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Analysis of ribonuclease-nucleotide interactions by quantitative affinity chromatography
Journal article   Open access   Peer reviewed

Analysis of ribonuclease-nucleotide interactions by quantitative affinity chromatography

I M Chaiken and H C Taylor
The Journal of biological chemistry, v 251(7), pp 2044-2048
10 Apr 1976
DOI: https://doi.org/10.1016/S0021-9258(17)33652-9
PMID: 1270420
url
https://doi.org/10.1016/s0021-9258(17)33652-9View
Published, Version of Record (VoR) Open
url
https://doi.org/10.1016/S0021-9258(17)33652-9View
Published, Version of Record (VoR) Open

Abstract

Binding Sites Binding, Competitive Chromatography, Affinity Kinetics Mathematics Osmolar Concentration Protein Binding Ribonucleases - isolation & purification Ribonucleases - metabolism Ribonucleotides - metabolism
Quantitative affinity chromatography on uridine-5'-(Sepharose-4-aminophenylphosphoryl)-2'(3')-phosphate was developed for the study of binding of ribonuclease species to nucleotide ligands. Elution of the native species ribonuclease-A and -S on the afffinity matrix in 0.4 M ammonium acetate, pH 5.2, containing various amounts of the soluble competing ligand 2'-cytidine monophosphate, reveals an inverse response of elution volume to concentration of soluble ligand. This response conforms to behavior expected for the competing binding equilibria enzyme-soluble ligand and enzyme-insoluble ligand. A-NALYSIS OF ELUTION DATA ALLOWS CALCULATION OF KI and KIM, the dissociation constants, respectively, for the soluble and insoluble protein-ligand complexes. The values of these chromatographically derived constants are similar to values of dissocation constants determined in solution by kinetics of inhibition by 2'-cytidine monophosphate and uridine-5'-(j-aminophenylphosphoryl)-2'(3')-phosphate. Successful competitive elution experiments with [p-F-Phe8]semisynthetic ribonuclease-S' and individual elution trials for [4-F-His12]semisynthetic ribonuclease-S' indicate the utility of the quantitative affinity chromatographic technique for determination of ligand binding properties of ribonuclease derivatives, including inactive species. Nonbiospecific aspects of the interaction of ribonuclease with the affinity matrix in ammonium acetate buffers of concentrations 0.1 M and below were noted, delinating limits of conditions allowing the biospecificity needed for ligand-binding analyses by competitive elution. The dependence of ribonuclease competitive elution behavior on the amount of protein eluted also was examined and related to theoretical considerations in the quantitative application of affinity chromatography.

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