`
`BIOCHEMICALS AND
`
`AFFINITY CHROMATOGRAPHY
`
`Edited by
`R. Bruce Dunlap
`Department of Chemistry.
`C
`1
`University of South
`are inu
`Columbia, South Carolina
`
`PLENUM PRESS ° NEW YORK AND LONDON
`
`Page 1 of 18
`Page 1 of18
`
`BD EXHIBIT 1019
`
`BD EXHIBIT 1019
`
`
`
`
`
`Library of Congress Cataloging in Publication Data
`
`Symposium on Affinity Chromatography and Immobilized Biochemicals, Char-
`leston, S. C., 1973.
`Immobilized biochemicals and affinity chromatography.
`
`(Advances in experimental medicine and biology, v. 42)
`Contains most of the papers presented at the symposium held Nov. 7-9 in
`conjunction with the Southeastern Regional American Chemical Society meeting.
`Includes bibliographical references.
`1. Biological chemistry — Technique - Congresses. 2. Affinity chromatography
`— Congresses. I. Dunlap, Robert Bruce, 1942-
`ed. II. Series. IDNLM: 1. Bio-
`chemistry — Congresses. 2. Chromatography, Affinity — Congresses W1 AD559 v.
`42 1974/ QD271 S986i 1973.]
`QP519.7.S9
`1973
`ISBN 0-306-39042-6
`
`574.1’92’028
`
`74-7471
`
`*5‘
`
`Proceedings of the -symposium on Affinity Chromatography and Iinniobilized
`Biochemicals held in Cllarleston, South Carolina, November 7 - 9, 1973
`
`© 1974 Plenum Press, New York
`A Division of Plenum Pulilisliiiig Corporation
`227 West 17th Street, New York, N.Y. 10011
`
`United Kingdom edition publislied by Plenum. Press, London
`' A Division of Plenum Publishing Company, Ltd.
`4-a Lower John Street, London WIR 3PD, England
`
`All rights reserved
`
`No part of this book may be reproduced, stored in a retrieval system, or transmitted,
`in any form or by any means, electronic, mechanical, photocopying, microfjlming,
`recording, or otherwise, without written pCl‘llllSSl0l1 from the Puhlislwr
`
`Printed in the United States of Anleflcu
`
`Page 2 of 18
`Page 2 of 18
`
`
`
`Contents
`
`PART ONE: AFFINITY CHROMATOGRAPHY
`
`Affinity Chromatography—~Old Problems and
`I
`I
`I
`I
`I
`I
`I
`O
`I
`I
`I
`I
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`3
`
`Indu Panthh,
`Steven C. Manah,
`and Pedro Cuatnecabaa
`
`New Approaches for the
`Affinity Chromatography.
`Preparation of Spacer Containing Derivatives and for
`Specific Isolation of Peptides .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`Meta witaheh
`
`Quantitative Parameters in Affinity Chromatography .
`
`A.H.Npwmmmn,P.Bmuwn,amiA.H.Rwm£
`
`Non—Specific Binding of Proteins by Substituted
`Agaroses n
`n
`I
`0
`u
`'
`1
`n
`1
`.
`o
`4
`0
`u
`I
`0
`u
`u
`A
`n
`
`n
`
`B. H. J. Hofiatee
`
`A Solid Phase Radioimmune Assay for Ornithine
`Transcarbamylase .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`Donatd L. Eéhenbaugh, Donafid Sena
`and Enic Jam2A
`
`o
`
`.
`
`0
`
`.
`
`.
`
`.
`
`I
`
`.
`
`.
`
`.
`
`o
`
`.
`
`.
`
`.
`
`u
`
`.
`
`.
`
`.
`
`u
`
`.
`
`15
`
`33
`
`61
`
`Purification of Acetylcholinesterase by
`Covalent Affinity Chromatography .
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`75
`
`Houatan F. V044, V. Abhani, and
`Inwin B. Wilson
`
`Cooperative Effects of AMP; ATP, and Fructose
`1,6-Diphosphate on the Specific Elution of Fructose
`l,6—Diphosphatase from Cellulose Phosphate .
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`85
`
`Jobeph Mendtcino and Huééein Ab0u—IAAa
`
`An Analysis of Affinity Chromatography Using
`Immobilised Alkyl Nucleotides
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`99
`
`P. D. G. Dean, D. B. Cnaven, M. J. Hahuey,
`and C. R. Laue
`
`M
`
`Page 3 of 18
`Page 3 of 18
`
`
`
`X
`
`Affinity Chromatography of Kinases and Dehydrogenases
`on Sephadex and Sepharose Dye Derivatives .
`.
`.
`.
`.
`.
`
`.
`
`Richa/Ld L. Ea/srmday and Inge/1. M. Eazyte/zday
`
`Affinity Chromatography of Thymidylate Synthetases Using
`5—Fluoro—2'-Deoxyuridine 5'-Phosphate Derivatives of
`'
`‘
`I
`I
`I
`I
`I
`I
`I
`I
`I
`O
`I
`I
`I
`I
`O
`I
`I
`O
`O
`
`I
`
`C
`
`.
`
`I
`
`John M. wmmeg,
`‘ and Thomcus D2/«Lbs
`
`Iuccnfea Jeiafaunica,
`
`The Biosynthesis of Riboflavin: Affinity Chromatography
`Purification of GTP~Ring—Opening Enzyme .
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`L. P/zezszton Me/mm and Cm/L22/5 M. Baugh
`
`CONTENTS
`
`123
`
`147
`
`0;
`
`.
`
`I
`
`.
`
`Purification of Tyrosine~Sensitive 3-Deoxy—
`DfArabino—heptulosonate—7~Phosphate and
`Tyrosyl—tRNA Synthetase on Agarose Carrying
`Carboxyl-Linked Tyrosine
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`157
`
`And/Law R. Gafliopo, Phéup S. K01‘/3«(',0p0bL€.0Z>,
`and Scott C. Mom
`
`Structural Requirement of Ligands for Affinity
`Chromatography Absorbents: Purification of
`Aldehyde and Xanthine Oxidases
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`Aflbwu“, E. Chu and starting Chaylzin
`
`PART TWO:
`
`IMMOBILIZED BIOCHEMICALS
`
`Immobilized Polynucleotides and Nucleic Acids .
`
`P. T. Gulham
`
`.
`
`.
`
`.
`
`.
`
`Immobilized Cofactors and Multi—Step Enzyme—Systems
`
`Ktau/5 M04 batch
`
`. Preparation, Characterization, and Applications of
`Enzymes Immobilized on Inorganic Supports .
`.
`.
`.
`
`.
`
`H. H. wec«ta,€,€
`
`Lactase Immobilized on Stainless Steel and Other
`
`Dense Metal and Metal Oxide Supports
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`M. Cha/2,80/5, R. w. Couglmun, B. R. Amen,
`E. K. Pa/ruchuiué, and F. X. Ha/szseflbengexa
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`165
`
`173
`
`187
`
`191
`
`213
`
`The Use of Membrane—Bound Enzymes in an Immobilized
`Enzyme Reactor
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`;
`
`235
`
`Clrzcutllexs C. W0/vtlméngzfon
`
`Page 4 of 18
`Page _4 of 18
`
`
`
`‘CONTENTS
`
`The Optimization of Porous Materials for Immobilized
`Enzyme Systems .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`David L. Eaton
`
`Water Encapsulated Enzymes in an Oil—Continuous
`Reactor: Kinetics and Reactivity .
`.
`.
`.
`.
`.
`
`.
`
`.
`
`R.
`
`I. Lecwxltt, F. X. Ryan, and U). P. Bwagez/.>
`
`Analysis of Reactions Catalyzed by Po1ysaccharide—
`Enzyme Derivatives in Packed Beds
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`M. H. Kayezs and F. E. Seme/I/slay
`
`The Preparation of Microenvironments for Bound
`Enzymes by Solid Phase Peptide Synthesis .
`.
`.
`
`.
`
`.
`
`.
`
`James B. Tccyliwz and Hcuwlid E.
`
`swam’/sgood
`
`Opfiimization of Activities of Immobilized Lysozyme,
`d—Chymotrypsin, and Lipase .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`Raihén Ddtzia and David F. OLEIA
`
`Chemical Modification of Mushroom Tyrosinase
`for Stabilization to Reaction Inactivation .
`
`.
`
`.
`
`.
`
`David Lextt/5 and T[’LQ.0d0/‘L0. Choose,
`
`J21.
`
`Chain Refolding and Subunit Interactions in
`Enzyme Molecules Covalently Bound to a Solid Matrix
`
`H. Rabemf Ho/1/ton and Hcuwlid E, Swot/Osgood
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`Immobilization of Lipase to Cyanogen Bromide
`Activated Polysaccharide Carriers
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`Pdufl Mefliué and BL-Chang Wang
`
`Use of Immobilized Enzymes for Synthetic Purposes
`
`.
`
`David L. Mcur/.» ha/£8
`
`o
`
`I
`
`9
`
`n
`
`0
`
`I
`
`a
`
`o
`
`u
`
`n
`
`I
`
`I
`
`o
`
`I
`
`0
`
`0
`
`u
`
`u
`
`a
`
`u
`
`0
`
`o
`
`I
`
`t
`
`xi
`
`241
`
`259
`
`269
`
`283
`
`293
`
`317
`
`329
`
`339
`
`345
`
`369
`
`Page 5 of 18
`Page 5 of 18
`
`
`
`IMMOBILIZED POLYNUCLEOTIDES AND NUCEEIC ACIDS
`
`P. T. Gilham
`
`Department of Biological Sciences
`
`Purdue University, West Lafayette, Indiana
`
`47907
`
`The use of immobilized nucleic acids and polynucleotides in
`the study of nucleic acids and their associated enzymes has become
`widespread in recent years. There are two main areas of applica-
`tion:
`the fractionation of nucleic acids and polynucleotides
`through base-paired complex formation and the isolation and puri-
`fication of nucleic acid—associated enzymes by affinity chromatog-
`raphy. There are now a number of methods for the preparation of
`immobilized polynucleotides and the potential uses of these mater-
`ials depend to some extent on the means by which the polymers are
`attached to the insoluble supports.
`
`METHODS OF IMMOBILIZATION
`
`the methods for the pre-
`indicated in the following table,
`As
`paration of immobilized polynucleotides can be broadly divided into
`two categories:
`those involving covalent
`linkages between the poly-
`mer.and the support, and those employing physical entrapment of the
`polymer within the support matrix.
`‘
`
`In those cases where covalent binding of the molecule is de-
`sirable,
`terminal attachment can be readily achieved by activation
`of the phosphomonoester group at the polynucleotide terminal in the
`presence of an insoluble polysaccharide. This reaction yields a
`product inhwhich the polynucleotide is connected to the support by
`a stable phosphodiester linkage.
`A second approach exploits the
`reactivity of the terminal dialdehyde group resulting from the peri—
`odate oxidation of ribopolynucleotides. Condensation of these dial~
`dehyde functions with supports containing primary amine or hydrazide
`
`173
`
`Page 6 of 18
`Page 6 of 18
`
`
`
`174
`
`P. T. GILHAM
`
`Table I
`
`1.
`
`Covalent Binding at the Polynucleotide Terminalso
`
`(a) Activation of terminal phosphate group.
`(b) Periodate oxidation of 3'—termina1s in RNA species.
`(c) Condensation of RNA 3'-terminals with supports con-
`taining dihydroxyboryl groups.
`‘ivan}
`
`2.
`
`Covalent Binding at Multiple Points.
`
`(a) Condensation with phosphocellulose.
`(b)4 Reaction with CNBr-activated supports.
`(c) UV—irradiation.
`
`3.
`
`Physical Entrapment.
`
`groups allows covalent attachment at the polynucleotide 3'—terminals.
`A third method arises from the discovery that the terminal cis diol
`groups in ribopolynucleotides are capable of forming specific cyclic
`complexes with supports containing covalently-bound dihydroxyboryl
`groups.
`The use of phosphocellulose and CNBr-activated supports
`permits the multiple point attachment of polynucleotides and,
`in the
`case of CNBr activation,the reaction mechanism appears to be similar
`to that operating in the immobilization of enzymes by this method.
`Immobilization induced by ultraviolet irradiation of polynucleotides
`probably results in multiple point attachment also, although the
`mechanism of the reaction is obscure.
`
`Covalent Attachment at Polynucleotide Terminals
`
`Insoluble supports containing small homopolynucleotides can be
`prepared by the chemical polymerization of the appropriate mono-
`nucleotide (1,2).
`For example,
`the treatment of thymidine 5'-phos-
`phate with dicyclohexylcarbodiimide in anhydrous solution produces
`thymidine oligonucleotides of the form, pdT—dTn—dT (n < 20).
`In
`the reaction mixture,
`the terminal phosphate groups are present in
`an activated form and the addition of dry cellulose to the mixture
`results in the condensation of these groups with the hydroxyl func-
`tions of the supporta
`In this case the oligonucleotides are bound
`to the support through stable phosphodiester linkages at their 5'-
`terminals.
`In an analogous way the polymerization of a nucleoside
`3'—phosphate yields immobilized oligonucleotides connected to the
`cellulose at their 3'-terminals“
`
`Page 7 of 18
`Page 7 of 18
`
`
`
`IMMOBILIZED POLYNUCLEOTlDES AND NUCELIC ACIDS
`
`*
`
`175
`
`Preformed polynucleotides from either synthetic or natural
`sources may also be bound to cellulose at one of their terminals (3).
`In this case,
`the reaction is carried out in aqueous solution and a
`water-soluble activating agent, N-cyc1ohexyl~N'~B-(4-methylmorpho—
`linium)ethylcarbodiimide,
`is used.
`The carbodiimide activates the
`terminal phosphomonoester group resulting in, again, a phosphodiester
`linkage to the support.
`The method was initially tested with
`mononucleotides, polynucleotides and tRNA (3), and it has recently
`been shown that,
`in the case of imobilized tRNA, practically all
`of the bound nucleotide material can be released by exposure to
`pancreatic ribonuclease (4), an observation that confirms the
`proposed terminal linkage to the support.
`
`The reactivity of the 3'~terminal cis diol group in poly-
`ribonucleotides and RNA can be exploited in two ways for the
`terminal binding of these molecules.
`'Periodate oxidation of the
`diol moiety to the corresponding dialdehyde yields a product that
`can undergo condensation with aminoethylcellulose, and the resulting
`linkage can then be stabilized by reduction with sodium boro-
`hydride (5).
`The binding in these products is thought to arise
`from the formation of a substituted morpholine ring structure that
`includes the nitrogen of the aminoethylcellulose and the five atoms
`that originally constituted the ribose ring of the terminal
`nucleoside.
`The assignment of this linkage is based on the
`structure of the product obtained from the borohydride reduction
`of the complex formed between periodate—oxidized adenosine 5‘-
`phosphate and methylamine (6). Supports containing hydrazide
`groups will react in a similar manner with periodate—oxidized
`polyribonucleotides to form reasonably stable linkages.
`For
`example, a mixture of agar and polyacrylic acid hydrazide has been
`used to immobilize tRNA in this way (7), and reduction with sodium
`borohydride has also been used to stabilize the linkage in a
`similar complex obtained from periodate—oxidized tRNA and hydra~
`zinyl-Sepharose (8).
`
`Another method for the terminal binding of polyribonucleotides
`arises out of the observation that polymers that possess terminal
`cis diol groups are capable of forming specific complexes with
`supports containing covalently-bound dihydroxyboryl groups (9-ll).
`N-EN‘-(m—Dihydroxyborylphenyl)succinamyl]aminoethylcellulose (9)
`prepared from the condensation of N-(m—dihydroxyborylphenyl)-
`succinamic acid with aminoethylcellulose forms cyclic boronate
`structures at pH 8-9 with molecules containing the ribonucleoside
`diol group.
`In certain applications immobilized polynucleotides
`prepared byfthis method may have some advantages over those
`mentioned above in that, at pH 6,
`the complex breaks down, allowing
`the recovery of the bound polynucleotide from the support and the
`substituted cellulose is then available for the binding of another
`polymer.
`
`Page 8 of 18
`Page 8 of 18
`
`
`
`176
`
`P, T. GILHAM
`
`Covalent Attachment at Multiple Points
`
`Some of the early attempts to immobilize nucleic acids and
`polynucleotides exploited an activated form of acetylated phospho-
`cellulose (12), Activation was achieved by treating the derivatized
`cellulose with dicyclohexylcarbodiimide in a non-aqueous solvent
`and it is presumed that the subsequent binding of the polynucleotide
`resulted from the formation of phosphodiester linkages between the
`support and the various hydroxyl groups on the polynucleotide chain,
`Thus, it was possible to bind the phosphocellulose to the glucosyl
`hydroxyl groups of phage T4 DNA (12) and to the 2‘-hydroxyl groups
`of various polynucleotides and RNA molecules (13).
`
`the immobilization technique that has been
`More recently,
`used so successfully for protein molecules and other ligands has
`been applied to polynucleotides. Single stranded RNA and DNA may
`be efficiently bound at pH 8 to cyanogen bromide—activated
`agarose (14) and, while the mechanism of the immobilization is not_
`known, it seems likely that binding results from the formation of
`covalent linkages consisting of isourea ether groups connecting
`the oxygen atoms of the polysaccharide hydroxyl groups to the
`nitrogen atoms of some of the nucleotide bases within the poly-
`nucleotide chains.
`It has been suggested that, if the condensation
`reaction with the activated polysaccharide is carried out at pH 6,
`polynucleotides may be immobilized by single point attachment at the
`terminal phosphate groups of the polynucleotide strands (15).
`However,
`in this study, control experiments involving polynucleotides
`that do not possess terminal phosphate groups were not reported,
`and a more recent investigation of the reaction with cyanogen
`bromide—activated agarose indicates that multi-point attachment of
`polynucleotides occurs also at pH 6 (16).
`
`Denatured DNA, RNA and polynucleotides are immobilized when
`exposed to UV—irradiation in the presence of inert supports.such
`as polyvinyl beads, cellulose, and nylon threads (17).
`In this
`procedure it is possible that no covalent
`linkages are formed
`between the support and the polynucleotide strands since,
`in the
`absence of the support,
`these polymers are capable of forming
`insoluble gels upon irradiation. These gels are presumably a
`consequence of the production of intermolecular cross-links
`resulting from pyrimidine-pyrimidine dimer formation and the
`supports may serve only to immobilize the insoluble products so
`formed. Alternatively,
`the induction of both inter— and intra-
`molecular cross-links of this type may be responsible for the
`formation of macrocyclic structures resulting in the physical
`entrapment of the polynucleotide strands within the support matrix.
`Binding could also result from UV-catalyzed addition reactions
`involving functional groups on the supports and the 5,6 double
`bonds in the pyrimidine moieties of the polynucleotide chains.
`Nevertheless,
`the method seems to have some general applicability
`
`Page 9 of 18
`Page 9 of 18
`
`
`
`IMMOBILIZED POLYNUCLEOTIDES AND NUCELIC ACIDS
`
`T
`
`177
`
`in that, by exposure to UV—irradiation, calf thymus DNA (18), viral
`RNA (19), and ribosomal RNA (20) have been bound to cellulose, and
`poly(U) has been immobilized on fiberglass filters (21).
`
`Physical Entrapment
`
`A number of methods for the physical immobilization of nucleic
`acids have been devised. With supports such as cellulose acetate
`(22), agar (22), and polyacrylamide (23) the nucleic acid is mixed
`with the support in a soluble state and this is followed by a treat-
`ment in which the support is rendered insoluble,
`thereby physically
`trapping the polynucleotide strands within the support matrix.
`For example,
`the DNA-cellulose acetate complex is formed by
`dissolving the two materials in an anhydrous solvent and then
`adding water to co-precipitate the two polymers, while immobili-
`zation in polyacrylamide gels is effected by the polymerization
`of acrylamide in the presence of the polynucleotide. Cellulose-
`DNA is prepared by simply drying a slurry of DNA and cellulose (24).
`Although these methods of immobilization have the advantage of
`V
`simplicity in preparation the complexes formed are subject to
`dissociation and are useful only for the immobilization of nucleic
`acids of large molecular weight where the rate of diffusion from
`the support matrix is relatively slow compared with the time taken
`to complete a particular chromatographic experiment.
`
`APPLICATIONS
`
`There are three main research areas in which immobilized
`
`polynucleotides have been exploited:
`
`l.
`
`2.
`
`3.
`
`Fractionation of polynucleotides and nucleic acids through
`base-paired complex formation.
`
`Study of the mechanism of enzymes involved in the synthesis
`or degradation of nucleic acids.
`
`Isolation of enzymes involved in the synthesis or degrada-
`tion of nucleic acids by affinity chromatography.
`
`Fractionation of Polynucleotides and Nucleic Acids
`
`The base¥pairing properties of cellulose containing terminally~
`linked thymfidine oligonucleotides was first demonstrated with a
`mixture of deoxyadenosine oligonucleotides of chain lengths,
`3-7 (1,2).
`The mixture was fractionated on a column of the
`oligo(dT)—cellulose using an elution procedure involving a step-
`wise temperature gradient.
`The temperature at which each oligo—
`
`Page 10 of 18
`Page 10 of18
`
`
`
`178 p
`
`P. T. GILHAM
`
`nucleotide could be eluted corresponded roughly to the dissociation
`temperature of the.complex formed in solution between the oligo-
`nucleotide and thymidine dodecanucleotide. Oligoribonucleotide
`fragments obtained from viral RNA could also be fractionated on the
`basis of their content of contiguous adenosine sequences by
`chromatography on oligo(dT)-cellulose using temperature gradient
`elution (25).
`
`The initial observation that oligo(dT)—cellulose coubd be
`used to specifically isolate A-rich polynucleotides from the total
`mammalian cellular RNA (26) has led to the widespread use'of the
`material for the isolation and study of messenger RNA species
`that contain covalently-linked poly(A) segments. Examples of the
`application of the method include the isolation of poly(A)—contain—
`ing nuclear RNA of HeLa cells (27),
`the purification of rabbit
`globin mRNA (28),
`the study of the i3 vivo and i3 vitro synthesis
`of po1y(A)-rich RNA by rat brain (29),
`the purification of the
`mRNA coding for a mouse immunoglobin L—chain (30),
`the purification
`of the 143 mRNA coding for the A2 chain of q~crystallin of calf
`lens (31), and the assay of the poly(A) content of yeast mRNA (32).
`
`in the preparation of oligo~
`It should be pointed out that,
`nucleotide—cel1uloses for the isolation of nucleic acids by the
`specific base—pairing mechanism,
`the commercial source of the
`cellulose used is of some importance. Oligo(dT)-cellulose
`synthesized from a cellulose preparation that differed from the
`type originally specified (2) has been shown to be no more effi-
`cient in the binding of poly(A)
`than the unsubstituted cellulose
`itself (33).
`A more recent study (34) of the binding of poly-
`nucleotides to various Celluloses has shown that some cellulose
`
`preparations are capable of binding considerable amounts of poly(A)
`and po1y(I) in the absence of derivatization with oligo(dT) or
`oligo(dC) and it was suggested that this non-specific binding
`probably results from a relatively high lignin content in these
`preparations.
`
`Celluloses containing polynucleotides that have been terminally
`linked by the water-soluble carbodiimide method have also been in-
`vestigated for their ability to selectively bind polynucleotides
`containing complementary sequendbs.
`A comprehensive study of the
`preparation and the binding properties of immobilized oligodeoxy—
`ribonucleotides of defined chain length and sequence has been
`carried out and the results indicate that such materials should
`
`be useful in the isolation of mRNA species containing particular
`oligonucleotide sequences (35,36).
`In another application,
`the
`RNA prepared from the DNA of the simian virus, SV40, has been
`attached to cellulose by the carbodiimide procedure and the product
`was shown to be capable of specifically absorbing that portion of
`fragmented SVAO DNA that contained sequences complementary to the
`
`Page 11 of 18
`Page 11 of18
`
`
`
`IMMOBILIZED POLYNUCLEOTIDES AND NUCELIC ACIDS
`
`*
`
`I79
`
`The technique has some potential as a
`immobilized RNA (37).
`general method for gene isolation since the specificity of the
`absorption could be maintained even in the presence of a large
`excess of bacterial DNA.
`
`immobilization via the periodate oxidation of the
`Covalent
`3'~terminals of polynucleotides has also been used for the isolation
`of complementary polynucleotides. Periodate oxidized E. coli 16S
`rRNA bound to an agarose derivative containing hydrazide groups
`was
`found to be effective in purifying complementary DNA chains
`from a mixture of fragments prepared from sheared E. coli DNA (38).
`A recent novel application involves the use of the complex formed
`between a periodate-oxidized tRNA species and a polyacrylamide gel
`containing hydrazide groups to specifically bind another tRNA
`The
`species possessing a complementary anticodon sequence (39).
`technique constitutes a new approach to the study of complementary
`anticodons as well as a method for the purification of certain
`tRNA species.
`
`The use of polynucleotides immobilized by multiple covalent
`linkages for the isolation of nucleic acids containing comple-
`mentary sequences was first reported by Bautz and Hall (12).
`Bacteriophage T4 DNA was attached to acetylated phosphocellulose
`and the resulting material was used to chromatographically separate
`T4-specific RNA from E, coli RNA. More recent applications of this
`separation technique have made use of supports containing poly-
`nucleotides that have been attached by the cyanogen bromide or UV-
`irradiation methods.
`Poly(U) attached to cyanogen bromide-activated
`agarose has been employed in the isolation of mRNA from KB-cells
`(40), and in the study of the poly(A) segments in HeLa mRNA (41,43).
`
`Myeloma cell mRNA containing»poly(A) sequences has been
`isolated by chromatography on polyvinyl beads containing UV-
`irradiated poly(U)
`(44), and the same absorbent has been used to
`effect a partial purification of tRNA3er from crude E. coli tRNA
`(45).
`In addition, po1y(U)-cellulose prepared by the UV-irradiation
`technique has found use in a number of studies concerned with the
`poly(A) sequences in viral and eukaryotic RNA species (2l,46,47),
`while ribosomal RNA-cellulose prepared by the same method has
`served as an absorbent in the partial purification of ribosomal
`RNA genes from E. subtilis (20). Merriam gt El.
`(48) have pre-
`pared, by the UV-irradiation method, a cellulose complex containing
`DNA from the bacteriophage, 0Xl74, and have used the material for
`the analysis of complementary strands in DNA preparations obtained
`from cells infected with the phage.
`W
`
`The immobilization of single—stranded DNA by physical entrap-
`ment in gels of cellulose acetate or agar produces materials that
`are capable of forming specific complexes with RNA molecules con-
`taining complementary sequences, and this property forms the basis
`
`Page 12 of 18
`Page 12 of 18
`
`
`
`180
`
`P. T. GILHAM
`
`o: a general method for the isolation of RNA species that possess
`sequences that are complementary to DNA from a particular source.
`The immobilization of DNA in agar has been subsequently applied to
`a number of studies on nucleic acids and the techniques of the
`preparation and uses of these materials have been reviewed (49).
`
`Mechanism of Enzyme Action
`
`‘I.-
`
`The methods used for the binding of polynucleotides by
`terminal covalent attachment
`to a support are such that the
`orientation of the bound molecules is known, and this property
`allows the use of these immobilized polymers in the study of the
`action of those enzymes that are associated with the synthesis or
`degradation of nucleic acids. O1igo(dT)-cellulose can be used as
`a substrate primer for terminal deoxynucleotidyl transferase to
`extend the immobilized oligo(dT) chain at its 3'-terminus with a
`covalently-linked po1y(dC) chain (50). This product together with
`soluble poly(dC)
`is capable of forming a bihelical structure with
`a common template-molecule, poly(dI), and the resulting complex
`may be used to assay and study the enzyme, polynucleotide ligase.
`Further studies along these lines have shown that oligo(dT)~
`cellulose serves also as a primer and template for E. coli DNA
`polymerase and as a template for RNA polymerase (51).
`For example,
`the synthesis of poly(dT)—poly(dA) by DNA polymerase in the presence
`of a complex formed between oligo(dT)—cellulose and oligo(dA) yields
`a product in which the poly(dT) chain is covalently bound to the
`cellulose and the poly(dA) chain is hydrogen—bonded to the poly(dT)
`chain.
`
`Polynucleotides with terminal phosphate groups have been
`linked to soluble polysaccharides such as Ficoll and dextran using
`the water—soluble carbodiimide technique, and the resulting macro-
`molecules have been shown to possess some novel properties in their
`use in the study of the action of the enzymes, deoxyribonuclease,
`polynucleotide kinase, and DNA polymerase (52).
`The water—soluble
`carbodiimide technique can also be applied to the attachment of
`fragmented DNA to an insoluble cross~linked dextran and the result-
`ing complex serves as a substrate for pancreatic deoxyribonuclease
`and as a template for RNA polymerase (53). Polynucleotides that
`have been linked at multiple points to cyanogen bromide—activated
`agarose also act as useful substrates in certain biochemical appli-
`cations. RNA-agarose prepared in this way forms the basis of an
`assay procedure for an endonuclease isolated from sea urchin
`embryos
`(16). This nuclease degrades RNA to large polynucleotides
`and is somewhat difficult to assay by the classical methods.
`Poly(I)—agarose and the base-paired complex that it forms with
`poly(C) have been prepared as potential reagents for the study of
`the mechanism of induction of host resistance to viral infection
`
`(15).
`
`Page 13 of 18
`Page 13 0f18
`
`
`
`IMMOBILIZED POLYNUCLEOTIDES AND NUCEUC ACIDS
`
`‘
`
`187
`
`Affinity Chromatography
`
`The method of purification of proteins that exploits their
`capacity to specifically bind to immobilized polynucleotides has
`found extensive application in the study of those enzymes that
`bind to DNA.
`Initial applications of the affinity chromatographic
`method in nucleic acid research have employed DNA physically
`immobilized in agarose (54) or cellulose (24), and DNA imobi—
`lized on cellulose by UV—irradiation (18). These chromatographic
`materials have been used for the purification of endonuclease I
`and exonucleases I and II (54), DNA polymerase (l8,24), RNA poly»
`merase (24), and the gene 32~protein of bacteriophage T4 (24).
`The methods for the preparation and use of DNA-cellulose together
`with other applications of the chromatographic method have been
`reviewed by Alberts and Herrick (55).
`DNA immobilized in poly-
`acrylamide gel has also been used for the purification of DNA
`polymerase (56). Many of the enzymes that are associated with the
`synthesis of nucleic acids possess the capacity to bind to single-
`stranded DNA and the use of single stranded DNA—agarose for the
`preparative purification of E, coli DNA polymerases I and II, RNA
`polymerase, exonuclease III, and bacteriophage T4 polynucleotide
`kinase has recently been described (57).
`
`The covalent attachment of DNA to agarose by the cyanogen
`bromide procedure also Phoduces materials that are suitable for
`affinity chromatography.
`DNA polymerase (14) and deoxyribonucleases
`from pancreas and spleen (58) have been purified by this method.
`In two other applications,
`the complex formed by the UV-irradiation
`immobilization of bacteriophage f2 RNA on cellulose has served in-
`the chromatographic purification of the poly(G) polymerase obtained
`from f2-infected E. coli cells (19), and oligo(dT)-cellulose, pre-
`pared by the carbodiimide procedure, has provided a purification
`method for the RNA-dependent DNA polymerase from RNA tumor
`viruses (59).
`
`Immobilized tRNA has been employed in a number of studies on
`the isolation of specific aminoacyl-tRNA synthetases.
`The
`chromatographic absorbent used in two of these studies was pre-
`pared by the periodate oxidation of a particular tRNA species and
`the subsequent covalent binding of the product
`to a support contain—
`ing hydrazide groups. Affinity chromatographic purifications have
`been effected with tRNAVa1 or tRNAlyS bound to a polyacrylhydra—
`zide—agar mixture (60), and with tRNAPhe attached to hydrazinyl-
`agarose (8).
`,In the latter case,
`the chromatographic method per-
`mitted thegisolation of yeast phenylalanyl—tRNA synthetase in a
`completelyhpure statea Aminoacyl—tRNA may be immobilized by the
`covalent binding of the amino group of the aminoacyl moiety to an
`activated support. E, coli iso1eucyl—tRNA can be bound to bromo—
`acetamidobutyl—agarose and the resulting complex serves as a
`chromatographic system for the purification of the corresponding
`tRNA synthetase (61).
`
`Page 14 of 18
`Page 14 of 18
`
`
`
`182
`
`P. T. GILHAM
`
`CONCLUSION
`
`It is apparent from the foregoing discussion that, for a
`particular study,
`the choice of the method of immobilization
`depends,
`to a large extent, on the nature of the subsequent experi-
`mental application of the immobilized polymer. As with the use of
`other immobilized substances some consideration should be given to
`the stability of the linkage to the support and the steriq‘avail—
`ability of the attached polynucleotide for specific interactions
`with molecules in solution.
`In certain applications involving
`the use of covalently immobilized polynucleotides the methods
`that produce terminal attachment would seem to offer some advan-
`tages over those employing multi—point attachment.
`In studies
`concerned with the assay and the mechanism of action of nucleic
`acid—associated enzymes the methods employing single point attach-
`ment at one of the polynucleotide terminals are desirable since
`they yield products in which the polarity of the attached polymer
`is known and in which there is likely to be the least steric inter-
`ference from the support matrix.
`In the isolation, by the base-
`pairing mechanism, of nucleic acids possessing homopolymer
`sequences, supports containing complementary homopolymers immobi-
`lized by single or multi—point attachment seem equally effective.
`In the future however, more sophisticated separations of nucleic
`acids based on the base-pairing of more complex complementary
`sequences will be attempted and,
`in these cases,
`the use of syn-
`thetic oligonucleotides of defined sequence, attached to a support
`at their terminals, may be preferred.
`In this regard, it is of
`interest that a study of the binding capacity of synthetic oligo—
`nucleotides bound to cellulose at their 5'-terminals by the water-
`soluble carbodiimide method indicates that the entire oligonucleo-
`tide,
`in each case,
`is apparently available for base-paired complex
`formation with its complementary nucleotide sequence (36)a
`
`Future applications of supports containing covalently—linked
`polynucleotides are likely to add more emphasis to the importance
`of the nature of the covalent
`linkage itself.
`In the case of multi-
`point imobilized polymers some applications may require a con-
`sideration of the number of attachment points per polynucleotide
`chain that are formed during the immobilization procedure.
`For
`example, RNA—agarose prepared by the cyanogen bromide procedure has
`been used to study an endonuclease that degrades RNA to large
`oligonucleot