Hydrophobic Immobilization of Enzymes and Polynucleotides on Trityl Agarose

Cashion, P.; Javed, Ali; Lentini, V.; Harrison, Dolores L.; J, Seeley; Sathe, Ganesh M.

doi:10.1007/978-1-4615-9290-7_46

Cited by 2 publications

(8 citation statements)

References 3 publications

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“…Similar patterns are seen (using 3H uridine as label) in RNA extracted from Hela cells, polio virus-infected Hela cells, WI38 human fibroblasts, both normal and SV-40 infected and murine liver (21). Sucrose gradient analysis of the non-binding HS RNA fractions generally show 3 peaks corresponding to 4-5S,18S and 28S rRNA.…”

Section: Resultssupporting

confidence: 60%

“…The resulting complex will bind to the tritylated column as efficiently as in figure 3B. Experiments on the binding of lyso-*zyme to 6'-trityl N-acetyl glucosamine, the latter being prebound to TS, qualitatively resembl-e those described in figure 3 (21). However the intrinsic binding constant between enzyme and monomeric sugar is too low (59) to give a useful retention of enzyme.…”

Section: Experiments Resultsmentioning

confidence: 81%

“…This, in turn, suggests corresponding differences in the mechanisms whereby poly A is bound by the respective columns. Hence, the similarity between dT cellulose and TRs in binding poly A at HS and releasing it at LS is probably only coincidental when viewed in the context of more detailed comparative experiments (21). The implication of these experiments is that, in contrast to the well known H bonded associations of dT.cellulose, poly A binds to TRs by means of distinctive hydrophobic, base-stacking forces.…”

Section: Resultsmentioning

confidence: 99%

“…A detailed comparison of poly A binding to dT cellulose and to TRs over a wide range of conditions including: different types of detergents (footnote 5, table 1), a variety of neutral Hofmeister-type salts, different pH and temperature conditions, shows pronounced differnces between the two (21). This, in turn, suggests corresponding differences in the mechanisms whereby poly A is bound by the respective columns.…”

Section: Resultsmentioning

confidence: 99%

“…TrCl, MTrCl and DMTrCl were from Aldrich Chemical Co.; cellulose powder (CF 11) was from Whatman; sepharose CL-4B was from Pharmacia, as were phenyl and n-octyl sepharoses. Neither of the latter derivatives (_40vM/ml) bound poly A (21). Most homoand hetero-polynucleotodes were from Sigma Chemical Co., otherwise, they were synthesized by us with PNP'ase as described below.…”

Section: Methodsmentioning

confidence: 99%

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Hydrophobic affinity chromatography of nucleic acids and proteins

et al. 1980

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5' tritylated oligonucleotides binding hydrophobically to low trityl cellulose/sepharose (< 15 microMTr/ml) retain their hydrogen-bonding specificities for complementary sequences. This, constitutes a novel mode of attaching affinity ligands to solid supports, is more convenient than existing methods, and proceeds with 100% yield. The salt, dielectric constant and temperature dependence of these non-covalently anchored ligands permits the isolation of a variety of RNAs including fibroin mRNA. Medium trityl sepharose (15-40 microM Tr/ml) has a high binding specificity for poly A and poly A containing mRNA, equivalent to dT cellulose. Most proteins, including nucleic acid enzymes, bind to these columns and retain enzymatic activity, thus mimicking enzymes attached covalently to solid phases. A number of in vivo counterparts to this hydrophobically determined specificity are noted, as are homologies to nitro-cellulose filters.

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Section: Resultssupporting

confidence: 60%

Section: Experiments Resultsmentioning

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Hydrophobic affinity chromatography of nucleic acids and proteins

et al. 1980

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The preparation and stability of immobilized polynucleotide kinase

Hutchinson

Collier

1987

Biotech & Bioengineering

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The binding of enzymes to insoluble supports has attracted considerable attention in recent years since immobilised enzymes frequently have superior properties compared with soluble enzymes, rendering them particularly useful for synthetic and analytical purposes. Many methods exist for the immobilization of enzymes,' but usually one of four methods have been used: 1) entrapment, 2) copolymerization, 3) covalent attachment, or 4) physical adsorption, e.g. by hydrophobic forces.We have been using bacteriophage T4 polynucleotide kinase (PNK) to prepare radioactivity labelled nucleotides and find that the immobilized enzyme offers several advantages over the soluble form, e.g., stability and ease of handling. We now report our investigations on the immobilization of PNK on various supports and the stabilities of these preparations. Preliminary studies on the hydrophobic immobilization of PNK have been reported2 but these investigations were part of a wider survey on the hydrophobic immobilization of enzymes and few details were published. MATERIALS AND METHODS MaterialsBacteriophage T4 polynucleotide kinase [E.C.2.1.7.78] was supplied as an aqueous solution (SO00 U/M1) containing bovine serum albumin [0.05% (w/v)] by Amersham International plc (Amersham England). Octyland phenyl-Sepharoses and cyanogen bromide-activated Sepharose were obtained from Pharmacia Ltd (Milton Keynes, England). The "C,-series" kit for hydrophobic chromatography, consisting of six I-mL bed volume columns containing agarose to which n-alkyl

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Hydrophobic Immobilization of Enzymes and Polynucleotides on Trityl Agarose

Cited by 2 publications

References 3 publications

Hydrophobic affinity chromatography of nucleic acids and proteins

Hydrophobic affinity chromatography of nucleic acids and proteins

The preparation and stability of immobilized polynucleotide kinase

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