerebrosidase effector

Grossmann and Lieflander

$b

=! 0 z

%W

s2:

i.;

F:

r 4

Steele and Smallman Landman and Dakshinamurti Junowicz and Paris Rafestin et al.

P

rl

z

s

: m 4

9

Grebner and Parikh Dawson et al. Banerjee and Basu Koshy et al. Bamberg ef al. Pokorny and Glaudemans (1975) Edwards et al. Norden and O'Brien Miller et QI. (1976)

Ho

(Continued on p. 258)

z

2 m

TABLE 11.1 (continued) Substances isolated

Affinity ligands -Acidic a-D-mannosidase (E.C. 3.2.1.24) D-Mannosylamine from human liver Adenine nucleotides Adenosine 3',5'-phosphate-dependent enzymes Dioxin (Na' + K3-adenosine-triphosphatases (E.C. 3.6.1.3) ---____-

-

.

___

-

-

Adenosine triphosphatases (mitochondrial) Adenosine triphosphatase inhibitor - from beef heart mitochondria Adenosine triphosphatase inhibitor protein Detergent solubilized Na+,K+-ATPase 64Purine 5'-ribosyltriphosphate)-4(E.C. 3.6.1.3) (1,3dinitrophenyl)thioether

S-Adenosyl-L-methionine decarboxylase from rat liver and sea urchin eggs Adenylate cyclase [ATP pyrophosphate. lyase (cyclizing), E.C. 4.6.1.1) Agarase from Littorina mandshurica Alginases a-Am ylase

Solid supports or immobilized affinity ligands -_._ CH-Sepharose 4B

References --

Apoglutamic-oxdoacetic transaminase (E.C. 2.6.1.1)

____

Phlhps et al.

Sepharose 4 8

Cuilford ct al.

Sepharose 4B with ethylenediamine or 3,3diaminodipropylainineor decameth ylenediamine Sepharose 4B CNBr-activated Sepharose 4B

Okarma et al.

- - - _.

_ I

Swanljung and Frigeri (1972) Swanljung and Frigeri (1974)

pAminophenylmercury(I1) acetate

Anderton et 01. Sepharose 4B pre-treated with epichlorohydrin and sodium borohydride with 3,3'-iminobispropylamineand with Nacetylhomocysteine thiolactone Manen and Russell (1974a, b) CNBr-activated Sepharose with ethylenediamine Cuatrecasas et al. (1975) Sepharose 4B

"Active" subunit of cholera toxin Agarose Alginic acids Starch

Sepharose 2B BioGel A-5m BioGel P-20 with hydrazine hydrate Starch

Glycogen Cycloheptaamy lose Protein inhibitors from wheat kernel

Starch granules AH-Sepharose 4B Epoxy-activated Sepharose 6B Sepharose 2B

pChlorornercuribenzoate

- from Clostridium acetobutilicurn - from germinated barley

- from triticale Amylases from Tenebrio molitor larvae and chicken pancreas &Amylase (sweet potato)

-

Cyclohexaamylose

N'4waminohexyl)pyridoxamine 5'-phosphate

Sepharose 6B with 1,4-bis(2,3epoxypropoxy )butane Sepharose 4B

Bennett et al. Usov and Miroshnikova Favorov Starkenstein Holmbergh Hockenhull and Herbert Schwimmer and Balls Dube and Nordin Tkachuk Silvanovich and Hill Buonocore et a l . Vretblad (1974a, b) Collier and Kohlhaw

Apo-tryptophanase

Pyridoxal5'-phosphate(3-Oimmobilized)-tryptophan complex

Apo-tyrosine phenol-lyase

Pyridoxal 5'-phosphate (3a-immobilized)

Arginase (L-arginine amidino hydrolase, E.C. 3.5.3.1) from Sacchuromyces cerevisine - from semipurified rat liver extract Arylsulphatase A (E.C. 3.1.6.1) Arylsulphatases

L-Arginine

L-Asparaginase(Ec 3.5.1.1) - from Proteus vulgaris Asparaginase and asparaginase modified with tetranitromethane

Antibodies to Lasparaginase N4w-Aminohexy1)-L-asparticacid

Anti-arginase antibody Psychonine sulphate 2-Nitroquinol sulphate

D-Asparagine - from Escherichia coli - from Escherichia coli and Erwinia Caratovora Aspartase (E.C. 4.3.1.1) - from Escherichia coli Aspartate pdecarboxylase(E.C. 4.1.1.12) Aspartate transcarbamylase (E.C. 2.1.3.2)

Bacteriolytic enzymes from crude preparations of animal and microbial origin Bacteriophage f2 replicase

Sepharose with hexamethylene diamine Ikeda et al. and O-bromoacetyl-lrl-hydroxysuccinimide Sepharose with hexamethylenHiamkte Ikeda et al. and O-bromoacetyl-N-hydroxysuccinimide Sepharose 6B with hexamethylenediPenninckx et al. amine and succinic anhydride Sepharose 4B Sepharose 4B pAminobenzamideethy1 derivative of Sepharose 4B Glass Sepharose 6B

Tarrab et al. Breslow and Sloan Agogbua and Wynn

s 3

F

r .c b

0

5

Weetall (1970) Chibata er al. (1974b) Tosa et al.

Aminoethylpolyacrylamide magnetic particles with 1,4-butanedioldiglycidyl ether Sepharose 6B

Kristiansen et aZ. (1970)

Sepharose 6B

Mardashev et al.

N4w-Aminohexy1)-L-asparticacid

Sepharose 6B

N4 w-Aminohexy1)-L-aspartic acid

Sepharose 6B

1,lO-Diaminodecaneand succinic anhydride or pyromellitic dianhydride

Sepharose 6B

Tosa et al. Chibata et al. (1974b) Chibata e f al. (1974b) Tosa et al. Yon (1974)

D-Asparagine with hexamethylenediamine Na46-Aminohexyl)-Dasparagine

U

2

5

M

Dunnill and Lilly

N43-Carboxypropionyl)aminodecane Sepharose 4B Lysozyme-lysate of Micrococcus CNBr-activated Sepharose 4B Zysodeikticus cell wall

Yon and Simmonds Yoshimoto et al.

Single-stranded f2 RNA

Fedoroff and Zinder

N

ul

Cellulose

(Continued on p . 260)

\o

TABLE 11.1 (continued) SGbstances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

References

Biosynthetic threonine deaminase [Lthreonine hydrolyase (deaminating), E.C. 4.2.1.161 Carbonic anhydrase (E.C. 4.2.1.1)

Valine and isoleucine-N-hexarnethyleneamine

Sepharose 4 9

Koerner et al.

Suiphanilamide Sulpham ylon (p-aminomethylbenzenesulphonamide) 2-Amino- 1,3,4-thiadiazole-5-sulphonamide p - [(2,4-Diaminophenyl)ao] benzenesulphonamide (Prontosil) Anti-catabolic dehydroquinase immunoglobulin

Sephadex G 1 5 0 Sepharose 4B

Falkbring et al. Whitney

Sepharose with NH,(CH,),,COOH

Champagnol

CM-Sephadex

Osborne and Tashian

Sepharose 4 8

Hautala et al.

rn-Aminopheny Itrimethylammonium L-Tryptophan 1,I-Diaminooctane Aniline n-Octylamine 2,4,6-Trinitrobenzene

CH-Sepharose 4B Sepharose 4B Sepharose 4B Sepharose 4B Sepharose 4 9 Sepharose 4B with hexamethylenediamine

Picard Sprossler and Lingens Imai and Sat0 Takemori et al. Wang and Kimura Kornbluth et al.

6-Immobilized adenosine analogue

Sepharose 4B with hexamethylenediamine Sepharose 4 9 with 3,3’diaminodipropylamine and succinic anhydride

Barry and O’Carra (1973a)

Adenosine N6-(6-Aminohexyl)-5’-AMP

Epoxyactivated Sepharose 6B 5’-AMP-Sepharose 4B

Schrader et al. Makarewicz and Stankiewicz

Isoleucine L-Valine

Sepharose 4B Sepharose 4B

Rahimi-Laridjani et al. Rimerman and Hatfield

- - isozymes Catabolic dehydroquinase (5dehydroquinate hydrolyase, E.C. 4.2.1.10) from Neurospora crassa Cholinesterase Chorismate mutase from Claviceps paspali Cytochrome P-450 - from bovine adrenocortical mitochondria Cytosolic NAD-linked glycerol-3phosphate dehydrogenase(E.C. 1.1.1.8) Deaminases Adenosine deaminase (adenosine aminohydrolase, E.C. 3.5.4.4) - - from calf spleen and intestinal mucosa, chicken duodena and erythrocytes - - from human erythrocytes AMP deaminase (E.C. 3.5.4.6) from human skeletal muscle Biosynthetic threonine deaminase ( L-threonine hydrolyase deaminating, E.C. 4.2.1.16)

6-Amino-9-(p-aminobenzyl)adenine

Rossi et al.

Guanine deaminase (rat liver)

9-@-p-Aminoethoxypheny1)guanine

Dehydrogenases Alcohol dehydrogenase (alcohol: NAD+ NAD oxidoreductase, E.C. 1.1.1.1) - - from brain NADP NAD+-N6-[N-(6-aminohexyl)acetamide] - - (Yeast) N6 -(6-Aminohexyl)-S'-AMP Alcohol dehydrogenase N6-(6-Aminohexyl)-S'-AMP Cibacron Blue - - from crude cottonseed extracts - - isozymes of horse liver N6-(6-Aminohexyl)-AMP - - steroid-active isozyme of horse liver Apo-phydroxybutyrate dehydrogenase NAD (E.C. 1.1.1.30) Anti-aspartokinase I-homoserine Aspartokinase I-homoserine dehydrodehydrogenase I antibody genase from Escherichiu coli - - - and I1 and aspartokinase 111 from Escherichiu coli Dehydrogenasesfrom yeast extract NADP+ - from a crude yeast extract - from crude extracts - (alcohol, glucosed-phosphate, glycer aldehyde-3-phosphate, isocitrate, lactate and malate) - contaminating cytochrome C oxidase preparations - and kinases Estradiol-17-pdehydrogenase (E.C. 1.1.1.62) of human placenta Estradiol-17pdehydrogenase - - from human placenta

Sepharose 4B

Baker and Siebeneick Siebeneick and Baker

Glass

Weibel et al.

Sepharose Sepharose 4B

Tabakoff and Von Wartburg Lindberg et al.

Sepharose Sepharose 4B Blue Sepharose 6B Sepharose 4B

Ohlsson et al. Comer et al. Lamkin and King Andersson et al. (1974) Andersson et al. (1975)

Sepharose 4B with 6-aminocaproic acid Grover and Hammes Sepharose 4B

=1 0 z e l

E

? s 0

0

::

r

4

*

0

ZI 4 m

Cowie et al. Truffa-Bachi et al.

Sepharose 4B with e-aminohexanoic acid Sepharose 4B Sepharose 4B

Craven et al. (197413) Lee er al.

Sepharose

Harvey et al.

NAD

AG-NAD, type 1

Holbrook et al.

N6-(6-Aminohexyl)-NAD+ Estrone hemisuccinate

CNBr-Activated Sepharose 4B Sepharose 4B with ethylenediamine

Craven et al. (1974a) Nicolas et al.

Estriol 16-hemisuscinate p-Hydroxymercuribenzoate Estrone

Sepharose 4B with 1,S-diaminopentane Chin and Warren Sepharose with ethylenediamine Nicolas (1974a) Sepharose 4B with aminocaproate Nicolas (1974b)

N6-(6-Arninohexyl)-S '-AMP 846-Aminohexy1)-amino-AMP or 8 46 aminohexy1)amino-NAD+ N6-(6-Aminohexyl)-5'-AMP or -NAD+

z*

Lowe et al. (1973b)

(Continued on p. 262)

z

0

c!

h,

z

TABLE 11.1 (continued) Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

References

Glucose 6-phosphate dehydrogenase (DGlucose 6-phosphate: NAD oxidoreductase. E.C. 1.1.1.49) Dehydrogenases (continued)

NADP

Cellulose Sepharose 4B with e-aminohexanoic acid Sepharose 4B with adipic acid dihydrazide Sepharose 4B with adipic or sebacic acid hydrazide Agarose-NADP Sepharose 4B Sepharose Sepharose 4B

Dean and Lowe De Flora et al.

Yoshida Biirgisser and Fauchere Kaplan era!. Lowe and Mosbach

Sepharose 4B

Brodelius et al.

Sepharose 4B

Lee and Kaplan

Sepharose 4B with bound L-glutamic acid 7-methyl ester Sepharose 4B with 6-hexanoic acid

Godinot et al.

Glucose-6-phosphate dehydrogenase - - from human erythrocyte

NADP

NADP+-N6-[N-(6-aminohexyl)acetamide] N646-Aminohexyl)adenosine 2‘,5’-bisphosphate

- - and glutamate dehydrogenase, glutathione reductase and 6-phosphogluconate dehydrogenase from crude Candida extract 8-(Aminohexyl)amino-TPN+ - - from yeast and human cry throcy tes Glutamate dehydrogenase (E.C. 1.4.1.3) GTP Glyceraldehyde 3-phosphate dehydrogenase (E.C. 1.2.1.12)

NAD+

6-Aminohexano y l-NAD' N646-Aminohexyl)-AMP Glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) D-3-Hydroxybutyrate dehydrogenase from crude extract of Rhodopseudo m n a s spheroides

1-Hexamethylenediamine analogue of DL-glycerol-3-phosphate 6-Phosphogluconic acid NAD+ e-Aminohexanoyl-NAD+

Benzamidohexyl derivative of Sepharose 4B Sepharose Sepharose 4B Sepharose 4B CNBr-activated agarose Sepharose 4B with 1,6-hexanediamine Sepharose 4B with 6-aminohexanoic acid Sepharose 4B

Lamed er al. (1973b) Wilchek and Lamed

Mosbach el al. (1972a, b) Barry and O’Carra (1973b) Hocking and Harris Comer et al. Mosbach (1974a) Mosbach et al. (1971) Mosbach ef al. (1972a) Holohan et al. McGinnis and de Vellis Lowe et al. (1973b) Mosbach (1974a)

15-Hydroxyprostaglandin dehydrogenase (15-hydroxyprostanoate oxidoreductase, E.C. 1.1.1.141) from dog lung 3a-Hydroxysteroid dehydrogenase ( 3 hydroxysteroid :NAD+-oxidoreductase, Ec. i.i.i.50) fromfieudomonas t estost eroni 7a-Hydroxysteroid dehydrogenase (E.C. 1.1.1.159) from Escherichia coli 17p-Hydroxysteroiddehydrogenase from Aeudomonas testosteroni 20p-Hydroxysteroid dehydrogenase (E.C. 1.1.1.53) Inosine 5'-monophosphate dehydrogenase (IMP: NAD oxidoreductase, E.C. 1.2.1.14) Isocitrate dehydrogenase L-Lactate dehydrogenase (E.C. 1.1.1.27) from ox heart - - from dogfish muscle - from human serum

NAD+

CH-Sepharose 4B

Ho and Towner

Glycocholic ~ acid

CNBr-activated Sepharose 4B with ethylenediamine

Aukrust et al.

Chenodeoxycholate

Sepharose 4B with ethylenediamine

MacDonald et al.

19-Nortestosterone-17-0hemisuccinate 1lor-Hydroxyprogesterone 1l-hemisuccinyl-bis-p-aminoethyl disulphide monoamide Guanosine 5'-monophosphate

Sepharose 4B with 3,3'diaminodipropylamine Agarose

Benson et al. (1974a)

Sepharose 4B with hexamethylenediamine and succinic anhydride

Krishnaiah

N6-(6-Aminohexyl)-adenosine5'monophosphate or -2' ,5'-bisphosphate N6-(6-Aminohexy1)-AMP

Sepharose 4B

Brodelius et al.

Sepharose

Ohlsson et al.

Sepharose 4B

Kaplan et al. Kaplan et al. Mosbach el al. (1972b)

-

- - (human)

846-Aminohexy1)amino-AMP

Sepharose 4B

P' 46-Aminohex-l-yl)-Pz45 'adenosy1)- Sepharose 4B pyrophosphate Sepharose 4B with hexamethylenedi6-Immobilized AMP analogue amine Sepharose 4B with 6-aminohexanoic NAD+ acid Sepharose 4B with hexamethylenediamine and diazotized p-aminobenzyl derivative

Sweet and Adau

Trayer et al. (1974b) Bachman and Lee Trayer et al. (1974b) Barry and O'Carra (1973a) Lowe et al. (1973a) Lowe and Dean (1973) Mosbach et 41. (1972b) Barry and O'Carra (1973b)

(Continued on p. 264)

m

TABLE 11.1 (continued)

P

Substances isolated

Affinity ligands

Solid supports or immobilized affinity b a n d s

References

Dehydrogenases (continuedj

E-A*iiinul*exanoyl-NAD+ Potassium oxalate

Sepliarose 4B Sepharose 4B with hexamethylenediamine Sepharose with hexamethylenediamine Aminohexyl-Sepharose 4B Sepharose Sepharose 4B

hlusbach ( 1 974,) Spielmann et al. (1973)

-

-

X from mouse testes

Potassium oxalate Oxamate

L-Lactate dehydrogenase from rat liver Blue Dextran and hepatoma Cibacron Blue F3G-A - - from kctobacillus casei Adenosine-5'-monophosphate - - isoenzymes N6-(6-Aminohexyl)-AMP Potassium oxalate

_ _ _ from rat tissue

Oxamate

Propyl lipoamide Lipoamide dehydrogenase (NADH: lipoamide oxidoreductase, E.C. 1.6.4.3) Lipoyl chloride from pig heart, yeast and Escherichia co li NAD' and lipoate - - isoenzymes from pig heart

Malate dehydrogenase

NAD+

NADP+-specific isocitrate dehydrogenase (threo-D-isocitrate: NADP' oxidoreductase (decarboxylating), E.C. 1.1.1.42) fromEscherichia coli - - - from Bombyx mori

NADP+

NADP (oxidized by periodate)

Sephadex G-200 N6-(6-Aminohexyl)-Sepharose Sepharose 4B Sepharose 4B with hexamethylenediamine Sepharose 4B with hexamethylenediamine Glass Aminoalkyl glass

Spielmann et al. (1976) Eventoff et al. Trommer and Becker Ryan and Vestling Gordon and Doelle Brodelius and Mosbach (1973a) Mosbach (1974b) O'Carra and Barry (1974) O'Carra et al. Don and Masters Scouten et al. Scouten

Visser and Strating Sepharose 4B with E-aminohexanoic acid and Sepharose 4B with ethylenediamine or L-lysine Sepharose 4B with hexamethylenediBarry and OCarra (1973b) amine and diazotized p-aminobenzyl derivative Agarose-hexane-nicotinamide Hy and Reeves adenine dinucleotide phosphate (Sigma) Sepharose 4B with adipic acid dihydrazide

Miake et al.

NADdependent cuglycerophosphate dehydrogenase NAD or NADP-liked dehydrogenases Nicotinamide nucleotidedependent dehydrogenases Pho sphogluconate dehydrogenase

Unsubstituted agarose

BioGel A-0.5 m

Bacchi et al.

NAD or NADP Chemically defined adenosine phosphate N6 -(6-Aminohexyl)adenosineS'-monophosphate or N'46-aminohexyl)adenosine 2',5'diphosphate Blue dextran

Cellulose Sepharose 4B

Lowe and Dean (1971) Trayer and Trayer (1974)

Sepharose 4B

Brodelius et al.

CNBr-activated Sepharose 4B

Thompson et al. (1975)

Sepharose 4B

Wermuth and Kaplan

Sepharose 4B with cadaverine dihydrochloride

Linder et al.

Agarose with ethylenediamine

Gauldie and Hillcoat

Sepharose with ethylenediamine Sepharose 4B with hexamethylenediamine Sepharose 4B with ethanolamine Sepharose 4B with eaminohexanoic acid Sepharose 4B

CheIlo et al. Kaufman

Proteins with the super-secondary structure called the dinucleotide fold (alcohol dehydrogenase, glyceraldehyde phosphate dehydrogenase, lactate dehydrogenase M, or H,, malate dehydrogenase, adenylate kinase, phosphoglycerate kinase, phosphoglyceromutase, ribonuclease, ferrocytochrome C) Pyridine nucleotide transhydrogenase 2'-AMP from Pseudomonasaerugimsa Oxaloacetate Succinate dehydrogenase (succinate : (acceptor)oxidoreductase, E.C. 1.3.99.1) from Micrococcus lysodeikticus Methotrexate Tetrahydrofolate dehydrogenase (5,6,7,8-tetrahydrofolate:NADP' oxidoreductase, E.C. 1.5.1.3) - from Llzlo leukaemia

L-Threonine dehydrogenase

Rero yllysine NAD+

Uridine diphosphate glucose dehydrogenase

pyrophosphate

P' -(6-Aminohex-l-yl)-PZ-(5'-uridyl)-

Pastore et al. Lowe et al. (1973b) Trayer and Trayer (1974)

Q\ t 4

(Continued on p. 266)

w QI m

TABLE 11.1 (continued) Substances isolated

Affinity ligands

Dehydrogenases (continued) Xanthine dehydrogenase (E.C. 1.2.1.37) Anti-xanthine dehydrogenase antibody from wild-type Drosophila melanogast er Deoxyribonuclease [deoxyribonucleate Single-stranded (heatdenatured) calf olionucleotidohydrolase, (E.C. 3.1.4.91 thymus DNA from bovine pancreas - from hog spleen Anti-hog spleen DNase I1 antibody - (Dnase) I1 from hog and bovine spleen RNA core - from testes and deferent ducts of the crab Gzncer pagurus (neutral) Lima bean protease inhibitor - through the removal of proteases - ATPdependent DNA Dicoumarol DTdiaphorase (E.C. 1.6.99.2) from rat liver Dopamine phydroxylase (3,rldihydroxy- Tyramine phenylethylamine, ascorbate :oxygen oxidoreductase, E.C. 1.14.17.1) Endo-p-(1-.4)AcetylgIuco~minidasefrom Chitin Staphylococcus aureus Endopolygalacturonase (poly-a-l,4-DPectic acid galacturonide glycanohydrolase, E.C. 3.2.1.15) from Aspergillus niger Enterokinase from human duodenal fluid p-Aminobenzamidine Enzymes acting on myo-inositol Enzymes of the metabolism of myoinositol phosphates C1-esterase from normal human serum Exoamylase from Pseudomoms stutzeri (1,4-a-D-glucan maltotetrahydrolase, E.C. 3.2.1.60)

Solid supports or immobilized affinity hgands

References

CNBr-activated Sepharose

Andres

Sepharose 4B

Schabort

Sepharose 2B Sepharose with c-aminocaproic acid

Schabort Ryder and Hodes Sabeur et al.

Sepharose 6B Agarose Sepharose 4B with bisamine propylamine and succinic anhydride p-Aminobenzoamidoethy 1-Sepharose 4B

Otsuka and Price Greth and Chevallier Rase et al.

Chitin

Leonenko et al.

Pectic acid cross-linked with epichlorh ydrin

Rexovi-Benkovi and TibenskL

Sepharose 4B with glycylglycine

DL4C[N-(ethylamino)aminomethyl]- Sepharose 4B with bound €-aminoepi-inositol Myo-inositol-2-phosphate

caproic acid Sepharose 4B with hexamethylenediamine Egg albumin in complex with antibody Sepharose 4B Dextrans Sephadex G-100

Aunis el al.

Grant and Hermon-Taylor (1976) Koller and HoffmannOstenhof Scheiner and Breitenbach Sumi et al. Dellweg et al.

m

X 9

5

r

rn

CA

% 2m % % z

z4

.e 0

2:

P

sr; 0

%

Flavin mononucleotide-dependent enzymes Riboflavin phosphate (NADPH-cytochromeC reductase, pyridoxine phosphate oxidase, glycolate oxidase) Flavokinase Flavin

Formiminotetrahydrofolate-cyclodeaminase (E.C. 4.3.1.4) a-L-Fucosidase @-1,4Galactanasefrom Bacillus subtilis

aGalactosidase (ceramide trihexosidase) from Cohn fraction IV-1 aGalactosidase (a-Dgalactoside galactohydrolase, E.C. 3.2.1.22)

a-and p-galactosidases

Tetmhydrofolate

Cellulose, cellulose phosphate or DEAE-cellulose

Arsenis and McCormick (1966)

CM-cellulose

Arsenis and McCormick (1964) Slavk et al. (1974)

Sepharose 4B with hexamethylenediamine Agaroser-aminocaproylfucosamine Bromoacetamidohexyl-Sepharose

Fucosamine Tetrasaccharide from the galactanase digest of the p-l,4galactan with p-(paminopheny1)ethylamine (reduced to secondary amine) Melibiose Sepharose with succinic anhydride p-Aminophenylmelibiose Affinose 202 Nr-Aminocaproyl-NraminocaproylSepharose 4B a-Dgalactopyranosylamine Sepharose 48 Nr-Aminocapro yla-Dgalactopyranosylamine or Nraminocaproyl-~ -~ N-caminocaproyliu-Dsalactop yranosylamine DGalactonoiu-lactone Sepharose 4B with bound benzidine Anti-pgalactoside antibody Sepharose 4B

Alhadeff el al. Labavitch et al.

Mapes and Sweeley (1972) Mapes and Sweeley (1973) Harpaz et al. Harpaz and Flowers

Kanfer et al. (1973) PGalactosidase (p-Dgalactoside galactoErickson and Steers hydrolase, E.C. 3.2.1.23) from Aerobacter cloaare - from Escherichia coli p-Aminophenyl-p-D-thiogalactopyrano-Sepharose 4B with 3-aminosuccinyl-3- Steers et al. side aminodipropylamine - from Aspergillus niger Woychik and Wondolowski Glass Sepharose 4B with 3,3’diaminodiSteers and Cuatrecasas propylamine and succinic anhydride Magnetic iron oxide with 3-aminoDunnill and Lilly propyltriethoxysilane and sebacic acid Sepharose 4B cross-linked with divinyl Robinson et al. (1974) - from Escherichia coli sulphone with 3,3diaminodipropylamine, succinic anhydride, ethylenediamine and succinic anhydride (Continued on p. 268)

t4 6,

TABLE 11.1 (continued) Substances isolated

00

Affinity ligands

pGalactosidase (continued) - from Aspergillus niger

Solid supports or immobilized affinity ligands

References

Baum

- wild type and some mutant proteins Glucoamylase (rabbit intestimal)

Antibody against &galactosidase Dextran

Akylamine CPG with malonic or azelaic acid Sepharose 4B Sephadex G-200

p-DGlucosidase Glucosylsphingosine p-Dglucosidase p-Glucuronidase (rat liver) (E.C. 3.2.1.31)

6 -DGluconolactone

Sepharose 4B with bound benzidine

p-Aminophen yl-Nacetyl-pDglucosaminide Saccharo-l,4-lactone

Sepharose 4B with 3,3'diaminodipropylamine and succinic anhydride Sepharose 4B with 3,3'diaminodipropylamine AH-Sepharose 4B CNBr-activated Sepharose 4 8

yGlutamy1 hydrolase Glycosidases from Takadiastase or soybean

Guanylate cyclase (GTP pyrophosphatelyase, E.C. 4.6.1.2) Hexosaminidases A and B (human) @2acetamido-2deoxy-Dglucoside acetamidodeoxy glucohydrolase, E.C. 3.2.1.30) 0-Hexosaminidase Hexosaminidases (pgalactosidase, 0-Nacetylglucosaminidase and a-mannosidase) . Histaminase (diamine oxidase, E.C. 1.4.3.6) from human pregnancy plasma Histidine decarboxylase (E.C. 4.1.1.22) from a mouse mastocytoma Histone phosphatase from human polymor phonuclear leucocytes

Hexa- and heptaglutamates Di-6-aminocaproyl-p-aminophenyl N-acetyl-i -thio-8-Dglucosaminide, p-Dglucoside, 0-Dgalactoside or a-D-mannoside Periodate-oxidized GTP

Melchers and Messer Sivakami and Radhakr ishnan Kanfer er al. (1974) Kanfer er al. (1974) Junowicz and Paris Harris et al. Silink et al. Mega and Matsushima

CNBr-activated Sepharose with adipic acid dihydrazide Sepharose 4B

Garbers

Antia-site bovine r g l o b u l i p-Aminobenzyl l-thio-2-acetamido-2deoxy-p-Dglucopyranoside ~Cadaverine (diaminopentane)

CNBr-activated Sepharose 4B Sepharose 4B with hexamethylenediamine treated with succinic anhydride Sepharose 4B

Vladutiu et al. Rafestin et al.

nOctyl glycidyl ether

Sepharose 4B

Hammar et al.

Histone

Sepharose 4B

Tsung et al.

2-Acetamido-N-(eaminocaproyl)-2deox y-p-Dglucopyrasylamine

Geiger et al.

Bayiin and Margolis

0 Q

P

ti 4

Hyaluronidase (hyaluronate glycanohydrolase, E.C. 3.2.1.35) from bull seminal plasma - (testicular) a-Isopropylmalate isomerase (yeast) (E.C. 4.2.1.33) A'-Ketosteroid isomerase from Pseudomonas testosteroni Kinases Adenosine 3',5'-monophosphatedependent protein kinase (adipose tissue) L-Arabinose kinases from Phaseolus aureus Creatine kinase (ATP: aeatine phosphotransferase, E.C. 2.7.3.2) Creatine phosphokinase (E.C. 2.7.3.2) Cyclic 3',5'-Amdependent protein kmase (E.C. 2.7.1.37) from rabbit skeletal muscle Cyclic GMPdependent protein kinase from calf lung Ethanolamine kinase and choline kinase (E.C. 2.7.1.32) from rat liver Glucokinase (rat hepatic) (E.C. 2.7.1.2)

- from a crude rat liver supernatant

Glycerolkinase (E.C. 2.7.1.30)

Concanavalin A

Sepharose 4B

Yang and Srivastava

Leucine or valine

Sepharose 6B Sepharose 4B

Balasubramanian et al. Bigelis and Umbarger

19-Nortestosterone 170-hemisuccinate

Sepharose 4B with 3,3'diaminodipropylamine

Benson et al. (1974a)

Histone or casein

Sepharose 2B

Corbin et al.

8+-Aminoethylthioadenosine triphosphate

Sepharose 4B with 3,3'diaminodipropylamine and succinic anhydride and N-hydroxysuccinimide Sepharose with ethylenediamine

Chan and Hassid

p-Mercuiibenzoate

Madeliin and Warren

pChloromercuribenzoate Casein Ns -(2-Aminoethyl)adenosine-3'3'mono phosphate Blue dextran

Aminoethyl-Sepharose Sepharose 4B Sepharose 4B

Boegman Reimann et al. Dills et al.

Sepharose 4B

Kobayashi and Fang

Choline

Epoxy-activated Sepharose 6B

Brophy and Vance

Glucosaminewith 6-aminohexanoic acid CNBractivated Sepharose 4B N-(6-Arninohexanoyl)-2-amino-Z Agarose deoxy-D-glucopyranose N-(e-Aminocaproyl)-2-amino-2-deoxy- Sepharose 4B Dglucop yranose 8-(6-Arninohexyl)amino-ADP Sepharose 4B ATP Sepharose 4B N-(6-Aminohexanoyl)-2-amino-2Sepharose 4B deoxy-Dglucose ATP Sepharose 4B with 6-aminohexanoic acid

Chesher et al. Holroyde and Trayer Barker et al. (1974a) Trayer et al. (19746) Azzar et al. Holroyde et al. Lowe et al. (1973a)

(Continued on p. 270)

TABLE 11.1 (continued) Substances isolated Kinases (continued) Kinases (pyruvate, creatine, glycero-, hexo- and 3-phosphoglycerate) Phosphofructokinase (E.C. 2.7.1.1 1) (yeast) - from brewer's yeast from thermophilic microorganisms

-

Affinity ligands

Solid supports or immobilized affiiity ligands

References

N6-(6-Aminohexyl)5'-AMP or -NADC

Sepharose

Harvey et al.

Blue dextran Cibacron Blue F3G-A

Polyacrylamide gel (cross-linked) Sephadex G-200 Sepharose 4B Sepharose 4B

Kopperschlager et al. Bohme et al. Tamaki and Hess Hengartner and Harris Comer et al. Ramadoss et al.

N646-Aminohexyl)-AMP

- (mammalian)

N6-[(6-Aminohexyl)carbamoylmethyl]-CNBr-activated Sepharose 4B

Phosphorylase kinase (E.C. 2.7.1.38)

ATP Phosphorylase b

CNBractivated SeDharose with 1-amino- Jennissen et al.

6-(bromoacetamido)hexane T, polynucleotide kinase from Escherichiu coli Protamine kinase Pyridoxal kinase (ATP :pyridoxal 5'phosphotransferase, E.C. 2.7.1.35) F'yruvate kinase (ATP: pyruvate phosphotransferase, E.C. 2.7.1.40) Succinate thiokinase from pig heart(Co Adependent enzyme) Thiamine pyrophosphokinase (E.C. 2.7.6.2) Thvmidine kinase (ATP: thvmidine 5 ' phosphotransferase, E.C. 2.7.1.75 from Escherichia coli - from rat colon adenocarcinoma @-Lactanuse(penicillin amidolactamhydrolase, E.C. 3.5.2.6) - from Bacillus cereus

Singlestranded DNA

4% agarose (for electrophoresis)

Schaller et al.

8-(6-Aminohexyl)amino cyclic AMP

Sepharose 4B BioGel A-l.5m

Jergil et al. Jergil and Mosbach Neary and Diven

Cibacron Blau

Sephadex (3-200

Roschlau and Hess

N646-Aminohexyl)-3' ,5'-ADP

Epoxy-activated Sepharose 6B

Barry et al.

Thiamine monophosphate

Sepharose 6B with ethylenediamine

Wakabayashi et al.

5'-Aminod 'deoxythymidine

Sepharose 4B

Rohde and Lezius

Thymidine-3'44-aminopheny l-

CH-Sepharose 4 8

Kowal and Markus

Indubiose ACA-314 Indubiose A4 Sepharose 4B with phloroglucinol, divinyl sulphone and ethylenediamine

Le Goffic et al. (1973a) Le Goffic et al. (1975) Coombe and George

phosphate) Ampicillin Cephalosporin c Methicillin

Lipases Lipase from Pseudomonas nuphitica var. Hydrophobic ligands lypolytim Lipoprotein lipase (EX. 3.1.1.34, Heparin glycerol ester hydrolase)

Phospholipase C (phosphatidylcholine choliiephosphohydrolase, E.C. 3.1.4.3) from Clostridium perfringens Triglyceride lipase Lipoxygenase (linoleate: 0, oxidoreductase, E.C. 1.13.11.12) Lysozyme (E.C. 3.2.1.17)

CNBr-activated Sepharose 4B

Kosugi and Suzuki

Sepharose 4B

Olivecrona er al. Egelrund and Olivecrona Iverius et al. Etienne er al. (1974) Etienne et al. (1976) Ganesan and Bass Takahashi er al. (1974b) Little et al.

Egg-yolk lipoprotein

Sepharose 4B

Heparin Linoleic acid

Sepharose Aminoethylagarose

Ganesan and Bass Grossman et al.

Chitin

Chitin

- from hen egg-white and turnip

Carboxymethyl chitin Deaminated chitin

Chitin-coated cellulose Carboxymethylchitin Glumchitin

Lysozyme-like enzymes Luciferase (bacterial)

Lysozyme lysate of bacterial cell wall p-Aminophenyl-N-acetyl-0-Dglucosaminide Chitin Flavin mononucleotide

Cherkasov and Kravchenko (1968) Pryme er al. Jensen and Kleppe Imoto and Yagishita (1973a) Imoto et al. Cherkasov and Kravchenko (1969) Yoshimoto and Tsuru Junowicz and Charm (1975)

-T4

Maleylacetone cis-trans-isomerase from Vibrio 01 a-Mannosidase (E.C. 3.2.1.24)

NADPdependent malic enzymes

CNBr-activated Sepharose 4B Sepharose 4B with 3,3’diaminodipropylamine and succinic anhydride Chitin-coated cellulose Sepharose 6B with 6-aminohexanoic acid Sepharose with N-(aminohexy1)Glutathione maleamate p-Aminobenzyl l-thio-2-acetamido-2- Sepharose 4B with hexamethylenedideox y-0-D-glucopyranoside amine and succinic anhydride CH-Sepharose 4B Mannosylamine N6-(6-Aminohexyl)adenosine-2’,5’di- Sepharose 4B phosphate

Imoto and Yagishita (1973b) Waters et al. Morrison et al. Rafestin et al. Robinson et al. (1975) Yeung and Carrico

(Continued on p . 272)

h)

I .

c

TABLE 11.1 (continued) Substances isolated

Neuraminidases Neuraminidase (sialidase, N-acetylneuraminate glycohydrolase, E.C. 3.2.1.18) from Vibrw cholerae, Oostridium perf~ngensand influenza virus - from Cbstridium perfringens - from Clostridiurn perfringens - from Vibrio cholerae

- from Clostridium perfringens Nicotinamide nucleot ide transhydrogenase (E.C. 1.6.1.1) from Pseudomonas aeruginosa Nucleases Endonuclease from mammalian cells Exonuclease I11 from Escherichia coli - 1 from Escherichia coli - from Staphylococcus aureus - from S.marcescens

-

(tobacco extracellular)

- S , from takadiastase

4 # #

Affinity ligands

Solid supports or immobilized affinity ligands

N-(4-Aminophenyl)oxamic acid

Sepharose 4B with glycylglycyltyrosine Ckatrecasas and llliano (197la, b)

2-Aminoethylamide of the 2-hydroxy- Sepharose 4B ethyl a-ketoside of Nacetylneuraminic acid or 2-amincethylaminocarbonylmethyl a-ketoside of N-acetylneuraminic acid Sepharose 4B a,-Acid glycoprotein containing glycosidically linked sialic acid N6-(6-Aminohexyl)adenosine-2‘,5’di- Sepharose 4B phosphate

DNA Single-stranded DNA Denaturated DNA 3’-(4-Aminophenylphosp hory1)thy midine-S’-phosphate Nucleic acids

Periodate-oxidized NADP Single-stranded DNA

Cellulose 4% agarose (for electrophoresis) Cellulose CNBr-activated Sepharose

References

Den et al. Rood and Wilkinson Holmquist

Geisow Hojeberg et al.

Caputo et al. Schaller et al. Ray et al. Wilchek and Gorecki (1974)

Sephadex G-75 with s-triazine chloride, Kurinenko et al. hexamethylenediamine and s-triazinechloride once again Janski and Oleson Sepharose 4B with adipic acid dihydrazide Slor Cross-linked copolymer of acrylamide

I

Staphylococcal nuclease - - treated under various conditions with the affinity labelling Old yellow enzyme (NADPH oxidoreductase, E.C. 1.6.99.1) Oxidases Aldehyde oxidase (E.C. 1.2.3.1)

- - from wild-type Drosophikr melanogaster Amine oxidase (monoamine :0, oxidoreductase (deaminating), E.C. 1.4.3.4) from bovine aorta - - from Aspergillus niger Cytochrome oxidase Galactose oxidase (Dgalactose: oxygen 6-oxidoreductase, E.C. 1.1.3.9) Glucose oxidase

- - from Penicillium vitale Glycolate oxidase Phenoloxidase from larvae of housefly Pyridoxine-5‘-phosphateoxidase Pyruvate oxidase (pyruvate: cytochrome b, oxidoreductase, E.C. 1.2.2.2) from Escherichia coli Xanthine oxidase

- - (milk) - - from rat liver or bovine milk 3-Oxosteroid A4 -As -isomerase (E.C. 5.3.3.1) from Pseudomonas testost eroni

3’-(4-amino-phenylphosphoryl)-deoxy Sepharose 4B thymidine 5’-phosphate

Cuatrecasas et al. (1968)

Thymidine 3’+aminophenyl phosphate)-S’-phosphate 4-Acetoxybenzoic acid

Cuatrecasas et al. (1969) Dunn and Chaiken (1974, 1975) Abramovitz and Massey

N-Benzyl-6-methylnicotinamide

Sepharose 4B BioCel A5m with hexamethylenediamine

Anti-aldehyde oxidase antibody

Sepharose 4B with a series of diamino- Chu and Chaykm alkanes CNBr-activated Sepharose Andres

Concanavalm A

Sepharose 4B

Shieh et al.

r A

0

r

50

2:

%

5

5

ij 5.

r r

4 5.

n el

w

Hexamethylenediamine Cytochrome C Agarose

CNBractivated Sepharose 4B CNBr-activated Sepharose 4B Sepharose 6B

Toraya et al. Ozawa et al. Hatton and Regoeczi (1976b)

s

v1

SJ

2z

c3

Concanavalin A

Specific antibodies Flavin mononucleotide NH, -(CH,), -NH-CO-CH, -NHC,H,-COOH Flavine mononucleotide Ethanolamine and thiamine p yrophosphates

Polymer of concanavalin A using glutaraldehyde as the polymerizing agent Sepharose 4B Cellulose Sepharose 4B DEAEcellulose Sepharose 4B with hexamethylenediamine and succinic anhydride

Sepharose 4B 9-(p-Aminoethoxyphenyl)guanine 3-1 1-H-Pyrazolo(3,4d)pyrimidin-4-y1- Sepharose 6B amino]-1-propyl-6-aminohexaoate Sepharose 4B 9-(p-p-Aminoethoxyphenyl)guanine 19-Nortestosterone-17-0-hemiSepharose 4B with diaminodipropylamine succinate

Avrameas and Guilbert

M

v1

Valiulis et ai. Kazarinoff et al. Shimoda et al. Kazarinoff et al. O’Brien et al.

Baker and Siebeneick Edmondson et al. Siebeneick and Baker Benson et al. (1974b)

(Continued on p. 274)

h)

4 W

h,

4 P

TABLE 11.l [continued) Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

References

Penicillinase from B, licheniformis Peroxidase (E.C. 1.1 1.1.7)

Cephalosporin c Concanavalin 4

Sepharose 4B Polymer of concanavalin A using glutaraldehyde as the polymerizing agent BioGel P-300 with glutaraldehyde Con A-Sepharose

Crane et al. Avral~~aas and Guilbert

Antibodies - (horseradish) Concanavalin A - (horseradish) Phenylalanine hydroxylase (E.C. 1.14.16.1) 2-Amino-6,7dimethy14-hydroxy-

CHSepharose 4B

Ternynck and Avrameas Wagner Brattain et al. Cotton

5,6,7,8-tetrahydropteridine - - from Mocaca irus L-Phenylalanine :tRNA ligase from E. coli

L-Phenylalanine

Concanavalin A Plasma-membrane enzymes in bile (alkaline phosphatase, 5'-nucleotidase, alkaline pho&hodiesterase I and Lleucine 6-naphthylamidase) Phosphatases Alkaline phosphatase (orthophosphoric L-Phenylalanine monoester phosphohydrolase, E.C. 3.1.3.1) from human placenta Anti-alkaline phosphatase antibody Alkaline phosphatase (human placental) Immunoglobuli fraction of the antiplacental alkaline phosphatase antisera - - (calf intestine) 4-(p-Aminophenylazo)phenylarsonic acid - - from human liver (1) Concanavalin A; (2) diazonium salt of 4-(p-aminophenylazo)phenylarsonic acid Concanavalin A 6-((3-Carboxy4-nitropheny1)thiol-9Membrane-bound F, adenosine triphosphatase (E.C. 3.6.1.3) from 0-D-ribofuranosyl Micrococcus Sp. ATCC 398E

Cotton and Grattan Sepharose 4B with E-aminocaproicacid Forrester and Hancock methyl ester, hydrazine and €aminocaproic acid methyl ester once again Sepharose 4B Holdsworth and Coleman

g* 5 r

m

V1

LJ

Sepharose 4B

Doellgast and Fishman

Sepharose 2B Sepharose 2B

Pitarra et al. Hoag et al.

Sepharose 4B with tyramine

Brenna et al.

(1) Sepharose 4B; (2) tyraminylSepharose 4B

Komoda and Sakagishi

Con A-Sepharose Sepharose 4 8 with iminobispropylaminyl-N-acetylhomocysteine

Trdpanier et al. Hulla et al.

v

2 i

2:

7 4 C3 3:

z

5 0

?2

Phosphorylase phosphatase (E.C. 3.1.3.17) Phosphoprotein phosphatase from bovine adrenal cortex Phosphodiesterase

Brandt et al.

Hexamethylenediamine

Sepharose

ATP

Sepharose 4B with E-aminocaproicacid Ullman and Periman

044-Aminopheny1)-0'-phenylthio-

Sepharose 4B

Glucan-synthesizingphosphorylase (E.C. 2.4.1.1) isozyme from Oscilhtoria princeps Glycogen phosphorylase (EC 2.4.1.1)

- - from human polymorphonuclear leukocytes - - from swine adipose tissue Maltodextrin phosphorylase from E. coli (E.C. 2.4.1.1) Phosphorylase b Polynucleotide phosphorylase (E.C. 2.7.7.8) from Escherichiu coli - - from Escherichia coli and Bacillus stearothermophilus Polygalacturonase Polymerases DNAdependent RNA polymerases from calf rhymus and rat liver - - - A from murine myeloma DNA-polymerase (E.C. 2.7.7.7) from avian myeloblastosis virus - - from Escherichia coli - - I from Escherichiu coli - - I from Escherichia coli

$

5

0

z

Eckstein and Frischauf

phosphate Phosphorylases ADPglucose pyrophosphorylase

t;

%

m,

P' 46-phospho-1-hexy1)-Pz46-amino-1- Sepharose 4B hexy1)pyrophosphate Concanavalin A Con A-Sepharose

Haugen et al.

0 L0

Fredrick

C,

m y iamine Alkanes 5'-AMP

Er-El et al. Shaltiel Sdrensen and Wang

2

F t4

Sepharose 4B Sepharose 4B 5'-AMP-Sephasose

N6-(6-Aminohexy1)adenosine-5'-mono-Sepharose 4B phosphate N-p-Sulphamylphenyl glycoside Sepharose 4B

Thanner et al.

Butylamine Polyadenylic acid

Sepharose Sepharose

Jennissen and Heilmeyer Lehrach and Scheit

p-Aminophenyl oligoxythymidylate

Sepharose 4B

Smith and Eaton

Sodium polygalacturonate

Sodium polygalacturonate cross-linked Foglietti et al. with epichlorohydrin

Denatured calf thymus DNA

Sepharose

Weaver et al.

Polyadenylic acid Polycytidylic acid

Sepharose Sepharose 4B

Hall and Smuckler Marcus er al.

Oligomer of deoxyribothymidylate DNA

Cellulose Sepharose 4B Cellulose

Jovin and Kornberg Amdt-Jovin et al. Uyemura and Lehman

b

=!

is

Miller et al. (1975)

L 1

(Continued on p. 276)

%

TABLE 11.1 (continued) Affinity liiands

Substances isolated

Polymerases (continued) - -- from Micrororcus

- - from chick embryo - - (nuclear and mitochondriai) - - of cellular and viral origin - - I and I1 from Escherichia coli Polyuridylic acid polymerase from tobacco leaves RNAdependent DNA polymerase

- - from RNA tumour viruses

RNA polymerase (E.C. 2.7.7.6) from Escherichia coli - - from Escherichia coli - - from Escherichia coli - - from Escherichia coli Prolyl hydroxylase [prolyl-glycyl-peptide, l-oxoglutarate: oxygen oxidoreductase(4hydroxylating), E.C. 1.14.1 1.21 Proteases, peptidases and their zymogens Acid proteinase from Aspergillus awamori (awamorin)

- - from Aspergillus saitoi

References

Acr ylamlde Sepharose 4B Cellulose

Litman Cavalieri and Carroll Poonian et al. Lynch et al.

Hydrazine-Sepharose

Chirikjian et al.

Cellulose CMcellulose Sepharose 4B 4% agarose (for electrophoresis) Sepharose 2B

Brunet al. Potuzak and Wintersberger Joseph et al. Schaller et al. Brishammar and Junti

Oligodeox ythymidylic acid (chain length 12- 18 nucleotides)

Cellulose

Gerwin and Milstien

Anti-polymerase antibody Single-stranded DNA

Sepharose 4B 4% agarose (for electrophoresis) 2% agarose Sephadex G-200 Sepharose 2B Sepharose 4B Sepharose 4B

Weissbach and Poonian Livingston et al. Schaller et al. Niisslein and Heyden Rickwood Arndt-Jovin et al. Sternbach et al. Tuderman

Sepharose 4B

Stepanov et al. (1974)

Sepharose 4B

Stepanov et al. (1975)

lrrtelrs

- - from HeLa cells - - 7.1 .s from regenerating liver nuclei and cytosol

-

Solid supports or immobilized affinity ligands

Pyran copolymer (= divinyl ether of maleic anhydride) Denatured DNA Polynucleotides Single-stranded DNA Yeast RNA

DNA Heparin Poly(L-proline)

Methyl esters of e-aminocapronyl-Dphenylalanine or of e-aminocapronylL-pheny lalanyl-D-phenylalanine Mono-N-DNP-hexamethylenediamine hydrochloride N,O-Dibenzyloxacarbonyl-L-tyrosine

Takeuchi et al.

- - from species of Mucor Acrosin (trypsin-like enzyme) from boar spermatozoa

D-Alanine carboxypeptidase

Concanavalin A Soybean trypsin inhibitor Gly -Gly -Tyr(0-benzyl) -Arg Benzamidine 6-Aminopenicillanicacid

Con A-Sepharose Bowman-Buk soybean trypsin inhibitor cellulose (Merck)

Rickert and McBride-Waren Fink er al. (1972)

Agarose-Gly-Gly-Tyr(0-benzy1)-Arg

Garner and Cullison Schleuning er al. Blumberg and Strominger (1972) Blumberg Blumberg and Strominger (1974) Gorecki ei al. (1974, 1975) Fujiwara and Tsuru

Aminoalkylcellulose Sepharose with 3,3’diaminopropylamine and succinic anhydride

- - from Bacillus subtilis - - from Escherichia coli Alkaline protease (E.C. 3.4.21.14) from Bacillus subtilis - - from Aspergillus oryzae Aminopeptidase from Streptomyces giseus - from Aeromonas proteolytica

p -Aminobenzylpenicillin Carbobenzoxy-L-phenylahyl-DLleucine Ovoinhibitor 1,CDiaminohexane

Sepharose 4B Sepharose 4B with triethylenetetramine and succinic anhydride Sepharose Sepharose 4B

N-(3-Amino-5-methyl-Z~xohexyl)-

Aminomethylcellulose

Kettner et al.

Spheron 300 with hexamethylenediamine Sepharose 4B with hexamethylenediamine

Turkovi et al. (1976b)

Sepharose 4B

Sato et al.

Sepharose 4B Aminoalkylsilylglass Aminoethylcellulose Sepharose 4B Sepharose 4B

Reeck et al. Robinson et al. (1971a) Uren Reeck et al. (1971a) Cuatrecasas et al. (1968)

Sepharose Aminoethylcellulose

Sokolovsky and Zisapel Uren

succinamic acid D-leucine

- from Aspergillus flavus Angiotensin I-converting enzyme (peptid yldipeptide hydrolase, E.C. 3.4.15.1) from rabbit lung Bacillus subtilis SO4 alkaline protease

Hippurylhistidylleucy1 OH

Microbial alkaline protease inhibitor SSI e-Amino-ncaproyl-D-tryptophan Carboxypeptidases A and B Carboxypeptidase A (peptidyl-L-amino- Glycyl-D-phenylalanine acid hydrolase, E.C. 3.4.12.2) e-Aminocaproyl-D-tryptophan Carboxypeptidase B (peptidyl-L-lysine L-Tyrosyl-D-tryptophan hydrolase, E.C. 3.4.12.3) D-Alanyl-L-arginine Glycyl-D-phenylalanine or glycyl-Darginine D-Tryptophan - from activated bovine pancreatic juice ~

t;

$

s

0 2:

% r

0

2

c)

F r 4

;P

c1

Feinstein and Gertler Vosbeck etal. (1973a, b)

Nishimura el al.

Sepharose 4B with eaminocaproic acid Sokolovsky (1974)

~~

(Continued on p. 278)

2

s

TABLE 11. I (continued) Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

References

Antiserum to carboxypeptidase G-1 1-Aminophenylmercury(11)acetate 1-Aminophenylmercury(I1)acetate Haemoglobin

Sepharose 2B Sepharose 4B Sepharose 4B Sepharose 4B or BioGel A-5m

Cornell and Charm Keilova' and TomiSek (1973) Keilova'and TomiSek (1975) Smith and Turk

e-Aminocaproyl-D-tryptophan methyl ester

Sepharose 4B

Cuatrecasas el al. (1968)

Sepharose 4B

Shaw et al. Brodelius and Mosbach (1973b) Tomlinson et al. Bergeron and De Medicis

Proteases (continued) -

G-1

Cathepsin B, (E.C. 3.4.22.1) Cathepsin C (E.C. 3.4.14.1) Cathepsin D (E.C. 3.4.23.5) from bovine spleen and thymus aChymotrypsin (E.C. 3.4.21.1) - from the papain digest of ch ymotr ypsinogen

N-(e-Aminocaproy1)-D-tryptophan methyl ester or phenyl-4-butylamine 4-Phenylbutylamine Glycyl-D-pheny lalanine Carbobenzoxy-L-pheny lalanine Benzylox ycarbonylglycyl-D-phen ylalanine or -D-leucine Benzylchloride

aChymotrypsin and trypsin from pancreas extract

N-Acryloyl-6-aminohexanol- and Nacryloyl-4-aminobutyryl derivatives of a-azaphenylalanine phenyl ester Turkey ovomucoid Trypsin inhibitor Microbial alkaline protease inhibitor s-s1 Soybean trypsin inhibitor

Sepharose 4B Aminoethylcellulose Sepharose 4B with triethylenetetramine Spheron 300 with hexamethylenediamine Sepharose 2B with 2,3dibromopropan-1-01 Polymers based o n polyacrylamide

Barker et al. (1974b)

Sepharose 2B Spheron 300 Sepharose 4B

Feinstein (1970b) Turkovi et al. (1973) Sat0 et al.

6%agarose

Porath

Tomlinson er al. Uren Fujiwara et 01. Turkovi et al. (1976a)

Chymotrypsinogen A and X, chymotrypsin, serum albumin, ovalbumin, p-lactoglobulin and lysozyme Chymotrypsin-like enzymes Clostridium histolyticum collagenase (clostridiopeptidase A, E.C. 3.4.24.3) Clostripain (clostridiopeptidase B, E.C. 3.4.22.8) from Clostridium histolyticum Collagenase A (clostridiopeptidase A, E.C. 3.4.24.3) from Clostridium histolyticum Collagenase (human skin, rheumatoid synovial and tadpole) - (vertebrate) - (mouse bone) Endogenous blackeyed pea protease Enterokinase (porcine, E.C. 3.4.21.9)

n-Alkylamines of varying chain length

Sepharose 4B

Hofstee (1973a, b)

4-Phenylbutylamine Alkali-treated elastin

Sepharose 4B Alkali-treated elastin

Stevenson and Laudman Serafini-Fracassiniet al.

Butylenediamine

Sepharose 4B

Kula et al.

Heptylenediamine

Sepharose 4B

Kula et al.

Collagen

Sepharose 4B

Bauer et al. (1971a, b)

Heparin Kunitz soybean inhibitor p-Aminobenzamidine

CNBr-activated Sepharose 4B Soybean-CMcellulose affinity resin Sepharose 4B with glycylglycine

- - free of intestinal aminopeptidase activity Ficin (EX. 3.4.22.3) Human gastric proteases (pepsinogen I group) Insulin-specifk protease Kallikrein and plasmin Kallikrein (E.C. 3.4.21.8) (porcine and human) - (porcine)

Concanavalin A

Con A-Sepharose

Eisen et al. Sakamoto et al. Gennis and Cantor Grant and Hermon-Taylor (1975) Barns and Elmslie

p-Aminophenylmercury(I1) acetate Anti-pepsinogen I group antibodies

Sepharose 4B-200 Sepharose 4B

Anderson and Hall Zoller et al.

Insulin Trypsin-kallikrein inhibitor Kunitz soybean inhibitor

Duckworth et al. Agarose Copolymer of maleic acid and ethylene Fritz et al. (1969a) CM-cellulose Fritz et al. (1972~)

Guanidinated trypsin-kallikrein inhibitor Carbobenzoxy-L-phenylalanyl-DLleucine Carbobenzoxy-L-phenylalanine

CM-cellulose

Fritz and Forg-Brey

Sepharose 4B with triethylenetetramine and succinic anhydride Sepharose 4B with triethylenetetramine AH-Sepharose 4B

Fujiwara and Tsuru

Neutral (E.C. 3.4.24.4) protease from Bacillus subtilis Neutral and alkaline proteases from Bacillus subtilis Neutral metalloendopeptidases from B. subtilis var. amybsacchariticus

Phosphoamidone

Fujiwara et al. Komiyama et al.

(Continued on p. 280)

TABLE 11.1 (continued)

__

Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

PrUteASeS (continued) - from Streptomyces griseus K-1 - from Clostridium histolyricurn - from Bacillus subtilis

Clycylleucine Acetyl-D-phenylalanine

Sepharose 4B with c-aminocaproic acid Sepharose 4B with ethylenediamine, succinic anhydride and triethylenetetramine Sepharose 4B with triethylenetetramine Sepharose 4B

- from Bacillus subtilis Papain (E.C. 3.4.22.2)

Glycyl-D-phenylalanine Gly cylglycy 1 4 0 - b e ny1)-L-t yrosy 1-Larginine p-Aminophenylmercury(I1)acetate

References

Komiyama et al. Sparrow and McQuade Pangburn et al.

Walsh et al. (1974) Blumberg et al. (1969,1970)

- from dried papaya latex

Glutathione-2-pyridyl disulphide

Sepharose 2B

Pepsin (E.C. 3.4.23.1) - (swine)

Poly-L-1ysine Methyl esters of e-aminocaprony1-Dphen ylalanine or of e-aminocaprony1-Lpheny lalany l-D-p henylalanine Pepsin inhibitor from Ascaris Iu mbricoides p-Aminophenylmercury(l1)acetate Mono-N-DNP-hexamethylenediamine hydrochloride Anti-pepsinogen I1 antibodies

Sepharose 4B Sepharose 4B

Sluyterman and Wijdenes (1970,1974) Brocklehurst et al. (1973, 1974) Nevaldine and Kassell Stepanov et al. (1974)

Sepharose 4B

Keilovi et al.

Sepharose 4B Sepharose 4B

Keilovi and Kostka (1975) Stepanov et 01. (1975)

Sepharose 4B

Matzku and Rapp

Sepharose 4B

Deutsch and Mertz (1970a, b)

BioGel P-300 with hydrazide

Summaria et al. (1972, 1973) Rickli and Cnendet

- (chicken) - from chicken forestomachs Pepsin and pepsinogen

Pepsinogen I1 group from gastric mucosal extract L-Ly sine monohydro chlor ide Plasminogen (zymogen of the proteolytic enzyme plasmin, E.C. 3.4.21.7) from human plasma - from human plasma fractions Ill and l1I2,+, pooled plasma, serum, plasma euglobulin - from human plasma - (human) DL-Lysine

Sepharose 4B

L-Lysine

Sepharose Sepharose 4B

Butyl p-aminobenzoate (Butesin) Butyl p-aminobenzoate

Sepharose 4B Sepharose 4B

Lysine

Sepharose

e-Aminocaproyl-L-alanyl-Lalany1-Lalaniie L-Arginine monohydrochloride e-Aminocaproyl-D-tryptophanmethyl ester Haemaglobin N-Acetyl-D-arginine Soybean trypsin inhibitor and ovomucoid

CNBr-activated Sepharose

Liu and Mertz Brockway and Castellino Walther et al. Nedkov Chibber et al. Chibber et al. Zolton et al. Zolton and Mertz Hatton and Regoeczi (1974,1975) Janoff

Sepharose 4B Sepharose

Suzuki and Takahashi Reeck and Neurath

Sepharose 4B AH-Sepharose 4B Polymer of affiiants using glutaraldehyde as the polymerizing agent

Chua and Bushuk Pacaud Avrameas and Guibert

Elastin Anti-prothrombin immunoglobulin fraction e-Aminocapro yl-D-phenylalanine methyl ester yOligo-Lglutamate Synthetic y-oligoglutamylpeptides Pepstatin (Nacylated pentapeptide from Actinomycetes with structure: isovaler yl-L-valyl-L-valyl4-amino-3hydroxyd-methylheptanoyl-l-alanyl4-amino-3-hydrox yd-methylheptanoic acid)

Cellulose Sepharose 4B

Legrand et al. Wallin and Prydz

Sepharose

Morihara and Tsuzuki

Sepharose 4B Sepharose 4B Aminoethyl BioGel P-150

Saini and Rosenberg (1973) Saini and Rosenberg (1974) Murakami et al.

- from plasma Plasminogen (human) - (bovine, human and sheep) - (rabbit) PMN lysosomal elastase Prekallikrein Procarboxypeptidase B from Botopterus aethiopicus Proteases from malted wheat flour Protease I1 from Escherichia culi Proteolytic enzymes from beef pancreatic extracts - from human blood platelets Prothrombin (bovine)

Aeudomonas aeruginosa elastase Pteroyl oligor-L%lutamyl endopeptidase from chick intestine Renin (E.C. 3.4.99.19)

Sepharose with hexamethylene diamine Devaux et al. (1973) Sepharose 4B with hexamethylenediDevaux et al. (1976) amine (or other spacers) (Continued on p. 282)

w

m

TABLE 11.1 (continued)

t 4

Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

Proteases (continued) - from hog kidney - (human and hog)

N-Hydroxysuccinyl-Pepstatin

Sepharose with hexamethylenediamine Corvol et al. Murakami and lnagami Sepharose 4B with 3,3’diaminodiMajstoravich et al. propylamine and succinic anhydride

- (hog) - from canine plasma Sorghum acid protease Stem bromelain Streptococcal proteinase Streptomyces griseus trypsin

Subtilisin-like enzyme from carboxypeptidase preparations Sulphydryl-protease from beans Thermolysin (E.C. 3.4.24.4 group)

Leucylleucylvalyltyrosine methyl ester and leucylleucylvalylphenylalanine methyl ester His- Pro -Phe- Leu-D-Leu-Val-Tyr Goat anti-renin rG globulins DGlutamyl-Dglutamic acid N-(c-Aminocaproy1)-D-tryptophan methyl ester p-Aminophenylmercury(l1) acetate 1,L-Diaminohexane Oligopeptides containing L-arginine as carboxyl termini obtained from a tryptic digest of protamine Tryptic digest of salmine Anti-subtilisin antibody Mercury derivative of methacrylanilide (Hg-MAA) Acet yl-D-phenylalanine

Glycy 1-D-phenylalanine

Thrombin (bovine, E.C. 3.4.21.5)

Carbobenzoxy-L-phenylalanyl-DLleucine Phosphoramidone (N-(a-L-rhamnopyranosylox yhydroxyphosphiny1)-Lleucyl-L-tr yptophan e-Aminocaproyl-p-chlorobenzamide HC1

-

References

CNBr-activated Sepharose 4B Sepharose 4B Sepharose 4B Sepharose 4B

Poulsen et al. Walsh et al. (1976) Virupaksha and Wallenfels Bobb

Sepharose 4B Sepharose 4B Sepharose 4B

Kortt and Liu Vosbeck et al. (1973) Kasai and lshii (1975)

Sepharose 4B Cellulose

Yokosawa et al. Stone and Williams

Hydroxyalkyl methacrylate gel with 15% MAA Sepharose 4B with triethylenetetramine, succinic anhydride and triethylenetetramine Sepharose 4B with triethylenetetramine Sepharose 4B with triethylenetetramine and succinic anhydride AH-Sepharose 4B

Turkovi et al. (1975)

BioCel 1.5 -m

P

Pangburn er al.

Walsh et al. (1974) Fujiwara and Tsuru Fujiwara et al. Komiyama et al.

Thompson and Davie

n

2: ;o

B

p-Aminobenzamidine m-Aminobenzamidine Thrombins (human, rabbit and bovine) - (human, E.C. 3.4.21.5) Thrombin-like enzyme from Bofhrops atmx snake venom IY- and P-trypsin (E.C. 3.4.21.4) Trypsin

- 6-

- P0-Trypsin (bovine, completely free of a-trypsin) - (a,P , $1 - (a,P )

-

from starfish Pisosrer brevispinus

- and related enzymes - in complex with dinitrophenylated soybean trypsin inhibitor

L-Lysine pChlorobenzylamine p-Aminobenzamidine p+'-aminophenoxypropoxy)benzamidine p-Aminophenylguanidine or p-aminobenzamidine or m-aminobenzamidine Ovomucoid Chicken ovoinhibitor Soybean trypsin inhibitor or potato inhibitor Soybean trypsin inhibitor (STI)

Ovomucoid Trypsin inhibitor (Kunitz inhibitor)

Lima bean inhibitor Peptides containing L-arginine in carboxyl termini Glycylglycy1-L-arginine Antidinotrophenyl antibodies

Agarose 15-m with e-aminohexanoic acid BioGel A-5 m with hexamethylenedim i n e and succinic anhydride Sepharose 4B Sepharose 4B with eaminocaproic acid Sepharose 4B with diaminodipropylaminosuccinate Sepharose 4B or cellulose

Schmer Hixson and Nishikawa (1973,1974) Hatton and Regoeczi (1976a) Thompson (1976) Holleman and Weiss

Jameson and Elmore (1971, 1974) BioGel A-5m with succinylated 1,6di- Hixson and Nishikawa aminohexane or Sepharose 4B with 6- (1973, 1974) aminohexanoic acid Feinstein (1970a, b) Sepharose 2B BioCel A-l5m Beardslee and Zahnley p-Diazobenzoylcellulose Mosolov and Lushnikova

STI cross-linked with Nethyl-5-phenyl- Bartling and Barker isoxazolium 3'-sulphonate (Woodward's reagent K) Phloroglucinol-hydroxylatedagarose Porath and Sundberg Sepharose 4B Liepnieks and Light Light and Liepnieks Sepharose 6B with diglycidyl ether Sepharose 4B Sepharose 4B Azido form of Sepharose 4B after oxidation with sodium periodate and reaction with adipic acid dihydrazide Sepharose Sepharose 4B

Sundberg and Porath Robinsonetal. (1971b) Chauvet and Acher (1973) Junowicz and Charm (1976)

Sepharose 4B Sepharose 4B

Kumazaki ef al. Wilchek and Gorecki (1973)

Gilliam and Kitto Kasai and Ishii (1972)

fcontinued on p. 284)

TABLE 11.1 (continued)

__

___

-

Substances isolated Proteases (continued) - and elastase Trypsin-like enzyme from Streptomyces paromomycinus (paromotrypsin) Urokinase (E.C. 3.4.99.26)

Protocollagen proline hydrolase Pyruvatecarboxylase (E.C. 6.4.1.1) from B. stemothermophilus Reductases Aldehyde reductase (E.C. 1.1.1.1) from brain Dihydrofolate reductase (T4bacteriophage specific) Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP' oxidoreductase, E.C. 1.5.1.3)

hl W

P

~

Affinity ligands

Solid supports or immobilized affinity ligands

References

Lima bean protease inhibitor Kunitz pancreatic trypsin inhibitor

Sepharose 4B Sepharose 4B

Lievaart and Stevenson Chauvet et al.

a-Benzylsulphonyl-p-aminophenylalanine

Maciag et al.

p-Aminobenzamidine HCI Reduced and carboxymethylated collagen Avidin

Sepharose 4B with hexamethylenedim i n e , succhic anhydride and hexamethylenediamine once again CHSepharose 4B BioGel A-5m

Holmberg er al. Berg and Prockop

Sepharose 4B

Libor et al.

NADP

Sepharose

Tabakoff and Von Wartburg

N'O -Formylaminopterin (4-amino-10formylpteroylglutamate) Methotrexate

BioCel P-150 with ethylenediamine

Erickson and Mathews

-

Sepharose 4B with 1,6-diaminohexane

Newbold and Harding Kaufman and Pierce Sepharose Nakamura and Littlefield Pter idine Dann et al. Alkylagarose Ametopterin Sepharose with hexamethylenediamine Whiteley et al. (1972) 10-Formylaminopterin Reddy and Rad Sepharose 4B with ethylenediamine Oxidized glutathione Harding Glutathione reductase from human Sepharose 4B Brodelius et al. lens, sheep retina and human red blood N6-(6-ArninohexyI)-adenosine-5'-mono- Sepharose 4B phosphate or N6-(6-aminohexy1)cell adenosine-2',5 'bisphosphate Yasukochi and Masters N6-(6-Aminohexyl)adenosine-2',5'-bis- 2',5'ADPSepharose 4B NADPH-cytochromeC (cytochrome phosphate P450) reductase (E.C.1.6.2.4) Flavin mononucleotide Kazarinoff et al. Cellulose Heimer et al. NADH Nitrate reductase [NAD(P)H:nitrate Sepharose oxidoreductase, E.C. 1.6.6.21

s

M

Nitrite reductase (ferredoxin-nitrite oxidoreductase, E.C. 1.6.6.4) from green spinach Phage-T4-induced ribonucleotide reductase Ribonucleoside diphosphate reductase from Escherichia coli Ribonucleotide reductase from Loctobacillus leichmannii - from Escherichia coli

-

- - from Lactobacihs leichmannii Reverse transcriptase from murine type C RNA tumour virus Ribonucleases 7 3-Aminotyrosyl ribonuclease (pancreatic) Ribonuclease A (bovine pancreatic, E.C. 3.1.4.22) - (bovine pancreatic) - (porcine pancreatic) - (tobacco) - T, - L from Aspergillus sp.

- from Escherichia coli - 111 from Escherichia coli - from human spleen - from human liver Isozymes of bovine pancreatic ribonuclease

Ferredoxin (spinach)

Sepharose 4B

Ida et al.

p-Aminophenyl ester of dATP

Sepharose 4B

Berglund and Eckstein (1972) Berglund

5'OTosyladenosine p-Aminophenyl ATP and paminophenyl dATP

Sepharose 4B-200 with cyanocobalamine-aminododecylamide Sepharose 4B

Yamada and Hogenkamp Berglund and Eckstein (1974)

P3~6-Aminohex-l-yl)deoxyguanosine-Sepharose 4B

Hoffmann and Blakley

5'-triphosphate Antibody

Sepharose 4B

Livingston

5'44-Aminophenylphosphory1)uridine42') 3'-pliosphate

Sepharose 4B

Gorecki et al. (1971)

5'-(4-Aminophenyl-phosphoryl)-

Sepharose 4B

Wilchek and Gorecki (1969)

uridine-2'( 3')-phosphate

5'44-Aminophenylphosp hory1)guanosine-2'( 3')-monophosphate 5'~4-Aminophenylphosphoryl)guanosine-2' ,( 3')-monophosphate 5'-AMP

DNA Double-stranded RNA Anti-human liver RNase serum Poly4G) (1) N4 -(6-Aminohexyl)cytidine-2'(3')monophosphoric acid; (2) concanavalin A

A~ZOW-APUP Sepharose 2B Glass with 1,5.dihydroxynaphthalene

Wilchek and Gorecki (1974) Wierenga et al. Jervis (1972, 1974) Suckling et al. Jervis and Pettit

Sepharose 4B with hexamethylenediamine

Horitsu et al.

8% agarose (for electrophoresis) Agarose-hexane-poly(1) .poly(C) CNBractivated Sepharose 4B CNBractivated Sepharose 4B (1) Activated CH-Sepharose 4B and (2) Con A-Sepharose

Weatherford et al. Dunn Neuwelt et al. Frank and Levy Baynes and Wold

(Continued on p. 286)

TABLE 11.1 (continued) Affinity ligands

Solid supports or immobilized affinity ligands

References

NADH

Sepharose 4B with 6-aminohexanoic acid

Brook and Large

Anti-streptokinase-yglobulin 19-Nortestosterone 17-0-hemisuccinate

Agarose Diaminodipropylamino-Sepharose 4B

Ionescu-Stoian and Schell Benson et al. (1974b)

Hexamethylenediamine

Sepharose 4B

- - (valyl)

Specific tRNA

Anthranilate synthase complex from Salmonella t y p h i m r i u m Chorismate synthase from Neurospora crassa Citrate synthase (E.C. 4.1.3.7) from pig heart - - from rat heart

L-Tryptophan

CNBr-activated Sepharose with adipic acid dihydrazide Sepharose 2B

Jakubowski and Pawelkiewicz (1973) Joyce and Knowles

Phosphate

Phosphocellulose

Substances isolated -

Secondary amine mono-oxygenase [secondary amine, NAD(P)H: oxygen oxidoreduc tase(Ndeaikylating), E.C. 1.14.99.91 from Pseudomonas aminovorans St repto kinase Steroid A-isomerase (3-oxosteroid A 4 - A s isomerase, E.C. 5.3.3.1) fromfieudomonas testosteroni Synthases and Synthetases Aminoacyl-tRNA synthetase

3-Deoxy-D-arabino-heptulosonate-7phosphate synthetase (E.C. 4.1.2.15) - isozymes from Saccharomyces cerevisiae Dihydropteroate synthase (E.C. 2.5.1.15) D-Erythrodihydroneopterintriphosphate synthetase from Lactobacillus plantarum Fatty acid synthetases from pigeon liver

Marcus (1974b) Cole and Gaertner

N6-[(6-Aminohexyl)carbamoylmethyl]-Sepharose 4B

Lindberg and Mosbach

ATP Periodate-oxidized ATP

Mukherjee and Srere

L-Tyrosine

Sulphonamide Periodate oxidized GTP

e-Aminocapro ylpantetheine

Sepharose 4B with adipic acid dihydrazide Sepharose 4 8

Chan and Takahashi (1969) Chan and Takahashi (1974) Takahashi and Chan

Sepharose 4B with di-(3-aminopropyl)- Suckling et al. amine Sepharose 4B with Eaminocaproic acid Jackson er al. methyl ester and hydrazine hydrate Sepharose

Qureshi et al.

Glutamine synthetase [L-glutamate: ammonia liase(ADP-for ming) , E.C. 6.3.1.21 from Escherichia coli - - from Neurospora crassa Glycogen synthase (UDP-glucose: glycogen a4glycosyltransferase, E.C. 2.4.1.11) - from muscle extract L-Isoleucyl t-RNA synthetase

a-Isopropylmalate synthase [a-isopropylmalate a-ketoisovalerate-lyase (CoA-acetylating),E.C. 4.1.3.121 Lactose synthase from human milk Leucyl-tRNA synthetase [ L-leucine: tRNA ligase(AMP), E.C. 6.1.1.41 Light and heavy enzyme of gramicidin S synthetase Methionyl-tRNA synthetase (E.C. 6.1.1.10) Myoinositol-phosphate synthase (1 L-myoinositol-1-phosphate lyase; isomerizing; E.C. 5.5.1.4) - from chicken erythrocytes and Lemna gib ba Phenylalanyl-tRNA synthetase - from Salmonella typhimurium

3-sn-Phosphatidyl-l’-snglycero-3‘phosphate synthetase from Bacillus licheniformis membranes A protein of lactose synthase (E.C. 2.4.1.22)

Pentamethylenediamine

Sepharose 4B

Shaltiel et al. (1975)

Anthranilic acid Concanavalin A Alkanes

CH-Sepharose 4B Con A-Sepharose Sepharose 4B

Palacios (1976) Sdlling and Wang Shaltiel

Tetramethylenediamine Nfert.-Butyloxycarbonyl-[ U-YI-Lisoleucinyl N“ -(2-aminoethyl)-5adenylate Isoleucyl-tRNA

Shaltiel and Er-El Rainey et al.

Leucine

Sepharose 4B Bio-Gel A-15 with 3,3’-iminobispropytamine and with succinic anhydride Sepharose 4B with tetramethylenediamine and bromoacetic ester of N-hydroxysuccinimide Sepharose

a-Lactalbumin tRNALeU

Sepharose 6B Sepharose 4B

Andrews Hayashi

Proline

Sepharose 4B with 3,3’diaminodipropylamine Sepharose 4B with hexamethylenediamine Sepharose 4B with 6-aminocaproic acid

Pass et al.

Mehionine NAD

Bartkowiak and Pawelkiewicz Doellgast and Kohlhaw (1972a, b)

RobertGero and Waller (1972,1974) Pittner et al.

Schwarcz et al. Remy et al. Schiller and Schechter

Oxidized cytidinephospho-sn-l,2diacylglycerol

Sepharose 4B with hydrazine Sepharose 4B with tetramethylenediamine Sepharose 4B with adipic acid dihydrazide

a-Lactalbumin

Sepharose 4B

Trayer et al. (1970)

tRNAPhe L-Phenylalanine

Larson et al.

(Continued on p. 288)

h)

00

TABLE 11.I (continuedl Substances isolated

00

Affinity ligands

Solid supports or immobilized affinity ligands

6-Carboxyethy1-7-oxo-8-D-ribityllumazine 6-p-Aminobenzamidohex yldeoxyuridine-5-phosphate

Sepharose 4B

Trayer and Hill Kulick et al.

Sepharose 4B-200

Danenberg et al.

2’-Deoxyuridine-5’46-p-amino-

Sepharose

Danenberg and Heidelberger

References

Synthases and Synthetases (continued) Riboflavin synthase (E.C. 2.5.1.9) Thymidylate synthetase - - from Luctobacillus casei

benzamido)hex ylphosphate

5-Fluoro-2’deoxyuridine-S‘-@amino- Sepharose 4B with hexamethylenedi-

Whiteley et a1. (1974)

phenyl phosphate) Tetrahydromethotrexate Heparin

Slavik et al. (1976) Elbein and Mitchell

- - from Escherichia coli B Trehalose phosphate synthetase from Mycobacterium smegmtis Tryptophan synthase (L-serine hydro- Indolepropionic acid lyase, adding indole, E.C. 4.2.1.20) Tryptophanyl-tRNA synthetase Tryptophan Tyrosyl-tRNA synthetase (E.C. 6.1 .l.1) tert-Butyloxycarbonyla-benzyl-Ltyrosine 4-nitrophenyl ester - from baker’s yeast

Tyrosine

Seryl-tRNA synthetase Valine- and lysine-specific synthetases

Seryl-tRNA Transfer RNA fractions enriched in r a l i e - and lysine-specific acceptor activities Unfractionated lupin tRNA

Val-, Trp-, Phe-, Leu- and Ile-tRNA synthetases Valyl-tRNA, tryptophanyl-tRNA, and seryl-tRNA synthetases from yellow lupin (Lupinus luteus) seeds Transferases

Periodate-oxidized lupin tRNA

amine and succinic anhydride Sepharose 4B with ethylenediamine Sepharose 4B CNBractivated Sepharose 4B with hexamethylene diamine Sepharosd4B with ethylenediamine Copolymer of maleic acid anhydride and butanediol divinyl ether with 1,6-hexanediamine Copolymer of maleic acid anhydride and butanediol divinyl ether with 1,6diaminohexane Sepharose 4B Polyacrylhydrazide agar gel

Wolf and Hoffmann

Sepharose 4B with hydrazine

Jakubowski and Pawelkiewicz (1975b) Jakubowski and Pawelkiewicz (1975a)

CNBr-activated Sepharose 4B with h ydrazine

Shimizu et al. Beikirch et al.

Beikirch el al.

Befort et al. Nelidova and Kisselev

Acetyltransferase from Escherichia coli Gentamicin C1 or kanamicin A (involved in gentamicin inactivation) Anthranilate phosphoribosyltransferase Anthranilic acid Aspartate aminotransferase (L-aspartate; Pyridoxal5'-phosphate 2-oxoglutarate aminotransferase, E.C. 2.6.1.1) L-Asparaginewith 3-ketoglutaric acid Pentamethylenediamine ATP:glutamine synthetase adenylyltransferase (EX. 2.7.7.42) from Escherichh coli

Indubiose 4A Sepharose 2B with hexamethylenediamine and succinic anhydride Sepharose 4B with hexamethylenediamine AHSepharose 4B Sepharose 4B

Catechol-O-methyltransferase

3,4-Dimethoxy-5-hydroxyphenylethyl-Sepharose 4B with diaminopropyl-

- - from rat liver

amine Catechol

Chloramphenicol acetyltransferase (E.C. 2.3.1.28) Choline acetyltransferase (acetylCoA: choline-0-acetyltransferase, E.C. 2.3.1.6) Cholinephosphate cytidyltransferase (E.C. 2.7.7.15) from rat liver Collagen galactosyltransferase Collagen glucosyltransferase Debranching enzyme (amylo-l,6glucosidase and oligo-l,4+1,4glucantransferase, E.C. 3.2.1.33 and E.C, 2.4.1.25) Galactosyltransferase (UDPgalactose: Nacetylglucosamine galactosyltransferase, E.C. 2.4.1.22)

amine and succinic anhydride Sepharose 4B with m-phenylenediamine after diazotisation Sepharose 4B Reduced chloramphenicol CNBr-Activated Sepharose with Chloramphenicol H,N-(CH,)n-COOH Antiserum polymerized with glutarAntiserum aldehyde NCarboxyphenyl4-(m-bromostyryl)- Aminoalkylsuccinylaminoalkylpyridinium chloride Sepharose 4B Epoxy-activated Sepharose 6B Glycerolphosphorylcholine Denatured citrate-soluble collagen

Sepharose 4B

UDP-glucoronic Tetramethylenediamine

AHSepharose 4B Sepharose 4B

P' -(6-Amino-l-hexyl)-P2~5'-uridine)- Sepharose 4B pyrophosphate or 6-amino-l-hexyl-2acetamido-2deoxy-D-glucopyranoside Sepharose 4B a-Lactalbumin

Le Goffic and Moreau Le Goffic et al. Marcus (1974a) Ryan and Fottrell

Schell et al. Shaltiel et al. (1975)

Borchardt et al. Gulliver and Wharton Guitard and Daigneault Zaidenzaig and Shaw Rossier et al. Husain and Mautner (1973a, b) Choy and Vance Risteli et al. Risteli et al. Anttinen and Kivirikko Taylor et al.

Barker et al. (1972)

Mawal et al. Trayer et al. (1974a)

-

(Continued on p. 290)

t 4 03

W

N

W 0

TABLE 11.1 (continued) Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

References

N-Acetylglucosamine p-Aminophenyl-N-acetyl-pDglucosamine Norleucine, UDP and a-lactalbumin

Sepharose 4 8 CNBr-activated Sepharose

Barker ef al. (1974a) Berger ef al.

Sepharose 4B or Sepharose 4B with hexamethylenediamine (UDP) Sepharose 4B with hexamethylenediamine Sepharose 4B

Geren ef al. Slavik et al. (1974)

Transferases (continued)

- from bovine skim milk Glutamate formiminotransferase (E.C. 2.1.2.5) yGlutamyltransferase (E.C. 2.3.2.2) from rat kidney Glycosyl transferases L-Histidinol-phosphate aminotransferase (E.C. 2.6.1.9) from Salmonella typhimurium Hyp0xanthine:guanine phosphoribosyl-transferase (E.C. 2.4.2.8) (HGPRTase) Lecithin: cholesterol acyltransferase Neomycin-phosphotransferase from Escherichia coli Nucleoside deoxyribosyltransferase (E.C. 2.4.2.6) Phosphate acetyltransferase (E.C. 2.3.1.8) RNA (guanine-7-)methyltransferase from HeLa cells Tyrosine aminotransferase (L-tyrosine: 2+xoglutarate amino transferase, E.C. 2.6.1.5)

Tetrahydrofolate Concanavalin A

Takahashi ef al. (1974a)

Uridine, adenine or guanosine nucleo t ide L-Histidinol phosphate Hexamethylene diamine

Sepharose 4B

Shaper et al.

Sepharose 4B Sepharose 4B

Shaltiel ef al. (1974) Henderson el al.

Anti-HGPRTase immunoglobulins

Sepharose 4B

Beaudet et al.

Highdensity lipoprotein Neomycin

Sepharose 2B CNBr-activated Sepharose 4B

Akanuma and Glomset Ganelin et al.

6-(p-Aminobenzylamino)purine

Sepharose with m-phenylenediamine

Cardinaud and Holguin

Coenzyme A

Sepharose 6B

Chibata ef al. (1974a)

DNA

Agarose

Ensinger and Moss

Pyridoxamine phosphate

Miller et al. (1972) Sepharose 4B with ethylene diamine and succinylated Sepharose 4B with 3,3'diaminopropyl- Thompson (1974) amine and succinic anhydride Sepharose 4B Schwartz and Rod& (1973,1974)

Xylosykransferase (UDP-D-xylose: core Proteoglycan (Smith degraded) or Core protein from cartllage protein 0-D-xylosyItransferase proteoglycan

trans-N-Deoxyribosylases (E.C. 2.4.2.6) I and I1 Transhydrogenase from Pseudomoms aerugirwsa Triacylglycerol lipase (E.C. 3.1.1.3) from human adipose tissue Tryptophan hydroxylase (tryptophan-5monooxygenase, E.C. 1.14.16.4) from rabbit hind brain Tryptophan hydroxylase (tryptophan-5monooxygenase, E.C. 1.14.16.4) from rabbit hind brain Tyrosinase from mushrooms - (mushroom) Tyrosine hydrolase (E.C. 1.14.16.2) - - from sheep brain Urease from Proteus morganii and from jack bean (E.C. 3.5.1.5)

p-D-Xylosidase (E. pumilus)

N6-(6-Aminohexyl)-AMP

Sepharose with bound m-phenylenedi- Holguin and Cardinaud amide Sepharose Kaplan et al.

Dioleoylglycerol

CHSepharose 4B

Verine et al.

6-Aminocaproyl-D-tryptophanmethyl ester

CNBr-activated Sepharose 4B

Widma et al.

6-Fluorotr yptophan

Sepharose 4B

Tong and Kaufman

p-Azophenol derivative Dihydroxyphenyl derivative 3-Iodot yrosine

Cellulose Sepharose 4B Sepharose 4B

Hydroxyurea

CNBr-activated Sepharose 4B with ethylenediamine and succinic anhydride, repeated twice Sepharose 2B after thiolation Sepharose 2B

Lerman (1953b) ONeil et al. Shiman Pollon Shobe and Brosseau

Carlsson et al. Claeyssens et al.

Sepharose 6B Sepharose

Mosolov et al. Summaria et al. (1976)

Sepharose 4B Sepharose

Plummer Ako et al. (1972b) Ako et al. (1974) Ako et al. (1972a) Yung and Trowbridge Spangenberg et al. Thompson et al. (1976;

6-bemylamino purine

2,2'-Dipyridyl disulphide p-Aminobenzyl-1-thio-p-D-xylopyranoside

ENZYME SUBUNITS AND MODIFIED DERIVATIVES Acyltrypsins Ovomucoid Affinity chromatography forms, 1 and 2, L-Lysine of human Glu- and Lys-plasminogens and plasmins Alkylated carboxypeptidase B L-Leucyl-D-arginine Anhydro-chymotrypsin Lima bean inhibitor Anhydro-trypsin a and 0-anhydro-trypsins

Anionic trypsin from the gastric juice Apoprotein from glyceraldehyde-3phosphate dehydrogenase (removal of NAD)

Soybean trypsin inhibitor Soybean trypsin inhibitor (Kunitz) Trasylol (natural trypsin inhibitor) Blue dextran

Sepharose 2B Sepharose 2B Sepharose 6B CNBractivated Sepharose

(Continued on p. 292)

t;

2

sz

h)

\o

TABLE 11.1 (continued)

N

Substances isolated

Affinity hgands

Solid supports or immobilized affinity ligands

References

Biotin-containing subunits of carboxylase t)l and B2 subunits of the reductase from DNA F mutant of Escherichia coli Catalytic and regulatory subunit fractions of protein kinase from human polymorphonuclear leucocytes Forms (a and b) of glycogen phosphor ylase Fractions of rabbit plasminogen PGalactosidase fragments

Biocytin (e-N-biotinyl-L-Iysine) dATP

Sepharose 4B Sepharo se

Lane e f al. Fuchs

Histone

Sepharose 4B

Tsung et al.

cr-Alkylamine

Sepharose 4B

Er-El and Shaltiel

L-Lysine p-Aminophenyl p-D-thiogalactopy-ranoside Concanavalin A

Sepharose 4B Affinose 202

Sodetz et al. Villarejo and Zabin

Sepharose

Wang and Bantle

Glycogen particle with adhering glycogen synthetase and synthetase phosphatase dhanidinated trypsin Modified RNA polymerase Molecular forms of acetylcholinesterase

- - of arginase from rat liver Monocarboxamidomethyl derivative of human erythrocyte carbonic anhydrase B Native, synthetic or oxindole-62 lysozyme Normal and mutant form of the B, subunit of Escherichia coli tryptophan synthase Protein B1 from ribonucleotide reductase from Escherichia coli Regulatory subunit of pig-brain histone kinase RNA polymerase haloenzyme Subunits of lombricine kinase (E.C. 2.7.3.5) from Lumbricus terrestris muscles

5

r

m

Chicken ovomucoid DNA 1-Methyl-9-[NY-(e-aminocaproyl)-yaminopropylamino] acridinium bromide hydrobromide L-Lysine p-Aminomethylbenzene sulphonamide N-Acetylglucosamine-p( 1 4 ) t r i saccharide Immunoglobulin G

Sepharose 4B Cellulose Sepharo se

Robinson et al. (1973) Dharmgrongartama et al. Dudaietal. (1972b)

Sepharose 4B Sepharose 4B

Tarrab et al. (1974) Whitney (1973)

Sepharose 4B

Cornelius et al.

3 4

Epichlorhydrin cross-linked desulphated-Sepharose 4B

Shannon and Mills

n 1: P

dATP

Sepharo se

Thelander

8-(~€arboxypropylthio)adenosine3',5'-cyclic phosphate DNA p-Aminophenylmercury(I1) acetate

Sepharose 4B with polylysine

Severin et al.

Cellulose Sepharose 4B

Mukai and Iida Der Terrossian et al.

cn

%

2

% % z 4

$2 2; 8P

ii<

- of phenylalanyl-tRNA synthetase - of pigeon liver fatty acid synthetase - of transcarboxylase (methylmalonylCoA pyruvate carboxytransferase, E.C. 2.1.3.1) GLYCOPROTEINS AND SACCHARIDES Blood group I glycoproteins Blood group substance A Brain-specifk glycoprotein from human white matter Cell surface glycoproteins

Concanavalin A-binding glycoproteins Copolymeric galactosaminoglycans

Dengue virus envelope glycoprotein from membranes of infected cells Galactomannan from the seeds of olssia ahta DCalactose from Phaeolus aureus seedlings Galactose-containingbiopolymers

pAminophenylmercury(I1) acetate Pantetheine

Sepharose 4B Sepharose with e-amino*-caproic acid

Murayama et al. Lornitzo ei a1.

Avidin

Sepharose 4B

Berger and Wood

t; 0 r 9

2 0

z

%

E Anti-I cold agglutinins Vicia cracca phytohaemagglutinin Antibodies against brain-specific proteins Lectin from L. culinaris (specifically for glucose- and mannose-related sugars) Lectin from R. communis (specifically for Dgalactose-related sugars) Concanavalin A Dermatan sulphate or dermatan sulphatg after partial oxidation with periodate Concanavalin A

Sepharose 4B Sepharose 2B Sepharose 4B

Feizi and Kabat Kristiansen (1974b) Brunngraber et al. (1974)

Sepharose 4B

Smart et al.

Sepharose 4B

Smart et al.

Con A-Sepharose CHSepharose 4B or AH-Sepharose 4B

Susz et al. Fransson

Con A-Sepharose

Stohlman et al.

a-DGalactopyranosyl-bindinglectin from Bandeiraeu simplicifolia 8-6-Aminoethylthioadenosinetriphosphate

Sepharose 4B

Ross et al.

Sepharose 4B with 3,3'diaminodipropylamine and succinic anhydride and Nhydroxysuccinimide Sepharose 4B

Chan and Hassid

Concanavalin A polymerized with glutaraldehyde Sepharose 4B

Donnelly and Goldstein

Galactose-specific lectin from Ricinus

Surolia et al.

wmmunis Glycogen

Concanavalin A

Wheat germ agglutinin Glycophorin A (sialoglycoprotein) from the human erythrocyte membrane Concanavalin A-Sepharose Glycoprotein and proteodermatan sulphate Concanavalin A from bovine achilles tendon Glycoproteins Glycopeptides obtained by proteolytic Sepharose 2B or 4B digestion of various glycoproteins

Kahane et al. Anderson N

Sepulcre and Mocza~

(Continued on p. 294)

W

W

h,

W P

TABLE 11.1 (continued) Substances isolated

- and neutral polysaccharides Glycoproteins from human serum - from human or rabbit serum

- from liver microsomal membranes Hog blood group substance Human brain-specific at glycoprotein '251-Labelled glucagon Lymphocyte plasma membrane glycoproteins [ ' Hl Mannose-labelled oligosaccharides from human diploid fibroblast Polysaccharides and glycoproteins

Affinity ligands

Solid supports or immobilized affinity ligands

References

Concanavalin A L-Valine or L-norleucine Concanavalin A

Kristiansen (1974a) Bussey et al. Aspberg and Porath Avrameas and Guilbert

2-H ydroxy-5-nitrobenzylated

Sepharose 4B Sepharose 4B Sepharose 2B Polymer of concanavalin A using glutaraldehyde as the polymerizing agent Con A-Sepharose Concanavalin A copolymerized with L-leucine-N-carboxyanhydrideor Sepharose 4B Sepharose 4B CNBr-activated Sepharose 4B

antibody Lens culinaris phytohaemagglutinin

Sepharose 4B

Hayman and Crumpton

Concanavalin A

Con A-Sepharose

Muramatsu et al.

Concanavalin A

Brain-specific antibodies

Warecka et al. Murphy et al. (1976)

8 4 (A

m

Concanavalin A

Proteoglycans from bovine achilles tendon Concanavalin A Collagen Serum proteins Lectin Soybean 1IS globulin Concanavalin A Synaptic plasma membrane glycoproteins Lens culinaris phytohaemagglutinin or wheat germ agglutinin Synaptic vesicle membrane glycoproteins Concanavalin A Lens culinuris phytohaemagglutinin Virus glycoproteins HORMONES Chorionic gonadotrophin (human) Estradiol

Winqvist et al. Lloyd

Concanavaiin A Antiestradiol antibody

Concanavalin A polymerized with glutaraldehyde Concanavalin A-Sepharose Sepharose 4B Sepharose 4B Con A-Sepharose 4B CNBr-activated Sepharose 4B

Anderson and L e e a w n Greenwald et al. Ersson and Porath Kitamura et al. Gurd and Mahler

Con A-Sepharose Sepharose 4B

Zanetta and Gombos Hayman et al.

Sepharose 6B Sepharose 4B with 3,3'diaminodipropylamine, succinic anhydride and N-hydroxysuccinimide ester

Donnelly and Goldstein

Dufau et al. (1972) Comoglio et al.

n LC P

0

5

4

0

Follicle-stimulatinghormone (human) High-molecular-weightforms of adrenocort icotropic hormone 'Z51-Labelledlysine vasopressin Insulin

Concanavalin A Concanavalin A

Sepharose 6B Concanavalin A-agarose (Sigma)

Dufau ef al. (1972) Eipper e f al.

Neurophysins Anti-insulin serum globulin fraction

Sepharose 4B Sepharose 2B

Long-acting thyroid stimulator Luteinizing hormone (human) - (ovine)

4 s thyroid proteins Concanavalin A Anti-luteinizing hormone immunoglobulin fraction Neurophysins

Sepharose 4B Sepharose 6B Sepharose 4B

Fressinaud ef al. Akanuma et al. (1970) Akanuma and Hayashi Smith Dufau ef al. (1972) Gospodarowicz

Antibodies against human pituitary prolactin Anti-human placental lactogen antibody Concanavalin A

Sepharose 4B

Pradelles e f 41. Pradelles and Jard Hwang ef al.

Sepharose 4B

Guyda and Friesen

Con A-Sepharose

Printz er al.

Anti-(huqn-chorionic gonadotrophin

Sepharose 4B

Closset ef al. (1974) Closset ef al. (1975)

Con A-Sepharose Sepharose 4B CNBr-activated Sepharose 4B

Murthy and Hercz Liener ef al. Laurell er al.

8-Lysine-vasopressin Prolactin (from human amniotic fluid) - (monkey) through the removal of growth hormone Serum prohormone of angiotensin (renin substrate) a- and @-subunitsof human luteinizing hormone

Sepharose 4B

or) and anti-(human luteinizing

hormone @) antibodies INHIBITORS or, -Antitrypsin (protease inhibitor) -

- from plasma

Concanavalin A

3Carboxy-4-nitrobenzenethiol K
Sepharose 4B

Myerowitz ef aI. (1972a)

selective removal of albumin - - from human serum Avidin

(&GI Antialbumin globulin Biotin Biocytin (= 6-N-biotinyl-L-lysine)

Sepharose 4B Cellulose Sepharose 4B

Basic trypsin inhibitor of bovine organs Binary complex of Bowman-Birk inhibitor and chymotrypsin Broad-specificity proteaseinhibitors from snails (Helix pomafh)

Trypsin Trypsin

Sepharose 4B Sepharose

Myerowitz ef al. (1972b) McCormick Cuatrecasas and Wilchek Bayer and Wilchek Kassell and Marciniszyn Seidl and Liener

Trypsin

Trypsin-CMcellulose

Tschesche and Diet1

a1-Antitrypsin from mouse serum through Anti-mouse albumin immunoglobulin

(Confinued on p. 296)

TABLE 11.1 (continued) Solid supports or immobilized affinity ligands

References

Sorenson and Scandalios Feinstein ( 1 97 1b) Avrameas and Guilbert

Deox yribonuclease

CNBr-activated Sepharose Sepharose Polymer of chymotrypsin using glutaraldehyde as the polymerizing agent Spheron 300 Sepharose 4B

Turkovi el al. (1973) Lindberg

Tryp sin Cathepsin D Deoxyribonuclease Trypsin

Sephazyrne trypsin Sepharose 4B Sepharose 4B Sepharose 6B

Hirschhauser and Kionke Keilovs and TomPKek (1976) Lindberg and Eriksson Belew et al.

Substances isolated

Affinity ligands

Catalase inhibitor from maize Chymotrypsin inhibitors - from soybeans, jack beans and 6 coli

Catalase Chymotrypsin

~

- from potatoes Deoxyribonuclease inhibitor from crude extract of calf thymus Human seminal plasma inhibitor Inhibitor of cathepsin D from potato juice - of deoxyribonuclease (calf thymus) - of trypsin and chymotrypsin from chick peas Modified trypsin inhibitor (which consists of two peptide chains linked by disulphide bridges) from maize Natural proteinase inhibitors

5

r Trypsin

lnsoluble trypsin resin

Hochstrasser et al. (1967)

cf

%

Trypsin, chymotrypsin or kallikrein Pro teinases

Chymotrypsin Trypsin Trypsin or trypsinogen

EMA Fritz el al. (1966, 1967) EMA or EMA with hexamethylenediFritz el al. (1968) amine and dimethylethylendiamine EMA or EMA with dimethylethylendi- Fritz ef al. (1969b) amine Sepharose 2B Feinstein (1971a) Sephadex G-200 Beeley and McCairns Sepharose 4B-200 Stewart and Doherty (1971)

Plasmhogen Trypsin

Sepharose 4B Cellulose

Moroi and Aoki Fritz etal. (1972a)

Trypsin

Cellulose- trypsin (Merck) Bio-Gel Trypsin- cellulo se

Tscheche et al. Hochstrasser et al. (1972) Fritz et 01. (1970)

Trypsin or plasmin Ovoinhibitor (chicken) Ovomucoid Peanut trypsin inhibitor and Kunitz soybean trypsin inhibitor a,-Plasmin inhibitor from human plasma Polyvalent isoinhibitors of trypsin, chymotrypsin, plasmin and kallikreins from sea anemone - - from snails - - from human bronchial secretion Protease inhibitors

m

X b

2 % % z!

2el

<

n

g

%

z

4

- - from human sperm plasma - - from sea anemones - - from chicken serum - - from Scopolia japonica cultured cells - - from black-eyed peas Ribonuclease inhibitor from rat liver Troponin I (inhibitory protein) from rabbit soleus muscle Trypsin inhibitor (Kunitz inhibitor) - - from lungs - - from the germs of wheat and rye - - from ground nuts (Arachis hypogaea and from potato (Sohnum fuberosum) - - from soybeans, jack beans and E. coli

CM-cellulose Sepharose 4B Trypsin and chymotrypsin Ribonuclease Troponin C

Sepharose 6B CMcellulose Sepharose 4B

Trypsin Chymotrypsin Trypsin

Sepharose 4B Spheron 300 Insoluble trypsin resin

Chauvet and Acher (1972) Turkovi et al. (1973) Hochstrasser and Werle Hochstrasser et al. (1969~1,b)

Polymer of trypsin using glutaraldehyde Avrameas and Guilbert as the polymerizing agent Sepharose 4B Eechovi Sepharose 6B Sundberg er al. Mosolov and Fedurkina Sepharose 4B Casati er al.

- - from cow colostrum - - from bean seeds - - from urine of pregnant women LECTINS N-Acetyl-Dglucosamine-specific wheat agglutinin Agglutination factor from Dictyostelium discoideum Agglutinin Agglutinins - from Abrus precatorius and Ricinus communis - from jack beans, wheat germ and soybean - from lima bean, soybean, wax bean and Bandeiraae simplicifolia - from Limulus polyphemus - from Sohnum tubsosum (STA-lectin)

Finketal. (1971) Wunderer et al. Barrett Sakato et al. Gennis and Cantor Gribnau et al. Syska et al.

2-Acetamido-N~eaminocaproyl)-2deoxyQ-Dglucopyranosylamine Agarose

Sepharose 4B

Lotan et al.

Sepharose 4B

Rosen et al.

Ovomucoid p-Aminophenylglucosides Cross-linkedgalactose

Kieselguhr Sepharose 4B Sepharose 4B

Burger Bloch and Burger (1974a) Olsnes er al.

Glycoproteins (peroxidase and ovomucoid) or derivatives of stromata Concanavalin A

Polymer of affinants using glutaraldehyde as the polymerizing agent Con A-Sepharose

Avrameas and Guilbert

Horse erythrocytes Formalinized horse erythrocytes Ovomucoid Sepharose 4B p-Aminobenzyll-thio-p-D-l,2dideoxy-Sepharose 4B pDglucop yranoside

Bessler and Goldstein Nowak and Barondes Delmotte er al. Delmotte et al. (Continued on p. 298)

h)

W

TABLE 11.1 (continued)

00

Substances isolated

Affinity ligands

Solid supports or immobilized affinity liands

References

Anti-A-agglutinin from Helix pomatia Anti-A-phytohaemagglutininfrom Vicia cracca

Dextran Blood group substance A

Sephadex G-200 Sepharose 2B with lysine or aminoethyl cellulose Sepharose 2B Gum arabic cross-linked with epichlorh ydrin Starch Tri-N-acetylchitotriose and starch cross-linked by epichlorhydrin Starch L-Fucose and starch cross-linked by epichlorhydrin Sepharose 6B The blood group substance was copolymerized with Ncarboxyanhydride of L-leucine Sephadex G-50

Ishiyama and Uhlenbruck Kristiansen et al. (1969;

Anti-B haemagglutinin from Streptomyces Gum arabic SP. Tri-N-acetylchitotriose Anti-H haemagglutinin Tii-N-acetylchito triose Anti-H haemagglutinin from eel serum

L-Fucose

Axinella agglutinin Blood group A specific lectin from Doliches biflorus

Agarose Blood group A substance

Concanavalin A

Cross-linked dextran gel

- from jack beans (Canavalia ensiformis) Fetuin Fetuin Favin N-(e-aminocaproyl-P-L-fucopyranosylL-Fucosa-binding proteins (anti-H lectin) from Lotus tetragonolobus seeds mine Guaran (= galactomannan) DGalactopyranosyl binding lectins Galactose-binding haemagglutinin Agarose DGalactose and N-acetyl-Dgalactosamine- Acid-treated agarose binding lectins Polymer of P-galactosyl structure Galactose-binding phytoagglutinins from Ricinus cornmunis, Momrdia charantia and Abrus precatorius Potassium melibionate a-DGalactosyl binding lectin Dextran Haemagglutinin from the common lentil (Lens culinaris)

Sundberg et al. Fujita et al. Matsumoto and Osawa (1972) Matsumoto and Osawa (1974a, b) Matsumoto and Osawa (1972) Matsumoto and Osawa (1974a, b) Maino et al. Etzler and Kabat

Sephadex G-100 Sepharose 4B Sepharose 4B Sepharose 4B

Agrawal and Goldstein Karlstam Olson and Liener Sela et al. (1975b) Sela et al. (1975b) Blumberg et al. (1 972)

Cross-linked guaran Sepharose 4B Acid-treated Sepharose 6B

Lonngren et al. GilboaGarber et al. Allen and Johnson (1976a)

Sepharose 4B

Tomita et al.

Aminoethyl-BioGel P-300 Sephadex G-100

Hayes and Goldstein Howard and Sage

m

* X

5r

m

cn

4) 5 m % % 2

5 4

0

3:

w

P 5 0 0

w

zx 4

- from lentil - from the pea - from the pea - from Phaseolus vulgaris - from Limulus polyphemus - from

Lotus tetragenolobus

- from Sophora japonica seeds - from Sophora japonica seeds

- I from Ulex europeus Iodinated wheat germ agglutinin Lectins - fractions from Ricinus cornmunis seeds - from Gragana arborescens (seeds) - from

small California white beans, Idaho red beans and white pea beans - from Ricinus communis (seeds) - from Vicia ervilkr - from Viciafa& - from Lima bean

Limulus haemagglutinin Limulus polyphemus haemagglutinii Maclura pornifera lectin Maleylated dimer of concanavalin

Sephadex (3-150

Thyroglobulin Sepharose 4B Glycoprotein from bovine submaxillary CNBr-activated Sepharose 4B mucin Hog A + H blood-group substance Hog A + H substance copolymerized with the N-carboxyanhydride of Lleucine DGalactose Cross-linked D-galactose gel Sepharose 6B A- and H-active hog gastric mucin A- and H-active gastric mucin copolymerized with N-carboxyanhydride of L-leucine a-and p-anomers of 6amino-1-hexylSepharose 4B L-fucopyranose Sepharose 4B Ovomucoid Various sugars Sepharose 4B p-Aminophenyl glycoside Sepharose 4B Agarose Sepharose 4B 2-Acetamido-O)-@aminophenyl)-2deoxy-0-D-galactopyranoside Sepharose 4B Fetuin Sepharose 4B AgarOSe Sepharose 6B D-Mannose Sephadex G-150 Dextran Type A blood group substances inType A blood group substance solubilized with leucine N-carboxyanhydride Sepharose 4B Fetuin Sepharose 4B Povinc submaxillary mucin Sepharose 4B with N,N'diaminodiDGalactosamine propylamine and succinic anhydride Cross-linked dextran gel Dextran Sephadex G-75

Tichl et al. Entlicher e l al. (1970) Entlicher and Kocourek Matsumoto and Osawa (1972) Oppenheim et al.

-

v1

0

r

2

E

%

Pereira and Kabat

Terao and Osawa Poretz er al.

.e ?-

0

Frost et al. Cuatrecasas (1973) Lis and Sharon Bloch and Burger (1974a) Lhermitte et al. Bloch et al.

=!

s

s

v)

1 ?-

2:

0

m

v1

Sela et al. (1975b)

Hsu et al. Fornstedt and Porath Allen and Johnson (1976b) Galbraith and Goldstein Sela et al. (1975b) Roche et al. Ulevitch et al.

Young

(Continued on p . 300)

h)

W W

W

0 0

TABLE 11.1 (continued) Substances isolated

Affinity liiands

Solid supports or immobilized affinity ligands

References

Mitogens from pokeweed roots (Phytohcm a mericona) Non-specific phytohaemagglutinin from Vicia cracco Phytohaemagglu tinins

Desialized human erythrocyte glycopeptide Dextran

Sepharose 4B

Yokoyama et al.

Sephadex G-100

Aspberg et al.

Acid-treated epichlorhydrin crosslinked disulphated Sepharose 6B AlkenylO-glycosides copolymerized with acrylamide and N,N’-methylenebisacrylamide Polyacrylamide gels

Ersson et al.

Acid-treated agarose Alkenyl-Oglycosides

- from Bauhinia purpurea seeds - from Maackia amurensis seeds

DGalactosides, D-glucosides, D-mannosides Ally1glycosides Cross-linked Dgalactose Porcine thyroglobulin glycopeptides Dextran Thyroglobulin Dextran

- from pea and lentil - from Phaseolus vulgaris Protectin Anti AHP from protein gland of Helix pomatia Rabbit liver lectin (binding protein specific Asialo-orosomucoid for asialoglycoproteins) Ricinus communis agglutinin Agarose Snail hemagglutinin Hog gastric mucin with A + H bloodgroup activities (hog A + H) N-e-aminocaproyl$-Dgahctop yranosylSoybean agglutinin amine Galactosamine Soybean lectin N-Acetyl-Dgalactosamine Arabinogalactan Tridacnin from the haemolymph of Tridacna maxima Wheat-germ agglutinin

Ovomucoid Chitin (= polymer of N-acetylglucosamine)

Hof?ejEiand Kocourek (19733

HofejXi and Kocourek (1 974a)

m

Acrylamide Sepharose 6B Sepharose 4B Sephadex (3-200, G-100or G-50 CNBr-activated Sepharose 4B Sephadex (3-200

HoYejEi and Kocourek (1 974b) Irimura and Osawa Kawaguchi et al.

5 r

Sepharose 4B

Hudgin et al.

BioGel A4.5 m Hog A + H copolymerized with Ncarboxyanhydride of L-leucine Sepharose 4B

Nicolson and Blaustein Hammarstrom and Kabat

CH-Sepharose Epoxy-activated Sepharose 6B Arabinogalactan copolymerized with N-carboxyanhydride of L-leucine mixed with Sepharose 4B Sepharose 4B Chitin

Entlicher et al. (1969) Felsted et al. Kuhnemund and Kohler

Gordon er al. Allen and Neuberger Vretblad (1976) Baldo and Uhlenbruck Le Vine et al. Bloch and Burger (1974b)

x

*

rn

Ln

2 2m z

z4

.e

0

3:

P

B

50 d P

i

4

Acetamido-N-(~-aminocaproyl)-2deoxy-p-Dglucopyranosylamine 6-Amino-l-hexyl-2-acetamido-2deoxy-p-Dglucopyranoside 6-Aminohexyl-2-acetamido-2-deoxyp-Dglucopyranoside Fetuin Asparaginyl-Nacetylglucosamine N-Acetyl-Dglucosamine LIPIDS Cholinergic proteolipid from nucleus caudatus of the cow Lipids from plasma NUCLElC ACIDS AND NUCLEOTIDES Adenosine, 5’-adenylate, polyadenylate and sRNA Adenosine 3‘:5’-monophosphate Adenosine monophosphate-rich polynucleotides Biologically active globin messenger RNA Biologically pure messenger RNA from a mouse myeloma Calf thymus DNA Coding strand of the ovalbumin gene Complementay DNA sequences Complementary RNA sequences Complementary strands in DNA preparations obtained from cells infected with the single stranded DNAcontaining bacteriophage ex174 Cytidine Deoxyadenosine nucleotides

p-Phenyltrimethylammonium

Sepharose 4B

Lis ei al.

Sepharose 4B

Shaper et al.

Polyacrylamide gels copolymerized with active esters (N-succinimidyl acxylate and N-phthalimidyl acrylate) Sepharose 4B Epichlorhydrin cross-linked desulphated Sepharose 4B Epoxy-activated Sepharose 6B

Schnaar and Lee

Sela et al. (1975b) Wang et al. Vretblad (1976)

Dodecylamine

Sephadex LH-20 with 3,3-iminobispropylamine Sepharose 4B

Deutsch et al.

5’-Thymidylate

Cellulose

Sander et al.

Specific cyclic AMP-binding proteins Polydeoxythymidylate oligomers

Sepharose 2B Cellulose

Fisch et al. Edmonds and Caramela

Oligothymidylic acid Antibodies (double antibody technique) Histone KAP Ovalbumin mRNA RNA DNA DNA

Cellulose Sepharose 4B

Aviv and Leder Schechter

Sepharose 4B Phosphocellulose Cellulose Cellulose Cellulose

Panusz et al. Woo et al. Shih and Martin (1974) Shih and Martin (1974) Merriam et af.

Guanosine

Amberlite CG 50/II with acid hydrazide Cellulose

Tuppy and Kiichler

Thymidine polyiiucleotide

Saracen0 and de Robertis

Gilhm (Continued on p. 302)

w

0

TABLE 11.1 (continued)

p3

Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

References

Deoxycytidine

Adenosine or guanosine

Tuppy and Kiichler

Deoxythymidylic acid oligomers obtained by polycondensation

Oligomers of deoxyadenylic acid covalently attached to poly(viny1 alcohol) Histones (calf and human) Concanavalin A RNA

Amberlite CG 50/11 with acid hydrazide DEAE-cellulose

Schott (1975)

Sepharose 4B Sepharose 4B Cellulose Sepharose 48-200 Glycosylex A

Ayad and Parker Edelman (1974) Shih and Martin (1973) Robberson and Davidson Edelman (1975)

Poly(U) Sephadex Oligo(dT)-cellulose type T-2 Cellulose DEAE-cellulose Cellulose Nitrocellulose Cellulose Oligo(dT)cellulose Cellulose Cellulose Olio(dT)cellulose

Flavell and Van den Berg Krystosek et al. Popov ei al. Pirro and Feldmann Jones and Parsons Bautz and Reilly Weissbach and Poonian Gielen et aI. Ginder et al. Forget et al. Gander et al.

Anti-Y antibodies

CNBr-activated Sepharose

Salomon et al.

Isoleucyl-tRNA synthetase Oligothymidine

Sepharose 4B Cellulose

Denburg and d e Luca Schechter

Oligo(deoxythymidy1ate) Oligothymidylate

Oligo(dT)cellulose Cellulose

Gozes et al. Swan et al.

DNA (calf and human)

- defined sequences - from Escherichia coli - through the removal of polysaccharide contamhiants Duplex DNA containing (dA.dT) clusters Eukaryotic messenger ribonucleic acids _ _ _ _ (rabbit globin) Fractions of cellular DNA - of yeast ribonucleic acids Genespecific messenger RNA Globin messenger RNA

Human globin messenger RNA Globin messenger ribonucleoprotein complexes from puromycindissociated duck and rabbit polyribosomes Isoaccepting tRNAPhe species containing Y base (highly modified guanosine derivative) Isoleucyl-tRNA Messenger RNA coding for a mouse immunoglobulin L-chain - - _ for tubulin and actin - - _ for a myeloma light chain

Concanavalin A Polyuridylic acid Olgodeoxythymidylic acid Polyuridylic acid Benzoyl chloride Guanine DNA Oligothymidylic acid Ohgodeoxythymidylic acid Oligodeoxythymidylic acid

- - from animal cell polysomes containing polyadenylic acid and KB cells - - from human fibroblasts - - from polysomes - - from rat lens tissue - - from rat liver polysomes Microsomal RNA Mononucleotides Non-helical polynucleotides

Nucleic acids Oligodeoxyribo- and oligoribonucleotides Oligonucleotides

- and

polynucleotides

Ovalbumin messenger ribonucleic acid Phage specific DNA fragments Phenylalanyl- and lysyl-sRNA Phenylalanine transfer ribonucleic acid from yeast (t-RNAPhe) Polyadenylic acid Polyadenylic acid-containing RNAs - containing RNA fraction - containing RNA from Escherichia coli - hn RNA from total nuclear RNA Polycytidylic acid Polyinosinic acid

Polyuridylic acid

Sepharose 4B

Olgodeoxythymid ylic acid

Cellulose Cellulose Cellulose Cellulose Cellulose

Adenosine Mercury(I1) acetate

Sephadex G 2 5 with l-allyloxy-2,3epoxypropane 3,6-Bis(acetatomercurimethyl)dioxane Sephadex with 2-aminomethylhydrogen sulphate and N-acetylhomocysteine thiolactone Chromophore analogue of actinomycin Sepharose 4B with hexamethylenediC, (D) amine Oligodeoxyribonucleotides of defined Cellulose structure Dihydroxyboryl derivative Dihydroxyboryl-substituted methacrylic acid polymer Poly(vinytalcoho1) with oligodeoxyDEAE-cellulose thymidine-5’-phosphate Cellulose Trityl or naphthylcarbamoyl derivatives Cellulose Oligodeoxythymidylic acid DEAECellulose Deoxyoligothymidine-5’-phosphate Cellulose Polydeoxyribonucleotides or polyribonucleotides Anti-tRNAPhe antibodies CNBr-activated Sepharose Short oligomers of polythymidylic acid Polythymidylate Polyuridilic acid Polydeoxythymidylic acid Oligodeoxythymidylic acid Polyinosinic acid Polycytidylic acid

Cellulose Cellulose Cellulose Cellulose Oligo(dT)-cellulose Oligo(dT)cellulose T2, T3 Acetylated phosphocellulose Acetylated phosphocellulose

Lindberg and Persson (1972,1974) Pestka et al. Pemberton et al. Cohen et al. Nardacci et al. Bosch et al. Gruenwedel and Fu Cerami

t; 0 9

r

2

0

Z

% r 0

z

(7

9

r

r 4

*

Seela

cl

=!

Astelland Smith (1971, 1972) Astell et al. Schott et al. (1973) Schott et al. (1974) Schott (1974b)

s

v)

H

9 2:

cl

Fridkin et al.

M rn

Rhoads Schott (1974a) Erhan et al. Fuchs et al. (1974) Edmonds et al. Armstrong et al. Sheldon et al. Mach et al. Srinivasan et al. Bantle et al. Adler and Rich Adler and Rich (Continued on p. 304)

W W 0

w 0

TABLE 11.1 (continued) Substances isolated

P

Affinity ligands

Solid supports or immobilized affinity ligands

References

Polyinosinic acid Polyriboadenylic acid Thymidine polynucleotide

Acetylated phosphocellulose Acetylated phosphocellulose Cellulose

Adler and Rich Adler and Rich Gilham and Robinson

Po ly cyt id y lic acid Polyinosinic acid Polyriboadenylic acid Polyriboadenylic acid Polyadenylic acid Polyadenylic acid

Acetylated phosphocellulose Acetylated phosphocellulose Acetylated phosphocellulose Acet y la ted phosp hocellulose Sepharose Methylene dianiline derivative of starch (S-MDA resin)

Adler and Rich Adler and Rich Adler and Rich Adler and Rich Yogo et al. Venkatesan et al.

Methylalbumin Polythymidylate

Kieselguh Cellulose

Kiss et al. Nakazato and Edmonds

Oligothymidylic acid

Cellulose

Burns and Williamson

Sepharose 2B Sepharose 6B

Gyenge et al. Spiridonova et al.

- genes from Bocillus subtilis 34-S ribonucleic acid subunits RNA RNA-DNA hybrids Soluble ribonucleic acid Specific transfer ribonucleic acid - substituted with dinitrophenyl groups SV40 messenger RNA Thymidine

Ribosomal proteins Ribosome proteins from Escherichia coli RNA Polyuridylic acid Concanavalin A Polyinosinic acid Benzoyl chloride Benzoyl chloride Antidinitrophenyl antibodies S V 4 0 DNA Adenosine or guanosine

Smith et al. Ihle et al. Edelman (1974) Coffin et al. Gillam et al. (1967) Gillam et al. (1968) Fuchs et al. (1971) Gilboa et al. Tuppy and Kuchler

Thyroglobulin messenger RNA

Polyuridylic acid

Cellulose Poly(U)Sepharose Sepharose 4B Sephadex G-10 D EAE-cellulo se DEAE-cellulose Sepharose Sepharose 4B Amberlite CG Sol11 with acid h ydrazide Sepharose 4B

Polynucleotides derived from the rib* nuclease digestion of the ribonucleic acid from the bromegrass mosaic virus Polyriboadenylic acid

Polyuridylic acid - fragment from polio[ "PIRF Poly U sequence (30-40 nucleotides) from nuclear and cytoplasmic extracts of HeLa cells Pyrimidine oligonucleotides Rapidly labelled polysome-bound ribonucleic acid Reticulocyte globin messenger ribonucleoprotein (mouse) Ribosomal RNA

Vassart et al.

0

X

Transfer RNA (yeast) - - species containing inosine - - substituted selectively with dinitrophenyl groups T, -specific RNA Uridine

Nucleoside derivatives Anti-inosine antibodies Antidinitrophenyl antibody

Cellulose Sepharose 4B CNBr-activated Sepharose

Morris Inouye et al. Fuchs et al. (1971)

DNA Guanosine

Bautz and Hall Tuppy and Kiichler

Wetekam et al. (1975) Jost Dziegielewski and Jakubowski

Viral specific RNA from SV40 infected cells Vitellogenin messenger RNA

SV40 DNA fragments

Cellulose Amberlite CG Sol11 with acid hydrazide Cellulose

Polyuridylic acid

Sepharose

Yellow lupin transfer RNA

Hexamethylenediamine

Sepharose 4B

PROTEINS, BINDING AND TRANSFER RECEFTORS Acetylcholine receptor I

- from denervated rat hemidiaphragms

- from the electroplax of a narcine from the Gulf of California Acetylcholine receptors from Torpedo culifornica

- from mammalian muscle - from muscle membrane N-Acetyl-D-glucosamine-binding proteins Adrenergic receptor

Sepharose 4B Methyl[(6-aminocaproyl-6'-aminocaproyl)-3-amino]pyridinium bromide [ N<6-aminocaproyl)-p-aminobenzoyl]- Sepharose 4B trimethylammonium bromide Sepharose treated with epichloriiydrin Cobratoxin and desulphated [ N<e-Aminohexanoyl)-3-aminopropyl] - Sepharose 2B trimethylammonium bromide hydrobromide [N<e-Aminohexanol)-3-aminopropyl]- Sepharose 2B trimethylammonium bromide hydrobromide (concn. 0.4 M) Trimethyl@-aminopheny1)ammonium Sepharose 4B with 3,3'diaminodipropylamine and succinic anhydride, chloride repeated twice CNBr-activated Sepharose 4B Eurabutoxin B Sepharose 4B with hexamethylenedip-Aminobenzyl 1-thio-2acetamido-2amine and succinic anhydride deoxy-p-D-glucop yranoside 4% agarose (Sepharose or BioCel) with L-Norepinephrine HCl 3,3'diaminodipropylamine and succinic anhydride

-

-

Shih and Khoury

Chaw Chang Brockes and Hall Schmidt and Raftery (1972)

Schmidt and Raftery (1973)

Dolly and Barnard

Merlie et al. Rafestin et al. O'Hara and Leflcowitz

(Continued on p. 306)

W

w

0

TABLE 11.1 (continued)

o\

Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

References

Albumin from plasma or serum - from serum

Blue dextran Succinic anhydride or octyl, decyl, dodecyl, octadecyl succinic anhydride Deoxycorticosterone hemisuccinate L-Arginine

Sepharose 4B Sepharose 4B with diaminopropane, -butane, -hexane, -octane, decane Sepharose 4B with ethylenediamine Sephadex G-25

Travis and PanneU Aslam et al.

Concanavalin A

Con A-Sepharose

Brunngraber el al. (1975)

8-(6-Aminohexyl)aminoadenosine3’,5’-monophosphate

CNBr-activated Sepharose

Ramseyer et al.

Avidin monomer

Sepharose 4B

Guchhait et al.

Norepinephrine

Lefkowitz et al.

Ong and Brady Pandian et al.

Aldosterone-binding macromolecules Amino acid-binding glycoproteins from Neurospora crassa Brain-specific proteins binding concanavalin A cAMP receptor proteins (subunits of cAMPdependent protein kinase and regulatory cAMP receptor subunit) Carboxyl carrier protein from Escherichia coli Cardiac padrenergic receptor protein

Cholinergic receptor protein from Electro phorus electricus

Quaternary ammonium salts

Cholinergic receptor protein(s) from Torpedo nobiliana I4C-Labelled receptor for luteinizing hormone and human chorionic gonadotropin Cobalamin binding proteins from gastric juice and serum - (intrinsic factor and transcobalamin I)

Neurotoxin

Sepharose 4B with 3,3‘diaminodipropylamine and succinic anhydride, repeated twice (30 A) Sepharose 2B with hexamethylenediamine and Nacetylhomocysteine thiolactone Sepharose 4B

Human chorionic gonadotropin

Sepharose 4B

Coenzyme A affmity protein Concanavalin A receptor from L cells Corticosteroid-binding globulin from human plasma

Ludens et al. (1972a, b) Stuart and de Busk

Olsen et al.

Hydroxocobalamin-albumin conjugate Bromoacetylcellulose

Olesen et al.

Hydroxocobalamin

Nex

Reduced form of coenzyme A Concanavalin A Cortisol

CNBr-activated Sepharose 4B with 3,3’diaminodipropylamine Sepharose 6B Con A-Sepharose Sepharose 4B

Matuo et al. (1974) Hunt et al. Trapp et al.

s”5

r m v1

M

Cortisol-bindingimmunoglobulin Cytokinin binding protein from tobacco leaves Cytoplasmic glucocorticoid-binding protein(s) from rat liver DNA-binding proteins from Escherichia

Cortisol hemisuccinate Cortisolsuccinylalbumin Benzyladenine

AH-Sepharose 4B CNBr-activated Sepharose 4B CNBr-activated Sepharose 4B

Gijzen Gijzen et al. Takegami and Yoshida

B

Cortisol hemisuccinate

Wong et al.

8

Single-stranded DNA

Sepharose 4B with 3,3'diaminodipropylamine 4% agarose (for electrophoresis)

DNA

Cellulose

Melero et al.

z

Schaller et al.

coIi

- - from crude extracts of clones and virus-transformed subclones of fibroblast - - from human, murine and man-mouse hybrid cell lines Ecdysone receptors from insect tissues Electrolectin (p-Dgalactoside binding protein from electric organ tissue of Electrophorus electricus) Estradiol receptors

- - from rat uterus

- from calf uterus - from calf uterine cytosol Folate-binding protein

m,

P

0

Et

0

z-

Agarose (Serva) or cellulose

Jost et al.

AH-Sepharose 4B Sepharose 6B with epichlorohydrin after alkaline and acid hydrolysis

Spindler et al. Teichberg et al.

3,17p-Dihydroxyestra-1,3,5(10)-trien'la-undecanoic acid Denatured calf thymus DNA Estradiol p-Aminobenzylestradiol 1'ID-Estradiol 17-hemisuccinate

Sepharose 4B with aminodipropylamine Cellulose Polyvinyl4N-phenylenemaleimide) p-Aminobenzylcellulose Sepharose 4B with poly(L-lysyl-D,Lalanine)

Truong et al.

7n-Estradiol derivative [E2-7dCH2) loCOO-1 Folic acid

Sepharose

1nokosterone-C-26-carboxylicacid Galactosyl residues

Glycoprotein receptors for concanavalinA Concanavalin A from pig lymphocyte plasma membrane Glucagon receptors of liver cell membranes Glucagon

Glucocorticoid receptor from hepatoma tissue culture cells - from rat liver

E0i

Deoxycorticosterone derivative DNA

Sepharose 6B with hexamethylenediamine Sepharose 4B

Yamamoto and Alberts Von Der Haar and Mueller Von Der Haar and Mueller Sica et al.

r r 4

*z

c3

t?

Parikh et al. Truong and Baulieu Salter et al. Allan et al.

Sepharose 2B with bound 3,3'diamino- Krug ef al. dipropylamine after reaction with p-nitrobenzo ylazide Bio-Gel A-50m with hexamethylenedi- Failla er al. amine Cellulose Eisen and Glinsmann (Continued on p. 308)

W

4 0

W

22

TABLE 1 1.1 (continued) Substances isolated

Affinity ligands

Solid supports or immobilized affinity ligands

References

Glucose-binding protein

Phloretin

Fannin and Diedrich

Gonadotropin receptors of rat testis Haemoglobin (human)

Human chorionic gonadotropin Haptoglobin Aniline or L-phenylalanine Decamethybnediamine or dodecamethylenediamine Alkanes Anti-human serum albumin antibodies Immunoglobulin G or M or F (ab'), fragment of immunoglobulin G Insulin

BioGel A-15m with 3,3'diaminodipropylamine and p-nitrobenzoylazide AffiCel 10 Sepharose 4B Sepharose 4B Sepharose 4B

Dufau et al. (1975 j Klein and Mihaesco Memoli and Doellgast Shaltiel er al. (1973)

Sepharose 4B Aminocellulose CNBr-activated Sepharose 4B

S haltiel Gallop et al. Guimezanes er al.

Cuatrecasas (1972)

Concanavalin A Capr y ly 1h y dr azide

Sepharose 4B with 3,3'diaminodipropylaminosuccinyl N-hydroxysuccinimide ester Agarose with 3,3'diaminodipropylamine and succinic anhydride Sepharose 4B Sepharose 4B

Cuatrecasas and Parikh Nishikawa and Bailon

Long-acting thyroid stimulator

Sepharose 4B

Smith

Folic acid

Waxman and Schreiber

Asialoorosomucoid

CNBractivated Sepharose 4B with hexamethylenediamine Sepharose 4B

L-Methionine

CH-Sepharose

Stepien

(8-Ly sine)-vasopressin (after reversibly protecting the or-amino group of vasopressin) (8-Lysine)-vasopressin Neurotoxin

CNBr-activated Sepharose 4B

Edgar and Hope

Sepharose 4B Epichlorohydrin cross-linked desulphated Sepharose 4B

Fr6noy et al. Karlsson et al.

Histidine-binding protein J from Salmonella typhimurium .- - from Salmonella typhimurium I 31 I-Labelled human serum albumin Immunoglobulin-binding factor from alloant ken-act ivated thymus cell Insulin receptor of liver cell membranes

- - from liver-cell membranes

&Lactoglo bulin, ovalbumin and bovine serum albumin Long-acting thyroid stimulator binding protein Low-molecular-weight binding protein from human milk Membrane receptor protein for asialoglycoproteins from rabbit liver Methionine binding protein from Aspergillus niduhns Methylated and unmodified neurophysins (bovine) Neurophysins Nicotin acetylcholine receptor

Cuatrecasas and Parikh

Pricer et al.

Oestradiol receptors

17-p-Estradiol 17-hemisuccinate

- - complex Penicillin-binding components from Bacillus subtilis membranes

DNA 6-Aminopenicillanicacid

Progesterone-binding components of chick Deoxycorticosterone hemisuccinate oviduct - globulin 17-Hemisuccinate of 19-nortestosterone Progesterone-receptor complex ATP Receptor proteins for concanavalin A from human erythocyte membranes Receptors for wheat germ agglutinin and the Ricinus com. lectins Retinol-binding protein RNA-binding proteins RNA polymerase-binding proteins Serum albumin -(hum) - (human) - (human) - (human) Sex hormone binding globulin

Soluble binding proteins from rat liver nuclei and cytoplasm Specific nuclear acceptor for estrogen receptor complexes

Agarose with 3,3’diaminodipropyb amine Cellulose Sepharose 4B with 3,3’diaminopropylamine and succinic anhydride

Sica et al.

0

r

>

3

Cheng et al. (1976)

ij

Concanavalin A Lectins

Sepharose 2B

Adair and Kornfeld

Prealbumin rRNA

Sepharose 4B CNBr-activated Sepharose 4B

Polyuridylic acid RNA polymerase Fatty acids Bilirubin Aniline or L-phenylalanine Anti-human serum antibody

CMCellulose Sepharose 4B Sepharose 4B with ethylenediamine AH-Sepharose 4B Sepharose 4B Thiolated and cross-linked antibody Aminocellulose Disulphide-linked antibodies with N-acetylhomocysteine thiolactone Sepharose 4B with 1,4diaminobutane Enzacryl AA

Vahlquist et al. Preobrazhensky and Elizarov Preobrazhensky et al. Ratner Peters et al. Hierowski and Brodersen Memoli and Doellgast Stephen et al. Gallop et al. Crook et al.

Nuclear fractions

LQ

Clemens and Kleinsmith Blumberg and Strominger (1972) Blumberg and Strominger (1974) Kuhn et al.

Sepharose 4B with bovine serum albumin Sepharose 4B with 3,3’diaminodipropylamine Sepharose 4B with adipic acid dihydrazide Con A-Sepharose 4B

Octyl succinic acid 3-Hemisuccinateof Sa-androstane3aJ7pdiol Potassium polyadenylate

0

z

%

E

@

13

*

r Moudgil and Toft Findlay

t; + 0

r!

is Y

Wichman and Andersson Hampl et al.

CNBractivated Sepharose 4B

Schweiger and Mazur (1974)

Sepharose 4B

Puca et al. (Continued on p . 31 0)

w

0 v)

W

TABLE 11.1 (continued)

6

. .-..

Substances isolated

Affinity ligands ~. .~ .

.

Solid supports or immobilized affinity ligands . .

Steroid-receptor complexes

Deoxyribonucleic acid

Cellulose

Testosterone-estradiol-binding globulin from human plasma Thiamine-binding protein from Escherichia coli Thyroxine-binding globulin from human serum - (human) Thyrotropin receptor Transcortin human

Sa-Androstane-3@,1 ’Ipdiol 3-hemisuccinate Thiamine pyrophosphate

Sepharose 4B with epichlorohydrin and azodianiline Sepharose 6 8 with ethylenediamine

L-Thyroxine

Sepharose 4 8

Thyrotropin Periodic acid oxidized corticosterone

Sepharose 4B Sepharose 4B with 3,3’diaminodipropylamine PAB cellulose (Bio-Rad) Sepharose with 3,3’diaminodipropylamine Sepharose 4B with 3,3’diaminodipropylamine

Uterine estradiol receptor Vitamin B,,-binding proteins

- - (human granulocyte vitamin BIZ-

binding protein and human plasma transcobalamin 11) - fxom human milk and saliva

- - from human gastric juice

Estradiol Vitamin B,, (monocarboxylic acid derivatives) Monocarboxyl derivatives of vitamin B,, Vitamin B,, Concanavalin A

PROTEINS AND PEPTIDES, SHCONTAINING Cysteine-containing histones pChloromercuribenzoate p-Mercuribenzoate Mercaptalbumin Glutathione-2-pyrid yl disulphide 3,6-Bis(acetatomercurimethyl)dioxane SH-co nt aining DNA-nuclea pro t eins SH-proteins p-Mercurianiline - (bovine serum albumin, ficin, p-lactoglobulin, ovalbumin, bovine haemoglobin) p-( Acet oxymer cur i)aniline Sulphydryl-containing peptides from insulin

Sepharose with 3,3‘diaminodipropylamine Sepharose Sepharose 4B with ethylenediamine Sepharose 4B with ethylenediamine Sepharose 2B Sephadex G-25 SH-Sephadex Copolymer of maleic anhydride and ethylene with p-mercurianiline in the presence of hexamethylenediamine Copolymer of maleic anhydride and ethylene

References - .., - - . Mainwaring and Irving Irving and Mainwaring Rosner and Smith

Matsuura et al. (1973, 1974) Pensky and Marshall (1969, 1974) Korcek and Tabachnik Tate er 01. Le Gaillard et al. Best-Belpomme er al. Allen et al. Allen and Majerus

C

v1

m

Burger and Allen Whitehead et al.

RuizCarrillo Ishikawa and Iwai Carlsson and Svenson Jellum and Edjarn Eldjarn and Jellum Liener

Liener and Li-Pen Chao

- proteins and peptides - peptides from parvalbumin, mercaptplbuminamiceruloplpsmin Tryptic peptide containing the~esseatirl thiolgroup of lombriciuekiarse

5,So-Dithbb~2&~te) Glutathioni~2-pyrldyldisulpbide

!%pharos-4B with 1 , W i h e x a n e Activated thiolsepharose 4B

Lin and Foster

Sephame 4B

Terrossianet al.

Egomv et al.

OTHER PROTEINS A protein from n o d nllrse shark serum ara c pmtem (reguhtory protein requid for expression of the L d i w s e opaon)

Sephadex 6 7 5 or mss-linked levangel HarisdasgLul et al. Sepharose4B Wilcox et al.

Sepharose4B

W h x and ctemetson

whatman CF 11=nulose

r Bacterialandphageproteins , Calf thymushistones Cationic proteins from human neutrophil gronul0Cyt~ C4 from a pseudogmObulin fraction of humanserum Chicken W-hite ovofhvopmtein

Cholinergicproteolipid from rat leg muscle Clottable fibrinogen from plasma

H-W

Sepharose 6B

carmichzel MizonetuI. (1974) Rindler-Ludwg and Braumteiner Patrick et al.

3Carboxymethylriboflavin

Sephamse 4B

Blankenhorn et al.

Sephadex LH-20 with 3,3-iminobkprowlamine Sepharose 6B

Llorente de Carlin and de Robertis Stemberger and Hormann (1975) Furie and Furie (1974) Stemberger and Hormann (1976) Egly er al.

Sepharose4B CNBrgctivnted Sepharose 4B

S'-phosphate pPhenyltr imethylammonium Thrombin-activated fibrinogen

Coagulant protein of Russell's viper venom Factor X (coagulation) Fibrinogen Cold-insolubleglobulin

Sepharose 4B Sepharose 6B

Cytoplasmic protein with high af€iity for dRNA Different myosins and active myosin fragments Dodecyl sulphatesolubilized proteins of the human erythrocyte membrane Ferritin (tumour)

RNA

Sepharose 2 8

Periodate-oxidied ATP

Sepharose 4B with sebacic acid (or 2B with adipic acid) dihydrazide Sepharose 4B with H,N(CH,),,NH,

Oplatka et ul. (1976)

Sepharose 4B

Marcus and Zinberg

a-Fetoproteins (human, dog and rabbit) - (human)

Succinic anhydride Antibody (IgC fractions of sheep antiserum to breast tumour fractions) Anti-human a-fetoprotein antibody a-Fetoprotein specific antibody

Simmonds and Yon

Nishi and Hirai Sepharose 6B P N ~ ~ ~ g l ~ ~ i n ~ l - h y d r ~ ~ y p ~Caron ~ p y let- al. Sepharose 4B (Continued on p. 312)

4

?i 5

s

2

zr3 *

W

c t3

TABLE 11.1 (continued)

~-

Substances isolated

Affinity lzgands

Solid supports or immobillzed affinity ligands

References

a-Fetoprotein (human) (continued)

Concanavalin A yClobulin YClobulin fraction from anti-human a-fetoprotein antiserum

Con-A-Sepharose Sepharose 4 8 CNBr-activated Sepharose 4B

Pag6 Cittanova er al. Porrester ef al.

Estradiol-l7p-monohemisuccinate Fibrinogen or fibrin monomer

Sepharose with diaminononane Sepharose 6B Sepharose 2B with adipic acid dihydrazide Sepharose 4B Sepharose 2B with adipic acid dihydrazide Sepharose 2B Sepharose 2B with adipic acid dihydrazide Sepharose 4B with ethylenediamine Sepharose 4B

Uriel et al. Heene and Matthias Lamed elal. (1973a)

RiuzCaxrillo and Alfrey Mizon et al. (1976)

Sepharose 6B

Yon (1972)

Sepharose 4B Sepharose 4B with adipic or sebacic acid hydrazide Glycosylex-A or Con A-Sepharose Sepharose 4B

Azuma ef al. Wilchek and Lamed

Sepharose 2B with sebacic acid dihydrazide Sepharose 4B Sepharose 4B

Lamed and Oplatka

- from human cord serum - (human, rat and mouse) Fibrinogen derivatives Heavy meromyosin

Heavy meromyosin double-headed and one-headed S-l Heavy mero myosin subfragment- 1 _ - _ reacted with thiol reagents Histone fraction F3 from calf thymus Histones Lipophilic proteins Lipoproteins from pig serum Meromyo sin

ATP 6-Aminohexan-1 -01 pyrophosphate ATP ATP

pChloromercuribenzoate n-Butylamine, nzhexylamine or nactylamine 1,lO-Diaminodecane and succinic anhydride Concanavalin A ATP

Concanavalin A Molecular variants of a, -fetoprotein MOPC 315, myeloma IgA protein (mouse) c-N-DNP-lysine which binds nitrophenyl ligands Myosin (chicken) ATP

- from rabbit skeletal muscle - from small amounts of muscle and nonmuscle tissue Nicotinic and muscarinic hydrophobic proteins

N646-Aminohexyl)-ADP 8-(6-Aminohexyl)amino-ADP or N6-(6-aminohexyl)-ADP Quaternary ammonium compound, p-phenyltrimethylammonium of recognized cholinergic character

Sephadex LH 20 with 3,3'diaminodiprop ylamine

Trayer et al. (1974b) Lamed and Oplatka Oplatka er al. (1975) Lamed e t a ! . (1976)

rn X

*

r m

v1

Smith and Kelleher Goetzl and Metzger

Trayer et al. (1974b) Trayer and Trayer (1975) Barrantes et al.

e 5 m e

“One-head” trinitrophenylated myosin and heavy meromyosin Papain digest of myosin P-450 haemeproteins from Rhizobiurn japonicurn Phosphorylated nucleolar non-histone proteins Plant chromatin Platelet microtubule protein Precursor head protein (P23) of bacteriophage T, Protein factor stimulating DNAdependent RNA synthesis from pea and corn shoots Protein fractions from postribosomal supernatants of rabbit reticulocyte and rat liver Proteins from cell-free extract of Escherichia coli - of rat liver nuclear 30s particles Protein species isolated from HeLa cell cytoplasm Reticulocyte [ ’HJnon-histone protein Ribosomal proteins RNase-resistant ribonucleoprotein fraction from HeLa polyribosomes Serotypeapecific globulin Soft-tissue collagens Specific p-aminobenmate “pick-up” protein Tropomyosin and troponin Venom coagulant protein o f Vipera russelli -

Sebacic or adipic dihydrazide ATP

Sepharose

Gadasi et al.

8-(6-Aminohexyl)amino-ADP rn-Aminophenobarbital

Sepharose 4B Sepharose 4B

Trayer et al. (1974b) Dus et al.

DNA

Cellulose

Bearden and Chandra

RNA-polymerase 7Globulin fraction of anti-tubulin serum Anti-P 23 antibody

Affi-Gel 10 AffiGel 10

Jalouzot et al. Ikeda and Steiner

CNBr-activated Sepharose 4B

Y anagida

2,4-Dichlorophenoxyacetyl~-Llysine

Sepharose 4B

Venis

Polyadenylic acid

Sepharose 4B

Fukami and Itano

L-Valine

Sepharose 4B

Rimerman and Hatfield

Polyuridylate Polyadenylate

Poly(U)-Sepharose4B Sepharose 4B

Schweiger and Mazur (1975) Blanchard et al.

Erythrocyte DNA RNA

Gadski and Chae Burrell and Horowitz

Polyuridylic acid

Cellulose Sepharose 4B with adipic acid dihydrazide CNBr-activated Sepharose 4B

Bacterial cells 2,2’dipyridyl disulphide p-Aminobenzoyl chloride

DE AE-cellulo se Thiol-activated Sepharose 4B Cellulose

McKinney and Thacker Sykes Toth-Martinez et al.

Factin Factor X equilibrated with lanthanide ions

Sepharose 4B Sepharose

Kondo et al. Furie and Furie (1975)

Kish and Pederson

~~~~

(Continued on p. 314)

W

TABLE 11.1 (continued) Substances isolated

P

Affinity ligands

SPECIFIC PEPTIDES AND AMINO ACIDS Affinity labelled peptides from the tryptic Nuclease digest of staphylococcal nuclease Anti-azobenzenearsonate antibodies Arsanilazotyrosyl-wntaining peptides Biotin-containingpeptides Avidin Anti-DNPantibodies Cysteine and methionine containing peptides (after reaction with P-bromoacetykN'-DNP-L-lysine) Degradation products of human fibrinogen Fibrinogen or fibrin monomei Dinitrophenyl(DNP)4abelled peptide from Anti-DNP antibodies antibody to DNP - peptides from a whole digest of heavy chains Anti-DNP antibodies DNP-lysyl-containingpeptide Fibrinogen Fragment D together with higher molecular weight materials from the plasmic digest of fibrinogen Antibodies Fragments of papaindigested human immunoglobulinG Glycopeptides obtained by Glycopeptides proteolytic digestion of various glycoproteins Concanadin A Glycopeptides (neutral) obtained from rat brain glycoproteins Heme peptide of cytochrome C Immunoglobulin polypeptide chains lmmunobgically active and structurally similv fragments from tryptic digestbn of protein A D- and L-isomers of &3,4-dihydroxyphenylplanine (DOPA)

Solid supports or immobilized affinity Iigands

References

Sepharose 4B

Wilchek (1970)

Sepharose 4B Sepharose 4 8 Sepharose 4B

Wilcheker al. (1971a) Bodanszky and Bodanszky Wilchek (1 974)

Sepharose 6B Sepharose 4B

Matthias et al. Givol et al. Givol and Rotman

Sepharose 4B CNBr-activated Sepharose 4B

Wilchek er al. (1971a) Collen er al. (1975)

BioGel P 300

Sapin er al.

Sepharose 2B or 4B

Sepulcre and Monar

Con A-Sepharose Glycosylex A Con A-Sepharose

Ogata et al.

Tomana et al. Krusius

~mmuwglobulinG

Sepharose 4B Sepharose 4B Sepharose 4B

Wilchek (1972,1974) Mannik and Stage Hjelm er al. (1975)

L--

Sephadex G-25 with cyanuric chloride

B a a & et al.

Concanavalin A SeNm albumin (bovine or human) Anuiy

P5 r m v)

NH,-Terminal activation peptide from plasminogen Nucleasederivatives (mixture of nuclease fragments) Nitrotyrosine-contaming peptides - - from the chymotryptic dkest of carhxypeptidase B Peptides containing tyrosine labelled with m-nitrobenzenediionium reagent - - modified tyrosine Peptide from the binding site of antidmitrophenyl antibody Plasmic fragment D Ribonuciease-S-peptidesynthesized by the solid-phase technique Synthetic fragments of ribonuclease Synthetic peptide analogues to fragment P, of staphylococcal nuclease Synthetic ribonuclease S-peptide derivative D- and L-tryptophan Tryptophancontaining peptides - - after modification with 2,4dinitrophenylsulphenyl chloride

Plasminmen B

Sepharose 4B

Wiman and Wall6n

3-(4-Aminophenylphosphoryl)deoxythymidine S'ghosphate Antibodies to nitrotyrosine

Sepharose 4B

Andria et al.

Sepharose

Helman and Givol Sokolovsky (1972)

Antidinitrophenyl antibodies

Sepharose 4B

F r a n k (1971) Bustin and Givol

Frana (1973)

*

r

r

4

Trombin-treated fibrinogen Ribonucled-protein

Sepharose 4B Sepharose 4B

Native fragment of ribonuclease Sepharose Fragment P, (residues 49 through 149) Sepharose 4B

Kudryk et al. Anf'i'in

b

Chaiken Ontjes and Anfimsen (1969b)

$ +I 5 0

=!

is v)

Ribonuclease S-protein Bovine serum albumin Deoxyoligothymidylic acid Antidinitrophenyl antibodies Sulphur monochloride

Tyrosine-containing peptides (after Anti-nitrotyrosine antibody or antiarsanilazo antibodies nitration or arsanylation) Uridine diphosphatelabelled peptide from RNase bovine pancreatic ribonuclease VIRUSES Aleutian mink disease virus

8

Aleutian disease antibody (found in the immunoglobulin G fraction) Immunoglobulin from chronically infected milk

Sepharose 4B Sepharose 4B-200 with ethylenediamine and with succinic anhydride DEAEcellulose Sepharose 4 B

Kato and Anfimsen Stewart and Doherty (1973)

ci

Ecstein et al. Wilchek and Miron

Rubinstein et al. Aminoethylated polyacrylamide with di-N-hydroxysuccinimide ester of 55'dithiobis-2-nitrobenzoic acid Sephose 4B Wilchek (1974) Sepharose 4B

Givol et al.

Sepharose 4B

Kenyon et al.

Sepharose 4B

Yoon et al.

W

(Continued on p. 316)

c. VI

w _.

c

TABLE 11.1 (continued)

m

Substances isolated

Affinity h a n d s

Solid supports or immobilized affinity liiands

References

Foot-and-mouth disease virus (FMDV) Influenza viruses

Tobacco mosaic virus

Anti-tobacco mosaic virus antibodies

Sepharose 4B Disulphide-linked yglobulin with N-acetylhomocysteine thiolactone Cellulose Antibodies cross-linked by glutaraldehyde p-Aminobenzylcellulose

Matheka and Mussgay Sweet et a!.

Murine type< virus p30 precursor protein Plant virus

FMDV antibodies yClobulin of rabbits immunized with influenza virus Single-stranded DNA Antibodies

Galvez

BioCel A-15 Sepharose 4B

Lazarides and Lindberg Rosen et al.

Phosphocellulose BioGel A-5m

Cole and Gaertner Plotz et al.

Con A-Sepharose

Pitlick

Agarose with 3,3’diaminodipropylamine or with albumin Agarose (Bio-Rad 0.5) Sepharose 4B with bound e-aminocaproic acid Sepharose 4B with bound native albumin Sepharose with e-aminocaproic acid Sepharose 4B with 1,5diaminopentane and succinic anhydride Sepharose 4B Sepharose 6B Sepharose 6B BioCel A-5m

Parikh and Cuatrecasas

Oroszian el ai. ( 1 976) Lapido and Zoeten

OTHERS Actin from chicken skeletal muscle Agglutination factor

Deoxyribonuclease 1 Polymer of Dgalactose and 3,6. anhydro-Lgalacto se Aromatic complex from Neurospora crassa Phosuhate Human serum albumin Bilirubin and other albumin-bound substances from plasma and blood Bovine brain tissue factor of coagulant Concanavalin A activity Gangliosides Cholera toxin from Vibrio cholerae Coagulation factor Diphtheria toxin

Heparin Nicotinamide adenine dinucleotide

Enterotoxin from Vibrio cholerae

Ganglioside

Factor 4 from human platelet Factor VII from bovine plasma

Heparin p-Aminobenzamidine

Factor IX from human plasma First component (Cl) of human complement

Heparin Human yglobulin IgG immunoglobulin rn-(m-Aminophenoxybutox y)benzamidine p-Aminophenyl9-D-thiogalactoside

gal-Repressor

Sepharose with 3,3’diaminodipropylnnrl

m-,-o;..h

--h.A-:A-

Gentry and Alexander Cukor et al. Cuatrecasas ef al. (1973) Levine and Wohl Jesty and Nemerson Rosenberg et al. Sing (1971) Sing (1974) Sing (1974) Parks et al.

Gibberellins orGlucosylated teichoic acid Group A meningococcal surface components (aqueous phase) High- and low-active heparin species Host factor required for RNA phage Q P RNA replication Human blood clotting factor X Human chorionic somato-mammotropin (HCS) Human glomerular basement membrane fractions ImniunologicaIly active fraction of carcinoembryonic antigen Interferon (human) - from human leukocytes and foreskin fibroblast cells - (human) - from human fibroblast - from human fibroblast

- from human leukocyte - (mouse) - (mouse) - (mouse) - from mouse peritoneal cells - from mouse L cell Intrinsic factor (human) - - (human and hog) and non-intrinsic factor (hog) - - (hog) Membrane-bound opiate binding sites

Specific antibodies to gibberellic acid Concanavalin A 4-Phen yltiutylamine

Sepharose 4B Sepharose 4B Sepharose

Fuchs and Gertman Doyle et al. Cheng et al. (1975)

Antithrombin Polyadenylic acid

Sepharose 4B Cellulose

Hook et al. Carmichael et al.

Blue Dextran 2000 Antibody to HCS

Bio-Gel A-1 5 Sepharose 4B

Vician and Tishkoff Weintraub

Anti-human glomerular basement membrane r G globulin Concanavalin A

CNBractivated Sepharose 4B

Mahieu and Winand

Sepharose 4B

Rogers et al.

Serum albumin Antibody to leukocyte interferon Globulins from rabbits immunized with these interferons Concanavalin A

Sepharose 4B Sepharose 4B Sepharose 4B

Huang et al. Anfinsen et al. Berg et al.

Con A-Sepharose

L- or D-tryptophan, L-phenylalanine, L-tyrosine L-Tryptophyl-L-tryptophan or L-tryptophyl-L-tyrosine Anti-interferon immunoglobulin

Agarose-L-tryptophan and analogues (Miles) Affi-Gel 10

Davey et al. (1974) Davey et al. (1976b) Sulkowski et al.

BioGel A-15m CNBr-activated Sepharose 4B Sepharose 4B CNBr-activated Sepharose 4B Bovine serum albumin eaminocaproic CNBr-activated Sepharose 4B and acid and Bandeiraen simplicifolia lectin CH-Sepharose4B Cellulose Hydroxocobalamin albumin 3,3'-Diaminodipropylamine-substituted Vitamin B,, Sepharose Sepharose with 3,3'diaminodipropylVitamin B,, (monocarboxylic acid derivatives) amine Sepharose with bromoethylamine Cobalamin Glass Lavorphanol or etorphine

Sulkowski et al. Sipe et al. @burn et al. Livingston Hajnicki et al. Davey et al. (1976a) Christensen et al. Allen and Mehlman Allen et al. Lien et al. Simon ~

(Continued on p . 31 8)

TABLE 11.1 (continued) Substances isolated Properdin from human serum - from human serum - (human) Rabbit reticulocyte initiation factors Rat secretory component in the free form RNASontaining preparations of Australia antigen Subunit of the first component of human complement Clq tRNA cistrons from Escherichlb coli Trp repressor from Escherichkr Cali Tubulin - from brain - without protein kinase activity

W r QD

Affinity ligands

Solid supports or immobilized affinity ba nds

References

Anti-human properdin antibodies

CNBr-activated Sepharose 4B

IgG fraction of antisera to human serum or to properdin Heparin Immunoglobulin G fraction of the antisecretory immunoglobulin A antiserum Immunoglobulin fraction of anti-Au antibody-containirtg serum Human Ipc

Sepharose 4B

Minta and Lepow Minta Konno and Huai

CNBr-activated Sepharose 48 Sepharose 4B

Waldman et al. Vaerman et al.

Sepharose 4 8

16zwiak and Kokielak

Sepharose

Sledge and Sing

Agarose BioGel A-150m Cellulose Sepharose 4B with ethylenediamine and p-nitrobenzoylazide Sepharose 48

Millmetal. (1974) Shimizu et al. Schmitt and Littauer Himnan et ul.

Sepharose 4 8

Sandoval and Cuatrecasas

Anti-dinitrophenyl antibody Q 84 pt ED DNA Colchicine A mixture of deacetylcolchicine and isodeacetylwlchicme (1) Colchicine derivatives; (2) casein

x”9

5r

s: U

=!I

3 %

NUCLEASE

319

graphy with additional purification procedures are far more numerous. General principles that must be observed for successful isolations were discussed in detail in Chapters 5 and 10, and the conditions should be modified in particular instances on the basis of these. In order to provide the reader with information on individual affinity ligands that have actually been used, or commercially available immobilized affinity ligands, commercial names are given in all instances if they have been published. A review of these, with an indication of their chemical natures and procedures, is given in Table 11.2. 11.2 RESOLUTION OF DGTRYPTOPHAN BY AFFINITY CHROMATOGRAPHY ON BOVINE SERUM ALBUMIN-AGAROSE COLUMN

The different types of bonds of some optical isomers on serum proteins have been elucidated, such as the binding of DL-tryptophan on bovine serum albumin (McMenamy and Oncley) and of DLaldosterone on corticosteroid-binding globulin (Varsano-Aharon and Ulick). Stewart and Doherty (1973) used affinity chromatography on bovine serum albumin attached to agarose for the resolution of Dbtryptophan. Of chromatographic columns packed with the bovine serum albumin attached directly to Sepharose 4B, with serum albumin attached to succinoylaminoethyl agarose or with defatted serum albumin (method according to Chen) attached in an analogous manner, the last method gave the best results. The course of this chromatography is shown in Fig. 11.1. It was shown that the material eluted with a borate buffer contained only D-tryptophan, whereas that eluted with acetic acid was L-tryptophan only. Evidently, affinity chromatography could be used in a similar manner for the resolution of other enantiomeric pairs. 11.3 SEMI-SYNTHETICNUCLEASE AND COMPLEMENTARY INTERACTION OF NUCLEASE FRAGMENTS

Staphylococcal nuclease is an enzyme that has been studied most widely, using affinity chromatography. A successful isolation of nuclease on Sepharose with attached 3<4-aminophenylphosphory1)deoxythymidine 5 '-phosphate (Sepharose-pdTp) was already described in 1968 (Cuatrecasas et d.).Using affinity chromatography of a tryptic hydrolysate of nuclease labelled with (deoxythymidine-3 ',5 '-diphosphate-aminopheny1)deoxythymidine 3 '-[ 14C] -bromoacetyl.p-aminophenylphosphateon Sepharose with attached nuclease, Wilchek (1970) obtained peptides of the active site. When carrying out a tryptic hydrolysis of nuclease in the presence of Ca" and nucleoside 3',5'-deoxythymidine diphosphate (pdTp), Ontjes and Anfinsen (1969a, b) obtained three fragments: a pentapeptide and two polypeptide fragments, nuclease-T<6-48) and nucleaseTx49-149) (see Fig. 11.2). The last two fragments were inactive and disordered when alone, but they associated reversibly with the formation of a non-covalently bound complex called nulcease-T'. Nuclease-T' had approximately 8%(1620 units/pmole) of the activity of the native enzyme (1 5,800 units/pmole), and its three-dimensional structure is similar to that of nuclease. The same results were then achieved even when the native nuclease-T(6-48) fragment was replaced with an analogous peptide prepared synthetically according

W

TABLE 11.2

N 0

COMMERCIAL NAMES OF SOLID SUPPORTS AND IMMOBILIZED AFFINITY LIGANDS MENTIONED IN TABLE 11.I Trade name

Nature

Producer

Activated CH-Sepharose 4B Activated-thiol Sepharose 4 8

CHSepharose 4B esterified with N-hydroxysuccinimide 2,2’-Dipyridyl disulphide and glutathione coupled to CNBr-activated Sepharose 4 8 N6-(6-Aminohexyl)adenosine-2’3’-bisphosphate covalently bound to Sepharose 4B using cyanogen bromide method Agarose-NH-(CH,),-NH-CO-(CH,), -COOH See Table 8.3 = AffiCsl 202 Fucosamine covalently bound to e-aminocaproylagarose

Pharmacia Fine Chemicals, Uppsala, Sweden Pharmacia Fine Chemicals, Uppsala, Sweden

Miles-Yeda, Rehovoth, Israel

5’-(4-Aminophenyl)uridine-2’,3’-phosphate coupled to

Miles Labs., Kankakee, IU., U.S.A.

Z‘,S’-ADP-Sepharose 4B

AF 201 AffiGel 10, 101,102,201,202,401,501 Affinose 202 Agarose-e-aminocaproylfucosamine (AF 5 , 7 , 1 0 and 11) Agarose-APUP

Pharmacia Fine Chemicals, Uppsala, Sweden‘

Affitron Corp., Costa Mesa, Calif., U.S.A. Bio-Rad Labs., Richmond, Calif., U.S.A.

agarOSe

Agarose-Gly -Gly -Tyr(O-benzyl)-&g Agarose-hexane-poly(1) .poly(C) Agarose-NADP AG-NAD, type 1 AH-Sepharose 4B Alkylamine CPC Amberlite CG 50/11 (= IRC-SO) 5‘ AMP-Sepharose 4B BioGel A-O.5m; 1.5m; 5m; 50m BioGel P-2, 150, 200, 300 Blue-Sepharose CH-Sepharose 4B

C1ycylglycyl-O-benzyl-L-tyrosyl-L-arghine coupled to agarose using the cyanogen bromide method Polyriboinosinic acid and polyribocytidylic acid bound to agarose (see Table 6.5) NADP coupled to agarose with adipic acid dihydrazide NAD+ coupled to agarose with hexanoic acid 1,6-Diaminohexane covalently bound to Sepharose 4B Alkylamine derivative of glass Weakly acidic polymethacrylic ion exchanger N6-(6-Aminohexyl)-5‘-AMP coupled to Sepharose 4B using the cyanogen bromide method Agarose gels with molecular weight exclusion limits of 0.5,1.5,5 and 50 lo6 Polyacrylamide gels with molecular weight exclusion limits of 2,150,200 and 300 lo3 Cibacron Blue F 3G-A covalently bound to Sepharose CLdB by the triazine coupling method 6-Aminohexanoic acid covalently bound to Sepharose 4B

-

-

Miles Labs., Kankakee, Ill., U.S.A. P.L. Biochemicals, Milwaukee, Wisc.,.U.S.A. P.L. Biochemicals, Milwaukee, Wisc., U.S.A. P.L. Biochemicals, Milwaukee, Wisc., U.S.A. Pharmacia Fine Chemicals, Uppsala, Sweden Pierce Chemical Co., Rockford, Ill., U.S.A. Rohm & Haas, Philadelphia, Pa., U.S.A. Pharmacia Fine Chemicals, Uppsala, Sweden Bio-Rad Labs., Richmond, Calif., U.S.A. Bio-Rad Labs., Richmond, Calif., U.S.A. Pharmacia Fine Chemicals, Uppsala, Sweden Pharmacia Fine Chemicals, Uppsala, Sweden

CNBr-activated Sepharose 4B Con A-agarose Con A-Sepharose Cuprophan Degalan, V 26 EMA Epoxy-activated Sepharose 6B Enzacryl AA Glycosylex A Indubiose A4; ACA-314 Insoluble trypsin resin Oligo(dT)-cellulose T2, T3 Poly(U) Sepharose 4B Sephadex G 2 5 , -50, -75, -100, -150, -200

Sephadex LH-20 Sepharose 2B, 4B, 6B Sepharose CL

Sephazyme trypsin Soybean-CM-cellulose affmity resin Spheron-300 Ultro-gel (AcA22, AcA34, AcA44)

Sepharose 4B activated with cyanogen bromide Concanavalin A attached to agarose Concanavalin A covalently bound to Sepharose 4B Cellulosic membrane Poly(methy1 methacrylate) plastic beads of average diameter 250 pm Copolymer of ethylene with maleic anhydride Sepharose 6B with 1,4-bis-2,3-epoxypropoxybutane Polyacrylamide gels containing aromatic acid residues Concanavalin A coupled to agarose Agarose Trypsin covalently bound to EMA Cellulose containing covalently bound oligodeoxythymidylic acid (length = 10-12 bases) Polyuridylic acid covalently bound to Sepharose 4B using the cyanogen bromide method Dextran cross-linked with epichlorhydrin with molecular weight exclusion limits of 5, 10,50, 100, 150 and 200.103 Hydroxypropylated Sephadex G-25 2%, 4%, 6% agarose Sepharose cross-linked by reaction with 2,3dibromopropanol and desulphated by alkaline hydrolysis under reducing conditions Trypsin covalently bound to Sepharose Soybean trypsin inhibitor covalently coupled to CMcellulose Hydroxyalkyl methacrylate gels with molecular weight exclusion limit of 300 000 Polyacrylamide-agarose beads

Pharmacia Fine Chemicals, Uppsala, Sweden Sigma Chemical Co., St. Louis, Mo. 63118, U.S.A. Pharmacia Fine Chemicals, Uppsala, Sweden Enka Glanzstoff, Munich, G.F.R. Degussa Wolfgang, Hanau am Main, Germany

sz % m

Monsanto Co., St. Louis, Mo.,U.S.A. Pharmacia Fine Chemicals, Uppsala, Sweden Koch-Light, Colnbrook, Great Britain Miles Labs., Elkhart, Ind., U.S.A., and Slough, Great Britain Industrie Francaise Biologique, GenneviUers, France Merck, Darmstadt, G.F.R. Collaborative Research, Waltham, Mass., U.S.A. Pharmacia Fine Chemicals, Uppsala, Sweden Pharmacia Fine Chemicals, Uppsala, Sweden

Pharmacia Fine Chemicals, Uppsala, Sweden Pharmacia Fine Chemicals, Uppsala, Sweden Pharmacia Fine Chemicals, Uppsala, Sweden

Pharmacia Fine Chemicals, Uppsala, Sweden Seravac Labs., Maidenhead, Great Britain Lachema, Brno, Czechoslovakia Realm Chem. Co., New Brunswick, N.J., U.S.A. LKB, 91400 Orsay, France

W c N

EXAMPLES OF USE OF AFFINITY CHROMATOGRAPHY

322

0

Lu

FRACTION

NUMBER

Fig. 11.1. Chromatography of DL-tryptophan on de-fatted bovine serum albumin-succinylaminoethylagarose (250 x 9 mm). DL-Tryptophan (500 nmole) dissolved in 0.1 ml of 0.1 Mborate buffer (pH 9.2) containing 1%(v/v) of dimethyl sulphoxide, was applied to the column (containing a total of 630 nmole of bovine acrum albumin). The column was eluted at 30 ml/h with borate buffer (no dimethyl sulphoxide) for 20 tubes then with 0.1 N acetic acid. The void volume was determined fxom the elution volume of dimethyl sulphoxide. Reproduced with permission from K.K. Stewart and R.F. Doherty,Proc. Nut Acad. Sci. US.,70 (1973) 2850-2852.

NUCLEASE

72

1

49 NUCLEASE-T-/49-149/

6

122

149 I

48 126 J

<

T

99

NUCLEASE-/99- 149/

133

149

49

NUCLEASE-/49-126/

NUCLEASE-/ 127-149/

gg 106

67

L

NUCLEASE- T - / 6 -481

NUCLEASE -/1- 126/

-

-

55

35

I

,

, , / ? t , , A

~

I

149

,/,/,,,,, t

1

126

[

1

127 149 t=====!

Fig. 11.2. Diagram of linear relationship of the amino acid sequences of nuclease and its fragments that yield productive complementation. The X-ray crystallographic study showed @-structuresbetween residues 12 and 35 and three a-helical parts between residues 55 and 67,99 and 106 and 122 and 133, which are indicated on the nuclease line. Nuclease-(127-149) does not bind to nuclease-(l-126). Reproduced with permission from G.Andria et ul., J. Bwl. Chem., 246 (1971) 7421-7428.

NUCLEASE

323

to Merrifield. During the purification of this synthetic 42 peptide, affinity chromatography was again of considerable help. The synthetic product was purified on Sepharose with attached fragment nuclease-T.(49-149). In a preliminary experiment first a small amount of native fragment nuc!ease-T(6-48) was chromatographed on this column, At p# 8 in the presence of calcium chloride and pdTp (necessary for the stabilization of the complex), sorption took place. The native fragment was then eluted with dilute acetic acid of pH 3, l e . , under conditions when dissociation of the complex takes place. On the basis of this experiment, the purification procedure for the synthetic fragment was established as fob lows. A 5-mg amount of synthetic peptide were applied on to the column ( 5 x 1 cm) in 0.0sM borate buffer of pH 8, containing 0.01 M calcium chloride and 0.001 M pdTp. Using the same buffer, some peptidic material was eluted that had the same composition and mobility as the native peptide on disc electrophoresis. However, it was not adsorbed in the column even after repeated chromatography. About 7% of the total material applied was then eluted with 0.001 M acetic acid of pH 3, This part did not differ significantly in its amino acid composition from the starting material. This peptide fragment was then used for complementation with nuclease-T.(49-149) fragments, where it could fully replace the native peptide. In this manner the semi-synthetic nuclease was prepared, The second example of a productive complementation was the complex of two other fragments, i.e., nuclease (1-126), prepared by limited tryptic hydrolysis of trifluoroacetylated nuclease and the cyanogen bromide fragment of nuclease (99-149). This complex possessed 10-12% of the enzymatic activity of native nuclease (Taniuchi), Both of the mentioned complexes were bound with Sepharose-pdTp. On Sepharose-pdTp a mixture of three fragments was bound by Andria et al.; 0.085 pmole of nuclease-T(6-48), 0.060 pmole of nuclease-T(49,50-126) [Nuclease-T(49,SO-126) is a mixture of the peptides nuclease-T(49-l26) and nuclease-T($B-l26)], 0.067 pmole of nuclease(99-149) and 0.01 M calcium chloride in 0,2ml of 0.1 Mammonium acetate solution of pH 8 were applied each time on a column of Sephrose-pdTp (4x 1 cm) at 4, 10,15 and 20°C. The amount of the complex eluted with 0.1 Maceticacid was greatly dependent on temperature; it was highest at 4OC and decreased with increasing temperature (see Fig. 10.1j. On the basis of amino acid analysis and two-dimensional peptide maps of tryptic hydrolysate, it was shown that the material is composed of equimolar amounts of nuclease-T.(6-48), , nuclease-T(49,50-126) and nuclease-(99-149). The specific activity of the complex was 15.3 units/pmol. The decrease in the amount of the sorbed complex observed with increasing temperature may be caused by the decreasing formation of the complex or the decrease in the affinity of the affinity figand for the eomplex, which is connected with the thermal instability of the enzymatic activity of the threefragment complex. Affinity chromatography of any combination of two of the three fragments mentioned does not indicate a sipificant amount of adsorbed material, even at 4%. At this temperature no adsorption of the three fragments wag observed in the absence of calcium, or when a mixture of nuclease fragments (1 -120) and nucieease (1 27-149) WIIB applied. From these results, it is evident that affinity chromatography may become an important method in the study of the complementation of peptide fragments of active proteins, prepared both by isolation from natural material and by synthesis.

3 24

EXAMPLES OF USE OF AFFINITY CHROMATOGRAPHY

1I .4 STUDY OF INTERACTIONS OF BIOLOGICALLY ACTIVE SUBSTANCES

Ihe use of affinity chromatography for the determination of dissociation constants for binary complexes of dehydrogenases and NADH was discussed in Chapter 2 . This method was developed recently by Brodelius and Mosbach (1976). The determination of dissociation constants on the basis of the elution volumes of the enzyme released from a column of immobilized inhibitor by variously concentrated solutions of free inhibitor is discussed in detail in Chapter 4, as is the study of the interaction of oligoadenylic acid with immobilized polyuridylic acid on the basis of the analysis of elution profiles or the interaction of nucleotides with peptides by means of template chromatography. A further example is the study of the interactions of human haemoglobin and its polypeptide chains with haptoglobulin bound t o agarose matrix (Tsapis et al.). Haptoglobulins (a2glycoproteins present in the blood plasma of many animal species) have a high binding affinity for haemoglobin. While the detailed three-dimensional structure of a haemoglobin molecule is known, far less information is available on the structure of haptoglobin. As regards the common binding sites, it is known that the heavy chains of haptoglobin contain four binding sites, viz., one pair of sites for each olD-haemoglobin dimer, while the binding affinity for the a-haemoglobin chain is substantially higher than that for the &chain. The study of the type of interaction between human haptoglobin covalently bound to Sepharose 4B and the human haemoglobin and p-chloromercuribenzoic acid-treated a- and @-chains was carried out in two ways. On the one hand, the binding was studied duringflowthrough chromatography, and on the other, equilibrium binding experiments with &+-chains were carried out. a+-and @chains are 14C-labelled chains prepared by using p-chlorom e r ~ u r i [ ' ~ Cbenzoic ] acid. The binding of haemoglobin and a+-and P'chains on to the immobilized haptoglobin (38 nmole of haptoglobin attached to 1 ml of Sepharose 4B) is shown in Fig. 1 1.3. The addition of increasing amounts of haemoglobin leads to a saturation plateau at a value of 1.5 mole of haemoglobin chain per mole of haptoglobin. In this respect it differs from the stoichiometry of the saturated complex of haptoglobin and haemoglobin in solution, which is 1 mole of haemoglobin or 4 mole of achain per mole of haptoglobin. Of the values obtained on immobilized haptoglobins only 0.39 mole of haemoglobin corresponds to 1 mole of haptoglobin. The difference in binding capacity was ascribed by Tsapis et al. both t o the denaturation of haptoglobin during the coupling to the solid support and to the impaired steric accessibility of the binding sites of haptoglobin by bonds too close to the agarose surface. The addition of &+-chains of a similar concentration results in smaller amounts of bound ol+-chains, and the saturation plateau is not attained even at a concentration of 0.60 mM. The binding of the P'chain is so weak that it is measurable at high concentrations only (0.50 and 0.73 mM). From these results, the same order of binding affinity for the immobilized haptoglobins follows as for the free haptoglobin, i.e., haemoglobin >&-chain > &chain. On the basis of saturation experiments, the value of 14.8 nmole of functionally active haptoglobin per 1 ml of bed (0.39 x 38 nmole) was calculated in equilibrium binding experiments with &+chains.The distribution of intrinsic association constants, K j , was expressed on the basis of Scatchard's relation: r/c = K,, - K ,

(11.1)

INTERACTIONS OF BIOLOGICALLY ACTIVE SUBSTANCES

325

I

LIGAND ADDED.mM

Fig. 11.3. Binding of human liganded haemoglobin (Hb) and haemogIobin a-and P-chains treated with p-chloromercuribenzoicacid (a*-and P*-chains) to Sepharose 4B substituted with haptoglobin, '1 (Aga-Hp). Aliquots of 1 ml of ligand were applied on 1 ml of Aga-Hp. The excess (non-bound) ligand was eluted with 100 mM potassium phosphate buffer (pH 7.4) containing 1 mM EDTA. Binding of Hb (x), &*-chains( 0 ) and p*
where r is the number of molecules of affinity ligand bound on to functionally active molecules of the immobilized haptoglobin at c, which is the concentration of affinity ligand in solution, and n is the maximum number of molecules of affinity ligand whch can be bound to the molecules of haptoglobin. Scatchard's plot of 14C-labelledcw+-chainsbinding on haptoglobin Sepharose 4B is shown in Fig. 11.4. Equilibrium experiments were carried out by addition of a 1-ml volume of 14C-labelled &+-chainsto 1-ml volumes of immobilized haptoglobin. The concentration of the affinity ligand of &+-chainsvaried within the range 1.66 pM-0.66 mM. After stirring for 30 min at 4"C, the suspensions were separated by centrifuging for 10 min at 500 rpm. The concentration of the &+chainsin solution was determined on the basis of the measurement of ''C-radioactivity. The amount of attached &+-chainswas calculated from the difference between the total amount of the &+-chainsadded and free &+-chainsmeasured in the solution. At the same time, corrections for the non-specific binding and the liquid trapping in the solid matrix were calculated, which were determined using the same amount of human immunoglobulin G bound to agarose under the same conditions. Assuming a Sipsian distribution of the intrinsic association constants K j , the heterogeneity index, a,of haptoglobin binding sites was calculated according to the equation log

(L=crIogKa+fflogc ) n-r.

(11.2)

The calculated a index was 0.72 and the average affinity constant, K a , was 3.6- lo4 I/mole.

326

EXAMPLES OF USE 01: AFFINlTY CHROMATOGRAPHY

I

a

Fig. 11A. Scntchtlrd pbf of "C-labelled rr*-blnding e n Aga-Hp (abbreviation8nu In Fig. 11.31, The procedure wan carried out a$ dereribed in the text. Insst: Siprirn reprewntntlon of the name results. Reproduced with permlsnion Prom A. TeapL @f d ,Eur. J. Bfochcm.,64 (1976) 369-372.

The shape of the Scatchard plot indicates heterogeneity of the binding sites of the immobilized haptoglobin for the &-chins.The population of the high-affinity binding sites with an extraplated affinity constant of K i = 5.260 10' l/mol is minor and the number of low-affinity binding sites with a constant Kg = 6.230 lo4 l/mol is major. Extrapolation to the r axis indicated that the maximum number of sites (n) was 4.3 and the number of Mghaffinity sites was 1.5. The maximum number of rites found for the immobilized haptoglobin is in agreement with the generally accepted value of four sites per haptoglobin molecule in solution. Hence the haptoglobin attached to agarose generally possesses the same binding properties for haemglobin and its a- and k h a i n r as in aoiution, and therefore it is a suitable agent for a detailed study of the mechanism of the interaction between the haptoglobin and hemoglobin and its subunits. Other uses of affinity chromatography include the study of the formation of complexes of arboxypeptidase I)with basic and aromatic analogues of amino acids (Ahnuma et al., 1971), the determination of the steric requirements for the binding of the substrate of the enzyme ant banilate 5-ghosphoribosyl pyrophosphte gbspbribosyl transferase on Sephrotw with attached anthranilate derivatives (Marcus and Balbinder), the study of histone-histene interactions (Mkon et al., 19749, the active centre of trypsinogen (Chauvet and Aeher, 19741, the interaction of progerterone-receptor complexes with nucleotides (Moudgil and Toft) and the binding sites of mammalian acetylcholinesterare (Hollunger and Niklasson).

MECHANISMS OF ENZYMATIC ACTION

321

11.5 STUDY OF THE MECHANISMS OF ENZYMATIC ACTION

In order to elucidate the stages of the ATP-splitting reaction catalysed by myosin or by its enzymatically active proteolytic fragments, Oplatka er al. (1975) attached on Sepharose 2B, through adipic acid hydrazide, [y-"P]ATP, non-labelled ATP, ADP or adenosine S'-(@,yimino)triphosphate [AMP-P(NH)P] . Using enzymatic digestion with papain, they prepared from myosin enzymatically active heavy meromyosin subfragment S-1 , from w h c h they then prepared its trinitrophenyl derivative. Fig. 11.5 shows the chromatography of the native and trinitrophenylated heavy meromyosin S-1 in the presence of either Mg2+ or EDTA on columns of agarose-ATP, agarose-ADP and agarose-AMPP(NH)P. It is evident that in all instances, except for agarose-ATP, the concentration of potassium chloride necessary for elution in the presence of Mg2+ was not higher than 0.2M and the difference in the behaviour between the native and dinitrophenylated heavy meromyosin S-1 derivative was not significant. With ATP columns, a higher concentration of potassium chloride was necessary for elution in the presence of Mg2+,viz., 0.5 M for the native and even 1.6 M for the trinitrophenylated heavy meromyosin S-1. Similar results were obtained when Mg2+ was replaced with Ca2+, with the difference that the concentration of potassium chloride necessary for elution was lower. Under all conditions the adsorption of both proteins on the column with AMP-P(NH)P was weaker then on the columns with ATP and ADP, and in the presence of Mg2' (or Ca2+) only part of the active protein was adsorbed. Incomplete adsorption on a sorbent with ADP in the presence of CaZf was also observed. Hence the presence of Mg2+ and Ca2+ increases the affinity of myosin and its derivatives to agarose-ATP significantly. However, it has no effect (in fact, causing a slight decrease), on the affinity to ADP and AMP-P(NH)P columns (especially in the presence of Ca"). Divalent cations accelerate simultaneously the cleavage of bound ATP both with native and with trinitrophenylated heavy meromyosin S-1 , w h c h was studied mainly on agarose with attached [y3'P]ATP. These results served as one of the important bases for the proposal of a scheme for the mechanism of cleavage of ATP with myosin. Oplatka et al. concluded that under the conditions at which the bound ATP is split, the protein is adsorbed not only on intact ATP, but also on the decomposition products of the latter. They used the name "dynamic" affinity chromatography for this type of chromatography on immobilized substrate under conditions favourable for an enzymatic reaction. They also assumed that the binding of enzymes under these conditions is determined by some kind of constant of the Michaelis-Menten type, rather than by the association constant of the enzyme and the intact substrate. Therefore, the dynamic affinity columns in which other substrates would be immobilized may evidently be useful for the detection of the modifications of the binding sites of other enzymes and for the separation and the isolation of various isoenzymes. The interaction between myosin and actin, which is the basis of the conversion of chemical energfinto mechanical energy in muscle, was investigated by means of affinity chromatography by Bottomley and Trayer. On Sepharose with attached G-actin, they followed the effect of the ionic strength on the specific and reversible binding of heavy meromyosin and myosin subfragment 1. The complexes formed between the derivatives of myosin and the immobilized G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphates, in both the presence and absence of Mg2+.On the other hand, how-

328

EXAMPLES OF USE OF AFFINITY CHROMATOGRAPHY

16

32 4 8

64

16

32 48

64

FRACTION NUMBER

Fig. 11.5. Effect of Mg2+on the behaviour of heavy meromyosin S-1 (solid line) and of trinitrophenylated heavy meromyosin S-1 (dotted line) on agarose-ATP, agarose-ADP and agarose-AMPP(NH)P columns. Amounts of 6-10 mg of protein were loaded on columns (130 x 9 mm) filled with agarose nucleotide, previously equilibrated with 30 mM potassium chloride, 10 mM imidazole, pH 7 (or borate, pH 8, for the ATP-P(NH)P columns) and either (A) 0.5 mM EDTA + 2 mM Mg”, or (B) 1mM EDTA. Washing with the initial equilibrating solution was followed with a potassium chloride gradient (dashed line), leaving the other ingredients at the same concentration. Fractions of 2 ml were collected. Hatched areas indicate the lack of ATPase activity. Reproduced with permission from A. Oplatka et d., Biochim. Biophys. Acta, 385 (1975) 20-27.

ever. both Sepharose-bound and free G-actin cause only a weak increase in Mg2+-stimulated ATPase activity of myosin. This enables a study of whether the complex formation between myosin and actin leads necessarily to the activation of ATPase to be carried out. Similarly, affinity chromatography offers new possibilities for the study of the conversion of protein kinase into cyclic AMP-independent form by chromatography on N6-caproyl3’,5‘-cyclic adenosine monophosphate-Sepharose (Wilchek et al., 197 1b). O’Carra and Barry (1972), using Sepharose with an attached oxamate derivative, proved that NADH induces the binding site for pyruvate in lactate dehydrogenase and also the formation of an “abortive” ternary enzyme-NAD+-pyruvate complex. In an analogous manner, affinity chromatography enabled Mawal et al. to detect enzyme-reactant complexes of galactosyl

FIBROBLAST AND LEUCOCYTE INTERFERONS

3 29

transferase. Doellgast and Kohlhaw (1972a, b) proved, by a study of the effect of the concentration of potassium phosphate on the binding of a-isopropylmalate synthetase on to L-leucine-Sepharose, that a high concentration of salts induces a conformational change involving the leucine binding site. Similar results were obtained by Rahimi-Laridjani et al. with biosynthetic threonine deaminase. The phosphorylation of soybean callus succinylCoA synthetase with immobilized adenosine triphosphate was studied by Wider de Xifra et d., while the re-naturation of agarose-bound aldolase after denaturation with 8 M urea was investigated by Chan. Affinity chromatography can also be used for the isolation of enzymes that take part in certain metabolic paths. For example, using N{waminohexyl)-L-aspartic acid-Sepharose 6B, Tosa et QZ. isolated enzymes involved in the metabolism of L-aspartic acid (asparaginase, aspartase and aspartate-Pdecarboxylase).

11.6 MOLECULAR STRUCTURE OF FIBROBLAST AND LEUCOCYTE INTERFERONS

INVESTIGATED WITH LECTIN AND HYDROPHOBIC CHROMATOGRAPHY Human fibroblast and leucocyte interferons, both polyinosinic-polycytidylic acidinduced and virus-induced, are different from the point of view of their antigenicity. For example, antibodies formed against the fibroblast interferon are not capable of neutralizing the leucocyte interferon. The antibodies that were formed against the preparations of the leucocyte interferon cross-react partly with the fibroblast interferon, whch, however, may be caused by the presence of fibroblast-type interferons in these leucocyte preparations. The sources of the difference of the antigenic behaviour of both interferons may be both in the differences in the primary structures of the interferon polypeptide chains and in the differences in the post-translation modification (glycosylation), or in a combination of both. As the protein surface reflects its primary structure and because hydrophobic amino acids on its surface determine their interactions with hydrophobic sorbents, Jankowski et al. decided to compare the behaviour of both interferons during various types of hydrophobic chromatography. The binding properties of human leucocyte and fibroblast interferons, induced both by polyinosinic-polycytidylic acid (rIn*rC,) and by Newcastle disease virus, are summarized in Table 11.3A. The chromatographic behaviour of all interferons was followed on a high-molecular-weight hydrophobic ligand (bovine serum albumin immobilized on cyanogen bromide-activated Sepharose 4B) and on two low-molecular-weight immobilized hydrophobic ligands (CHSepharose 4B and AHSepharose 4B). Chromatography on serum albumin-agarose was carried out on columns (20 x 0.9 cm) equilibrated with phosphate-buffered saline (0.02 M sodium phosphate, pH 7.4,0.15 M sodium chloride) at room temperature. A sample of interferon (5 ml) containing 125,000 interferon units (or 1 ml of sample with 14,000 units) was applied on the column and washed out with the equilibration buffer. Most of the protein, but only a small part of the interferon activity, was eluted in the first peak. The latter was eluted only when 0.02 Msodium phosphate solution of pH 7.4, containing 1 M sodium chloride and 33% (v/v) of ethylene glycol, was applied. Fractions of 1.3 ml each were collected at a flow-rate of 30 ml. cm-* h-' (Huang et al.). Chromatography of interferons on CHSepharose (Jankowski et al.) was carried out in

W W

TABLE 11.3

0

BINDING OF HUMAN LEUKOCYTE AND FIBROBLAST INTERFERONS ON IMMOBILIZED HYDROPHOBIC LIGANDS AND IMMOBILIZED LECTINS Chromatographic system

Sugar specificity

Interferon source and inducer* Leukocyte rln-rCn

Leukocyte virus

Fibroblast rIn-rCn

Fibroblast virus

(A) immobilized hydrophobic ligands Bovine serum albumin-agarose CH-Sepharose 4B AH-Sepharose 4B -

-

(B) immobilized lectins Horseshoe crab

Lotus Concanavalin A Bondeiraea simplicifolia Soybean lectin Wheatgerm lectin

N-Acety lneuraminic acid L-Fucose D-Mannose a-and p-Dgalactose N-Acetylgalactosamine N-Acety lglucosamine

*+, Complete binding; f,partial binding; -, no binding.

%

FIBROBLAST AND LEUCOCYTE INTERFERONS

331

the following manner. Fibroblast interferon was dialysed against phosphate-buffered saline for 16 h at 4”C, then 1 ml of sample containing 12,000 interferon units was applied on to a column (20 x 0.9 cm) equilibrated with the dialysis solution at room temperature. This buffer eluted a protein fraction without interferon activity; 11,500 units of interferon activity were eluted with 0.02 M phosphate buffer of pH 7.4, containing 1 M sodium chloride and 50%(v/v) of ethylene glycol. Leucocyte interferon was dialysed against a 0.02 M phosphate buffer of pH 7.4 at 4°C for 16 h. An 8-ml sample containing 4800 units of interferon activity was applied on a 20 x 0.9 cm column equilibrated with the same buffer at room temperature. In the first peak, eluted with the equilibration buffer, 97% of the applied protein and 83%of the applied interferon activity were eluted. Elution with a 0.02 M phosphate buffer of pH 7.4 containing 0.15 Msodium chloride, then containing 1 Msodium chloride and finally containing 1 M sodium chloride t 50% ethylene glycol, did not bring about a further elution of the interferon activity. Chromatography on AHSepharose was similar. The same sample of fibroblast interferon ( 1 2,000 units) was applied on a column (20 x 0.9 cm) equilibrated with a phosphatebuffered saline at room temperature. The first peak contained 100% of the applied protein and 72% of the applied interferon activity. The remaining activity was eluted with a 0.02 M phosphate buffer of pH 7.4, containing 1 M sodium chloride. A 2.5-ml volume of dialysed leucocyte interferon (2750 units) was applied on a column (1 5 x 0.9 cm) equilibrated with a phosphate-buffered saline at room temperature. The first fraction contained half of the 73% of the interferon activity obtained and 81%of the applied protein. The remaining activity was eluted with 0.02 M phosphate buffer of pH 7.4 containing 1 M sodium chloride. From the above results, it follows that fibroblast interferon is more hydrophobic than leucocyte interferon. In order to compare the post-translation modification of interferons, Jankowslu et al. also characterized both interferons on the basis of their binding properties for various immobilized lectins. The results are summarized in Table 11.3B. From the lack of binding of human leucocyte interferons to lectins distinguishing equally terminal sugar residues (horseshoe crab, Lotus, Bandeiraea) just as internal residues (wheat germ, soybean, concanavalin A) it can be concluded that these sugar moieties are not present on interferon. Hence, it can be concluded that this interferon is not a glycoprotein. In contrast, human fibroblast interferon is a glycoprotein. On the basis of the binding to horseshoe crab lectin, it can be assumed that it contains a sialic acid residue. Further, L-fucose also exists in a form accessible for binding by specific lectins. On the basis of the data given in Table 11.3, it seems that the differences in molecular structure can be attributed primarily to the type of cells in which the interferon was induced. The differences based on the induction signal (virus or rI,* rC,) in the same cell seem to be of a much weaker character. Affinity chromatography was also used by Di Prisco and Casola for the detection of structural differences. Using specific immunoadsorbents they detected structural differences between nuclear and mitochondria1 glutamate dehydrogenase. Wiman and Wallen investigated by means of affinity chromatography the structural relationship between the “glutamic acid” (A) and “lysine” (B) forms of human plasminogen, and their interaction with the NH2-terminal activation peptide. They chromatographed the NH, -terminal activation peptide on Sepharose with covalently bound plasminogen B, 0.27 pmole of t h s

332

EXAMPLES OF USE OF AFFINITY CHROMATOGRAPHY

peptide being adsorbed on 1.O pmole of immobilized plasminogen B. The adsorbed peptide could be eluted with a buffer containing 0.005 M 6-aminohexanoic acid. Its specific influence on the interaction can be deduced from the sufficiently low concentration. If the NH2 -terminal activation peptide was applied on to Sepharose with attached plasimogen A, only 0.10 pmole was adsorbed. In comparison with plasminogen B-Sepharose, this means a binding capacity of about 35%. In an effort to establish which part of the activation peptide is responsible for the specific interaction, Wiman and WallCn applied the tryptic digest of 1 pmole of the activation peptide on to a column of plasminogen-B-Sepharose. Among the five peptides formed was sorbed, after this tryptic hydrolysis, only the heptapeptide Ala-Phe-Glu-Tyr-HisSer-Lys which is located at the position 41 -5 1 of the total of 81 residues of the N-terminal activation peptide. Hence this peptide is responsible for the interaction, which, it seems, is of great importance for the conformational state of the plasminogen molecule.

11.7 IMMUNOASSAY

An extremely sensitive and selective immunoassay is used for the determination of the concentration of molecules against which specific antibodies can be prepared. The immobilization either of the investigated substance or of the antibody led to a considerable simplification of the immunoassay (radioimmunoassay, enzyme immunoassay and fluorescent antibody techniques). 11.7.1 Solid-phase radioimmunoassay

The classical immunoassay, described by Yalow and Berson, is based on the ability of the unlabelled antigen to compete with the antigen, labelled with a radioactive isotope, in binding to a limited number of binding sites of the antibody. Hence, the process consists in the inhibition of the binding of pure labelled antigen on to the antibody under the effect of the unlabelled antigen that is to be determined. When the system is in equilibrium, the radioactivity bound to the antibodies is separated from the unbound radioactivity, and then one or the other is determined. On the basis of this measurement, the amount of the unlabelled antigen in the unknown sample can be determined. The sensitivity of the radioimimunoassay is within the concentration range 10-11-10-17 mole and therefore it permits the quantitative determination of most of the various biologically active compounds that occur in biological systems in nanogram and picogram amounts. The method was substantially simplified by the utilization of the binding of antibodies on to solid supports (Catt et al.;Wide and Porath). The use of antisera attached covalently to agarose, cellulose and Sephadex in radioimmunoassay systems for proteins and haptens was investigated by Bolton and Hunter. They demonstrated that the covalent binding on the insoluble matrix did not affect the antiserum unfavourably. If a decreased sensitivity of the assay was observed, then it was caused merely by steric hindrance of high-molecular-weight antigens. Bogdanove and Strash reported on possible errors in radioimmunoassay, caused by the escape of radioiodine. Radioimmunoassay is becoming increasingly widely used, primarily in the field of drugs,

IMMUNOASSAY

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hormones, chemotherapeutic agents, antibiotics, immunoglobulin, etc., as is evident from reviews by, for example, Landon and Moffat, Broughton and Strong and Cleeland et al. and from a book edited by Gupta and Abraham (1977). An example of the application of radioimmunoassay in the solid phase is the determination of ornithine transcarbamylase synthesized in a coupled transcription-translation system, directed by the arg F gene carried on the specialized transducing bacteriophage (Eshenbaugh et al.). Anti-ornithine transcarbamylase antibody, bound covalently to Sepharose 4B (0.1 ml), was added to the solution to be assayed in a total volume of 0.5 ml, adjusted to 0.050 M phosphate, 0.15 M sodium chloride, pH 7.2. The assay mixture was mixed with continuous gentle rotation on a New Brunswick rotor drum at 5 rpm in an incubator at 33°C for 60 min, diluted with 5 ml of assay buffer, centrifuged at 500 g for 3 min and the supernatant was then discarded. The Sepharose was transferred to a Whatman CF/C 25 mm filter and washed with 50 ml of assay buffer. The radioactivity was determined using Beckman ReadySolv VI scintillation fluid in a Beckman LS 230 liquid scintillation counter. The method described is rapid, sensitive and specific of ornithme transcarbamylase and can be used in studies aimed at the elucidation of the control mechanism of the arginine synthetic pathway, carried out in vitro. 1 1.7.2 Enzyme-linkedimmunosorbent assay (ELSA)

Engvall et al. used the principle of the radioimmunoassay described for the determination of rabbit immunoglobulin G, with the difference that instead of the labelling of antigens with radioactive isotope they conjugated it with the enzyme, using alkaline phosphatase. The basis of the competitive immunoassay is the separation of the free and antibody-bound antigen, so that the antigen can be determined quantitatively. T h s was facilitated by the fixation of the antibody onto the solid phase by physical adsorption on polystyrene tubes. The immunoassay was carried out in the following manner. Polystyrene tubes (80 x 11 mm) were each coated with 1 ml of a solution of antibody. The antibody solutions were diluted with 0.1 M sodium carbonate buffer of pH 9.8 or with phosphate-buffered saline. The tubes with the coating solution were allowed to stand at 37OC for 3 h and then stored in the same solution in the cold. Before determination, a suitable number of tubes was washed with0.9% sodium chloride solution containing 0.05% of Tween 20. A 0.5-ml volume of one of the following was placed in each tube: (1) the standard solution of immunoglobulin G , ( 2 ) the unknown sample, or (3) the buffer alone. This was followed by the addition of 0.1 ml of a dilute conjugate of alkaline phosphatase and immunoglobulin G (prepared by coupling with glutaraldehyde). All dilutions were carried out with a phosphate-buffered saline containing 1% human serum albumin and 0.02%sodium azide. The tubes were incubated at room temperature overnight (16 h) in a roller drum, so that the surface coated with the antibody was covered with the 0 . 6 4 volume present in the tubes. The tubes were then washed three times with a sodium chloride-Tween solution. The amount of the enzyme-linked immunoglobulin G bound to the tube coated with the antibody was determined by adding 1 ml of 0.05 Msodium carbonate buffer (pH 9.8) containing 1 mg/ml of p-nitrophenyl phosphate and 1 d of magnesium chloride. After 30 min the reaction was stopped by the addition

3 34

EXAMPLES OF USE OF AFFINITY CHROMATOGRAPHY

of 0.1 ml of 1 M sodium hydroxide solution. On the basis of the absorbance measured at 400 nm, a standard graph was constructed by plotting the enzyme activity (increase in absorbance per unit time) of individual samples against their content of standard immunoglobulin G. Eazymoimmunoassay for the determination of soluble antigens with at least two antibody-combining sites was described by Maiolini and Masseyeff. This non-competitive sandwich method comprizes three steps: (1 j the antigen to be determined reacts with antibody-coated cellulose immunosorbent ;( 2 ) the enzyme-labelled antibody is incubated with the antigen bound to the solid phase; ( 3 ) the enzymatic activity of the immunosorbent is then measured. Examples of the application of this method were demonstrated in the determination of a-fetoprotein. The use of enzyme-linked immunoassay enabled Kato et al. to determine an amount of human immunoglobulin G as low as 3 fmole. A review on the quantitative enzyme immunoassay was published by Scharpk et al. 11.7.3 Microfluorimetric immunoassay i n order t o circumvent some of the disadvantages of radioimmunoassay, such as the instability of the radioactive labelling or the denaturation of proteins during the labelling, Haaijman and Bloemmen employed a fluorescent instead of a radioactive antiserum or antigen. They studied two types of fluorescent immunoassay systems: fluorescence inhbition immunoassay and fluorescence immunocompetition assay. The former system was studied with the use of human immunoglobulin G and mouse immunoglobulin M and the latter with ovalbumin. They used fluorescein or tetramethylrhodamine isothiocyanate for labelling. These methods were ten times less sensitive than the radioimmunoassay. For the study of the distribution of tetanus antibodies in various classes of immunogiobulin, Hernandez et al. developed a simple immunofluorescent method. A 0.02-ml volume of a suspension of antigen, containing 10 mg of hydrated tetanus-toxicoid-agarose, were mixed with 0.2 ml of diluted (usually 1 : 10) serum to be tested and incubated at room temperature for 1 h. After washing the beads three times with a barbital buffer of pH 7.2, containing 1 M sodium chloride, 0.2 ml of rabbit anti-human immunoglobulin M, A or G was added, then both the incubation and the washing were repeated. Finally, 0.2 mi of fluorescein-labelled goat anti-rabbit immunoglobulin was added and the incubation and washing were repeated. After the last wash, the sediment was placed on a slide and observed in blue light under a fluorescence microscope. The presence of specific antibodies was shown by brilliantly green beads with fluorescent borders.

11.8 SPECIFIC REMOVAL OF BOVINE SERUM ALBUMIN (BSA) ANTIBODIES IN VWO BY EXTRA-CORPOREAL CIRCULATION OVER BSA IMMOBILIZED ON NYLON MICROCAPSULES It seems to have been proved that antibodies and immune complexes may cause some diseases. The therapy of many of these immunologically mediated illness was dependent on the use of immunosuppressive drugs which suppress host immunity non-specifically. Specific elimination from the circulation of immune substances that are pathological in the disease

SPECIFIC REMOVAL OF BSA-ANTIBODIES IN VIVO

335

might represent another therapeutic approach. Schenkein et al. eliminated antibodies against BSA from the plasma of positively immunized rabbits by letting their blood circulate through an immunoadsorbent prepared by binding BSA to bromoacetylcellulose. Using a similar system in a similar manner, DNA antibodies were eliminated specifically from the blood of actively immunized rabbits (Terman et al., 1974, 1975). Immunoadsorbents for the elimination of serum hepatitis antigen from blood, blood plasma and plasma products were prepared by Charm and Wong by coupling goat serum with the anti-serum hepatitis antigen-antibody on Sepharose 2B after activation with cyanogen bromide. The elimination of substances from the blood by affinity chromatography was studied further by Plotz et al. and Scharschmidt et al.,who employed albumin attached to BioCel A5m for the elimination of bilirubin and other albumin-bound substances. Terman et al. (1976b) used BSA immobilized on a microcapsule for the elimination of antibodies against BSA from canine blood. The microcapsule chambers were composed of a 8 x 2.5 cm I.D. glass cylinder with a 40-mesh stainless-steel screen at its ends. This chamber was filled with approximately 400 microcapsules of diameter 1.O-1.1 mm and then connected with a Travenol roller pump using polyethylene tubes. The capsules were washed with 2 1 of water of 30°C for 60 min, hydrolysed with 500 ml of 3 M hydrochloric acid by circulating it for 60 min at 30°C and a flow-rate 30 ml/min and then washed with water. A 12.5% solution of glutaraldehyde in 0.1 M borate buffer of pH 8.3 was circulated at 30°C for 15 min at a flow-rate of 30 ml/min, then the capsules were washed with 0.1 M borate buffer of pH 8.3 at 30°C. After the exchange of the tubes, a solution of 200 mg of BSA with 0.2 pg of [1Z51]BSAadded as a marker in 0.9% sodium chloride solution was circulated through the capsules for 30-60 min. Finally, 200 ml of 10 pMNazSz05 solution were circulated through the capsules at a flow-rate of 200 ml/min. After washing with 0.9% sodium chloride solution, when the wash water did not contain any ''I (approximately 500 ml), the capsules were transferred in solution into a siliconized chamber for in vivo use. In this manner, 32.1-34.5 mg of BSA were bound to microcapsules. A scheme of the extracorporeal system in vivo is shown in Fig. 11.6. The dogs were anesthetized with sodium pentobarbital and the femoral artery and vein were canulated with wide-bore polyethylene tubing. Sodium heparin (3 mg/kg) was injected intravenously and the femoral artery and venous catheters were connected to a Travenol roller and then to the microcapsule chamber. The latter was coated with silicone before use. The insertion of a three-way stopcock permitted the interruption of the blood flow through the chamber at 7-1 2-min intervals, so as to prevent the microcapsules from impeding the flow by clogging the screens. The chamber was connected with a bubble trap and then to the femoral vein. The heparinized blood was circulated through the extracorporeal system for 20 min before the injection of antibodies. The flow-rate was kept at 200 ml/min during the whole procedure. Blood samples were withdrawn from the venous line and sampled on antibodies at various intervals. The rabbit antiserum against BSA and human serum albumin (HSA) was infused simultaneously into the anaesthetized dogs, using a dilution at w h c h the antiserum binds 95% of their corresponding antigens. For the anti-BSA this was a 1 :520 dilution and for the anti-HSA a 1 : 270 dilution. After 12-min equilibration, the BSA microspheres were introduced into the extracorporeal circulation. The specific reduction of BSA-binding antibodies in the four dogs investigated was fastest during the first 15 min after the introduc-

3 36

EXAMPLES OF USE OF AFFINITY CHROMATOGRAPHY

FEMORAL FEMOR

I1

ROLL€ R P U M P

Fig. 11.6. Schematic representation of the in vivo extracorporeal circulation system. Reproduced with permission from D.S. Terman eral., J. Immunol., 116 (1976) 1337-1341.

tion of BSA microcapsules, whle after the next 60 min the drop was much slower. In the same time interval the anti-HSA binding remained constant. In order to prove that the observed reduction in the BSA binding is not caused by the setting free of BSA from microcapsules during the experiments, the vital organs and the serum of the dogs were analysed for the presence of '''1 at the end of the experiments. No significant radioactivity was found in the organs in the serum of the dogs. A haematocrit and a leucocyte count in the dogs were carried out before and after the extracorporeal circulation through the BSA microcapsules and had almost identical values. Only one of the four dogs displayed a reduction in leucocyte count. After the termination of the experiments, the microcapsules were analysed for the content of thrombotic materials or cellular debris, which, however, could not be proved. Hence, it seems that tins use of immunoadsorbents could in future be applied in medicine. It has become evident that the nylon microspheres have certain properties that make them suitable for use in extracorporeal systems: their large surface area, permitting covalent binding of the necessary amount of proteins; they have a minimum toxicity towards the host, which can be attributed to the chemical inertness of the microcapsules; and finally, their structural stability reduces the possibility of embolization.

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Vladutiu, G.D., Carmody, P.J. and Rattazzi, M.C.,Prep. Eiochem., 5 (1975) 147-159. Von der Haar, B. and Mueller, G.C.,Eiochirn. Eiophys. A c f a , 176 (1969) 626-631. Vosbeck, K., Chow, K.F. and Awad, W.M., Fed. Proc. Fed.Arner. Socs. Exp. Eiol., 32 (1973a) 504. Vosbeck, K.D., Chow, K.F. and Awad. W.M., J. Biol. Chern., 248 (1973b) 6029-6034. Voss, H.F., Ashani, Y . and Wilson, I.B.,Methods Enzymol., 34 (1974) 581-591. Vretblad, P., FEES L e f f . ,47 (1974a) 86-89. Vretblad, P., Ewchem. Soc. Trans., 2 (1974b) 1327-1328. Vretblad, P., Biochim. Eiophys. Acta, 434 (1976) 169-176. Wagner, M., Acta Eiol. Med. Ger., 34 (1975) 1429-1431. Wakabayashi, Y.. Iwashima, A. and Nose, Y.,Eiochim Eiophys. Acta, 429 (1976) 1085-1089. Waldman, A.A., Marx, G. and Goldstein, J.,Proc. Nut. Acad. Sci. U.S., 72 (1975) 2352-2356. Wallach, D.F.H.,Methods Enzymol., 34 (1974) 171-177. Wallach, D.F.H., Kranz, B., Ferber, E. and Fischer, H., FEES Lefr., 21 (1972) 29-33. Wallin, R. and Prydz, H., FEES Lett., 51 (1975) 191-194. Walsh, K.A., Burstein, Y. and Pangburn, M.K., Methods Enzymol., 34 (1974) 435-440. Walsh, R., Mason, D.T., Tonkon, M.J. and WikmanCoffelt, J.,Prep. Eiochem., 6 (1976) 177-191. Walther, P.J., Hill, R.L. and McKee, P.A., J. Eiol. Chern., 250 (1975) 5926-5933. Wang, P. and Bantle, G., Ewchem Soc. Trans., 2 (1974) 1315. Wang, H.P. and Kimura, T., J. Eiol. Chern., 251 (1976) 6068-6074. Wang, R., Sevier, E.D., David,G.S. and Reisfeld, R.A., J. Chromafogr., 114 (1975) 223-226. Warecka, K., Moller, H.J., Vogel, H.M. and Tripatzis, I.,J. Neurochem., 19 (1972) 719-725. Waters, C.A., Murphy, J.R. and Hastings, J.W., Eiochem Eiophys. Res. Commun., 57 (1974) 1152-1158. Waxman, S. and Schreiber, C., Biochemistry, 14 (1975) 5422-5428. Weatherford, S.C., Weisberg, L.S., Achord, D.T. and Apirion, D., Eiochem. Eiophys. Res. Commun., 49 (1972) 1307- 1315. Weaver, R.F., Blatti, S.P. and Rutter, W.J.,Proc. Nut. Acad. Sci. U.S.,68 (1971) 2994-2999. Webb, T. and Lapresle, C.,Eiochern. J., 91 (1964) 24-31. Weetall, H.H., J. Eacferiol.,93 (1967) 1876-1880. Weetall, H.H., Eiochem. J., 117 (1970) 257-261. Weetall, H.H. and Weliky, N., Nature (London), 204 (1964) 896-897. Weibel, M.K., Doyle, E.R., Humphrey, A.E. and Bright, H.J.,Eiofechnol.Eioeng., S3, (1972) 167-171. Weinstein, Y., Wilchek, M. and Givol, D., Biochern Ewphys. Res. Comrnun., 35 (1969) 694-701. Weintraub, B.D., Biochern Eiophys. Res. Commun., 39 (1970) 83-89. Weissbach, A. and Poonian, M.,Methods Enzymol., 34 (1974) 463-475. Weliky, N., Weetall, H.H., Gilden, R.V. and Campbell, D.H., Irnmunochemistry, 1 (1964) 219-229. Wermuth, B. and Kaplan, N.O., Arch. Eiochem. Eiophys., 176 (1976) 136-143. Wetekam, W., Mullinix, K.P., Deeley, R.G., Kronenberg, H.M., Eldridge, J.D., Meyers, M. and Goldberger. R.F.,Proc. Nut. Acad. Sci. U.S., 72 (1975) 3364-3368. Whitehead, J.S., Weitz, M.A. and Kim, Y.S., Proc. Soc. Exp. Biol. Med., 148 (1975) 777-779. Whiteley, J.M., Jackson, R.G., MeU, G.P., Drais, J.H. and Huennekens, F.M., Arch. Ewchern. Eiophys., lSO(1972) 15-22. Whiteley, J.M., Jerkunica, 1. and Deits, T., Biochemistry, 13 (1974) 2044-2050. Whitney, P.L., J. E w l . Chern., 248 (1973) 2785-2789. Whitney, P.L.,Anal. Eiochem., 57 (1974) 467-476. Wichman, A. and Andersson, L.O., Biochim Eiophys. Acta, 372 (1974) 218-224. Wide, L. and Porath, J.,Eiochim Eiophys. A c f a , 130 (1966) 257-260. Wider de Xifra, E.A., Mendiara, S. and Batlle, A.M., FEES L e f t . , 27 (1972) 275-278. Widmer, F., Mutus, B., Murthy, J.R., Snieckus, V.A. and Viswanatha, T., Life Sci., 17 (1975) 1297-1 302, Wierenga, R.K., Huizinga, J.D., Gaastra, W., Welling, G.W. and Beinterna, J.J., FEES Lett., 31 (1973) 181- 185. Wigzell, H. and Anderson, B., J. Exp. Med., 129 (1969) 23-36. Wilchek, M., FEES L e f t . ,7 (1970) 161-163. Wilchek, M., Anal. Eiochem., 49 (1972) 572-575.

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365

Chapter 12

Immobilized enzymes Techniques involving the use of immobilized biological molecules have found uses in many fields (Mosbach, 1976). A number of examples have shown how biologically active substances bound to solid matrices have found an important use in affinity chromatography. The study and use of enzymes bound to solid matrices is closely connected with affinity chromatography. The progress achieved in enzyme immobilization technology parallels that achieved in affinity chromatography. The study of immobilized enzymes is one of the most dynamic areas in presentday enzymology. Immobilized enzymes are also the affinity ligands that have been most studied and therefore a separate chapter is devoted to them.

12.1 CLASSIFICATION OF IMMOBILIZED ENZYMES At the Conference on Enzyme Engineering, held in 1971 in Henniker (U.S.A.), a committee of experts recommended individual names for variously modified enzyme systems (Sundaram et d.),such as the term “immobilized” instead of “insplubilized”, “matrixbound”, “solid-supported” and other similar terms. In general, four main categories of immobilized enzymes can be differentiated: (1) enzymes’immobilized by physical or chemical sorption; (2) enzymes covalently attached to soluble or insoluble polymers; (3) cross-linked enzyme molecules, for example using suitable bifunctional reagents; and (4) enzymes entrapped in gels, membranes or microcapsules. The enzymes can be classified according to the following scheme: Enzymes

I “Modjfied“ I

‘Immobilized enzymes

I

“ Native“

’

I Entrapped

r ‘

Matrix-entrapped (ENT)

I

Microencipsulated (MEC)

Adsorbed (ADS)

Covalently bound ( C VB )

Most of the immobilized enzyme systems, though not necessarily all of them, fit into this scheme. It was recommended that the symbols ENT, MEC, ADS and CVB should be inserted between the names of the carrier and the enzyme, thus characterizing any of the four classes of immobilized enzymes. Thus, for example, trypsin covalently bound to carboxymethylcellulose should be indicated as “CM-celluloseCVB-trypsin”, nylonencapsulated urease as “nylon-MEC-urease” and glucoamylase entrapped in polyacrylamide gel as “acrylamide-gel-ENT-glucoamylase”.

366

IMMOBILIZED ENZYMES

As regards the kinetic parameters of these enzymes, the committee recommended expressing their activity as an initial reaction rate (in micromoles per minute) per gram of dry immobilized enzyme preparation, measured under clearly specified conditions. For the activity of enzymes bound to surfaces (for example, tubes, sheets and membranes), the committee suggested expressing the activity as the initial reaction rate (in micromoles per minute) per unit area of the covered surface, measured under clearly specified conditions. The committee also recommended indicating (a) the conditions of drying of the immobilized enzyme preparations; (b) the content of protein in a dry preparation; and (c) the specific activity of the enzyme prior to immobilization. The measurement of the kinetic constants of immobilized enzymes does not give true kinetic constants which are equivalent to those obtained in homogeneous solutions, because physical factors, such as diffusion, have a major influence. For t h s reason, the rate and binding constants should be referred to as apparent V,, [= Vm,(app)], and apparent K , [= K,(app.)]. K,(app) is then defined as the substrate concentration at which the reaction velocity corresponds to half V,,(app). Other kinetic constants should also be presented as the respective apparent constants. When indicating the stability of immobilized enzymes, it is essential to give all specific conditions of its measurement. The last recommendation of the committee concerns the information that should be included in the report o n a new system for covalent coupling of enzymes. These are (a) the number of reactive groups occurring in unit weight of the carrier and (b) the maximum amount of bound small molecules, just as for high-molecular-weight molecules (proteins), per specific weight of the carrier (polymer).

12.2 ATTACHMENT OF ENZYMES TO SOLID SUPPORTS AND ACTIVITY OF IMMOBILIZED ENZYMES At present, the most commonly used method for the immobilization of enzymes is their covalent binding t o solid supports. The requirements of the properties of solid supports, a review of them, and a number of methods for the coupling of proteins are discussed in detail in Chapter 8. Some methods suitable for the characterization of immobilized enzymes are given in Chapter 9. In contrast to their use in affinity chromatography, glass supports are more often used for the immobilization of enzymes, and a series of ceramic supports seem potentially to be useful. Much information, with numerous examples of immobilized enzymes, their properties and uses, is available in a series of reviews and monographs (Berezin et ul., 1976; Goldman et ul., Messing; Mosbach, 1977; Salmona et ul., Wingard, Wiseman). The effects of attachment and of the solid support can easily be evaluated in immobilized enzymes on the basis of their determined enzymatic activities. They are most commonly expressed as relative activities, which means the ratio of the activity of the immobilized enzyme referred to 1 g of dry material t o the activity calculated by multiplication of the amount of the enzyme in milligrams bound to 1 g of dry material by its activity in solution. Examples of the relative activities of enzymes bound to various supports by dif-

367

ATTACHMENT TO SOLID SUPPORTS AND ACTIVITY

ferent methods, together with an indication of the efficiency of the binding, are given in Table 12.1 (Schnapp and Shalitin). The difference between the kmetic properties of the free and the covalently bound enzyme is influenced by a number of effects: (a) Effects of dif-sion limitations. By analogy with nonenzymatic heterogeneous catalysis, where the rate of diffusion of the reactants towards the active surface of the catalyst plays an important role in the determination of the kinetic reaction, in catalysis by immobilized enzymes the rate of diffusion of the substrate into the binding site of the enzyme also considerably affects the apparent kinetic parameters of the bound enzyme system. The surface of the solid support with covalently attached enzyme in aqueous suspension is surrounded by an unstirred layer of solvent (Goldman et d.).In an enzymatic reaction, a concentration gradient of substrate is established across the unstirred layer. The saturation of the immobilized enzyme occurs at a substrate concentration which is higher than that required for the saturation of the corresponding native enzyme in solution. This leads to an increase in the apparent Michaelis constant, K,(app). (b) Steric effects. The covalent attachment of the enzyme on the surface of the solid support may lead to steric restrictions of the availability to predominantly high-molecularweight substrates. Thus, for example, covalently attached proteolytic enzymes in most instances possess an esterase relative activity higher than the protease activity (Goldstein et al., 1970). Steric hindrance also results in inaccessibility of some bonds in proteins for the proteolytic cleavage by immobilized proteases, thus causing a change in their specificity toTABLE 12.1 EFFICIENCY OF BINDING -

Derivative*

Means of enzyme binding*

% of binding

Seph-CHT Seph-CHT Cell-(CH,) ,NH-CHT PA-NH(CH,),NH-CHT PA-NH(CH,),NH-CHT PA-NH(CH,),NH-CHT PA-NHNH--CHT PA-CHT Glass-NH-CHT Glass-NH-CHT Glass-NH-TR Glass-NH-SUB Glass-NH-SUB Seph-SUB

CNBr KIO, GA EDC CNBr CNBr CNBr NaNO, CNBr GA CNBr CNBr GA CNBr

50 100 70 10 40 25 25 40 65 100 100

60 75

90

% relative activity** 25-40 6 6 5 30 8 8

30 35 10 22 100 100 40

*Abbreviations: Seph, Sepharose; Cell-(CH,),NH,, aminoethylcellulose, PA-NH,, polyacrylamide; GA, glutaraldehyde; EDC, l-ethyl-3-( 3-dimethylaminopropy1)carbodiimide;CHT, a-chymotrypsin; TR, trypsin; SUB, subtilisin. **Relative to the activity of free enzyme. The activity of chymotrypsin and subtilisin was measured in a pH-stat using acetyltyrosine ethyl ester (pH 8) as substrate, and that of trypsin using benzoylarginine ethyl ester (pH 8).

368

IMMOBILIZED ENZYMES

wards lugh-molecular-weight substrates. Thus, for example, Ong et al. cleaved pepsinogen with both free and immobilized trypsin (attached to a copolymer of ethylene and maleic anhydride). Both hydrolysates differed in their chromatographic pattern on Sephadex G-25 and in peptide maps of individual peaks. In addition to the steric effect, the polyelectrolytic nature of the immobilized enzyme derivative also played a role, because electrostatic interactions between the charged solid support and the protein substrate may occur on it. (c) Effects of chemical modification of enzymes. The covalent attachment of the enzymes to a polymeric carrier affects the kinetic behaviour of the bound enzyme as a consequence of the alteration in its net charge, of the nearest neighbour effects on the region of the active site, and of the perturbation in intramolecular interactions (Goldman et al.). These effects are analogous to a chemical modification of the enzyme by low-molecularweight reagents. Thus, for example, acetylation and succinylation of chymotrypsin results in a shift of the pH optimum of the activity curve towards more alkaline pH values compared with native chymotrypsin. However, t h s shift is observed only at low ionic strengths and it can be prevented at high ionic strengths. This phenomenon is probably due to the increase in the net negative charge of the protein resulting from the blocking of the amino groups and the corresponding increase in the pK, value of the imidazole moiety of histidine in the active site of chymotrypsin. The effects of chemical modification can be easily explained in systems in whch a support was used without any electric charge. With a polyelectrolytic carrier the effects of a chemical modification result from the much higher electrostatic effects due to the charged carrier. ( d ) Micro-environmental effects.The eifects of the microenvironment on the kinetic behaviour of immobilized enzymes were studied predominantly with enzymes attached to polyelectrolytic carriers forming by their charges an electrostatic field. The effects of polyanionic and polycationic carriers on the kinetic properties of proteolytic enzymes have been most investigated. In addition to the mentioned effect of the electrostatic field of polyelectrolytic carriers, further effects also arise during the formation of the micro-environment, such as the effect of the carrier on the dielectric constant of the immobilized enzyme phase or the effect on the local solub'ility of substrates or the products. The microenvironmental effects are further due to the enzymatic activity proper, i.e., during the catalysis varying local concentrations of substrate, product, protons, effectors, etc., are produced. A few examples of these studies will be given later. How, and to what extent, some properties of the carriers or the type of attachment can affect the activity of enzymes will be shown with several practical examples. One of the most important factors is the content of the reactive groups. Datta and Ollis investigated the dependence of the specific activity of immobilized cuchymotrypsin on the concentration of hydrazide groups on the surface of BioGel P-2 beads (Fig. 12.1). They found that the dependence shows a sharp maximum. With chymotrypsin, lysozyme and lipase, they followed the changes in the specific activity not only after their immobilization, but simultaneously after treatment of soluble proteins with soluble analogues of the same chemical groups as were used to bind the protein to the support surface. In all instances they obtained a correlation between the behaviour of the modified enzymes in their soluble state and in their immobilized forms. When coupling papain on poly(viny1 alcohol) carriers, Manecke and Vogt (1975) followed the amount of bound papain as a function of the concentration of the reactive diazonium groups on the carrier in millimoles. From Table 12.2,

ATTACHMENT TO SOLID SUPPORTS AND ACTIVITY

I

369

I

a

5

m

c2 =?

t

c> I-

2

0

I

I

I

2

I

I

3

4

5

HYDRAZIDE GROUPS, mmole/g.dry

wt

Fig. 12.1. Specific activity of immobilized a-chymotrypsin versus concentration of surface hydrazide groups on Bio-Gel P-2 beads. Reproduced with permission from R. Datta and D.F. Ollis, Advun. Exp. Med. Biol.,42 (1974) 293-315.

it is evident that the amount of the attached papain passes through a maximum, while the relative activity steadily decreases with increasing amount of reactive groups on the carrier. One of the reasons may be the unfavourable effect of the multiple attachment of the enzyme molecules on a carrier that contains an excessively high content of reactive groups. A similar result was also obtained with immobilized pepsin by Valentovi et al. A preparation TABLE 12.2 DEPENDENCE OF THE AMOUNT OF BOUND PAPAIN ON THE CONTENT OF REACTIVE GROUPS OF THE CARRIER PREPARED BY REACTING POLY(VINYL ALCOHOL) (CROSSLINKED WITH 5% TEREPHTHALALDEHYDE AND WITH 2-(m-AMINOPHENYL)-1,3-DIOXOLANE) Reactive groups per gram of carrier (mmole)

Bound papain Relative activity of per gram of carrier* (mg) immobilized papain** (%)

1.8 1.6 1.4 1.1 0.6 0.3

615 635 685 335 310 300

1.1 9.5 12.0 14.8 20.8 21.5

*Coupling conditions: 1 M NaHCO,, 4"C, 15 h. **Substrate: benzoyl-Larginine ethyl ester, 0.05 M,pH 6.O,3O0C.

370

IMMOBILIZED ENZYMES

containing 13 mg of bound pepsin per gram of dry carrier had a relative proteolytic activity of 92.8%, that with the content of 46.8 mg/g had 65.7% relative activity, that with a 50.8 mg/g content had 45.3% relative activity and that with a 65 mg/g content had 37.8% relative activity. The nature of the carrier and the type of attachment does not, however, affect only the activity, but also has a considerable effect on its dependence, for example, on temperature and pH. The effect of the carrier on the optimal temperature for trypsincatalysed hydrolysis of casein is evident from Fig. 12.2 (Brummer er d.). While a temperature below 40°C is optimal for free trypsin, that for trypsin bound to a copolymer of ethylene and maleic anhydride is lower than 6OoC,and for trypsin bound to CM-cellulose it is higher than 70°C. The effects of ionizable groups of the carrier on the pH dependence of activity for a free chymotrypsin, its polyanionic derivative (i.e.,chymotrypsin bound on the copolymer of ethylene and maleic anhydride) and its polycationic derivative (i.e.,polyornithine-bound chymotrypsin) are represented in Fig. 12.3 (Goldstein and Katchalsh). The activity was

20

40

do

TEMPERATURE .‘%

I

00 PH

Fig. 12.2. Temperature-activity curves for hydrolysis of casein by free and bound trypsin (bovine). The reaction mixtures contained 10 pg of free or 100 pg of carrier-bound trypsin in 1.6 ml of phosphate buffer, pH 7.5.0, Free trypsin; 0 , trypsin bound to CM-cellulose; 0 , trypsin bound to maleic copolymer. Reproduced with permission from W. Briimmer et al., Eur. J. Bwchem., 25 (1972) 129-135.

Fig. 12.3. pH-activity curves at low ionic strength ( r / 2 = 0.001) for chymotrypsin (01, a polyanionic ethylene-maleic acid copolymer, derivative of chymotrypsin (0)and a polycationic, poly-L-ornithyl, derivative of chymotrypsin (A), using acetyl-L-tyrosine ethyl ester as substrate. Reproduced with permission from L. Goldstein and E. Katchalski, Z.Anal. Chem., 243 (1968) 375-396.

ATTACHMENT TO SOLID SUPPORTS AND ACTIVITY

371

determined by means of N-acetyl-L-tyrosine ethyl ester at low ionic strength. The information on the ionic strength is important in this instance, because the variation of activity with pH for chymotrypsin and trypsin and its derivatives is dependent on ionic strength (Goldstein et al., 1964). The value of the pH optimum shift of esterolysis in dependence on the carrier charge was also calculated theoretically, according to the equation ApH = pH’

- pHo = 0.43 ZE $ / k T

(12.1)

where ApH is the difference between the local pH in the phase polyelectrolyte-enzyme (pH’) and the pH of the external liquid (pH’), J/ is the electrolytic potential in the region of the particle of the charged immobilized enzyme, e is the charge on the proton, z is the valency, which is unity for hydrogen ions, k is Boltzmann’s constant and T is absolute temperature. Theoretical results, calculated on the basis of this equation, were in good agreement with the experimental results. The phenomena described are attributed to the electrostatic carrier-induced effects. These lead to an uneven distribution of the hydrogen and the hydroxyl ions between the polyelectrolyte “immobilized enzyme phase” and the bulk solution, causing a lower “local pH’ in the domain of an anionic enzyme derivative and a higher “local pH’ in the region of a cationic enzyme derivative (Goldstein). From eqn. 12.1, it follows that an attachment to a neutral carrier should not result in a pH shift. Indeed, the proteolytic activity versus pH curves for both free and hydroxyalkylmethacrylate gel-bound chymotrypsin, for example, have equal optima in both instances (Turkova et al.). However, this is not so with esterase activities. When acetyltyrosine ethyl ester was used as a substrate, a shift of the optimal pH of esterolysis to higher, alkaline values took place, even with chymotrypsin bound to t h s neutral carrier. The same results were also obtained by Axdn and Ernback, who studied chymotrypsin bound to Sephadex and Sepharose. They demonstrated that this shift is only an apparent one, owing to the accumulation of the cleavage products of acetyltyrosine in the proximity of gel surface, and thus the occurrence of a pH difference between the gel surface and the surrounding solution. Hence, the shift is only apparent, which they confirmed by means of an internal indicator, and it is also evidently dependent on the amount of the attached enzyme. Konecng and Slanicka (1975a, b) further demonstrated that, in contrast to the free enzyme, the activity of the immobilized enzyme also depends on the concentration of the buffer, and the pH-activity profile of the immobilized enzyme at a given buffer concentration depends considerably on the pK value of the buffer. Fig. 12.4 shows the dependence of the activity of the esterase fromBacilZus subtilis, attached by means of glutaraldehyde on to alkylamine-zirconia-clad controlled-pore glass CPG-550, on pH in various buffers, and in the case of phosphate buffer even at various concentrations. They discussed the results obtained in detail in connection with the equations of Engasser and Horvath, and derived simple equations for internal pH (or internal pH gradient). The changes of internal pH are generally dependent on external pH, buffer concentration and its pK, and it greatly affects the determined K,(app) values. In order to differentiate the effects of the chemical nature of the carrier from steric and diffusion effects, Svensson prepared analogous soluble and insoluble derivatives of subtilisin by binding the enzyme on to cyanogen bromide-activated DEAEdextran and DEAESephadex. As is evident from Table 12.3, DEAEdextran-subtilisin displayed

372

IMMOBILIZED ENZYMES

I

8

7

10

9

PH Fig. 12.4. Activity of the glass-bound esterase from Bacillus subtilis as a function of pH in 10 mM phosphate (l),Tris (2) and borate (3) and in 2 M p h o s p h a t e (4). Reproduced with permission from J. Konecny and J. Slanicka, Biochim Biophys. Acta, 403 (1975) 573-578.

optimal pH and K , values for the hydrolysis of ester that were similar to those of subtilisin. In contrast, the pH versus activity profile obtained on DEAESephadexsubtilisin displayed a shift to the alkaline pH region and the Km(app) values were increased. which were decreased by modification. The Table 12.3 also gives the rate constants, k, modification, but much more the immobilization, leads to a considerable decrease in subtilisin activity on the high-molecular-weight substrate casein. While the activity of free TABLE 12.3 KINETIC PARAMETERS FOR HYDROLYSIS OF N-ACETYL-L-TYROSINE ETHYL ESTER BY DEAE-DEXTRAN-SUBTILISIN,DEAESEPHADEX-SUBTILISIN AND SUBTILISIN AT DIFFERENT IONIC STRENGTHS The kinetic parameters were calculated, using linear regression analysis, from Lineweaver-Burke plots of activities determined at the appropriate pH optima and using ester concentrations ranging from 0.0025 to 0.025 M.The ionic strength was adjusted b y addition of KCI. Material

Ionic pH strength

K , or Km(app)

kcat (sec-')

(mM)

DEAE-Dextran-subtilisin

1.0 0.125

8.4 8.0

33 38

410 320

Subt ilisin

1 .o 0.125

8.5 8.2

26 35

880 520

DEAE-Sephadex- sub t ilisin

1 .O 0.125

9.0 9.0

110 105

210 150

ATTACHMENT TO SOLID SUPPORTS AND ACTIVITY

313

subtilisin was higher than 400 pmole of H+ released per minute per micromole of enzyme, after the attachment to DEAEdextran it decreased to a value lower than 200, and after attachment to DEAESephadex even to 20. In order to be able to follow the effect of the charged medium on the properties of immobilized proteases Goldstein prepared a hydrophilic anionic and cationic carrier. He attached hydrazine or p,p’diaminodiphenylmethane(MDA) on a copolymer of ethylene and maleic anhydride (EMA): CO.NH-NH2 COOH

EMA-hydrazide resin CO.NH-Q--CH,~NH, COOH

CO.NHQCH,ONH COOH

Ethylene-maleic anhydride copolymer ( E M A )

€MA-MDA resin

From anionic polymers, EMA-hydrazide and EMA-MDA resins he prepared a cationic polymer by substituting carboxyl groups with N,N-dimethyl-l,3-propanediamine(in the presence of dicyclohexylcarbodiimide, DDC):

; ;1

lcOx cox

H,N.CH,.CH,.CH,N(CH,)2

C0.NH.CH2.CH2.CH,.N(CH3),

~

DCC

C0.NH.CH,.CH,.CH,.N(CH3), Anionic resin

Cationic resin

X = -NH-NH, -N H ~ C H , ~ N H ,

After converting the cationic and anionic carriers to polymeric diazonium salts or acylazides, he attached trypsin, chymotrypsin, subtilisin Novo and Carlsberg and papain. The immobilization of all enzymes brought about shifts of the optimal pH to the alkaline pH region, but the shifts were virtually identical for anionic and cationic carriers, and were independent of the ionic strength of the medium. In order to interpret these data, an electrostatic model is not sufficient and other influences, mainly the presence of substrate, must also be taken into consideration. After a very short period of coupling for trypsin binding on hydroxyalkylmethacrylate gel (4 mg of attached enzyme per gram of dry gel), various micro-environments of trypsin were obtained by blocking the remaining reactive groups with glycine, ethanolamine or ethylenediamine (Turkovd er al., unpublished work). In no instance was a shift in the optimal pH of activity determined by means of benzoyl-DL-arginine-p-nitroanilide observed,

314

IMMOBILIZED ENZYMES

and the K,(app) values determined with the same substrate for immobilized trypsin after and 0.91 treatment with glycine, ethanolamine or ethylenediamine were 1.1 1 M f o r native trypsin. 1.5. M,respectively, compared with a K , value of 0.93. During the determination of the K,(app) values, their considerable dependence on the amount of the immobilized enzyme should be borne in mind, as is evident from Table 12.4, where the determined Michaelis constants of native trypsin and of trypsin bound to a poly(vinyl alcohol) carrier at various trypsin concentrations are listed (Manecke and Vogt, 1975, 1976). The effect of hydrophobicity on enzymatic activity was studied by Johansson and Mosbach. Alcohol dehydrogenase was immobilized on matrices of various degrees of hydrophobicity (using acrylamide-methylmethacrylate copolymer). The K,(app) values for n-butanol as substrate were shfted to lower values, proportionally to the hydrophobic character of the copolymeric preparation on to which the enzyme was bound.

-

TABLE 12.4 MICHAELIS CONSTANTS OF NATIVE TRYPSIN AND IMMOBILIZED TRYPSIN Carrier: poly(viny1 alcohol), 5% crosslinked, 1.4 mmole of reactive groups per gram. Substrate: benzoylarginine-p-nitranilide, pH 7.8,25"C Bound trypsin K,(app) (mmole/dm3) per gram of carrier (mg) 86.5 273 32 1 400 Native trypsin - ___ -

0.3 1.4 1.7 3.1 2.7

12.3 STABILITY OF IMMOBILIZED ENZYMES As for the activities of immobilized enzymes, their stability is also dependent to a considerable extent (in addition to the properties of the enzyme itself) on the nature of the solid support and the type of bond between it and the matrix. In general, it can be stated that the supports containing hydrophobic groups can denature proteins, for example, analogously to the denaturation by hydrophobic solvents. However, the proximity of the hydrophobic carrier need not directly and immediately denature the enzyme, but it can inactivate itslowly during storage, or it may increase its sensitivity to heating, change in pH or denaturing agents. The positive or negative charge on the hydrophilic carrier can both increase and decrease the stability of the attached enzyme as a result of the effect of electrostatic interactions between the attached protein and the carrier. With proteolytic enzymes their covalent bond to the solid support mostly substantially increases their stability because it prevents interactions among the individual molecules of the enzyme, and thus their autodigestion.

12.3.1 Stability during storage

In a review, Goldman et ul. (1971) described a number of enzymes that did not lose any

375

STABILITY

of their activity during storage at 4°C for several months, after they had been bound to a copolymer of ethylene with maleic anhydride or to CMcellulose or Sepharose. The freezedried preparations of these immobilized enzymes retained their activity during prolonged storage not only at 4"C, but also at room temperature. The enzymes bound to p-aminobenzylcellulose, a copolymer of leucine with p-aminophenylalanine or the starch derivative dialdehyde starch-methylenedianiline (mentioned in Section 8.2.2) can also be stored in the cold for several months. However, they are almost completely inactivated by lyophilization or drying in air, probably owing to the hydrophobic nature of the carrier. The enzymes attached to a copolymer of methacrylic acid and the fluoroanilide of methacrylic acid usually lose their activity during a few weeks' storage at 4°C. Goldstein (1973) followed the stability of several immobilized proteases. An aqueous suspension of anionic EMA-MDA derivatives (see Section 12.2) of chymotrypsin and subtilisin Carlsberg and Novo did not significantly lose their activity on storage at 4°C for 3-4 months. The suspensions of EMA-MDA, papain and trypsin could be stored under the same conditions for up to 8 months without a decrease in activity. Corresponding cationic EMAMDA derivatives lost 20-30% of their activity under identical conditions. EMAhydrazide derivatives, both anionic and cationic, of all five enzymes displayed an extremely high stability during storage. Even after storage for 1 year at 4°C in the presence of bactericidal agents the suspension did not lose any of its activity. The enzymatic activity which was retained in various EMA-MDA and EMA-hydrazide derivatives after their lyophdization is given in Table 12.5. During storage of the lyophilized powders in a desiccator at 25°C for 1 year no decrease in activity was observed. TABLE 12.5 RECOVERY OF ENZYMIC ACTIVITY ON LYOPHILIZATION Enzyme

Trypsin Chymotrypsin Subtilisin Novo Subtilisin Carlsberg

EMA-MDA resin

EMA-hydrazide resin

Anionic (%) Cationic (%)

Anionic (%) Cationic (%)

76 30 60 27

100

46 8 5 22

50 70 70

93 91 98 86

12.3.2 Dependence stability on pH

The effect of pH on the stability of anionic and cationic EMA-hydrazide derivatives of chymotrypsin, trypsin and subtilisin Novo after incubation at 37°C for 30 min is shown in Fig. 12.5. In relation to the corresponding native enzymes, all anionic EMA-hydrazide, as well as unmentioned anionic EMA-MDA derivatives, displayed an increased stability at alkaline pH. Increased stabilities in the acidic region were observed for cationic EMAhydrazide and EMA-MDA derivatives. Modification of stability patterns as a function of pH can be attributed to the effects of local pH created from the redistribution of hydrogen and hydroxyl ions in the domain of immobilized polyelectrolyte enzyme derivatives.

IMMOBILIZED ENZYMES

3 76

PH

Fig. 12.5. Effect of pH on the stability of anionic and cationic EMA-hydrazide derivatives of chymotrypsin (A) and trypsin (B) and subtilisin Novo (C). 0 , Native enzyme; A , anionic EMA-hydrazide derivative; *, cationic EMA-hydrazide derivative. The test solutions (0.5 mi) in the appropriate buffer, containing enzyme or immobilized enzyme derivative (about 15 esterase units per millilitre) were incubated at 37°C for 30 min; 0.1-ml aliquots were withdrawn and the residual esterase activity was determined by standard procedures. The following buffer solutions were used to cover the pH range investigated: pH 3.0,0.05 M citrate; pH 4-5,0.1 M acetate; pH 6-9,0.05 M phosphate; pH 10-10.7, 0.05 M carbonate. Reproduced with permission from L. Goldstein, Biochim Biophys. Actu, 315 (1973) 1-17.

12.3.3 Thermal stability The thermal stability of anionic and cationic EMA-MDA and EMA-hydrazide derivatives is shown in Fig. 12.6. All anionic EMA-hydrazide derivatives are more stable than the corresponding native enzymes. Anionic EMA-MDA derivatives were less stable than the corresponding EMA-hydrazide derivatives in all instances. In all instances a decrease in the thermal stability of cationic derivatives was found, in contrast to the anionic analogues. Cationic EMA-MDA derivatives were less thermostable than the native enzymes. These re-

377

STABILITY

TEMPERATURE OC

Fig. 12.6. Temperature stability of chymotrypsin (A), trypsin (B) and subtilisin Novo (C) and of their anionic and cationic EMA-MDA and EMA-hydrazide derivatives. 0,Native enzyme; A, anionic EMAhydrazide derivative; A, cationic EMA-hydrazide derivative; 0,anionic EMA-MDA derivative; 0 , cationic EMA-MDA derivative. The test samples (0.5 ml) containing enzyme or immobilized enzyme derivative (about 15 esterase units per millilitre) in a buffer of pH of optimal stability (see Fig. 12.5) were incubated at the specified temperature for 15 min; 0.1-ml aliquots were withdrawn and the residual activity was determined by standard procedures at 25°C. Reproduced with permission from L. Goldstein, Biochim. Biophys. Acta, 315 (1973) 1-17.

sults are in agreement with the common view of the effect of the chemical nature of various solid supports. EMA-MDA resins contain bulky aromatic groups (see Section 12.2) and they are therefore more hydrophobic than the corresponding EMA-hydrazide resins. It has thus been proved experimentally that the thermal stability and the stability after lyophilization of many immobilized enzymes is qualitatively dependent on the chemical nature of the solid support. Even the decrease in thermal stability of cationic derivatives could be attributed to the presence of additional hydrophobic residues, -(CH2)3 - side-chains and dimethylamino terminal groups. If a decrease in thermal stability is brought about by the immobilization of the enzyme, then this phenomenon may be explained by the decrease in the probability of the return to the native conformation of the enzyme after previous thermal perturbation caused by the modification of the enzyme. 12.3.4 Stability against denaturing agents Immobilized enzymes often display an increased stability towards denaturing agents.

IMMOBILIZED ENZYMES

378

Fig. 12.7 shows the course of the irreversible denaturation of native and immobilized chymotrypsin in 6 M urea (Schnapp and Shalitin). The free enzyme is inactivated completely within 90 min, while the immobilized derivatives retain their activity even after 24 h, depending on the solid carrier used.

MINUTES

Fig. 12.7. Irreversible denaturation of chymotrypsin derivatives in 6 Murea, 0.1 M phosphate buffer, pH 8,2SoC. Enzyme concentration, 0.2 mg/ml. 0 , Native enzyme; *, chymotrypsin bound to cyanogen bromide-activated Sepharose; 0,chymotrypsin bound to aminopropylglass via cyanogen bromide activation; 0 , chymotrypsin bound to poly(8aminooctyl)acrylamide via cyanogen bromide activation. Reproduced with permission from J . Schnapp and Y. Shalitin, Biochem Biophys. Res. Commun., 70 (1976) 8-14.

12.3.5 Increase of stability

Weetall and Mason followed the stability of immobilized papain as a function of different methods of coupling on glass during continuous catalysis. In flow-through column reactors they hydrolysed 1% casein solution at 45°C and constant flow-rate, and they determined the operational half-lives ([%) of individual enzyme derivatives. While the half-life of papain attached to alkylamino-glass using the carbodiimide method was 1.9 days, it was 7.2 days on arylamine-glass after attachment by the azocoupling method, and even 35 days on ZrOz -coated glass, bound by the amidecoupling method. The stability of immobilized enzymes can be increased by their modification before attachment to the solid support. Thus, for example, Kershengol'ts e[ ai. attached peroxideinert protein-albumin oligomers (peroxidase pre-treatment with glutaraldehyde in the presence of inert proteins and serum albumin) on Sepharose and obtained highly active preparations with a thermal stability of up to 500 times higher than that of non-modified peroxidase covalently bound to Sepharose. Marshall and Rabinowitz demonstrated a considerably increased stability of soluble enzyme-carbohydrate conjugates which they

APPLICATION

3 79

prepared by attachment of trypsin and a-and 0-amylases to cyanogen bromide-activated dextran. It can therefore be concluded that the stability of enzymes covalently bound to solid supports is determined not only by the physical or chemical nature of the carrier, but also by the character of the chemical modification of the enzyme caused by the covalent attachment on the carrier. 12.4 APPLICATION OF IMMOBILIZED ENZYMES 12.4.1 Affinity ligands

A number of examples of the applications of immobilized enzymes in affinity chromatography have been given in Chapter 11. Table 1 1.1 lists enzymes functioning as affinity ligands for the isolation of a number of substances. In Section 11.3 the use of immobilized nuclease for the isolation of a labelled peptide from its active site by affinity chromatography of the hydrolysate of inhibited nuclease is discussed. Quantitative affinity chromatography permits the study of the interactions of immobilized enzymes with their inhibitors, cofactors and other substances forming specific complexes with enzymes. 12.4.2 Study of stabilized enzyme molecules and of their subunits

Immobilization permits the study of enzymes under conditions that would usually cause their aggregation. The immobilization of chymotrypsin on glass enabled Tanizawa and Bender to study the effect of aprotic dipolar organic solvents on the kinetics of chymotrypsincatalysed hydrolyses under the conditions leading, in the case of free chymotrypsin, to its aggregation. During this investigation it was found that the apparent Michaelis constant, K,(app), and the deacylation rate constant, k3(app), are considerably dependent on the concentration of the organic solvent. The behaviour of the soluble and the immobilized acid phosphatase in aqueous-organic solvents was studied by Wan and Horvath. They put forward several reasons for this study. Firstly, it permits further experiments leading to a better understanding of the mechanism of enzymatic activity. Further, it offers an opportunity to follow the effect of temperature below O'C, whch is impossible in aqueous solutions. As in vivo the membrane-bound enzymes usually occur in a lipophlic microenvironment, the study of the enzyme action in aqueous-organic solvents or on matrices with hydrophobic moieties can contribute to the elucidation of the behaviour of enzymes in their natural environment. Enzymatic reactions in organic solvents can also be utilized for substrates with low solubilities in water. Wan and Horvath, who investigated the decomposition of p-nitrophenylphosphate by acid phosphatase in various aqueous-organic mixtures, generally demonstrated a higher activity of the immobilized enzymes in comparison with the free enzymes. In 1 :1 (v/v) mixtures of various solvents with citrate buffers of various pH values, it was found that the enzymatic activity depends rather on the pH value of the aqueous buffer component than on the pH of the aqueous-organic mixture, as measured with a glass-calomel electrode. From this, it can be concluded that immobilized enzymes are less exposed to organic solvents, because the true concentration of water is higher in the closest proximity of the immobilized

380

IMMOBILIZED ENZYMES

protein molecules than in the bulk of the solution. In addition, the covalent inter- and perhaps even intra-molecular bonds may stabilize the structure of the enzyme. Glutamate dehydrogenase is a hexameric enzyme with a molecular weight of 336,000. In the presence of ADP it forms linear aggregates with a molecular weight exceeding 2 . lo6, possessing very hgh activity. In order to decide whether the increased activity observed is a consequence of aggregate formation, or whether ADP acts as an alosteric modulator or effector directly on the “monomeric” form, while aggregation is then a secondary phenomenon only, Horton et al. attached the “monomeric” form of glutamate dehydrogenase on porous glass beads. They found that in the presence of ADP even in this instance an increase in activity takes place, so that the increase is independent of association. The immobilization of proteolytic enzymes prevents their autodigestion (and also aggregation) and thus permits their study even under conditions when rapid autolysis would occur in the case of free enzymes. An example is the study of heat-induced conformational transitions of immobilized a-and Ptrypsin in the range 20-75°C (Gabel and Kasche). Immobilization can also be utilized for the prevention of spontaneous association between the subunits of oligomeric proteins. Ths makes it possible to determine, for example, whether the subunit form of the enzyme is catalytically active. If it is, then the comparison of the enzymatic properties with the properties of the corresponding immobilized oligomer may afford valuable information on the effect of the interactions of subunits on the function of the enzyme. An example is the work of Chan et al., who immobilized muscle aldolase under conditions when only the attachment of one of the four subunits took place. Using guanidine hydrochloride, they dissociated enzyme molecules attached to the solid support and eluted the column so that only covalently bound unfolded subunits remained. Elimination of the dissociation reagent led to the re-folding of the immobilized subunit. When using subtle dissociation reagents, the immobilized monomer possessed the same activity as the tetramer. Immobilization also permitted Berezin et d . (1974) to regulate the catalytic activity of the immobilized enzymes mechanically. After immobilization of trypsin on an elastic carrier, as for example nylon, it was found that a decrease in enzymatic activity takes place after the stretching of the nylon fibre. The reason may consist in the deformation of the protein molecules. In view of the small volume of the enzyme molecules, mechanical studies can hardly be carried out in other than immobilized form. 12.4.3 Models of biological systems

Nowadays, it is generally accepted that only few enzymes exist in vivo actually as a free protein in aqueous medium, and that most of them exist bound to membranes or solidstate assemblies or are present in a gel-like surrounding. According to Kempner and Miller, for example, virtually all intraceliular enzymes of the alga Euglena gracilis are associated with particulate fractions of the cell. The enzymes attached to well characterized synthetic carriers may serve as simple models of biological systems that occur in living cells. It is true that synthetic polymeric matrices do not mimic the in vivo situation accurately, but the study of these models is an important stage in regarding enzymatic catalysis as heterogeneous catalysis (Mosbach, 1976). In contrast to natural membranes, polymeric matrices possess certain advantages. Firstly,

APPLICATION

381

they are mechanically more stable. The well defined cheniical structure of the matrices permits the study of the effect of a single parameter only, such as the effect of hydrophobicity or the effect of charged particles on the enzyme action. It is also possible to study the micro-environmental effect of the matrix and also the effects caused by different local concentrations of substrate, product, protons, effectors, etc., which are formed under the catalytic activity of enzymes or neighbouring enzyme molecules. In Sections 12.2 and 12.3 the effect of the micro-environment on the activity and stability of the immobilized enzymes was discussed in detail. The effect caused by the matrix can be distinguished only with difficulty from the micro-environmental effects caused by the proper enzymatic reaction of both the enzyme itself and of other enzymes in the environment. As many enzymes produce or utilize protons, it is possible that such action could modify other enzyme activities and thus regulate metabolic pathways. Gestrelius et al. ( 1973) investigated a model system containing entrapped hexokinase, glucose oxidase and trypsin. At pH 8.6 (optimal for hexokinase), 15% of the added glucose was phosphorylated and the remainder oxidized by glucose oxidase (optimal pH 6.6). After addition of trypsin substrate, this was cleaved with the production of protons entering the micro-environment of two glucose-utilizing enzymes. Although the external pH was kept constant at 8.6, the acidification of the microenvironment led to a decrease in hexokinase activity with a simultaneous increase in glucose oxidase activity, so that all of the glucose was oxidized. Immobilization of two sequentially working enzymes on the same matrix may serve as a simple mode for the situation in vivo where the enzymes are arranged in consecutive series on membranes or within gel-like structures. The first such two-step enzymatic system was described by Mosbach and Mattiasson: hexokinase glucose

A

ATP

c

glucose 6-phosphate

ADP

glucose 6-phosphate dehydrogenase NADPnNADPH

t

-

6-phosphogluconolactone

H+

The enzymes were immobilized by a covalent bond with cyanogen-bromideactivated Sepharose or by entrapment within cross-linked polyacrylamide. The rate of the overall reaction catalysed by enzymes coupled together on one carrier was compared with the rate obtained when an amount of free enzymes equivalent in activity (units per volume of incubation solution) to those of the enzymes of the immobilized system was used. A further reference system was composed from individual enzymes immobilized on separate polymeric particles. All systems reached the lag phase earlier than the steady-state level of the production of NADPH. The system in which the enzymes were immobilized on a common matrix, however, achieved the steady state earlier than the corresponding soluble system or a system of enzymes immobilized on separate particles. Finally, the steady-state rates of all three systems achieved were equal. From this, it is evident that in an immobilized system the product from the reaction catalysed by the first enzyme is availablein a higher concentration in the environment of the second enzyme, than in the case corresponding to a free system. This can be attributed both to the dose proximity of the enzyme

382

IMMOBILIZED ENZYMES

molecules in an immobilized state (through immobilization, a concentration of different enzymes relative t o each other can be obtained that would be virtually impossible to accomplishin free solution) and to the impeded diffusion of the intermediate, caused by the Nernst diffusion layer present around the enzyme-polymer particles in stirred solutions (Mosbach, 1976). Thus the subsequent enzyme in the sequence operates much more effectively, and increases the overall rate of the reaction. This twoenzyme system was then extended to a three-enzyme system (Mattiason and Mosbach) including P-galactosidase, hexokinase and glucose &phosphate dehydrogenase. Again, the immobilized system was much more effective in the initial phase than the corresponding soluble system. The so-called “artificial enzyme membranes” are another example of interesting model systems. For example, hexokinase and phosphatase cross-linked with an inert protein form membranes that are capable of acting as a glucose pump. The multi-step enzyme systems are not only theoretically but also practically important. Bouin et aZ. demonstrated that for a scaled-up production of gluconic acid from glucose it is more advantageous to employ the twoenzyme system glucose oxidase-catalase, immobilized together on the same particles, than when they are immobilized on separate particles. In this instance the advantage gained was the local enrichment of the catalase substrate, oxygen. 12.4.4 Application of immobilized enzymes

Immobilized enzymes find applications mainly in the following four areas (Mosbach, 1976): ( 1) enzyme technology, also referred to as enzyme engineering; ( 2 ) analytical studies; ( 3 ) medical studies; and (4) organic chemistry. Immobilization of enzymes produces specific heterogeneous catalysts that can be used repeatedly if they are sufficiently stable. The insolubilized enzyme can be separated from the reaction mixture by filtration or centrifugation. With a column arrangement continuous catalysis is possible, which permits the automation of catalysed processes. The simultaneous or consecutive action of several enzyme can easily be achieved by mixing or layering the packings. The reactions catalysed by immobilized enzymes usually require less space and they are more easily controlled. By attachment to a suitable support they often become more stable in comparison with the free enzymes, as already discussed in detail in Section 12.3. In medical applications encapsulation or entrapping of enzymes may prevent immunological reactions. Ever more attention is given to the application of immobilized enzymes on an industrial scale. For example, the industrial production of L-methionine from a racemic mixture of acetyl-DL-amino acid may be mentioned, in which immobilized aminoacylase is employed (Tosa etal.).The capacity of a fully automated process in an enzyme reactor has been stated to be 700 kg of L-methionine per day, at 60% of the cost of a conventional batchwise process based on the use of a native enzyme (Mosbach, 1976). Another example is the preparation of 6-aminopenicillinic acid from penicillin using immobilized penicillinamidase (Lagerlof et d.).

“SYNTHETIC BIOCHEMISTRY”

383

The most important commercial application of immobilized enzymes in the food industry is probably the production of high-fructose corn syrups, where immobilized glucose isomerase converting D-glUCOSe into D-fructose is used. In the process used by Clinton, Corn Processing, U.S.A., a 93% glucose solution is isomerized to about 42% fructose with a current capacity of 500. lo6 lb per year. The product competes economically with inverted sugar produced from sugar-cane and sugar-beet. Another industrial application of immobilized enzymes is the production of high-fructose syrup from starch, using a threeenzyme process involving a-amylase, glucoamylase and glucosoisomerase. According to the advertized output of lo9 lb in 1974, this is evidently the largest volume of immobilized enzymes used (Mosbach, 1976). A number of reviews of applications of immobilized enzymes have been published (Berezin et al., 1976; Messing; Mosbach, 1977; Pye and Wingard; Wingard; Wiseman). In analytical studies and in connection with immobilized enzymes, enzyme electrodes (Guilbault, 1971), enzyme thermistors (Mosbach et al.) and enzymes bound covalently on polystyrene or nylon in an automated analysis (Hornby et al.; Sundaram and Hornby) should be mentioned. For example, Guilbault (1976) used enzyme electrodes for the determination of glucose, urea, L-amino acids, galactose, acetylcholine and dehydrogenases. Enzymes bound t o capillary reactors were used in connection with a Technicon AutoAnalyzer for the analysis of various substrates, such as glucose, urea and uric acid (Weetall). Goodson et al. described the use of immobilized cholinesterase for monitoring air and water for the detection of enzyme inhibitors, such as pesticides. The system is characterized by unusual sensitivity. For example, paraoxon, an organophosphate, can be detected at a level of 0.1 ppm in air and water. In medicine, mainly encapsulated enzymes are used, such as microencapsulated urease for a decrease in the urea level in blood (Chang et al.) or asparaginase for a decrease in the level of L-asparagine (Chang, 1971). Reviews of applications of bound enzymes in medicine have been published by Chang (1972), Weetall and others. The last application of immobilized enzymes, mentioned by Mosbach (1976), is in organic chemistry in r e s e m h laboratories for various small-scale syntheses. An example is the synthesis of porphobilinogen from two molecules of Gaminolevulinic acid under catalysis of immobilized 6-aminolevulinic acid dehydrogenase (Gurne and Shemin). In view of numerous advantages, even in this field development in the use of immobilized enzymes can be expected in the near future (Jones).

12.5 “SYNTHETIC BIOCHEMISTRY” In Section 12.4 it was shown that the attachment of enzymes to a solid matrix opens up many possibilities for research in many directions. It can already be seen today that research will not be limited to the exploitation of natural enzymes only and to the imitation of metabolic pathways that occur in nature. By creating various microenvironments for enzyme molecules, and the possibility of their modification, conditions are formed for gaining highly effective catalysts that possess properties different from those of native enzymes. The formation of new “artificial” cells, achieving complete metabolic arrangements and cycles, is also not precluded. For tlus independent stage in the preparation of artificial

IMMOBILIZED ENZYMES

3 84

biological systems, Mosbach ( 1 976) used the name “synthetic biochemistry”. In Fig. 12.8 an example is given from this so-called “synthetic biochemistry”. Gestrelius et al. (1975) attached alcohol dehydrogenase covalently to a carrier that already contained covalently bound general ligand NAD’. In this manner they obtained an enzyme which, in contrast to the native enzyme, did not require a free coenzyme for its activity. The regeneration of a reduced coenzyme formed after oxidation of ethanol to acetaldehyde was obtained by the simultaneous addition of an “alternative” substrate, lactaldehyde. It is probable that for other oxidoreductases a similar pair of substrates might be used (or maybe even replaced with artificial electron acceptors and/or donors) for the regeneration of the coenzyme. This last example is a clear demonstration of the close relationshp between affinity chromatography and the immobilization of enzymes.

/

CH3CH,0H

CL

CH3CH(OH)CH20H

Fig. 12.8. Schematic diagram of the active alcohol dehydrogenase NAD(H)-Sepharose complex. The enzyme and the NADH analogue, N6-[(6-aminohexyl)carbamoylmethyl]-NADH were simultaneously coupled to a CNBractivated matrix, under conditions permitting the formation of enzyme-coenzyme complexes. The coenzyme was regenerated in situ by the coupled oxidoreduction between the two alternative substrates for alcohol dehydrogenase, viz., ethanol and lactaldehyde. The results show that these immobilized enzyme-coenzyme complexes are responsible for almost all of the internal activity of the preparations, i.e., the alcohol dehydrogenase activity in the absence of free coenzyme. Reproduced with permission from S . Gestrelius et al., Eur. J. Biochem., 57 (1975) 529-535.

REFERENCES Axdn, R. and Ernback, S., Eur. J. Biochern., 18 (1971) 351-360. Berezin, I.V., Antonov, V.K. and Martinek, K . (Editors), Immobilizovannye Fermenty, Bd 1,2; Izdatelstvo Moskovskogo Universiteta, Moscow, 1976. Berezin, I.V., Klibanov, A.M.and Martinek, K.,Biochirn Biophys. Acta, 364 (1974) 193-199. Bouin, J.C., Atallah, M.T.and Hultin, H.O..Methods Enzymol., 44 (1977) 478-488. Briimmer, W.,Hennrich, N., Klockow, M.,Lang, H.and Orth, H.D., Eur. J. Bwchem., 25 (1972) 129-135.

REFERENCES

385

Chan, W.W.C., Mort, J.S.,Chong, D.K.K. and MacDonald, P.D.M.,J. Biol. Chem., 248 (1973) 2778-2784. Chang, T.M.S., Nature (London), 229 (1971) 117-118. Chang, T.M.S., Artificial Cells, Charles C. Thomas, Publisher, Springfield, Ill., 1972, pp. 1-207. Chang, T.M.S., Gonda, A., Derks, J.H. andMalove, N., Duns. Amer. Soc. ArtiJ Int. Organs, 17 (1971) 246-251. Datta, R. and Ollis, D.F.,Advan. Exp. Med. Biol., 42 (1974) 293-315. Engasser, J.M. and Horvath, C., Biochim. Biophys. Acta, 358 (1974) 178-192. Gabel, D. and Kasche, V., Biochem. Biophys Res. Commun., 48 (1972) 1011-1018. Gestrelius, S., Mansson, M.O. and Mosbach, K., Eur. J. Biochem., 57 (1975) 529-535. Gestrelius, S., Mattiasson, B. and Mosbach, K., Eur. J. Biochem., 36 (1973) 89-96. Goldman, R., Goldstein, L. and Katchalski, E., in Stark, G.R. (Editor), Biochemical Applications of Reactions on Solid Supports, Academic Press, New York, 1971, pp. 1-78. Goldstein, L., Biochim Biophys. Acta, 315 (1973) 1-17. Goldstein, L. and Katchalski, E., 2. Anal. Chem., 243 (1968) 375-396. Goldstein, L., Levin, Y. and Katchalski, E., Biochemistry, 3 (1964) 1913-1919. Goldstein, L., Pecht, M., Blumberg, S., Atlas, D. and Levin, Y., Biochemistry, 9 (1970) 2322-2334. Goodson, L.H., Jacobs, W.B. and Davis, A.W.,Anal. Biochem., 51 (1973) 362-367. Guilbault, G.G.,Aue Appl. Chem., 25 (1971) 727-740. Guilbault, G.G., Handbook o f Enzymatic Methods ofAnalysis, Marcel Dekker, New York, 1976, pp. 445-543. Gurne, D. and Shemin, D., Science, 180 (1973) 1188-1190. Hornby, W.E., Fillipuson, H. and McDonald, A., FEBS Lett., 9 (1970j 8-10. Horton, H.R., Swaisgood, H.E. and Mosbach, K., Biochem. Biophys. Res. Commun., 61 (1974) 1118-1124. Johansson, A.C. and Mosbach, K., Biochim Biophys. Acta, 370 (1974) 339-353. Jones, J.B., in Jones, J.B., Sik, C.J. and Perlman, D. (Editors), Applications of Biochemical Systems in Organic Chemistry, Wiley, New York, 1976, pp. 1-46. Kempner, E.S. and Miller, J.H.,Exp. Cell. Res., 51 (1968) 150-156. Kershengol’ts, B.M., Ugarova, N.N. and Berezin, I.V., Bioorg. Khim., 2 (1976) 264-272. Konecny, J. and Slanicka, J., Biochim Biophys. Acta,403 (1975a) 573-578. Konecny, J. and Slanicka, J., Pathol. Microbiol., 42 (1975b) 245-247. Lagerlof, E., Nathorst-Westfeldt, L., Ekstrom, B. and Sjoberg, B., Methods Enzymol., 44 (1977) 759-768. Manecke, G. and Vogt, H.G., International Symposium on Analysis and Control of Immobilized Enzyme Systems, Compiegne, fiance, 19 75. Manecke, G. and Vogt, H.G., Makromol. Chem., 177 (19763 725-739. Marshall, J.J. and Rabinowitz, M.L., Arch. Biochem Biophys., 167 (1975) 777-779. Mattiasson, B. and Mosbach, K., Biochim Biophys. Acta, 235 (1971) 253-257. Messing, R.A. (Editor), Immobilized Enzymes for Industrial Reuctors, Academic Press, New York, 1975. Mosbach, K., FEBS Lett., Suppl., 62 (1976) E80-E95. Mosbach, K. (Editor), Methods Enzymol., 44 (1977) 1-999. Mosbach, K., Danielsson, B., Borgerud, A. and Scott, M., Biochim Biophys. Acta, 403 (1975) 256-265. Mosbach, K. and Mattiasson, B., Acta Chem S a n d . , 24 (1970) 2093-2100. Ong, E.B., Tsang, Y. and Perlmann, E.G.,J. Biol. Chem., 241 (1966) 5661-5666. Pye, E.K. and Wingard, L.B., Jr. (Editor), Enzyme Engineering, Vol. 2, Plenum Press, New York, London, 1974. Salmona, M., Saronio, C. and Garattini, S . (Editors), Insolubilized Enzymes, Raven Press, New York, 1974. Schnapp, J. and Shalitin, Y., Biochem. Biophys. Res. Commun., 70 (19763 8-14. Sundaram, P.V. and Hornby, W.E., FEBS Lett., 10 (1970) 325-327. Sundaram, P.V., Pye, E.K., Chang, T.M.S., Edwards, V.H., Humphrey, A.E., Kaplan, N.O.,Katchalski,E., Levin, Y.,Lilly, M.D., Manecke, G., Mosbach, K., Patchornik, A., Porath, J. Weetall, H.H. and Wingard, L.B., Jr., Biotechnol. Bioeng. Syrnp., No. 3 (1972) 15-18.

IMMOBILIZED ENZYMES

3 86

Svensson, B., Biochim Bwphys. Acta, 429 (1976) 954-963. Tanizawa, K.and Bender, M.L., J. Biol. Chem., 249 (1974) 2130-2134. Tosa, T., Mori, T., Fuse, N. and Chibata, I., Enzymobgia, 31 (1966) 225-238. Turkova', J., Huba'lkova', O., Kfivikkovri, M. and coupek, J.,Eiochim Biophys. Acta, 322 (1973) 1-9. Turkovri, J., VanEurovi, D. and Saber, M., unpublished data. ValentovP, O., Turkovsi, J., Lapka, R., Zima, J. and eoupek, J., Biochim Biophys. Acta, 403 (1975) 192-196.

Wan, H. and Horvath, C., Biochim Biophys Acta, 410 (1975) 135-144. Weetall, H.H., in Messing, R.A. (Editor), Immobilized Enzymes for Industrial Reuctors, Academic Press, New York, 1975, pp. 201-226. Weetall, H.H. and Mason, R.D., Bwtechnol. Eioeng., 15 (1973) 455-466. Wingard, L.B., Jr. (Editor), Enzyme Engineering, Interscience, New York, 1972. Wiseman, A. (Editor), Handbook of Enzyme Bwtechnobgy, Ellis Honvood, Chichester, 1975.

387

Subject Index* *** A

Acetamide, N,N’-ethylene-, bis-iodo- 177 Acetylcholinesterase, study of binding site 326 Acid-base titration, of polar affinity ligands

212 Activation peptide, study 332 Active-site titration, of immobilized proteases

216-219

__- , recirculation system 21 7 Adipic acid dihydrazide, as spacer 94,327 Adipimidate dimethyl, bifunctional reagent

177 Adsorption, see also Sorption Adsorption isotherm, 27-29,45,46,51,52,

90

__-

,Langmuir 90

AF 201 320 AffiGel 160,162,320 AffiGel 10,preparation 172 Affi-Gel 100 189, Affmants, see also Affinity liiands Affinant, group-specific 89 Affinity chromatography, biospecific 7-1 1 _-- ,determination of small amounts of antibodies 8 ---,history 7-11 ---,principle 7-11 _-- , statistical theory 33 _-- ,term 1,2 --- ,theory on the basis of the equilibrium model 13-34 Affinity density perturbation 140-144 _-- ,principle 141 Affinity electrophoresis 144-147 Affinity elution 7, 139, 140 __- , effect of affinity ligand concentration

140

-_- ,polymers used 139 Affinity gel, copolymerization of alkenyl 0glycosides with acrylamide 144 Affinity gel filtration 8,53,54

Affinity ligand(s) 7,379,380 --_ ,alteration by immobilization 239 --_ ,bonding on sorbent 5 1-87 --_ ,choice 89-129 --_ ,competitive 77-80 --_ ,concentration 17,36,55,62-67 -__ ,---, critical 66 __- ,---, intragel 65 ---,---,optimal 63 --_ ,density 41 --_ ,distribution 64 --_ ,high-molecular-weight, attachment to support 61 --_ ,immobilized, characterization 203-222 ---,---, commercially available 118, 119,

320,321

-__ ,__- ,determination on basis of nitrogen 214

___ ,---, effect of heterogenity 237-239 --_ ,---,enzyme interaction 63

---,---, labelled, determination on basis of radioactivity 215 polar, determination by acid-base titration 212 _-_ ,leakage, see Leakage, affinity ligand _ _ _ , saturation 17,18,26 --_ , selectivity 91 --_ ,used 246-318 Affinity systems, with macroligands 91,92 --_ ,with small ligands 91 Affinose 320 Agarox 159-173 --_ ,alkaline desulphurkation 169 --_ , alkyl- 131 __- ,w-aminoalkyl- 13 1 --_ ,E-aminocaproylfucosine 11 8,320 --_ ,E-aminocaproyl-D-tryptophanmethyl ester 118 ---,p-aminophenylmercury (11) acetate 118 _ _ _ ,5’-(4aminophenyl)phosphoryluridine 2‘(3’)-phosphate 118 ---,anti-BSA 118 --- ,anti-human IgG 11 8 --- ,APUP 320

__- ,---,

*Compiled by H.Be8vdva‘Fovd. *For some general terms which occur throughout the book,reference is given only to the page where the particular term is explained or t o the chapter or paragraph dealing with the term in question.

388

--_ ,arsanilic acid 118 ---,attachment by means of benzoquinone 173,174 ---,attachment by means of bifunctional oxiranes 169 ---, attachment by means of cyanogen bromide activation 161-169 --_ , attachment by means of N-hydroxysuccinimide esters 172 --- , attachment by means of thioldisulphide interchange 170, 171 ---,capacity of binding 163 - - _ , casein 118 ---, concavalin A 118-122,321 ---,cyanogen bromide activated, coupling of affinants 161-169 --_ ,- _ _ , positive charge containing 132 ---,---, with alkyl- or arylamines 241 ---,cysteamine 125 --- ,derivatives 159-173 --_ ,---, detergent-like 163 ---,diaminodipropylaminosuccinyl-N-pheny1alanyl-insulin 105 --_ ,DNA 93,122 --_ ,fetuin 115 ,gangliosidediaminopropylamino 115 --- ,(glutathione-2-pyridyl disulphide) conjugate 106, 107 --- ,glygly-tyr(0BZ)-arg 118 --- ,hemoglobin 125 --- , hexane-adenosine 123 --- ,hexane-coenzyme A 122 --- ,hexane-nicotinamide adenine dinucleotide 122 --- ,hexane-nucleotide 123 ---, hexane-poly(I)-poly(C) 320 --- ,hexane-thiol 125 --. __ ,hydrazido-, polyacrylic, preparation 186, 187 ---,hydrocarbon coated 131 --_ , L-lysine 118 . ._. - ,mercaptohydroxypropyl ether 170 --_ ,mixed disulphide derivative 170 --- ,NADP 94,320 --- ,negative charge containing 132 ---,non-specific sorption 169 --- , oxirane activated, bis-carboxymethylamino groups containing 148 , oxirane groups introduction 169 --_ ,L-phenylalanine 118 ,polynucleotide 124 ---,SAG (AgoCel) 160 ---,SBA 118 --- , solubilization 21 1 ---, solubilized, spectra 21 1 --- , stability 160 - - _ , succinylated aminoalkyl- 172

---

--I

SUBJECT INDEX

--_ ,thyroxine 118 -_- ,triiodothyronine

118 118,125 _-- ,D-tryptophan 118 --_ ,L-tryptophan 118 -_- , L-tyrosine 118 _ _ _ ,with caprylic hydrazide 134 AG-NAD, type 1 320 Albumin-agarose chromatography 329 Alcohol dehydrogenase, binding 01) in relation to temperature 74 Allosteric effector 95 Amino acids, copolymers as support 181 _-- ,immobilized, determination after acid hydrolysis 313 -__ ,modification, general 108 --- - _ _ , selective 108 - _ _ ,_-_ ,sitedirected 108 c-Aminocaproic acid, immobilized, determination after acid hydrolysis 213 _ _ _ ,---, determination by titration or on basis of nitrogen 166 --- ,Sepharose attached 183,329 _ _ - , spacer 57, 231 Amino groups, acid-base titration 205 _-- ,fluorescamine test 207, 208 _ _ - ,reaction with 2-hydroxy-1-naphthaldehyde 206 - _ _ ,reductive methylation determination 213 Amphiphilic gels 135 Antibodies, anti-DNP, use for peptide isolation 110 _ _ _ ,anti-nitrotyrosyl 110 - _ _ ,binding site heterogenity 95-97 -_- ,elimination from blood 334-336 --- ,elution, effect of antiserum titration value 98 _-- , immobilized, isolation of interferons 98 -__ ,interaction with antigens 95 Antigens, isolation 98 Aquapak 182 Anenius plot 73 Association constant, intrinsic (Ki) 324, 325 Association process 35 Azide assay 206,207

--_ ,trypsin inhibitor

B Batchwiseadsorption 14, 15, 24,67, 231 Beads, coated with biospecific reagent 116 Bemamidine, 4-amino- 126 Benzene sulphonate, 2,4,6-trinitro-, Na salt, colour test 207 Benzoic acid, h i t r o - , 5,Ydithiobis- 209 -_- ,--_ ,---, SH active-site titration 219 Bemoquinone, attachment on to agarose 173,

.^_

SUBJECT INDEX

“Binding” (6) 47,49,57,65,66,68-70 74 Binding efficiency 367 Binding isotherm 27-29,45,46, 51,52,90 Binding proteins, isolation 103-1 06 ---, properties 106 Binding site, complementarity of 60 --_ ,high affinity, population 326 -__ ,orientation of 68 Bioaffinity chromatography 1 , 7 Bioelution 7 BioGel, A 160,320 ---,CM 174 -_- , P 174 ---,P-2 174 --_ ,---, aminoethyl and hydrazide derivatives 174 ---,PdO 174 --_ ,---, aminoethyl and hydrazide derivatives 174 BioGlass 182 Biologically active substances, interaction 324326 Biological system, artificial 384 Bis(3-aminopropyl) amine 80 Bis-diazobenzidine, bifunctional reagent 177 Bismaleinimide, N,N‘-l, 3phenylene, bifunctional reagent 177 3,6-Bis(mercurimethyl) dioxan, bifunctional reagent 177 Bisoxirane, bifunctional reagent 169,177 --_ ,crosslinking of agarose carbohydrate chains 169 Blocking, active groups remaining 187 ---,unreacted carbonyl groups, elimination 189 Borohydride of sodium, reduction with 159 - _ _ ,unreacted aldehyde groups, elimination 189 Bovine serum albumin 133 -_- ,microspheres 335 Bromoacetylation 185 Bromoacetyl bromide, cellulose 155 BSA, see Bovine serum albumin

--_ , effect of temperature

C Capsules, for elimination of antibodies by extracorporeal circulation 334, 335 Carbodiimide 154,183, 184 ---,l-cyclohexyl-3-(2-morpholinoethyl)-, methop-toluene sulphonate 184 ---,dicyclohexyl- 172,373 -_- , lethyl-3-(3dimethylaminopropyl)-,hydrochloride 184 ---, promoted method, estradiol-Sepharose preparation 184

389

---,---, scheme of attachment 183 Carbohydrates, coupling 200 ---,polymers, commercially available 99 ---, immobilized, determination after hydr olysis 214 Carbonyl groups, acid-base titration 205 Carboxyl group, reaction with glycine 206 Catalysis, heterogenous 367 Cells, fibre fractionation 116 --_ ,isolation 116,117 --_ ,lymphoid 116 ---,topology 144 Cellulose 153-15 7 --_ ,bromoacetyf bromide 155 ---,commercially available 153, 154 --_ ,DEAE- 133 ---,---, benzoylated 135 ---,derivatives 153-157 ---,---, binding capacity 114 ---,DNA 122 ---,general considerations 197 --_,oligodeoxythymidylic acid, specifity for polyadenylic acid containing molecules 127 ---,oligo(dT) 124,126,321 ---,oligothymidylic acid 111 --_ ,succinylated aminododecyl- 126 ---,---, with 3-aminobenzylboric acid 114, 127 ---, succinylated aminohexyl-,with trypsin inhibitor 127 ---, treatment with sodium hydrogen sulphite 111 Chelate forming sorbents 147,148 Cholera toxin 114,115 Cholinesterase, immobilized, detection of pee ticides 383 Chymotrypsin, immobilized, active-site titration 21 8 ---,---, specific activity 369 CMC, see Carbodiimide, l-cyclohexyl-3-(2-morpholinoethyl) metho-p-toluenesulphonate Coenzyme, immobilized 93,94 Cofactors, isolation 95 Coliphage K29 144 Column, capacity 66 --_,efficiency 68,69 --- ,equilibration time 69 ---,geometry 66 ---, ligand concentration 66 ---,total ligand amount 66 ---,working (operating) capacity 36,41 Column chromatographic results, simulation of 24 -26 Column chromatography 68 --_ ,cooperative adsorption 29-33 --- ,on bovine serum albumin-agarose 319 Competitive effect 77, 78

390

Complex formation, scheme of competitive, non-competitive and uncompetitive effect 77,78 “Compound affinity” 86, 241 Concave gradient 9 Conformational adaptation 68 Conformational changes, determination using fluorescence method 219 “Conformational occlusion” 60 Cooperative adsorption, column chromatography 29-33 - , chromatograms, characteristic features 3 1-33 Cooperative bonding, theory 27-33 Cooperative elution 44-46 Copper chelate 149 -- - -, complex with histidine and cysteine 147 Coupling, diazo-, stability 200 --- ,of aminogroups 154, 155, 159, 161, 169,172,173,175,176,179-184 -_- ,of carboxylgroups 161, 162, 180, 181, 183, 184 --- ,of hydroxyl groups 155,161,169 --_ ,of phenolic hydroxyl groups 155, 174, 175,181 - _ _ , o f thiolgroups 161, 162, 169, 170, 174 --_ ,multiple 200 Coupling procedure, general considerations 198-200 --_ ,review 199 Covalent affinity chromatography 107, 137, 138 CPG 182 --_ ,alkylamine 320 Cuprophan 321 Curtius azide method 154 Cyanogen bromide, required properties 166 Cyanogen bromide activation, by use of pH meter 164, 165 _ _ _ ,effect of cyanogen bromide amount 166 _ _ _ , effect of temperature 166, 167 - - _ ,in concentrated phosphate buffer 165, 166 __ - , with acetonitrile solution 166 Cysteine, complex with zinc and copper 147 D DDC, see Carbodiimide, dicyclohexylDebye-HUckel theory 81-86 “Deforming buffers” 7, 132 Degalan V 26 321 Density perturbant 141 Desorption, by use of chaotropic ions 236

SUBJECT INDEX

_ _ - , b y use of quanidine salts 236 _ _ _ ,by use of x e a 236 _-- , effect of ionic strength 80-86, -_- , effect of pH 235 -__ ,effect of temperature 234 ---,practice 233-237

236

Detachment, of alcohol- and thiolesters 237 _-- ,of azo bond 237 Detergents, use for elution 243 Dextran gels 158, 159 --- , commercially available 158 ---, stability 158 Dianiline, methylene- 15 7 Diazonium groups, attachment on to cellulose 155 Diazotization capacity 158 Difference analysis 210 Diffusion 70 -_- ,Eddy 70 _-- ,longitudinal 70 ---,restricted 70 Dihydroxyboryl derivatives, isolation of nucleotides and tRNA 113, 114 -__ , isolation of sugars, nucleosides and serine proteases 127 Diisocyanate hexamethylene 177 Diphenyl, p,p’ difluoro-m,m’ -dinitre, bifunctional reagent I77 Dephenylmethane, p , p ’ -diamino- 373 Dipropylamine, diamino-, ninhydrin colorimetric determination 215 Z,Z‘-Dipyridyl disulphide 106, 170, 210’ 4,4'-Dip yridy l disulphide 2 10 Displacer 91 Dissociation constant, binary complexes of dehydrogenase isoenzymes with NADH 9, 10,324 -_- ,complex of chymotrypsin with anti-lysine-Spheron 41 -_- ,complex of chymotrypsin with N-benzyloxy-carbonylglycyl-phenylalanine-NH, Spheron 41 _ _ _ ,complex of nuclease with pdTpAPSepharose 39 -_- ,complex of ribonuclease-S-protein with Agarose-S-peptide 47 _-- ,complex of trypsin with p-aminobenzamidine-NH, -Spheron 44 ---,complex of trypsin with glycyl-glycyl-iarginine-Sepharose 43 -_- ,comparison of different determinations 39,44 _-- ,determination, by direct titration 47 _ _ - ,---,by elution analysis 35-41 _ _ - ,---,by frontal analysis 41-44 ---,--_ ,use of affinity electrophoresis 145,147

SUBJECT INDEX

---,effect of immobilization 39

_ _ _ ,effect of immobilized affinity ligand concentration 36

_ _ _ ,effect of ionic strength 44, 80, 81 _ _ _ ,for complex of enzyme with immobilized affinity ligand (KL) 13,36,5 1

_ _ _ ,for complex of enzyme with soluble inhibitor (K,) 13,22,26,36 sugar complex 146 Distribution constant, effect of temperature 71-75 Di-substrate enzymic reaction 93 Divinyl sulphone, bifunctional reagent 177 Dodecylamine 114 Dry weight determination 204

_ _ _ ,lectin-bound

E

EDC, see Carbodiimide, l-ethyl-3-(3-dimethylaminopropyl); hydrochloride Effector, allosteric 95 Electrophoresis, affinity, see Affinity electrophoresis Elemental analysis 214 ELISA, see Enzyme-linked immunosorbent assay Elman's reagent 209 Eluents, chaotropic 91 Elution, affinity, see Affinity elution _ _ _ ,after chemical modification of immobilized affinity ligand 91,98 __- ,biospecific 7,19,139 -__ , b y change in pH 93,235 _ _ _ ,conditions 232-237 ---,cooperative 44-46 ---,effect of soluble affiant concentration 38-44,234 _ _ _ ,effect of varying gradient type 234 __- ,from ion exchangers 140 _ _ _ ,gradient 233 _ _ _ , specific by substrate, see Affinity elution _ _ _ ,stepwise 233 _ _ _ ,with affinity ligand solution 19,77-80, 91,93,233 _ _ _ ,with detergents 243 _ _ _ ,with different salt concentration 136 _ _ _ ,with KC1 gradient 95, 236 _ _ _ ,with pH gradient 95,235 _ _ _ ,with pulse of 5 mhf ATP-Mg" 95 _ _ _ ,with pulse of 5 mhf NADH 95,234 ---,with solutions of salts 237 Elution of enzyme, by medium changing 19 ---,following multiple washes 21, 22 ---,recovery 20,24 ---,without washing out 19-21 Elution profie, analysis 44 _ _ _ ,concentration dependence 30

391

---,temperature dependence 29 Elution ratio, relative 48 Elution volume, relative 48 EMA, see Ethylene-maleic anhydride copolymer Enthalpy, adsorption 72 Enthropy, adsorption 72 ---,translantation 54 Enzacryl(s) --_ ,AA 174,321 _ _ _ ,AH 174 _ _ _ ,enzymes bound 126 ---,polyacetal 174 ---, polythiol 174 _ _ _ ,polythiolactone 174 Enzite 119 Enzymatic action study 327-329 Enzyme(s) ---,binding, fractional 17 ---,electrode 383 _ _ _ ,elution, see Elution of enzyme _ _ _ ,engineering 2 , 3 -__ ,immobilized, see Immobilized enzyme _ _ _ ,in metabolic paths 329 ---,isolation 92-95 ---,multiple attachment 369 ---, recovery 20,24 --_ ,thermistor 383 Enzyme-liked immunosorbent assay 333,334 Epoxide groups, agarose 169,170 -__ ,glycidyl methacrylate gels 180 Equilibration time 67-71 Equilibrium-based limitation 17, 18, 26, 55 Equilibrium concentration constant (K,) 82 Equilibrium conditions 70 Equilibrium constant, see also Dissociation constant Equilibrium constant, effect of ionic strength 80-86 Equilibrium model, adsorption with a fixed binding constant 13-19 --_ ,elution by change in K , 19-22 _ _ _ ,elution by competitive inhibitor 19, 2224 Equilibrium ratio constant, effect of ionic strength 83 Erythrocytes, binding 117 Esterolysis, pH optimum 371 Ethanol, 2-amino- 187 _ _ _ ,2-mercapto- 187 Ethylene-maleic anhydride copolymer 173, 174,321,373 _ _ _ ,hydrazide 373 Exclusion, secondary 68 Extension arm, see Spacer Extracorporeal system, scheme 335, 336

392

F FAD, immobilized, determination 21 1 Flow-rate 67-71, 231 Fluorescamine test 207,208 Fluorescein technique 222 Fluorescence cell 220 Fluorescence method, conformational changes, study 219 Fluorescence microscopy, distribution of immobilized proteins 221 Fluorescence spectra 220 Fluorescent immunoassay 334 Fluorochrome fluorescent isothiocyanate, labelling of enzymes 221 Fucopyranosyl derivatives 10 1 Fucosylex 120 G

p-Galactosidase, effect of concentration of various anions on sorption 135,136 G e W , amphiphilic 135 -__ ,porosity 53-56 “General ligand” 89, 90 Glass beads, commercially available 182 --_ ,derivatives 180, 181 --_ , methods of attachment 181 --_ ,silanization 181 --_ , stability 180 Glucagon, receptor 104 Glucosamine, blocking of 187 Glutaraldehyde, bifunctional reagent 177 --_ , coupling of proteins 176, Glycaminosylex 120 Glycine, blocking of 187 Glycoproteins, isolation 101-103 --_ , terminal sugar 101 Glycosides, alkenyla-, copolymerization with acrylamide 100 Glycosylex A 120,321 Gradient, concave 9

H Half-live 79 Half-time, calculation 192, 195 Haptens, isolation of antibodies 95 Haptoglobulins, study of interactions 324 Heat adsorption 71 Hemoglobin, and a-and P-chains, interaction with haptogfobin Sephatose 324, 325 Heterogenity index 325 HETP (height equivalent to theoretical plate) 68 Hexamethylene diisocyanate 177

SUBJECT INDEX Hexamethylenediamine, Sepharose attached 183,331 _ _ _ , spacer 57 High-performance affinity chromatography 111,112 Histidine, complex with zinc and copper 147 Histone-histone interaction 3 26 Hofmeister’s series 135, 229 Hormones, interaction with complementary receptors 104 _ _ _ , isolation 114, 115 Hydrazide, polyacrylic, preparation 186, 187 Hydrazide groups, acid-base titration 205 Hydrophobic arm 131 Hydrophobic chromatography 131-136, 237, 24 1 _ _ _ , o f interferons 329,330 Hydrophobic interaction 133, 229 _ _ _ ,“pure” 135 Hydrophobic salting-out chromatography, use of non-ionic amphiphilic gels 134 Hydroxylamine, cleavage of thiol ester bond 184,200 Hysteresis effect 85 I

Immobilization, effect on dissociation constant 39,41,60 ---, modification of affinity ligands 240 Immobilized enzymes 3,118,365-384 _ _ _ , activity 366-374 --_ , application 379-383 _ _ _ ,---, industrial 382,383 - _ _ ,---, medical 382 _ _ _ ,attachment to solid support 366-374 _ _ _ , classification 365,366 _ _ _ ,effect of carrier chemical nature 371 - _ _ , effect of nature and concentration of buffer 371 _ _ _ ,effect of support hydrophobicity 374 _ _ _ ,ionizable groups 370 _-_ ,model of biological system 380 _-_ ,relative activities 366 _ _ _ ,stability 374-379 _ _ _ ,---, against denaturing agents 377, 378 _ _ _ ,---,effect of pH 375, 376 _ _ _ ,---, effect of support hydrophobicity 3 74 -3 7 7 _ _ _ ,---, effect of temperature 375-377 _ _ _ ,---,increase 198,378,379 _ _ _ ,---, lyophilization 375 _ _ _ , steric restriction 367 _ _ _ , surface reactive groups, concentration of carrier 369 _ _ _ ,thiol-disulphide interchange 170-172 Immunoadsorbent 97

SUBJECT INDEX

-__ ,application in medicine 336 Immunoaffinity chromatography 95-99 Immunoassay 332-334 _ _ _ ,microfluorimetric 334 Immunofluorescent method, distribution of antibodies 334 Incubation time, effect on sorbent capacity 71 Indubiose A4 321 Inhibitors, isolation 95 Interaction, hydrophobic, non-specific 186 ---, of biologically active substance 324326 - _ _ , of peptides with nucleotides 48 Interferons 98 Interferon binding, study with hydrophobic ligand and lecitin chromatography 329, 330 Ion exchanger, as affinity polymer 139 Ionic strength, in sorption and desorption 75-77,80-86,228-230,235 -__ ,linear gradient 235 Isourea, 0-acyl- intermediate 184 K K , = Concentration equilibrium constant 82 KI = Dissociation constant for enzyme-soluble inhibitor complex 13,22,26,36 Ki = Association constant, intrinsic 324, 325 K L = Dissociation constant for enzyme-immobilized affinity ligand complex 13,36, 51 K,(app) values, dependence of immobilized enzyme amount 374 Kjeldahl method 214

L Leakage, affinity ligand 189-191 -_- , examples 191 --_ ,function of time and pH 190 -_- , function of used carrier 191 --_ , half-time calculation 192, 195 --_ ,mathematical approach 191-195 __- ,supressed 191 Leaving group 137 Le Chatelier’s principle 71 Lectins, classification according t o affinities 102,103 --_ ,immobilized 101 -__ ,isolation 99-101 ---,properties 99 Lectin chromatography, interferons 329, 330 Linkage, azo-, detached with sodium dithionate 200 Lipids, isolation 114, 115

393

M Malonimidate diethyl, bifunctional reagent 177 MDA, see Diphenylmethane, p,p‘ -diaminoMembrane, fractionation of fragments 141 _ _ _ , topology 144 Mercogel 182 Mercury, use in affinity chromatography 106 Meromyosin, heavy 328 Metal chelate affinity chromatography 147149 Metal chelate gel, see Chelate forming sorbents Metal ions, heavy 147 Methacrylate gels, glycidyl-, a-amino group binding, pH dependence 180 ---,---, carbonyl group binding, pH dependence 180 -__ ,_ _ _ , reaction of e-amino group 180 --_ ,hydroxyalkyl-, characterization 178 --_ ,__- ,commercially available 17 8 --_ ,---, mercury methacrylanilide derivative 106 --_ ,- --, p-nitrophenyl ester derivatives, capacity determination 209 ---,---,---, coupling as function of pH and concentration 178-180 --_ ,---,---, unreacted groups, elimination 189 ---,---,preparation 178 --_,---, proteins binding by using active esters 178 ---,---, stability 178 4-Methylumbellifer yl p-(N,N,N-trimethylammonium) cinnamate 21 8 Microenvironment 3,198,368 “Mock affinity system”’ 80 MUTMAC, see 4-Methylumbelliferyl p-(N,N,Ntrimethylammonium) cinnamate

N NAD(+) derivatives, sorption of glyceraldehyde 3-phosphate dehydrogenase 84 NADH, concave gradient 9 --_ ,immobilized, determination 211 Newton-Raphson procedure 192 Nickel oxide 181 Ninhydrin colorimetry 215 Nitrogen determination 214 Non-biospecific interaction 39-44, 86 Noncompetitive effect 77,78 Non-specific effects 80-86 Non-specific sorption 241, 242 ---,effect of ionic strength 80, 81 NPAC gel, see Methacrylate gels, hydroxyalkyl-, p-nitrophenol ester derivatives NPGB, see p’quanidin benzoate, p-nitrophenyl-,

394

Nuclease, fragments, complementary interaction 319 ---, semi-synthetic 319 Nucleic acids, bound to inert matrices 111 - _ _ ,coupling 155,200 --_ ,isolation 111-114- _ _ ,periodate oxidation 15: _ _ _ ,sequence study 111 Nucleic acid bases, high-performance affinity chromatography 111 , 1 1 2 Nucleosides, high-performance affinity chromatography 111,112 Nucleosides immobilized, determination using phosphorus analysis 213,214 Nucleotides, attachment to support 60, 11 1, 200 --_ , interaction with enzyme, determination of nature 61 _ _ - ,interaction with peptides 4 8 _ _ - ,isolation 111-114 Nucleotides immobilized, capacity in relation to ligand concentration 18 _ _ _ ,determination after hydrolysis 213, 214 Nylon 181 0

Oligoadenylic acid, cooperative elution 29, 30 Oligo(dT)-~llulose 124,126,321 Operating capacity 38,41 Organophosphates, immobilized 137 Ovalbumin, hydrophobic interaction 133 Oxirane groups, blocking 170 - _ _ ,determination 208 _ - _ ,of agarose, alkylamine linkages 169 _ _ _ ,_ _ _ ,etherlinkagrs 169 --- ,_ _ - , thioether linkages 169 P Papain, immobilized, stability 198 Partition function, calculation 27 Peak trajectories 32,46 Peptides, affinity labelled 108 ---,_ _ _ , isolation 108, 109 _ _ _ ,cysteine containing 110 _ _ _ , histidine containing 110 - _ _ ,immobilized, determination after acid hydrolysis 2 13 --- ,lysine containing 110 --- ,methionine containing 110 _ _ _ ,SH groups containing, isolation 106108 _ _ _ , specific, isolation 108-1 11 _ _ _ ,synthetic purification 323 _-_ ,tryptophan containing 110 Periodate oxidation, attachment of nucleic acids 155

SUBJECT INDEX

_ _ _ ,attachment of proteins 159 Pf = Purification factor 20 pH effect on affinity 75-77 Phenol, 2,4-disulphonyl chloride, bifunctional reagent 177 Phosphofluoridate 0,O-diethyl 137 Phosphofructokinase, binding (p) in relation to temperature 74 Phosphorus determination 2 14 Phosphorylase b, isolation, effect of hydrocarbon chain length 131 Plasminogen B-Sepharose, activation peptide study 332 Plate theory 27-33,68 Poissin distribution 194 Polyacrylamide gel, affinity electrophoresis 144,145 _ _ _ ,p-aminobenzamidoethyl derivatives, preparation 175 - _ _ ,aminoethyl derivatives, preparation 175 _ _ _ ,bifunctional derivatives 177 _ _ _ ,characterization 174, 175 _ _ _ ,commercially available 174 _ _ _ ,coupling of proteins, after activation with nitrousacid 175,176 _ _ _ ,_ _ _ ,after activation with thiophosgene 176 ---,_ _ _ ,by using glutaraldehyde 176 _ _ _ ,hydrazide derivatives, preparation 175 _-_ ,stability 174 _ _ _ ,with bound polysaccharide 145 Polyacrylic acid, with aliphatic amines, fractionation of tRNA 135 Polydiazonium salts, coupling on t o S-MDA 158 Polystyrene matrices 181 Poragel 182 Propane-l,3-diol, 2-amino-2-hydroxymethyl-, blocking of 187 2-Propanol, 1,3diamino-, spacer 185 Propioimidate, 3-mercapto- 170 Proteases, immobilized, active-site titration 216-219 Proteins, affinity labelled, isolation 108, 109 _ _ _ ,coupling 200 _ _ _ ,immobilized, conformational changes 219-221 _ _ _ ,---, determination, colorimetric methods 211,215 -- -,- --,- --,fluorimetric analysis 21 1 _-_ ,---,---, from amino acid analysis after acid hydrolysis 21 1,213 - --,- --,---, on basis of elemental analysis 214 ---,- --,---,on basis of tryptophan content 215,216 _ _ _ ,_ _ - ,---, protein balance before and after binding 210, 211, 213

SUBJECT INDEX

-_ -,---,- --,spectrophotometricaly 21 1 --_ ,---, distribution determination, by fluorescent microscopy 221

-_ -,-_ _ ,---, b y radiography 222

_ _ _ ,irreversibility of binding on to specific sorbents 242

__- ,lipophilic 134 _ _ _ ,non-homogenity of binding 242 -_- ,non-specific sorption 24 1 _ _ _ ,SH groups containing, isolation 106-108 _ _ _ ,suppression of non-specific sorption 241 -__ ,transport, isolation 103-1 06 Purification factor (Pf)20 Pyridinium-2-aldoxime, N-methyl- 137 2-Pyrrolidone, N-methyl 166

Q p'quanidin benzoate, p-nitrophenyl- 217 Quantitative affinity chromatography 35-44 R R = Fraction enzyme recovery Radioactive affinants immobilized, determination 215 Radiography, distribution of immobilized proteins 222 Radioimmunoassay, solid-phase 332,333 Receptors, complementary, interaction with hormones 104 _ _ _ ,glucagon 104 _-- ,isolation 103-106 ---,membrane structure 104 Residual negatively charged groups, determination 204 Resins, coupled with thymine 112 Resolution, ability 68 __- ,of enzyme mixture, effect of ionic strestrength 235 _ _ _ ,-__ ,effectof pH 235 _-- ,---, effect of temperature 234, 235 _ _ _ , of optical isomers 319 -__ ,of D,L-tryptophan 319 mRNA, isolation 111 tRNA aminoacyl synthetase, affinity elution 140

S Saccharides, isolation 103 Salting-out chromatography, hydrophobic, use of non-ionic amphiphilic gels 134 Salting-out effect 134 Sample volume 230 Saturation effect, optimal conditions 240, 241

395

Scatchard plot 326 Scatchard's relation 3 24 Schiff s base 157 Sephadex 99,321 ---, B D 121 ---,LH 321 Sepharose _ _ _ ,AH 156,183,331 ---, w-aminoalkyl- 133 ___ ,N6-w-aminoalkyl-AMP- 5 7 ---, e-aminocaproic acid, see Sepharose, CH ---, N6-(6-aminohexyl)-S'-AMP 60, 63,65, 95,96 ---,---, effect of ligand concentration on capacity 63 _ _ _ ,---, effect of pH on capacity 76 _ _ _ ,---, effect of temperature on binding 73 _ _ _ ,---,effect of temperature on capacity 72 _ _ _ ,P1-(6-aminohexyl)-P2-(5'adenosine) pyrophosphate 6 1 -_- ,_ _ _ ,interaction with myokinase 65 _ _ _ ,6-aminohexyl-NAD+, titration graphs 156 _ _ _ ,6-aminopenicillanic substituted 138 _ _ _ ,5'-AMP, commercially available 120 _ _ _ ,blocking of cyanogen activated groups 189,241 ---,Blue 320 ---,bromoacetamidoethyl 184 __- ,CH 183,329 __- ,_ _ _ ,activated 189 _ _ _ , C L 160,321 _ _ _ ,---, general considerations 197 __- ,comparison of properties 166-169 _ _ _ ,concanavalin A 103,120,321 ---,---, electrophoresis 145 ---, crosslinked benzylated dibromopropanol, degree of substitution 209 _ _ _ ,cyanogen bromide activated affinant leakage 163 _ _ _ ,---,N, ,N, disubstituted guadinines 163 _ _ _ ,---, hydrazide binding 186 ---,---, multi-poly-DL-alanyl-polylysine 186 __- ,_ _ _ ,polylysine 186 ___ ,---, scheme of amine attachment 163 ---, epoxy-activated 169, 170 ---, estradiol, preparation 184 - _ _ ,hexamethylenediamine, see Sepharose AH _ _ _ ,hydrazido derivatives 186-188 _-- ,hydrazido-, polyacrylic preparation 187 ---, methylamine 135,136 ---, nuclease 109

SUBJECT INDEX

396

---, pdTp 319,323 _-- ,pdTpAP, see Sepharose, thymidine-3'@aminophenyl phosphate) Sphosphate

--- ,plasminogen B, activation peptide study 332 -- - , specific sorbent for lectins 99 _ _ _ , N-substituted isourea conjugates 132, 163 _ _ _ ,_ _ _ ,replacing with guanidine bond 163 ,---,stability 163 _-- , substituted with nalkyl amines 242 ---,substituted with 4-phenyl-n-butylamine 24 2 --_ ,succinylaminoethyl 184 -__ ,sulphanylaminebond determination 214 _ _ _ ,sulphydryl 184 -__ , thiol activated, attachment method 171 _-_ ,---,degree of substitution 171 -__ ,---, inactivated enzyme elimination 171 _ _ _ ,_ _ _ ,preparation 171 --_ ,---.stability 171 __- ,thymidine-3'-(p-aminophenylphosphate) 5'-phosphate 36,38 - _ _ ,tyrosyl 184 _-- ,with attached antibodies 98 _ _ _ ,with attached anti-nitrotyrosyl antibodies 110 _ _ _ ,with attached glucagon 98 --_ ,with bonded bis(3aminopropyl) amine 80 ---,with complex of alcohol dehydrogenase and NAD(H) 384 Sepharose2B 321 -__ ,NH(CH, CONH(CH, )3 N+(CH3&,effect of ligand concentration 67 Sepharose 4B 321 --- , 2',5'-ADP 320 _ _ _ ,AH 161,320 -__ ,5'-AMP 320 _ _ _ , bound with anti-ornithine transcarbamylase antibody 333 --_ ,CH 320 _ _ _ ,_ _ _ ,activated 161,320 -__ ,_ _ _ ,---, conditions for binding 172 -__ ,-- -,-- -, preparation 172 _ _ _ ,CL 120 --_ ,---,octyl- 121 _ _ _ ,---, phenyl- 121 _ _ _ ,---,protein A 120 --_ ,cyanogen bromide activated 161, 321 --_ ,lysine 121 -__ ,Pob'(U) 121,321 _ _ _ , substituted with 3,3'diaminodipropylm i n e 81 --_ , thiol activated 161,320 -I_

_ _ _ ,trimethylenediamine

136

--_ ,with caprylic hydrazide 133 Sepharose 6B 321 ___ , benzyl ether 135 ___ , cyanogen bromide activated 161 --- ,ECD, treated with acid 99, 100 -__ ,epoxy activated 161,321 _ _ _ ,_ _ - ,stability of coupled glycylleucine 170 Sepharose 6MB, wheat germ lectin 120 Sephazyme trypsin 321 Serum proteins, fractionation 103 Sipsian distribution 325 S-MDA, see Starch dialdehyde, methylenedianiline Sorbent, capacity 17 --_ ,chelate forming 147 - _ _ ,dilution 37,56,63 --_ ,effect of concentration 67 _ _ _ ,effectiveness 60 -__ ,for nucleotides and RNA 113, 127 -__ ,general considerations 195-197 _ _ _ ,group-specific 89-92 --_ ,highly specific 89-92 _ _ _ ,hydrophobic 135 - _ _ ,immobilized nucleotide capacity 18 _ _ _ ,mixed ionic and hydrophobic 133 _ _ _ ,operating (working) capacity 36, 38,41 -__ ,regeneration 243 -__ ,saturation capacity 55 _ _ _ ,specific 2 _ _ _ ,---, affmity ligand leakage 190 --_ ,---, in containers with semi-permeable walls 232 ---,storage 243 _ _ _ ,washing 189 Sorption 225 -243 _ _ _ ,biospecific 1 --- ,capacity, determination 204 _ _ _ ,concentration of substance to be isolated 55,67,71, 231 -__ ,effect of affinity ligand concentration 14,15, 133 _ _ _ ,effect of buffer concentration 230 _ _ _ ,effect of different eluent systems 79 _ _ _ ,effect of flow-rate 231 _ _ _ ,effect o f individual ions 229 --_ ,effect of ionic strength 14,80-86, 228-230 -_-,effect of pH 14,226-228 - _ _ ,effect of temperature 71-75, 225226 - _ _ ,energy 52,74 _ _ _ ,fixed binding constant, equilibrium model 13-19 _ _ _ ,irreversible 240 -__ ,mixed ionic-hydrophobic 135

SUBJECT INDEX

-_- ,non-specific 60,241,242 ---,- _ _ ,determination 203,204 ---,---, effect of ionic strength 80, 81 --_ ,---, of proteins 229,241

-_- ,of ATPase to immobilized ATP inhibitor, effect of Mg*+ 230

-__ ,of proteins on alkyl-Sepharose 229 -__ ,optimal conditions of complex formation 225

---,practice 230-232 ---, standard enthalpy 72

--_ ,standard enthropy 72 - _ _ ,time of contact 84,23 1 Soybean-CH-cellulose affinity resin 321 Spacer(s) 56-60,182-184 ---,1,4-bis(2,3-epoxypropoxy) butane 169 -_- ,effect of length 57-59 --_ ,general considerations 195-198 --- ,hydrazide 163 ---,hydrophobic 80,185 ---,hydrophylic 80, 185 ---,_-- ,example 186 -_-,-_- ,synthesis 185 ---,non-specific sorption origin 183 ---,polyvalent 186 ---, synthesis 183 -_- ,used 246 -3 18 Spacing effect 56-60 Specific activity, effect of ionic strength 229 Specific complex, quantitative evaluation 3549 Spectroscopic methods, determination of immobilized affinity ligands 210-212 ---,H-NMRspectroscopy, determination of substitution degree 212 Spheron(s), characterization and preparation 178 ---,cyanogen bromide activation 178 ---,immobilized, amounts of chymotrypsin and glycin as function of specific surface areas 152 ---,with attached chymotrypsin 95 ---,ZGly-D-Phe-NH, 92 Spheron 300, commercially available 321 Splitting, ATP, study by use of affinity chromatography 327 ---,of thioester bond, with hydroxylamine 138 Starch dialdehyde, commercially available 158 ---,methylenedianiline 157, 158 Steric accessibility 53-60 Steric hindrance 68 Steric modification, by desorption 236 Steric requirements 326 Structural differences, detection 33 1 Styragel 182

397

Suberimidate dimethyl, bifunctional reagent 177 Succinic anhydride 80 Succinimide ester, N-hydroxy-, attachment on to agarose 172 Sugars, methods for binding 169, 200 Sulpher determination 214 Sulphydryl goups, determination 209, 210 Sumstar 190 158 Supports, characterization 203-222 ---,solid, blocking of unreacted groups 187-1 89 ---,---, characteristics 151-15 3 __- ,-__ ,choice 196 ---,---,porosity 53 ---,used 246-318 “Synthetic biochemistry” 383,384

T Temperature effect on adsorption 71-75, 225,226,235 Template chromatography 48, 113 Tertiary structure, disturbance of 62 Thermodynamic parameters, determination of 46 Thiol ester bond 184,200 ---,cleaved with hydroxylamine 138, 184 Thiolation of enzymes 170,171, 174 Thymine, resins 112 Thymocytes, binding 117 TNBC colour test, see Benzene sulphonate, 2, 4,6-trinitro-, Na salt colour test Topographical studies 111 Translation enthropy 54 Triazine, 2amino-4,6dichloro- 154 Triazine method, protein binding 154, 171 Trypsin, complex with DNP soybean trypsin inhibitor 98 ---,immobilized, active-site titration 217 ---,---, temperature-activity curve 370 Trypsin resin insoluble 321 Trypsinogen, active centre 326 U Ultro-gel 321 Uncompetitive effect 77, 78 Urease, microencapsulated 383

V Vinyl copolymers 181 Vinyl groups, determination 209 Viruses, isolation 116,117 Vitamins 105

398

SUBJECT INDEX

W

2

Washing steps, calculation of efficiency 20, 21

Zinc, complex with histidine and cysteine 147

Woodward’sK 171 Working capacity, see Operating capacity

399

List of compounds chromatographed* A Acetylcholine, see Choline, acetylAcetyl CoA apocarboxylase 257 Acetylglucosaminidase 266 Acetyltransferase 289 Acrosin 277 Actin 316 Adenosine 301 --_ ,deoxy-, nucleotides 301 Adenosine diphosphate-glucose pyrophosphorylase 275 Adenosine monophosphates ---,cyclic, receptor proteins 306 --_ ,3',5'-cyclic monophosphate 301 ---,---, dependent enzymes 258 Adenosine triphosphatase 259,274 Adenosyl-methionine decarboxylase 258 Adenylate cyclase 258 ADPglucose pyrophosphorylase, see Adenosine diphosphate-glucose pyrophosphorylase Adrenergic receptor 305,306 Adrenocorticotropic hormone 295 Agarase 258 Agglutination factor 316 Agglutinin 297 --_ ,N-acetyl-glucosamine-specific 297 --_ ,anti-A 298 ---,anti-soybean y-globulin 251 ---,axinella 298 ---,wheat-germ 300 Alanine carboxypeptidase 271 Albumin 306 . ---,from serum, see Serum albumin --_ ,human 252 Albumin-bound substances 316 Alcohol dehydrogenase 95, 261 ---,from yeast 48, 235 Aldehyde oxidase 273 Aldehyde reductase 284 Aldolase 142 Aldosterone-binding macromolecules 306 Alginase 258 Amine mono-oxygenase, secondary 286 Amine oxidase 273 Amino acids, sp~,cific 314, 315 Amino acid-binding glycoproteins 306 *Compiled by H. BeEvGovil.

Aminoacyl-tRNA synthetase 142, 146, 286 Aminopeptidase 277 AMP, see Adenosine monophosphates Amylase 142 ---,a- 258 ---,P- 258 Angiotensin, serum prohormone 295 Angiotensin I-converting enzyme 277 Anhydrase, carbonic 260 Anthranilate phosphoribosyl-transferase 289 Anthranilate synthase 286 Antibodies 246-252 ---,anti-glucagon 98 ---,antisera to a-fetoprotein 251 ---,anti-ShigelZa sonnei sera 25 1 ---,anti-thyroglobulin, autoantibodies 251 ---,a,-anti-trypsin 295 Antigen($ 252 ---,carcinoembryonic, immunologically active fraction 317 Apoglutamic-oxaloacetic transaminase 258 Apo-p-hydroxybutyrate dehydrogenase 26 1 Apotryptophanase 258 Apotyrosine phenol-lyase 258 Arabinose kinase 269 Arginase 259 Arylsulphatase 259 Asparaginase 259 Aspartase 259 Aspartate aminotransferase 289 Aspartate p-decarboxylase 259 Aspartate transcarbamylase 259 Aspartokinase I-homoserine deh ydrogenase 26 1 Avidin 106, 295

B Bacteriophage 254 ---,f2 replicase 259 ---,T, 313 Bilirubin 316 Binding proteins 306-308 Biotin 255 Blood group substance 294 ---, A 293 Bromelain 282 Butyrate, 3-hydroxy-, dehydrogenase 262

400

C Carboxylase, subunits 292 Carboxypeptidase, A 277 --_ , B 277 Catalase, inhibitor 296 Catecho1-O-methyltransferase 289 Cathepsin 278 --_ ,D, inhibitor 296 Cells 253-255 ---, organelles 253-255 Chloramphenicol acetyltransferase 289 Choline, acetyl-, esterase 66,137, 256 - _ _ ,_ _ _ ,receptor 66, 305 - _ _ ,_ _ _ ,transferase 289 ---, esterase 260 --_ ,kina= 269 --_ ,phosphate cytidyltransferase 289 Cholinergic proteolipid 31 1 Cholinergic receptor 306 Chorismate mutase 260 Chorismate synthase 286 Chromatin 313 Chymotrypsin 92,233,237 ---, Q- 56,278 ---,anhydro- 291 --_ , inhibitor 95, 296 Chymotrypsin-like enzymes 279 Chymotrypsinogen 279 Citrate synthase 286 Clostr idiopep tidase 27 9 Clostripain 279 Clotting factor X,human blood 299 Coagulation factor 316 CoenzymeA 255 --_ ,affinity protein 306 Cofactors 255 Collagen 313 ---,galactosyltransferase 289 ---, glycosyltransferase 289 Collagenase 279 Complement human, component C1 316 Concanavalin A 298 --_ ,receptor 306 Creatine kinase 269 Cytidine 301 Cytochrome, C 284 --_,oxidase 273 --_,P-450 260

LIST OF COMPOUNDS CHROMATOGRAPHED

Deoxy compounds see also under parent names 3-Deoxy-arabino-heptulosonate-7-phosphate synthease 286 -__ ,isoenzymes 286 Deoxycytidine 302 Deoxyribonuclease 266 -__ ,inhibitor 296 Deoxyribonucleic acid 302 -__ ,nucleoproteins, SH-containing 310 _ _ _ ,polymerase 275,276 trans-Deoxyribosylase 291 Diaphorase-DT 266 Dihydrofolate reductase 284 Dihydropteroate synthase 286 DNA, see Deoxyribonucleic acid DOPA, see Phenylalanine, 3,4dihydroxyDopamine p-hydroxylase 266

E Ecdysone, receptor 307 Elastase 281 Endonuclease 272 Endopolygalacmronase 266 Enterokinase 266,279 Enterotoxin 3 16 Enzymes _ _ _ ,acting on myo-inositol 266 _ _ _ ,ADP-dependent 258 _ _ _ ,bacteriolytic 259 _ _ _ ,chymotripsin-like 279 _ _ _ ,FMNdependent 267 -__ ,malic, NADPdependent 27 1 _ _ _ ,modified derivatives 291-293 _ _ _ ,plasma membrane 274 --_ ,proteolytic 281 --_ ,subunits 291-293 Erythrocytes 254 --_ ,membrane proteins, labelled with trinitrobenzene 252 Erythrodihydroneopterin triphosphate synthetase 286 Esterase, C1 266 Estradiol 294 _ _ _ ,17pdehydrogenase 261,265 _ _ _ ,receptor 307,309,310 Ethanolamine kinase 269 Exoamylase 266 Exonuclease 272

D F

Dearninases 260, 261 Debranching enzyme 289 DehydIOgeMseS 261-266 --_ ,isoenzyrnes 261 Dehydroquinase 260

FAD, see Flavine, adenine dinucbotide Fatty acid synthetases 286 Favin 298 Ferritin 311

LIST OF COMPOUNDS CHROMATOGRAPHED

aPetoproteins 3 11 -__ ,antisera 251 Fibrinogen 31 1 __- ,derivatives 3 12 Ficin 279 Flagelae 254 Flavine, adenine dinucleotide 255 _ _ _ ,mononucleotide, enzymes 267 Flavokinase 267 FMN, see Flavine, mononucleotide, enzymes Follicle-stimulating hormone, see Hormone, follicle stimulating Formiminotetrahydrofolatecyclodeaminase 267 Fructose 1,6diphosphatase 142 Fucosa-binding proteins 298 a-Fucosidase 267

G @1,4Gaiactanase 267 Galactomannan 293 Galactosaminoglycans 293 Galactose 293 __- ,in biopolymers 293 - _ _ ,oxidase 273 Galactosidase(s) 267,268 -__ , a - 267 -_- , P - 267 __- ,_-_ ,acid 257 -__ ,---,fragments 292 -_- ,---, from Escherichia coli 81 Galactosyl transferase 289 Gibberellins 3 17 Globulin, a1corticosteroid binding 306 -_- ,y- 252 _ _ _ ,_ _ _ ,anti-soybean agglutinin 251 - _ _ ,from French bean 25 1 -_- ,sex hormone binding 309 -__ ,soybean 11s 294 Glucagon, immunoreactivity 252 -__ , 'Z5Wabelled 294 ---,receptor 307 Glucoamylase 268 Glucocorticoid, receptor 307 Glucokinase 62,269 Glucosamine, Nacetyl-, binding proteins 305 PGlucosaminidase, Nacetyl- 257 Glucose, oxidase 273 ---,6-phosphate, dehydrogenase 48, 143, 26 1 ---,---,---, NADPH-bound 94 ---, phosphate isomerase 142 PGlucosidase 26 8 PGlucuronidase 268 Glutamate dehydrogenase 262 G1utamate.formiminotansferase 290 Glutamine synthetase 287

40 1

TGlutamyl hydrokse 268 TGlutamyltransferase 290 Glutathione reductase 48,284 Glyceraldehyde 3-phosphate dehy drogenase 26 2 Glycerokinase 235,269 Glycerol 3-phosphate dehydrogenase 262 ---, cytosolic NAD-linked 260 Glycogen 293 -_- ,phosphorylase 275 ---,synthase 287 Glycolate oxidase 273 Glycopeptides 314 Glycophorin A 293 Glycoprotein(s) 293,294 -_- ,amino acid-binding 306 ---,receptor 307 Glycosidase(s1 268 ---,acid 257 Glycosyl transferase 290 Gonadotropin, chorionic 294 ---,---, receptor 306 ---,receptors 308 Gramicidin S synthetase 287 Guanylate cyclase 268

H Haptens 252 Head protein precursor 3 13 Hemagglutinin(s) 298-300 ---,anti-B 298 Heme peptide 314 Hemoprotein, P-450 313 Hexokinase 235 Hexosamididase, A 268 -_- ,B 268 Histaminase 268 Histidine decarboxylase 268 Histidinol-phosphate aminotransferase 290 Histones 311 ---,cysteine-containing 3 10 ---,kinase, regulatory subunit of pig-brain 292 -_- ,phosphatase 268 Hormone($ 294,295 ---,follicle stimulating 295 ---,luteinizing 295 ---,---,receptor 306 Hyaluronidase 269 Hypoxanthine-quanine phosphoribosyl-transferase 290 1

Ileucyl-tRNA synthetase 288

402

Immunoglobulin(s) ---,A,human 252 ---,anti$, -microglobulin 25 1 ---,anti-prothrombin 251 ---,antitoxic components, type A or B 251 --_ ,binding factor 308 --_ ,carbohydrate-specific 25 1 --_,cortisol-binding 307 --_ ,from human serum 25 1 --_ ,from serum 253 --_ ,G, 12sI-labelled 251 --_ ,J-chain 252 --_ , lymphocyte-surface 253 --- ,M,human 252 Inhibitor(s) 295-297 --_ ,seminal plasma 296 lnitiation factor 318 lnosine 5'-monophosphate dehydrogenase 263 Insulin 61,295 --_ ,receptor 105,308 hterferon(s) 98, 317,330 Intrinsic factor 3 1 7 Isocitrate dehydrogenase 142,263 --_ , NADP-specific 264 Iso-inhibitors, polyvalent 296 Isoleucyl-tRNA synthetase 287 a-Isopropylmalate isomerase 269 a-lsopropylmalate synthase 287

K Kallikrein 279 A5-Ketosteroid isomerase 269 Kinase(s) 269, 270 --_ ,subunits 292

L 0-Lactamase 270 Lactate deydrogenase 235, 263, 264 --_,isoenzymes 8 , 9 , 2 6 4 .&Lactoglobulin 308 Lactose synthase 287 Lecithin cholesterol acyltransferase 290 Lectin(s) 29 7-30 1 --_ ,blood group A, specific 298 Leucyl-tRNA synthetase 287,288 Lipases 271 Lipoamide dehydrogenase 264 --_ ,isoenzymes 264 Lipids 301 Lipoprotein 31 2 ---,lipase 271 Lipoxygenase 271 Luciferase 271 Luteinizing hormone, see Hormone, luteinizing Lymphocytes 253,254 --_ , membrane vesicles 255

LIST OF COMPOUNDS CHROMATOGRAPHED

Lysergide 253 Lysozyme 233,271 M

-Macroglobulin 25 3 Malate dehydrogenase 48,264 Maleylacetone isomerase 271 Malic enzymes, NADPdependent 271 Maltodextrin phosphorylase 275 Mannose, H-labelled, polysaccharides 294 a-Mannosidase 271 - _ _ ,acidic 258 Membrane(s) 255 --- ,plasma enzymes 274 --_ ,proteins from erythrocytes 252 _ _ - ,protein receptor 308 Mercaptalbumin 310 Meromyosin 312 _ _ _ ,heavy 312 Methionyl-tRNA synthetase 287 Mononucleotides 303 Myoinositol, acting enzymes 266 __- ,phosphate synthase 287 Myosine 311,312 N NADPH-oxidoreductase 273 Neomycin phosphotransferase 290 Neuraminidases 272 Neurophysins 308 Nicotin, acetylcholine receptor 308 Nicotinamide nucleotide, dependent dehydrogenase 235 --_ ,trynshydrogenase 27 2 Nitrate reductase 284 Nitrite reductase 285 Nuclease(s) 38, 143,226,272 -__ ,from Staphylococcus 273 Nucleic acid($ 301 -305 -__ ,bases 111 Nucleoside deoxyribosyltransferase 290 Nucleotides 111,301-305

0 Old yellow enzyme 273 Oligonucleotides 303 Opiate, membrane-bound binding sites 317 Orotate phosphoribosyltransferase 143 Orotidylate decarboxylase 143 Ovalbumin 253,308 ---, fluorescent 252 Ovoflavoprotein 3 11 Ovoinhibitor 296 Ovomucoid 296

LIST OF COMPOUNDS CHROMATOGRAPHED

Oxidases 273 34xosteroid isomerase 273 P Papain 280 Penicillin, binding components 138 Penicillinase 274 Pepsin 280 Pepsinogen 280 Peptidases 216-284 Peptide($ --_ ,affinity labelled 314 ---,cysteine containing 314 --_ , DNP-Iysyl-containing 314 ---,from binding site of antidinitrophenyl antibody 315 ---, methionine containing 314 ---,nitrotyrosine containing 110, 315 ---,ribonuclease-S-, synthesized by solidphase technique 315 --_ ,SH-containing 107,310,311 -__ ,specific 314,315 ---, synthetic 315,323 --_ ,tryptophan containing 315 -__ , tyrosine containing 315 Peroxidase 274 PhageT4 255 Phenoloxidase 273 Phenylalanine, 3,4-dihydroxy-, D.L-isomers 314 ---,hydroxylase 274 ---,tRNA liiase 274 Phenylafanyl-tRNA synthetase 287, 288 ---, subunits 293 Phosphatase(s) 274 --_ ,alkaline 274 Phosphate acetyl-transferase 290 3-Phosphatidyl-1'glycero-3'-phosphate synthetase 287 Phosphodiesterase 275 Phosphofructokinase 95 Phosphogluconate dehydrogenase 265 Phosphoglycerate kinase 143 Phospholipase C 271 Phosphoprotein phosphatase 275 Phosphorylases 275 -__ ,glucan-synthesizing 275 ---,kina= 270 ---, phosphatase 275 Phytoagglutinins 99 Phytohemagglutinin 300 ---,anti-A 298 Plasma factors VII 316 Plasma-membrane enzymes 274 a,-Plasmin inhibitor 296 Plasminogen 280

403

---,HN,-terminal activation peptide 315 GPolyadenylic acid 303 Polycytidylic acid 303 Polygalacturonase 275 Polyinosinic acid 303 Polymerases 275 Polynucleotide(s) ---,adenosine monophosphate-rich 301 ---,phosphorylase 275 ---,T, kinase 270 Polyriboadenylic acid 304 Polyribosomes 255 Polysaccharides 294 Polysomes 254,255 Polyuridylic acid 304 ---,polymerase 276 Prekallikrein 281 Procarboxypeptidase 281 Prolactin 295 Prolyl hydroxylase 276 Properdin 31 8 Prostaglandin, 15-hydroxy, dehydrogenase 26 3 Protamine b a s e 270 Protease($ 276-284 ---,acid 282 ---,alkaline 277 ---,inhibitors 295,296 ---,neutral 279 ---,SH, from beans 106 Protectin Anti-AH, 300 Proteins 305-310 ---,A, from Staphylococcus aureus 253 ---, a r a C 310 ---,bacterial 311 ---,binding 306-308 ---,carboxyl carrier 306 ---,coagulant 311,313 --_ ,cytoplasmic 31 1 ---,factor 313 ---,from erythrocyte membrane 252 ---,---, trinitrobenzene labelled 252 ---,from serum see Serum proteins ---,lipophilic 3 12 ---,membrane receptor 308 ---,phage 311 ---,receptor, binding 305-310 ---,---,transfer 305-310 ---,ribosomal 313 ___ ,SHzontaining 310,311 Proteinase, acid 276 ---,streptococcal 282 Proteoglycans 294 Proteolipid cholinergic 301 Prothrombin 281 Protocollagen proline hydrolase 284 Pteroyl oligo9-glutamyl endopeptidase 281

404

Pyridine nucleotide transhydrogenase 265 Pyridoxal kinase 270 Pyridoxine S’-phosphate oxidase 273 Pyrimidine oligonucleotides 304 Pyrophosphatese 143 Pyruvate enzymes --_ ,carboxylase 284 _-_ , kinase 143,270 _-_ ,oxidase 273

R Receptor($ 306-310 ---, acetylcholine 305 ,adrenergic 306 --_ ,CAMPproteins 306 --_ ,binding proteins 305-310 ---,cholinergic 306 --- , concanavalin A 306 ---, ecdysone 307 --_ ,estradiol 307,309, 310 --- ,glucagon 307 --- ,glycoprotein 307 --_ , gonadotropin 307 --- ,human chorionic gonadotropin 306 --- ,insulin 307 --- ,luteinizing hormone 306 --- ,membrane proteins 308 --- , nicotin acetylcholine 307 --- , steroid complex 310 ---,thyrotropin 310 ---, transfer proteins 305-310 Reductases 284,285 --- ,subunits 292 Renin 281 Repressor(ga1) 3 16 Repressor(trp) 3 18 Reverse transcriptase 285 Riboflavin, 5’-phosphate 256 ---,synthase 288 Ribonuclease(s) 285 _--, inhibitor 297 --_ ,isoenzymes 285 ---,S-peptide, synthesized by solid-phase technique 3 15 --- ,synthetic fragments 315 Ribonucleoside diphosphate reductase 285 Ribonucleotide reductase 285 Ribosomes 255 RNA 111,304 --_ ,microsomal 303 ,polymerase 93, 275, 276 --- ,_ _ - , from Escherichia coli 235 --- ,(quanine-7)methyltransferase 290 --- ,ribosomal 304 _-- ,saluble 304 mRNA 301-303

LIST OF COMPOUNDS CHROMATOGRAPHED

tRNA 302,303,305 -_- ,cistrons 318

S Saccharides 293, 294 Seminal plasma inhibitor 296 Serum albumin 309 --_ ,bovine 308 Serum proteins 184, 294 Seryl-tRNA synthetase 288 Somato mammotropin, chorionic 317 Steroid(s) -_- ,3-hydroxy-, dehydrogenase 263 --_ , isomerase 286 -__ ,AS-keto-, isomerase 269 - _ _ ,h x o - , isomerase 273 - _ _ ,receptor complex 3 10 Stxeptokinase 286 Subtilisin-like enzyme 282 Succinate enzymes -__ ,dehydrogenase 265 -__ ,thiokinase 270 Sulphydryl-protease 282 Synthases 286-288 Synthetases 286-288 _ - _ ,acid, subunits 293

T Teichoic acid 103 --_ ,aglucosylated 3 17 Tetrahydrofolate dehydrogenase 265 Thermolysin 282 Thiamine pyrophosphokinase 270 Thioredoxin 253 Threonine, deaminase 260 -__ , dehydrogenase 265 Thrombin 282 Thrombin-like enzyme 283 Thymidine 304 --_ , kinase 270 Thymidyllate synthetase 288 Thymocytes 254, 255 Thyroid stimulator 295 Thyrotropin receptor 310 Toxin, cholera 3 16 --_ ,diphtheria 316 -__ ,from Vibrio cholerae 115 Transcarboxylase, subunits 293 Transcortin 3 10 Transferases 289, 290 Transhydrogenase 29 1 Trehalose phosphate synthetase 288 Triacylglycerol lipase 291 Tridacnin 300 Triglyceride lipase 27 1

LIST OF COMPOUNDS CHROMATOGRAPHED

405

Tropomyosin 313 Troponin 313 _ _ _ , I 291 Trypsin 233,231,283 -_- ,acyl- 291 _ _ _ ,inhibitor 297 - _ - ,Streptomyces griseus 282 Trypsin-like enzyme 284 Tryptophan 315,319 -__ ,hydroxylase 291 --_ , synthase 288 --_ ,_ _ _ ,subunit 292 Tryptophanyl-tRNA synthetase 288 Tubulin 318 Tyrosinase 291 Tyrosine, aminotransferase 290 ---,hydrolase 291 Tyrosyl-tRNA synthetase 143,288

Xanthine, dehydrogenase 266 ---,oxidase 273 p-Xy!osidase 291 Xylosyltransferase 290

U

Z

Urease 291 Uridine 305 ---,diphosphate, glucose dehydrogenase 26 5 Urokinase 284

Zymogens 276-284

V

Valyl-tRNA synthetase 288 Vasopressin(1ys) 295 --_ , I-labelled 295 Virudes) 315,316 ---,aleutian mink disease 315 --_ ,influenza 316 --_ ,plant 316 --_ ,tobacco mosaic 316 Vitellogenin 253

X

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