Journal of Chromatography Library — Volume 12
`
`AFFINITY
`CHROMATOGRAPHY
`
`Jaroslava Turkova
`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
`
`

`

`ELSEVIER SCIENTIFIC PUBLISHING COMPANY
`335 Jan van Galenstraat
`P.O, Box 211, Amsterdam, The Netherlands
`
`Distributors for the United States and Canada;
`
`ca
`
`ELSEVIER NORTH-HOLLANDINC.
`52, Vanderbilt Avenue
`New York, N.Y, 10017
`
`!
`
`a!
`ar
`
`
`
`Library 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.A35187
`S47". 3h9'2
`78-825
`ISBN O-U44-W1605-6
`
`II. Series.
`
`ISBN :0-444-41605-6 (Vol.12)
`ISBN:0-444-41616-1 (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
`
`

`

`Contents
`
`Acknowledgements.
`
`Introduction .
`1,
`References
`.
`.
`
`+.
`
`.
`
`«
`
`.
`
`+
`
`.
`
`«#
`
`.»
`
`«
`
`5
`
`2, The principle, me? and use ofaffinity nomeED,
`
`References
`
`.
`
`.
`
`.
`
`Uo an
`
`ye
`
`pee
`
`a er:
`
`yt
`
`é
`3, Theory of affinity chromatography
`;
`:
`3.1 Theoretical guidelines deduced on the ibaaof the equiliriuns satel
`3.1.1 Equilibrium modelfor adsorption with a fixed binding constant.
`3.1.2 Equilibrium model for elution by a change in Bis
`tas Abe
`3.1.3 Equilibrium model for elution by a competitive inhibitor
`3.1.4 Simulation of,cope.oornarnetens results .
`3,1.5 Conclusion! *.
`g's
`oy
`Pe
`3.1.6 List ofsymbols vised
`'
`'
`3,2 Theory of cooperative,bonding within the plate theory
`3.2.1 Isotherm of binding ofoligoadenylic acid to polyuridylic adid
`3.2.2 Cooperative adsorption column chromatography
`3.2.3
`Charactéristic features of cooperative adsorption chromatograms
`3.2.4 Mist of symbols used
`,
`3.3 Statistical theory of chromatceriphyapplied tia tfnity chromatography
`
`2
`
`og
`
`ow
`
`ye
`
`Me) Ad
`
`References
`
`Ix
`
`r
`
`«
`
`33
`
`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
`.
`4.3 Cooperative elution of oligoadenylic acid in immobilized polyuridylic aia
`
`35
`
`6. 2. ee 8
`.
`chromatography
`4,3.1 List of symbols used
`
`8 ee
`
`4.4 Other methods for the quantitative evaluates of siésactions with immobilized affinity
`
`ligands
`References
`
`.
`
`.
`
`.
`.
`=.
`w bie
`
`.
`5. General considerations on affinant—sorbent bonding .
`5.1 Steric accessibility.
`.
`.
`Gee Gh ai
`5.2 Conformation of attached affinant
`5.3 Concentration of the affinant on the matrix
`;
`‘
`5.4 Concentration of proteins, equilibration time and flow-rate ;
`5.5 Effectof temperature.
`2.
`.«
`«©
`©
`2
`+
`«©
`4
`5,6 Effect of pH andionicstrength .
`.
`Moan
`4G
`5,7 Elution with competitive affinity tleands
`5.8 Non-specific effects
`5.8.1 Effect of ionic streretttonmemapenitie setntion
`§,8.2 Extended Debye—Hiickel theory applied to the study of the abgendence af 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
`
`4
`
`.
`
`.
`
`*
`
`*
`
`.
`
`+
`
`.
`
`4
`
`81
`86
`87
`
`Page 3 of 53
`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
`mn
`6.3
`Immunoaffinity chromatography .
`.
`;
`6.4
`Isolation of lectins, glycoproteins and iccharides
`6.5
`Isolation of receptors, binding and transport proteins.
`6.6
`Isolation of —SH proteins and peptides
`:
`6.7
`Isolation of specific peptides
`: a
`6.8
`Isolation of nucleic acids and nivelGotides:es oe
`be
`6.9
`Isolation of lipids, hormones and other substances
`.
`6.10 Isolation of cells and viruses.
`‘
`6.11 Commercially available insoluble affinants
`References
`
`so
`.
`
`oe ae
`os
`
`89
`89
`92
`95
`99
`103
`106
`108
`111
`114
`116
`118
`127
`
`131
`131
`137
`139
`140
`144
`147
`149
`
`.
`
`*
`
`7. Hydrophobic chromatography, covalent affinity chromatography, affinity elution and
`+
`.
`.
`.
`related methods.
`.
`.
`De)
`ie Qe
`ge
`ee
`at
`7.1 Hydrophobic enioutacananty
`fy oy ery
`jae
`7.2 Covalent affinity See
`é
`‘
`7.3 Affinity elution
`.
`.
`er
`Sie
`5
`Ase
`7.4 Affinity density pertiirbation
`Sra?
`de KO de
`CaN
`7.5 Affinity electrophoresis
`.
`.
`ae
`xf oe.
`7.6 Metal chelate affinity chromatography
`c=
`x
`
`References
`
`1
`
`.
`
`4 «4
`
`ae amen
`
`lat
`
`A
`
`de
`
`8. Solid matrix supports and the most used methods of binding .
`8.1 Required characteristics
`
`8.2 Survey ofthe most common solid supportsnd soupling procedure
`Pec,
`%
`8.2.1 Cellulose and its derivatives
`.
`8.2.2 Dialdehyde sinochmethylenidianiting (S—MDA)
`. EN ¢
`8.2.3 Dextran gels .
`oy bh Re
`te Beh
`8.2.4 Agarose andits Sesivatives, ae
`2
`8.2.5 Copolymer of ethylene and maleic anhivdride
`oP
`ap
`8.2.6 Polyacrylamide supports and their derivatives
`.
`.
`8.2.7 Hydroxyalkyl methacrylate gels
`.
`8.2.8 Glassandits derivatives
`1.
`.
`i
`«©
`8
`©
`«©
`8.2.9 Othersupports .
`.
`1
`5
`8
`2.
`he ee
`8.3 Spacers
`.
`.
`pe
`eld
`8.4 Blocking of sindeatted woUDS. Re)
`aha, Bee ao
`8.5 Leakage of the coupled affinant
`.
`8.6 General considerations in the choice of soshenits, piauets anilsgaplicn sail Stacking
`procedures
`.
`6
`6 ee ee ee
`.
`*
`“
`+
`References,
`2.
`65 Ge FS la ok we ue Be le ee
`
`*
`
`9. Characterization of supports and immobilized affinity ligands
`9,1 Methods for the determination of non-specific sorption .
`9.1.1 Determination of adsorption capacity
`.
`.
`9.1.2 Determination of residual negatively charged itp
`9,2 Determination of activatable and active groups
`9.2.1 Determination of carboxyl,hydrazide and amino Hoeson the heals of acidebas
`titration .
`.
`ees
`eee
`6
`9.2.1.1 Dry weight determination
`re ee te Te
`9.2.1.2 Determination of carboxyl groups .
`Ph
`9.2.1.3 Determination of hydrazide groups
`.
`.
`.
`.
`9.2.1.4 Determination of aliphaticamino groups
`9.2.2 Determination of the content of free carboxyl groups
`
`203
`203
`204
`204
`204
`
`204
`204
`205
`205
`205
`
`206
`
`Page 4 of 53
`Page 4 of 53
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`

`

`CONTENTS
`
`Vil
`
`9.2,3 Determination of free amino groups in polymers on the basis of the
`sete
`ae’
`condensation reaction with PyareTapas eked es.
`Procedure for azide assay
`.
`.
`Se Pk
`hm ee eG
`9.2.4
`92.5 The sodium 2,4,6-ininftscbendenesulbhonate colour‘test
`©
`a
`. 207
`Fluorescamine test for the rapid detection of trace amounts of amineerate . 207
`9.2.6
`9.2.7 Determination of oxirane groups .
`. 208
`9,2.8 Determination of the capacity ofpotttrophencl ester derinatives of HeboKy-
`alkyl methacrylate (NPAC) gels.
`. 209
`9.2.9 Determination of the degree of substitution of tenzylated dibromopropancl
`crosslinked Sepharose
`2
`6 eee 8 Ht HH eH HS 209
`9.2.10 Determination of vinylgroups.
`.
`-
`6
`8
`ete ee et 209
`9,2.11 Determination of sulphydryl groups
`.
`.
`- wm Re ans
`in S08
`9,3 Methodsfor the determination of immobilized affinity ligands
`hoe bee go wh
`#
`9.3.1 Differenceanalysis
`.
`-
`2
`©
`8
`8
`+
`8
`8
`oe
`hm
`oH
`HoH HH 210
`a
`9.3.2 Spectroscopic methods
`.
`.
`.
`mee
`ah
`ten
`&
`Boece
`we
`eve ele
`_ 9.3.3 Determination by meansof acid—base titration ce
`>»
`» 212
`9.3.4 Determination of immobilized proteins, peptides, amino acids, nucleotides,
`carbohydrates and other substances after liberation by acid, alkaline or
`enzymatic hydrolysis.
`.
`.
`ye B13
`9.3.4.1 Determination of ‘rommdibilizedamino acids,wogtldes and proteins
`pec nals
`9.3.4.2 Determination of nucleotides .
`.
`6
`6 ee 8 ee Hh es 213
`9.3.4.3 Determination of carbohydrate
`.
`.
`.
`«
`Yolo
`ae aed
`9.3.5 Determination of the amount ofbound affinant on the toad of‘elemental
`“ao arte
`analysis.
`-
`oe ot a hae
`ae
`215
`9,3.6 Determination of labelledaffinity ligands yopar
`x
`9.3.7 Determination of immobilized diamingdivrepylamine by sigiivdiin soledineiee: 215
`9.3.8 Determination of immobilized proteins on the basis of tryptophan content
`.
`. 215
`9.4 Active-site titration of immobilized proteases.
`-
`Cice
`g. Bgl
`ia oe oe DE
`9.5 Study of conformational changes of immobilized proteins eo 2S ete So, BY
`9.6 Studies of the distribution of proteins bound to solid supports.
`.
`+
`+
`+
`5
`+ 221
`RaPArenbee:
` “E.
`oye.
`ate
`& ak koe Per Bye eta ep Re
`
`+ 225
`=
`-
`-
`-
`.
`»
`10, General considerations on sorption, elution and non-specific binding
`ah eee
`fe Gere ved
`10.1 Sorptionconditions
`.
`.
`ad Alay
`Go
`dy BS
`tert
`om
`ee
`10.1.1 Effect of temperature, pH and salts Sa Pe ORME
`10.1.2 Practice ofsorption
`2
`2
`4
`8
`8 ee eH 330
`10.2 Conditions forelution .
`6
`6
`6
`8 ee ee 232
`10.2.1 Practice of desorption.
`.
`el
`thot
`gey 1 BES
`10.2.2 Effect of the heterogeneity ahthe immobilized‘affinants . yd ana
`ow
`oa EF
`10.2.3 Establishment of optimal conditions and saturation effect
`.
`.-
`»
`-
`+
`+ 240
`10.3 Non-specific sorption
`.
`.
`.
`POW aS ee dn 2a
`10.4 Regeneration and storage of affinity salurans
`sme
`ed
`ote
`Fey
`Ay
`liebe
`bb
`beg
`GNEAS
`BecGOe'\
`o
`cece Gwe.
`F
`pe ae tet Sed Oba |
`Bp
`ER!
`Sy BG
`
`11, Examples of the use of affinity chromatography . «6 +
`©
`5
`oe
`fos
`hos
`ors 245
`11.1 Isolation of biologically active substances
`.
`.
`~
`+»
`« 245
`11.2 Resolution ofDL-tryptophan by affinity chromatographyon‘ovineserum‘albumin—
`«
`SEO
`agarose column .
`.
`>
`ow
`« 319
`11.3 Semi-synthetic nuclease‘and cataplementanyditaraction of nuclease fragments
`»
`11.4 Study of interactions of biologically active substances.
`«©
`6
`4
`©
`©
`+
`to 5 324
`11.5 Study of the mechanism of enzymaticaction
`.
`.
`.
`- 327
`11.6 Molecular structure of fibroblast and leucocyte interferons investigated with lectin
`and hydrophobic PRSSE «ters
`aw
`ae
`ste We Oe
`oe
`If
`wey
`Eh Bee
`11.7 Immunoassay
`.
`.
`.-
`a, ee et oe Ba we
`a
`epee HR
`
`Page 5 of 53
`Page 5 of 53
`
`

`

`VII
`
`CONTENTS
`
`09
`11.7.1 Solid-phase radicimmunoassay
`.
`fa!
`git
`ten
`2
`11.7.2 Enzyme-linked immunosorbent ‘that (ELISA) ft ue
`11.8 Specific removal of bovine serum albumin (BSA) antibodies in vivo10 by seibiesseoepaneal
`circulation over BSA immobilized on nylon microcapsules.
`.
`1
`+ +
`References
`
`. 332
`
`334
`
`. 334
`. 336
`
`11.7.3 Microfluorimetric immunoassay .
`
`oe
`12. Immobilized enzymes...
`Lew
`12.1 Classification of inimobitized snaynies
`12.2 Attachment of enzymesto solid supports and activity of immobilized srizysies
`12.3 Stability of immobilized enzymes.
`hv
`er
`i
`6
`12,3,1 Stability during storage
`6
`Me eee
`12.3.2 Dependence of stability on pH .
`5
`12.3.3 Thermal stability
`.
`.
`fom ¥
`12.3.4 Stability against denaditeine agents
`12.3.5 Increase of stability
`.
`.
`2.
`.
`12.4 Application of immobilized enzymes.
`12.4.1 Affinity ligands,
`.
`.
`12.4.2 Study ofstabilized enzpine siinldoules gna of hats wiibailts
`
`6
`
`Sa es
`#
`«©
`#
`8
`
`+
`
`©
`
`=
`
`am
`
`,
`
`12.4.3 Models of biological systems .
`12.4.4 Application of immobilized enzymes
`12.5 “Synthetic biochemistry”
`References
`.
`.
`.
`‘
`
`Subjectindex
`
`.
`
`-
`
`©
`
`©
`
`8
`
`t
`
`8
`
`eo
`
`tos
`
`List of compounds chromatographed 6 ee te ts
`
`;
`
`. 365
`. 365
`. 366
`. 374
`. 374
`2 375
`3
`4o20
`. 377
`- 378
`. 379
`- 379
`» 379
`- 380
`» 382
`» 383
`- 384
`
`. 387
`
`. 399
`
`Sse
`
`Page 6 of 53
`Page 6 of 53
`
`

`

`Chapter 2
`
`Theprinciple, history and use of affinity chromatography
`
`Affinity chromatography (or, more exactly, bioaffinity or biospecific affinity
`chromatography) is based on the exceptionalability of biologically active substances to
`bind specifically and reversibly other substances, generally called ligands or affinity
`ligands (Lowe and Dean) or simplyaffinants (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 ofthis 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 a-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 ef al. were the first
`to use this principle, in 1951, for the isolation of antibodies on a columnofcellulose
`with covalently attached antigen. Affinity chromatography was fizst used in the isolation
`of enzymes in 1953 by Lerman, whoisolated tyrosinase on a column of cellulose with
`
`MATRIX
`
`fin< are A
`curren
`|
`
`*DEFORMING
`
`B
`
`mr
`
`Cc 4
`
`[reg
`
`:
`
`Fig. 2.1. Diagrammatic 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 et al, Methods Enzymol.,
`34 (1974) 108-126,
`
`Page 7 of 53
`Page 7 of 53
`
`

`

`8
`
`PRINCIPLE, HISTORY AND USE OF AFFINITY CHROMATOGRAPHY
`
`ethereally bound resorcinol residues. In subsequentyears 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 development of this method. A milestonein this
`development was the methodof attachmentofaffinant to agarose activated with cyanogen
`bromide, developed by Porath and co-workers (Axén and Ermback; Axén etai.; 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 ef al. successfully employed affinity chromatographyfor the isolation
`of nuclease, chymotrypsin and carboxypeptidase A. This study, in which the term affinity
`chromatography was used for thefirst 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 ofaffinity chromatographyis not limited to the isolation of
`biologically active substances. As early as 1960 Yagi et al. described a quantitative deter-
`mination of small amounts of antibodies by meansofsolid carriers with bonded antigens.
`The use of solid carriers in radioimmunoassaysis discussed in detail in Section 11.7. Im-
`mobilized oligomers of polythymidylic acid were used by Edmondse¢ai. for the quanti-
`tative determination of polyadenylic acid. The use ofaffinity gel filtration as a micro-
`scale method for rapid determinations of apparent molecular weights of dehydrogenases,
`based on their exclusion from gel filtration medium ofvarious pore sizes, was described
`by Lowe and Dean.
`By its nature, affinity chromatographyis ideal for the study ofinteractions 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 et 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. Akanumaefal. employed this
`methodfor the study ofthe 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 forminga “dead-end” complex with enzyme—NAD*
`complex rather than with enzyme—NADHcomplex,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 meansofaffinity chromatography by Jankowskiet al.
`Forthe separation of isoenzymesof lactate dehydrogenase, Brodelius and Mosbach
`(1973) used Sepharose with an attached AMP analogue; five separated peaksofiso-
`enzymes could be eluted by increasing the NADH concentration, as shownin Fig. 2.2.
`The separation has been interpreted as a result of the differences in dissociation constants
`(Kdiss) for the binary enzyme—NADHcomplex. Brodelius and Mosbach (1974) subse-
`
`Page 8 of 53
`Page 8 of 53
`
`

`

`PRINCIPLE, HISTORY AND USE OF AFFINITY CHROMATOGRAPHY
`
`9
`
`
`
`
`
`LDHACTIVITY,2A,,,/min-mi
`
`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 @-mercaptoethanol and 1 M
`sodium chloride was applied to an AMP-analogue—Sepharose column (140 X 6 mm, containing 2,5 ¢
`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 mi
`NADHin the same buffer, containing 1 mf 6-mercaptoethanol, Fractions of 1 ml were collected at
`the rate of 3.4 mi/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 Kajs, values and the elution
`concentration of NADH. The linearity indicates that in the given case the dissociation
`constants for the enzyme—NADHcomplex playagreater 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
`NADHonthe basis of the determination of elution concentrations of NADH.Nodif-
`ferences in Kgiss 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 advantageis 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
`
`Page 9 of 53
`Page 9 of 53
`
`

`

`PRINCIPLE, HISTORY AND USE OF AFFINITY CHROMATOGRAPHY
`
`RABBIT M,
`
`10
`
`Kuiss,aM
`
`0
`
`O BOVINE M,
`
`0.
`
`0.2
`
`:
`
`03
`
`04
`
`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. Soe. Trans., 2 (1974) 1308-1310.
`
`'
`
`bound inhibitors and with the soluble inhibitors employed. This methodis discussed in
`greater detail in Chapter 4. The great advantage of this methodis that when using the
`same inhibitor for the immobilization and the elution, direct conclusions can be drawn
`about the effects of the bonds andthe supporton the interaction being studied, from the
`agreement between or the difference in the dissociation constants determined, Hence the
`methodofaffinity 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.
`Axén, R, and Emnback, S., Eur. J. Biochem., 18 (1971) 351-360.
`Axén, 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., Biochem. Soc. Trans., 2 (1974) 1308-1310.
`Campbell, D.H., Luescher, E.L. and Lerman,L.S., Proc. Nat. Acad, Sci. US., 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.
`Denbuzrg, J. and De Luca, M., Proe. Nat. Aced. Sci. U.S., 67 (1970) 1057-1062.
`Edmonds, M., Vaughan, M.H. 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)
`1124-1130.
`
`Page 10 of 53
`Page 10 of 53
`
`

`

`REFERENCES
`
`li
`
`Kasche, V., Arch, Biochem, Biophys., 173 (1976) 269-272.
`Lerman, L.S., Proc. Nat, Acad. Sci, ULS., 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., Axén, 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, J. and Bayer, E., Biochemistry, 14 (1975) 5541-5548.
`Starkenstein, E., Biochem. Z., 24 (1910) 210-218.
`Yagi, Y., Engel, K. and Pressman, D., . Immunol., 85 (1960) 375-386.
`
`Page 11 of 53
`Page 11 of 53
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`

`

`Page 12 of 53
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`

`

`89
`
`Chapter 6
`
`Choice of affinity ligands for attachment
`
`6.1 HIGHLY SPECIFIC AND GROUP-SPECIFIC MATRICES
`
`A compoundis 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 ofaffinants 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 enzymeis 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. Notall 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 mustfirst 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 increasesif,
`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 enzymesrelated to the metabolism of aspartic acid show group-specific adsorption
`affinity to N-(«-aminohexyl)-L-aspartic acid—Sepharose. On this immobilized affinant,
`asparaginase, aspartase, aspartate-6-decarboxylase and asparaginase modified with tetra-
`nitromethane (Tosaetal.) 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 andif 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 enzymeeither 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 chromatographyis their
`
`Page 13 of 53
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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 showsaffinity 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 10°7——-107° ™,
`Enzymes 2 and 3 have affinity for sorbents with dissociation constants of about 107° M,
`and enzyme 4 shows a very weak affinity with a dissociation constant of > 107? M.
`For the generalized Langmuir adsorption isotherm
`=. Ky koC
`I+k, C;
`
`(6,1)
`
`qi
`
`where q;is the specific amount of the adsorbed substance i, C; is concentration and k,
`and kz are constants, For low concentrations of C;, eqn. 6.1 reduces to qj = k,k2C;, and
`for high concentrations of C; to qj = ky. In general we can write
`
`(6.2)
`ai = 1G)"
`where n = 0-1. It then follows that when the concentration of the ligandis sufficiently
`high, so that the adsorbent capacity is not a limiting factor, the specific amount ofthe
`adsorbed substancei, q;, is dependent on its concentration in the mobile phase, C;, and
`
`ABSORBED(q) a,
`
`AMOUNT
`
`ENZYME CONCENTRATION (C)
`
`Fig. 6.1. Adsorption isotherms for four enzymes interacting with a single immobilized affinity ligand,
`Reproduced with permission from C.R, Lowe and P.D.G, Dean, Affinity Chromatography, Wiley, New
`York, London, 1974, p. 91.
`
`Page 14 of 53
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`

`

`SPECIFIC MATRICES
`
`91
`
`not onits affinity towards the attached affinant. For a sample containing equimolar
`amounts of four enzymes, the amount of each of them adsorbed will be q1, 42,3 and qa.
`In displacement elution, using a concentration D of the displacer, enzymes 1, 2 and 3 with
`concentrations C,, C, and C3 will be eluted. Enzyme4 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 enzymeofhigh affinity is added. If the
`capacity of the adsorbentis exceeded, enzymeswill appear in the retention volumeof the
`eluate with both a high and a low affinity, ie,, not only those which are weakly adsorbed.
`This consequenceis 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 enzymescan be specifically excluded. Further, an inhibitor or
`otherligands 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 methodsof elution. A
`knowledge ofinhibitors or substrates of various enzymes can beutilized 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 ofthe solid support
`(Fritz et al.). Proteolytic enzymes boundto 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 attachmentof,
`for example, hexamethylenediamine and dimethylethylenediamine, the polyamphoteric
`derivative formed becamesuitable even forthe isolation of inhibitors with lowerisoelectric
`points.
`Asis 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 immobilizedaffinity ligand (Murphy
`et al.}. For example, the elution of anti-glucagon antibodies from a column of immobilized
`glucagon can be achieved under milder conditionsif 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-5-nitrobenzyl bromide, tetranitromethane or
`hydrogen peroxide.
`O’Carra recommendsdifferentiating 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 leading to an
`
`Page 15 of 53
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`

`92
`
`AFFINITY LIGANDS
`
`irreversible loss of activity, and for them the method of attachmentis usually not un-
`ambiguously 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 11,1,
`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 to Spheron via
`
`ABSORBANCE,
`
`260nm 0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`420
`
`FRACTION NUMBER
`
`Fig. 6.2. Chromatography of crude pancreatic extract on Z-Gly-D-Phe-NH,—Spheron. A 100-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 to a final pH of 8.0). Fractions (6 ml) were collected at 20-min intervals, —, Absorbanceat
`280 nmj--++++ PH. (a) Contaminants and trypsin; (b) chymotrypsin; (c) complex of chymotrypsin
`with lung trypsin inhibitor. In A the arrow i