Affinity chromatography of trypsin and related enzymes. V. Basic studies of quantitative affinity chromatography

Journal of Biochemistry
Kenichi Kasai, S Ishii

Abstract

A detailed study of the quantitative affinity chromatography of trypsin [EC 3.4.21.4] is reported here. Frontal chromatography using an enzyme solution of very low concentration on an affinity adsorbent gave the dissociation constant of the enzyme-immobilized ligand complex (Kd). Kd values determined under various conditions enabled us to discuss in detail the interaction of trypsin and affinity adsorbents (mainly Gly-Gly-Arg Sepharose). The pH dependence of Kd was consistent with that of the interaction of trypsin and product-type compounds. The effects of changes in temperature, ionic strength, dielectric constant, etc., were also studied. The Ki values of soluble competitive inhibitors can be determined by analysis of their effects on the elution volume of the enzyme. The values obtained were in good agreement with those obtained by kinetic analysis. The present method proved to be useful as a general procedure to investigate the interaction of a protein and a specific ligand.

References

Jun 1, 1984·Applied Biochemistry and Biotechnology·B M Dunn
Aug 2, 2001·Glycobiology·A Kobata
Aug 30, 2014·Proceedings of the Japan Academy. Series B, Physical and Biological Sciences·Kenichi Kasai
Jun 1, 1992·Journal of Molecular Recognition : JMR·N P Burton, C R Lowe
Apr 1, 1988·Journal of Molecular Recognition : JMR·T KumazakiS Ishii

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