Affinity chromatography of trypsin and related enzymes. IV. Quantitative comparison of affinity adsorbents containing various arginine peptides

Journal of Biochemistry
M NishikataS I Ishii

Abstract

In order to study the mechanism of substrate binding of trypsin by affinity chromatography, we synthesized various L-arginine-terminated oligopeptides having different chain length and amino acid sequences, and immobilized them on agarose gel. The interaction of beta-trypsin with these adsorbents was studied by a quantitative affinity chromatographic procedure which gave the dissociation constant (Kd) of the trypsin-immobilized ligand complex. This procedure proved to be very useful and to give information equivalent to that obtained by kinetic procedures. The contribution of the amino acid residue at P2 of the ligands to the affinity was studied by using tripeptide (Gly-X-Arg) Sepharoses, and alanine was found to be more effective than glycine or valine. This conclusion was supported by a kinetic experiment in which Ki values of the corresponding soluble tripeptides (Ac-Gly-X-Arg) were determined. A significant decrease in Kd was observed when the ligand was elongated from dipeptide to tripeptide. However, Kd decreased only slightly when the ligand was elongated further. This suggests that a tripeptide is sufficiently long as a ligand. On the basis of these results, the mode of substrate binding of trypsin is discussed.

Citations

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
Jun 1, 1984·Applied Biochemistry and Biotechnology·B M Dunn

Related Concepts

Arginine hydrochloride
Chromatography, Affinity
Mathematics
Oligopeptides
Trypure

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