IMMOBILIZED
`BIOCHEMICALS AND
`AFFINITY CHROMATOGRAPHY
`
`Edited by
`R. Bruce Dunlap
`
`Department of Chemistry
`University of South Carolina
`Columbia, South Carolina
`
`PLENUM PRESS • NEW YORK AND LONDON
`
`Page 1 of 18
`
`HOLOGIC EXHIBIT 1019
`Hologic v. Enzo
`
`

`
`Library of Congress Cataloging in Publication Data
`
`Symposium on Affinity Chromatography and Immobilized Biochemicals, Char(cid:173)
`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.
`L Biological chemistry- Technique- Congresses. 2. Affinity chromatography
`- Congresses. I. Dunlap, Robert Bruce, 1942-
`ed. II. Series. [DNLM: 1. Bio.
`chemistry- Congresses. 2. Chromatography, Affinity- Congresses WI ADS 59 v.
`42 1974 I QD271 S986i 1973.]
`QP519.7.S9 1973
`ISBN 0-306-39042-6
`
`574.1'92'028
`
`74-7471
`
`Proceedings of the symposium on Affinity Chromatography and Immobilized
`Biochemicals held in Charleston, South Carolina, November 7. 9, 1973
`
`© 1974 Plenum Press, New York
`A Division of Plenum Publishing Corporation
`227 West 17th Street, New York, N.Y. 10011
`
`United Kingdom edition published hy Plemun.Press, London
`· A Division of Plenum Publishing Company, Ltd.
`4a 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 hy any means, eledronic, mechanical, photocopying, mierofilming,
`recording, or otherwise, without written permission from the Publisher
`
`Printed in the United States of Amel'icu
`
`Page 2 of 18
`
`

`
`Contents
`
`PART ONE: AFFINITY CHROMATOGRAPHY
`
`Affinity Chromatography--Old Problems and
`New Approaches .
`,
`.
`,
`,
`.
`.
`.
`.
`.
`,
`,
`Steven C. M~ch, Indu P~Qh,
`and Pedho Cuatnecaoao
`
`,
`
`.
`
`.
`
`.
`
`,
`
`.
`
`.
`
`.
`
`.
`
`3
`
`Affinity Chromatography. New Approaches for the
`Preparation of Spacer Containing Derivatives and for
`Specific Isolation of Peptides .
`.
`.
`.
`.
`,
`,
`Mu!r. WilcheQ
`
`.
`
`.
`
`.
`
`.
`
`Quantitative Parameters in Affinity Chromatography .
`A. H. NMh-[Qawa, P. Bailon, and A. H. Ramel.
`
`Non-Specific Binding of Proteins by Substituted
`Agaroses
`.
`,
`.
`.
`.
`.
`. . . . . . . . . . . . . .
`B. H. J. Hon~tee
`
`A Solid Phase Radioimmune Assay for Ornithine
`Transcarbamylase .
`,
`.
`,
`.
`Vona£d L. E6henbaugh, Vona£d Se~
`and EJU.c Jamv.,
`
`Purification of Acetylcholinesterase by
`Covalent Affinity Chromatography .
`.
`Ho~ton F. Vo~~, Y. A~ha~, and
`IJWJ)_n B. Will on
`
`Cooperative Effects of AMP; ATP, and Fructose
`1,6-Diphosphate on the Specific Elution of Fructose
`1,6-Diphosphatase from Cellulose Phosphate . . ,
`Jo~eph Mend)_Q)_no and H~~un Abou-I~~a
`
`An Analy~is of Affinity Chromatography Using
`.
`.
`.
`.
`Immobilised Alkyl Nucleotides
`.
`.
`.
`.
`.
`P. V. G. Dean, V. B. CJr.aven, M. J. H~vey,
`and c. R. Lowe
`
`ix
`
`15
`
`33
`
`43
`
`61
`
`75
`
`85
`
`99
`
`Page 3 of 18
`
`

`
`X
`
`CONTENTS
`
`Affinity Chromatography of Kinases and Dehydrogenases
`on Sephadex and Sepharose Dye Derivatives • . • .
`. •
`Uc.hMd L. EcuteJr..day an.d In.geJr.. M. EcuteJr..day
`
`Affinity Chromatography of Thymidylate Synthetases Using
`5-Fluoro-2'-Deoxyuridine 5 1 -Phosphate Derivatives of
`Sepharose .
`.
`.
`.
`.
`.
`.
`.
`. • .
`.
`.
`.
`.
`, • • . • • . • . •
`John. M. Whitely, Ivan.ba JeJr..kun.ica,
`· an.d Thomcu VU:t:.!.:,
`
`The Biosynthesis of Riboflavin: Affinity Chromatography
`Purification of GTP-Ring-Opening Enzyme
`.
`• . • .
`.
`L. PJr.eJ.Jton. MeJr..ceJr.. an.d Ch(url.eJ.J M. Baugh
`
`Purification of Tyrosine-Sensitive 3-Deoxy(cid:173)
`D-Arabino-heptulosonate-7-Phosphate and
`Tyrosyl-tRNA Synthetase on Agarose Carrying
`Carboxyl-Linked Tyrosine
`• • • .
`.
`.
`. • .
`An.d!r.0W R. Gattopo, P~p S. Kot.¢~opouto~,
`an.d Scott C. Moh!r.
`
`Structural Requirement of Ligands for Affinity
`Chromatography Absorbents: Purification of
`Aldehyde and Xanthine Oxidases
`. • .
`.
`AlbeJr..t E. Chu and St~ng Chay~n.
`
`PART TWO:
`
`IMMOBILIZED BIOCHEMICALS
`
`123
`
`135
`
`147
`
`157
`
`165
`
`Immobilized Polynucleotides and Nucleic Acids .
`P. T. Gilham
`
`.
`
`.
`
`.
`
`. • .
`
`173
`
`Immobilized Cofactors and Multi-Step Enzyme-Systems . • .
`
`.
`
`187
`
`Kla~ Mo~bac.h
`
`Preparation, Characterization, and Applications of
`Enzymes Immobilized on Inorganic Supports . • .
`H. H. Wee;tatt
`
`Lactase Immobilized on Stainless Steel and Other
`Dense Metal and Metal Oxide Supports . . . . . •
`M. Cha.Jr.leJ.J, R. W. Coug~n, B. R. Allen.,
`E. K. PMuc.hu~, and F. X. Hcu~etbeJr..geJr..
`
`191
`
`213
`
`The Use of Membrane-Bound Enzymes in an Immobilized
`. • .
`, • .
`.
`.
`.
`.
`.
`.
`Enzyme Reactor
`Cha.Jr.leJ.J C. Wo!dhln.gto n.
`
`.
`
`.
`
`.
`
`.
`
`235.
`
`Page 4 of 18
`
`

`
`CONTENTS
`
`xi
`
`The Optimization of Porous Materials for Immobilized
`Enzyme Systems • • • • • • • • .
`. •
`• ,
`. • . • • .
`Vav,Ld L. Ea;ton.
`
`.
`
`.
`
`241
`
`Water Encapsulated Enzymes in an Oil-Continuous
`Reactor: Kinetics and Reactivity
`.
`, .•
`R. 1. Lea.v,Ltt, F. X. Ryan., an.d W. P. B~g~~
`
`Analysis of Reactions Catalyzed by Polysaccharide(cid:173)
`Enzyme Derivatives in Packed Beds
`• .
`.
`.
`.
`M. H. Key~ an.d F. E. SemeMk.y
`
`259
`
`269
`
`The Preparation of Microenvironments for Bound
`• . • • .
`Enzymes by Solid Phase Peptide Synthesis
`Jam~ B. Tay.tOJt an.d Hcuw.td E. Sw~good
`
`. •
`
`283
`
`Optimization of Activities of Immobilized Lysozyme,
`a-Chymotrypsin, and Lipase .
`.
`. •
`. . . . • •
`Ra;t!Un. Va;t;ta an.d Vav,Ld F. 0~
`
`Chemical Modification of Mushroom Tyrosinase
`for Stabilization to Reaction Inactivation .
`Vav,Ld Lero an.d Theodo!Le ChMe, J11..
`
`Chain Refolding and Subunit Interactions in
`Enzyme Molecules Covalently Bound to a Solid Matrix
`H. Robe!l.t Hofl.ton. an.d Ha11.o.td E. sw~good
`
`Immobilization of Lipase to Cyanogen Bromide
`Activated Polysaccharide Carriers
`Pau.t Me.t,L~ an.d B,L-Chon.g Wan.g
`"
`Use of Immobilized Enzymes for Synthetic Purposes
`Vav,Ld L. MaM haU
`
`Index
`
`1
`
`1
`
`t
`
`I
`
`I
`
`I
`
`a
`
`0
`
`I
`
`0
`
`I
`
`I
`
`0
`
`I
`
`I
`
`I
`
`I
`
`0
`
`I
`
`I
`
`I
`
`0
`
`I
`
`I
`
`I
`
`0
`
`0
`
`293
`
`317
`
`329
`
`33'9
`
`345
`
`369
`
`Page 5 of 18
`
`

`
`IMMOBILIZED POLYNUCLEOTIDES AND NUCLEIC 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(cid:173)
`tion:
`the fractionation of nucleic acids and polynucleotides
`through base-paired complex formation and the isolation and puri(cid:173)
`fication of nucleic acid-associated enzymes by affinity chromatog(cid:173)
`raphy. There are now a number of methods for the preparation of
`immobilized polynucleotides and the potential uses of these mater(cid:173)
`ials depend to some extent on the means by which the polymers are
`attached to the insoluble supports.
`
`METHODS OF IMMOBILIZATION
`
`As indicated in the following table, the methods for the pre(cid:173)
`paration of immobilized polynucleotides can be broadly divided into
`two categories:
`those involving covalent linkages between the poly(cid:173)
`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(cid:173)
`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 in~which 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(cid:173)
`odate oxidation of ribopolynucleotides. Condensation of these dial(cid:173)
`dehyde functions with supports containing primary amine or hydrazide
`
`173
`
`Page 6 of 18
`
`

`
`174
`
`P. T. GilHAM
`
`1.
`
`Covalent Binding at the Polynucleotide Terminals,
`
`Table I
`
`(a) Activation of terminal phosphate group.
`(b) Periodate oxidation of 3'-terminals in RNA species.
`(c) Condensation of RNA 3'-terminals with supports con-
`taining dihydroxyboryl groups.
`
`2.
`
`Covalent Binding at Multiple Points.
`
`(a) Condensation with phosphocellulose,
`(b). 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(cid:173)
`nucleotide (1,2). For example, the treatment of thymidine 5 1 -phos(cid:173)
`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(cid:173)
`tions of the support.
`In this case the oligonucleotides are bound
`to the support through stable phosphodiester linkages at their 5 1
`-
`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
`
`

`
`IMMOBILIZED POl YNUCLEOTIDES 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-cyclohexyl-N'-~-(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 immobilized 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(cid:173)
`ribonucleotides and RNA can be exploited in two ways for the
`terminal binding of these molecules. Periodate oxidation of the
`dial moiety to the corresponding dialdehyde yields a product that
`can undergo condensation with aminoethylcellulose, and the ~esulting
`linkage can then be stabilized by reduction with sodium bore(cid:173)
`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 1
`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(cid:173)
`zinyl-Sepharose (8).
`
`-
`
`Another method for the terminal binding of polyribonucleotides
`arises out of the observation that polymers that possess terminal
`cis dial groups are capable of forming specific complexes with
`supports containing covalently-bound dihydroxyboryl groups (9-11).
`N-CN'-(m-Dihydroxyborylphenyl)succinamyl)aminoethylcellulose (9)
`prepared from the condensation of N-(m-dihydroxyborylphenyl)(cid:173)
`succinamic acid with aminoethylcellulose forms cyclic boronate
`structures at pH 8-9 with molecules containing the ribonucleoside
`dial group.
`In certain applications immobiHzed polynucleotides
`prepared b~ this method may have some advantages over those
`mentioned a\ove 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
`
`

`
`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(cid:173)
`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 polynucleot~de 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).
`
`More recently, the immobilization technique that has been
`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(cid:173)
`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(cid:173)
`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 matri.x.
`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
`
`

`
`IMMOBILIZED POLYNUCLEOTIDES AND NUCELIC ACIDS
`
`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(cid:173)
`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(cid:173)
`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
`·
`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 ~romatographic experiment.
`
`APPLICATIONS
`
`There are three main research areas in which immobilized
`polynucleotides have been exploited:
`
`1.
`
`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(cid:173)
`linked thy~adine 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(cid:173)
`wise temperature gradient. The temperature at which each oligo-
`
`Page 10 of 18
`
`

`
`178
`
`P. T. GILHAM
`
`nucleotide could be eluted corresponded roughly to the dissociation
`temperature of the complex formed in solution between the oligo(cid:173)
`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 caul~ 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(cid:173)
`ing nuclear RNA of HeLa cells (27), the purification of rabbit
`globin mRNA (28), the study of the in vivo and in vitro synthesis
`of poly(A)-rich RNA by rat brain (29), the purification of the
`mRNA coding for a mouse immunoglobin L-chain (30), the purification
`of the 14s mRNA coding for the A2 chain of ~-crystallin of calf
`lens (31), and the assay of the poly(A) content of yeast mRNA (32).
`
`It should be pointed out that, in the preparation of oligo(cid:173)
`nucleotide-celluloses 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(cid:173)
`cient in the binding of poly(A) than the unsubstituted cellulose
`itself (33). A more recent study (34) of the binding of poly(cid:173)
`nucleotides to various celluloses has shown that some cellulose
`preparations are capable of binding considerable amounts of poly(A)
`and poly(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(cid:173)
`vestigated for their ability to selectively bind polynucleotides
`containing complementary sequen~s. A comprehensive study of the
`preparation and the binding properties of immobilized oligodeoxy(cid:173)
`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 SV40 DNA that contained sequences complementary to the
`
`Page 11 of 18
`
`

`
`IMMOBILIZED POL YNUCLEOTIDES AND NUCELIC ACIDS
`
`179
`
`immobilized RNA (37). The technique has some potential as a
`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.
`
`Covalent immobilization via the periodate oxidation of the
`3'-terminals of polynucleotides has also been used for the isolation
`of complementary polynucleotides. Periodate oxidized E. coli 168
`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
`species possessing a complementary anticodon sequence (39). The
`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(cid:173)
`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 ~· coli RNA. More recent applications of this
`separation technique have made use of supports containing poly(cid:173)
`nucleotides that have been attached by the cyanogen bromide or uv(cid:173)
`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(cid:173)
`irradiated poly(U) (44), and the same absorbent has been used to
`effect a partial purification of tRNAser from crude ~· coli tRNA
`(45).
`In addition, poly(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 (21,46,47),
`while ribosomal RNA-cellulose prepared by the same method has
`served as an absorbent in the partial purification of ribosomal
`RNA genes from B. subtilis (20). Merriam et al. (48) have pre(cid:173)
`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.
`'{
`The immobilization of single-stranded DNA by physical entrap(cid:173)
`ment in gels of cellulose acetate or agar produces materials that
`are capable of forming specific complexes with RNA molecules con(cid:173)
`taining complementary sequences, and this property forms the basis
`
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`

`
`180
`
`P. T. GILHAM
`
`of 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
`
`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. Oligo(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 poly(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)(cid:173)
`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(cid:173)
`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(cid:173)
`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(cid:173)
`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
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`

`
`IMMOBILIZED POLYNUCLEOTIDES AND NUCELIC ACIDS
`
`181
`
`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 immobi(cid:173)
`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 (18,24), RNA poly(cid:173)
`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(cid:173)
`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(cid:173)
`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 produces 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(cid:173)
`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(cid:173)
`pared by the periodate oxidation of a particular tRNA species and
`the subsequent covalent binding of the product to a support contain(cid:173)
`ing hydrazide groups. Affinity chromatographic purifications have
`been effected with tRNAval or tRNAlys bound to a polyacrylhydra(cid:173)
`zide-agar mixture (60), and with tRNAphe attached to hydrazinyl(cid:173)
`agarose (8).
`In the latter case, the chromatographic method per(cid:173)
`mitted the.isolation of yeast phenylalanyl-tRNA synthetase in a
`completely'{pure state, Aminoacyl-tRNA may be immobilized by the
`covalent binding of the amino group of the aminoacyl moiety to an
`activated support. !· coli isoleucyl-tRNA can be bound to bromo(cid:173)
`acetamidobutyl-agarose and the resulting complex serves as a
`chromatographic system for the purification of the corresponding
`tRNA synthetase (61).
`
`Page 14 of 18
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`

`
`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(cid:173)
`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 steric~avail­
`ability of the attached polynucleotide for specific inter~ctions
`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(cid:173)
`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(cid:173)
`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(cid:173)
`ference from the support matrix.
`In the isolation, by the base(cid:173)
`pairing mechanism, of nucleic acids possessing homopolymer
`sequences, supports containing complementary homopolymers immobi(cid:173)
`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(cid:173)
`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(cid:173)
`nucleotides bound to cellulose at their 5 1 -terminals by the water(cid:173)
`soluble carbodiimide method indicates that the entire oligonucleo(cid:173)
`tide, in each cas