Journal of Chromatography Library — Volume 12
`
`AFFINITY
`
`CHROMATOGRAPHY
`
`Jaroslava Turkové
`Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy ofSciences,
`
`Prague
`
`
`
`ELSEVIER SCIENTIFIC PUBLISHING COMPANY
`AMSTERDAM -— OXFORD — NEW YORK 1978
`
`CUREVAC EX2005
`CUREVAC EX2005
`Page 1 of 53
`Page 1 of 53
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`

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`ELSEVIER SCIENTIFIC PUBLISHING COMPANY
`335 Jan van Galenstraat
`P.0. Box 211, Amsterdam, The Netherlands
`
`Distributors for the United States and Canada:
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`.-
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`_
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`ELSEVIER NORTH-HOLLAND INC.
`52, Vanderbilt Avenue
`New York, N.Y. 10017
`
`.
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`. "."
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`‘
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`
`
`Lihrary of Congress Cataloging in Publication Data
`
`Turkové, Jaroslava.
`Affinity chromatography.
`
`(Journal of chromatography library ; v. 12)
`Includes bibliographical references.
`1. Affinity chromatography.
`I. Title.
`QP519.9.A35T37
`BHT' .3149'2
`78—815
`ISBN O—hhh-h1605—6
`
`II. Series.
`
`ISBN:O-444-41605-6 (Vol.12)
`ISBNzo-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, mechan-
`ical, photocopying, recording or otherwise, without the prior written permission
`of the publisher, Elsevier Scientific Publishing Company, P.O. Box 330,
`Amsterdam, The Netherlands
`
`Printed in The Netherlands
`
`Page 2 of 53
`Page 2 of 53
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`

`

`Contents
`
`Acknowledgements.
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`Introduction .
`1.
`References
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`2. The principle, history and use of affinity chromatography
`1
`References
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`3. Theory of affinity chromatography
`.
`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 elut1on by a change'1n KL .
`3.1.3 Equilibrium model for elution by a competitive inhibitor
`3.1.4 Simulation of-colprnn chromatographic results.
`3.1.5 Conclusiont':
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`3 1.6 List of symbols used
`3.2 Theory of cooperatiVebonding within the plate theory
`3.2.1 Isotherm of binding of oligoadenylic acid to polyuridylic acid
`.
`3.2.2 Cooperative adsorption column chromatography
`32.3
`haracteristic features of cooperative adsorption chromatograms
`3.2.4
`ist of symbols used
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`3. 3 Statistical theory of chromatography applied to affinity chromatography.
`References
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`4. Application of affinity 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
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`4. 3 Cooperative elution of oligoadenylic acid1n immobilized polyuridylic acid
`chromatography
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`4.3.1 List of symbols used
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`4.4 Other methods for the quantitative evaluation of interactions with immobilized affinity
`ligands
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`References
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`'
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`5. General considerations on affinant—sorbent bending
`5.1 Steric accessibility.
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`5.2 Conformation of attached affinant
`5.3 Concentration of the affinant on the matrix
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`5.4 Concentration of proteins, equilibration time and flow-rate .
`5.5 Effect of temperature.
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`5.6 Effect of pH andionic strength .
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`5.7 Elution with competitive affinity ligands
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`5.8 Non-specific effects
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`5.8.1 Effect of1onic strength on non-specific sorption
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`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
`5.8.2.1 List of symbols used
`,
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`References
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`13
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`19
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`Page 3 of 53
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`VI
`
`CONTENTS
`
`6. Choice of affinity ligands for attachment
`6.1 Highly specific and group-specific matrices
`6.2
`Isolation of enzymes, inhibitors and cofactors
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`6.3
`Immunoaffinity chromatography .
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`6.4
`Isolation of lectins, glycoproteins and saccharides
`6.5
`Isolation of receptors, binding and transport proteins
`6.6
`Isolation of —SH proteins and peptides
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`6.7
`Isolation of specific peptides
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`6.8
`Isolation of nucleic acids and nucleotides .
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`6.9
`Isolation of lipids, hormones and other substances
`6.10 Isolation of cells and viruses
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`6.11 Commercially available insoluble affinants
`References
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`127
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`7. Hydrophobic chromatography, covalent affinity chromatography, affinity eiution and
`related methods.
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`7.1 Hydrophobic chromatography
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`7.2 Covalent affinity chromatography
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`7 3 Affinity elution
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`’7.4 Affinity density perturbation
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`7.5 Affinity electrophoresis
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`7.6 Metal chelate affinity chromatography.
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`References
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`8. Solid matrix supports and the most used methods of binding .
`8.1 Required characteristics
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`8.2 Survey of the most common solid supports and coupling procedures
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`8.2.1 Cellulose and its derivatives
`8.2.2 Dialdehyde starch—methylenedianiline (8—MDA)
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`8.2.3 Dextran gels.
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`8.2.4 Agarose and its derivatives
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`8.2.5 Copolymer of ethylene and maleic anhydride
`8.2.6 Polyacryiamide supports and their derivatives
`8.2.7 Hydroxyalkyl methacrylate gels
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`8.2.8 Glass and its derivatives
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`8. 2 9 Other supports .
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`83 Spacers
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`8.4 Blocking of unreacted groups
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`8 .5 Leakage of the coupled affinant.
`8.6 General considerationsin the choice of sorbents, spacers and coupling and blocking
`procedures
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`9. Characterization of supports and immobilized affinity ligands
`203
`9.1 Methods for the determination of non-specific sorption .
`203
`9.1.1 Determination of adsorption capacity
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`204
`9.1.2 Determination of residual negatively charged groups
`204
`9. 2 Determination of activatable and active groups
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`9.2.1 Determination of carboxyl, hydrazide and amino groups on the basis of acid—base
`titration .
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`9.2.1.1 Dry weight determination
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`9.2.1.2 Determination of carboxyl groups .
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`9.2.1.3 Determination of hydrazide groups
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`9.2.1.4 Determination of aliphatic amino groups
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`9.2.2 Determination of the content of free carboxyl groups
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`204
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`Page 4 of 53
`Page 4 of 53
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`

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`CONTENTS
`
`VII
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`.1 206
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`9.2.3 Determination of free amino groups in polymers on the basis of the
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`condensation reaction with 2-hydroxy-l-naphtha1dehyde .
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`Procedure for azide assay
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`9.2.4
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`9.2.5 The sodium 2,,46-trinitrcbenzenesulphonate colour test
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`9.2.6
`Fluorescamine test for the rapid detection of trace amounts of amino groups
`9.2.7 Determination of oxirane groups
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`9.2.8 Determination of the capacity of p-nitrophenol ester derivatives of hydroxy-
`alkyl methacrylate (NPAC) gels
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`9.2.9 Determination of the degree of substitution of benzylated dibromopropanol
`crosslinked Sepharose
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`9.2.10 Determination of vinyl groups
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`9.2.11 Determination of sulphydryl groups
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`9.3 Methods for the determination of immobilized affinity ligands
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`9.3.1 Difference analysis
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`9.3.2 Spectroscopic methods
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`_ 9.3.3 Determination by means of acid—base titration
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`9.3.4 Determination of immobilized proteins, peptides,aamino acids, nucleotides,
`carbohydrates and other substances after liberation by acid, alkaline or
`enzymatic hydrolysis.
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`9.3.4 1 Determination of immobilizedamino acids, peptides and proteins
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`9. 3.4.2 Determination of nucleotides .
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`9.3.4. 3 Determination of carbohydrate
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`9.3.5 Determination of the amount of bound affinant on the basis of elemental
`. 214
`analysis
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`9.3.6 Determination of labelled affinity ligands .
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`9.3.7 Determination of immobilized diaminodipropylamine by ninhydrin colorimetry
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`9. 3.8 Determination of immobilized proteins on the basis of tryptophan content
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`9.4 Active-site titration of immobilized proteases
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`9.5 Study of conformational changes of immobilized proteins .
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`9.6 Studies of the distribution of proteins bound to solid supports
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`References.........................222
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`10. General considerations on sorption, elution and non-specific binding
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`10.1 Sorption conditions
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`10.1.1 Effect of temperature, pH and salts .
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`10.1.2 Practice of sorption
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`10.2 Conditions for elution .
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`10.2 1 Practice of desorption .
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`10.2.2 Effect of the heterogeneity of the immobilized affinants.
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`10.2. 3 Establishment of optimal conditions and saturation effect
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`10. 3 Non-specific sorption
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`104 Regeneration and storage of affinity columns
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`References.........................243
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`l 1. Examples of the use of affinity chromatography .
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`11.1 Isolation of biologically active substances
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`11.2 Resolution of DL--tryptophan by affinity chromatography on bovine serum albumin—
`agarose column .
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`11.3 Semi-synthetic nuclease and complementary interaction of nucleaSe fragments.
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`11.4 Study of interactions of biologically active substances...
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`11.5 Study of the mechanism of enzymatic action
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`11.6 Molecular structure of fibroblast and leucocyte interferons investigated with lectin
`and hydrophobic chromatography
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`11.7 Immunoassay
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`Page 5 of 53
`Page 5 of 53
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`VIII
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`CONTENTS
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`11.7.1 Solid-phase radioimmunoassay
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`11. 7.2 Enzyme-linked immunosorbent assay (ELISA)
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`11. '7. 3 M1crofluorrmetr1c immunoassay .
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`334
`11.8 Specific removal of bovine serum albumin (BSA) antibodies in vivo by extra--corporea1
`circulation DVel‘ BSA immobilized on nylon microcapsules
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`References
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`12. Immobilized enzymes.
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`12.1 Classification of immobilized enzymes
`12.2 Attachment of enzymes to solid supports and activity of immobil12ed enzymes
`12.3 Stability of immobilized enzymes.
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`12.3.1 Stability during storage
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`12. 3 2 Dependence of stability on pH .
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`12. 3. 3 Thermal stability
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`12.3.4 Stability against denaturing agents
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`12.3.5 Increase of stability
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`12.4 Application of immobilized enzymes.
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`12.4.1 Affinity ligands.
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`12.4.2 Study of stabilized enzyme molecules and of their subunits .
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`12 4. 3 Models of biological systems .
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`12.4.4 Application of immobilized enzymes
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`12.5 “Synthetic biochemistry" .
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`References
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`3’14
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`Subject index
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`List of compounds chromatographed
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`

`

`Chap ter 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 er £21.,
`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 or-amylase by 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 at. 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 195 3 by lerman, who isolated tyrosinase on a column of cellulose with
`
`g”
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`MATFHX
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`)
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`A
`”EDEFOHMING
`BUFFE/i"
`VK
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`Hafifl
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`Fig. 2.1. Diagrammatie representations of (A) biospecific adsorption; (B) elution by a “deforming
`buffer”; (C) bioelution with a soluble, competitive counter ligand. IN, immobilized inhibitor; C,
`competitive counter ligand. Reproduced with permission from P. O'Carra 21‘ UL, Methods Enzymol.,
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`34 (1974) 108—126.
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`\
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`Page 7 of 53
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`

`8
`
`PRINCIPLE, HISTORY AND 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 affinant. Non-specific adsorption was often observed
`when supports with hydrophobic or ionogenic groups were used. The last few years, how-
`ever, have witnessed an extensive deve10pment of this method. A milestone in this
`development was the method of attachment of affinant to agarose activated with cyanogen
`bromide, developed by Porath and co-workers (Axén and Ernback; Axén at 421.; Porath at
`01.). 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 er a1. 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 11.1 in Chapter 11.
`However, the use of affinity chromatography is not limited to the isolation of
`biologically active substances. As early as 1960 Yagi er al. described a quantitative deter-
`mination 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. Im-
`mobilized oligomers of polythymidylic acid were used by Edmonds et al. for the quanti-
`tative determination of polyadenylic acid. The use of affinity gel filtration as a micro-
`scale 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 bio-
`chemical 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 er al.) 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 er a]. 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 enzymeuNAD+
`complex rather than with enzymenNADH 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 iso-
`enzyrnes 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-
`
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`

`

`PRINCIPLE, HISTORY AND USE OF AFFINITY CHROMATOGRAPHY
`
`9
`
`0.5
`
`P.L
`
`LDHACTIVITY.«Amimin~m| Qto
`
`
`
`
`
`NADH,mM
`
`50
`
`100
`ELUTION VOLUME,mI
`
`150
`
`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 mM fl-mercaptoethanol and 1 M
`sodium chloride was applied to an AMP-analogueHSepharose 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 m1 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 B—mercaptoethanol. Fractions of 1 ml were collected at
`the rate of 3.4 ml/h. Reproduced with permission from P. Brodeh‘us 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. N0 dif-
`
`ferences 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 ad-
`vantage of using affinity chromatography for the determination of the dissociation con-
`stants 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 inhibi-
`tion 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
`
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`

`

`10
`
`PRINCIPLE, HISTORY AND USE OF AFFINITY CHROMATOGRAPHY
`
`RABBIT M,
`
`O BOVINE M,
`
`BOVINE H‘
`
`PIG H.
`
`o
`
`0.1
`
`0.2
`
`-
`
`. 0.3
`
`0.4
`
`ELUTING CONCENTRATION OF NADH,mM
`
`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.
`
`1
`
`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 inhibitdr 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 fonthe elucidation
`of the effect of the micro-environment on the formation of these complexes.
`
`REFERENCES
`
`Akanurna,.H., Kasuga, A., Akanuma, T. and Yamasaki, M., Biochem. Biophys. Res. Commun., 45
`(1971) 27—33.
`Axén, R. and Ernback, S.,Eur. J. Biochem., 18 (1971) 351—360.
`Axén, R., Porath, I. and Ernback, 5., Nature (London), 214 (1967) 1302—1304.
`Brodelius, P. and Mosbach, K., FEBS Lett., 35 (1973) 223—226.
`Brodelius, P. and Mosbach, K.,Biochem. Soc. Trans, 2 (1974) 1308—1310.
`Campbell, D.H., Luescher, EL. and Lerman, L.S., Proc. Nat. Acad. Sci. U.S., 37 (1951) 575—578.
`Cuatrecasas, P. and Anfinsen, C.B., Methods Enzymol., 22 (1971) 345—378.
`Cuatrecasas, P., Wilchek, M. and Anfinsen, C.B., Proc. Nat. Acad. Sci. U.S., 61 (1968) 636—643.
`Delaney, M. and O’Carra, P., Biochem. Soc. Trans” 2 (1974) 1311.
`Denburg, J. and De Luca, M., Proc. Nat. Acad. Sci. U.S. , 67 (1970) 1057—1062.
`Edmonds, M., Vaughan, M.I-I. and Nakazato, H., Proc. Nat. 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)
`1 124— 1 130.
`
`Page 10 of 53
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`

`

`REFERENCES
`
`11
`
`Kasahe, V., Arch. Biochem. Biophys., 173 (1976) 269—272.
`Lerman, L.S.,Proc. Nat. Acad. Sci. U.S., 39 (1953) 232—236.
`Lowe, C.R. and Dean, P.D.F., FEBS Lett., 18 (1971) 31—34.
`O'Carra, P., Barry, 5. and Griffin, T., Methods Enzymol., 34 (1974) 108—126.
`Porath, 1., Axén, R. and Emback, 3., Nature (London), 215 (1967) 1491—1492.
`Rainer, RH. and Walsh, A., Chromatographia, 4 (1971) 578—5 87.
`Schott, H., Eckstein, 1-1., Gatfield, I. and Bayer, E., Biochemistry, 14 (1975) 5541—5548.
`Starkepstein, E., Biochem. Z., 24 (1910) 210-218.
`Yagi, Y., Engel, K. and Pressman, D., J. Immunol., 85 (1960) 375—386.
`
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`

`Page 12 of 53
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`

`89
`
`Chapter 6
`
`Choice of affinity ligands for attachment
`
`6.] 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, anti-
`bodies, 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 macro-
`molecules 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-weight affinity 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—(w-aminohexyl)-L-aspartic acid—Sepharose. On this immobilized affinant,
`asparaginase, aspartase, aspartate-fi-decarboxylase and asparaginase modified with tetra-
`nitromethane (Tosa et at.) 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-phosphate dehydrogenase, 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
`
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`

`90
`
`AFFINITY LIGANDS
`
`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 to 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 104—10"8 M.
`Enzymes 2 and 3have affinity for sorbents with dissociation constants of about 10'5 M,
`and enzyme 4 shows a very weak affinity with a dissociation constant of >10'3 M.
`For the generalized Langmuir adsorption isotherm
`
`_ [€116ng
`
`(6-1)
`
`qr
`
`l+k1Ci
`
`where q,- is the specific amount of the adsorbed substance 1‘, C,- is concentration and k1
`and k; are constants. For low concentrations of Of, eqn. 6.1 reduces to q,- = klkg Cg, and
`for high concentrations of C,- to q,- = k;. In general we can write
`
`q.- = fro)“
`
`(6:2)
`
`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 1', (If, is dependent on its concentration in the mobile phase, 0,, and
`
`DISPLACEH,D
`
`ABSORBED(q)
`
`AMOUNT
`
`‘44
`
`ENZY-M E CONCENTRATION (Ci)
`
`Fig. 6.1. Adsorption isotherms for four enzymes interacting with a single immobilized aff‘mity ligand.
`Reproduced with permission from C.R. Lowe and P.D.G. Dean, Affim'ty Chromatography, Wiley, New
`York, London, 1974, p. 91.
`
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`

`

`SPECIFIC MATRICES
`
`9 1
`
`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 ql, £12, dig and £14.
`In displacement elution, using a concentration D of the displacer, enzymes 1, 2 and 3 with
`concentrations Cl, C2 and C3 will 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, 129., 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
`er (11.). 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-S-nitrobenzyl 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 be-
`tween them and the surface of the solid support. High-molecular-weight affinants are pre-
`dominantly proteins or nucleic acids. They often undergo denaturation lea