Kinetic Characterization of Affinity Chromatography Purified Clostripain

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Abstract

The cysteine protease clostripain, purified by affinity chromatography on a large scale, shows very high activity against BAEE using the titrimetric standard assay. Furthermore, titration of the active site with the irreversible inhibitor tosyl-lysyl-chloromethane resulted in a more than two times higher specific activity compared with literature data (Porter et al. (1971)J. Biol. Chem.246, 7675-7682). Based on the molar enzyme concentration determined, the hydrolysis kinetics of the standard substrate BAEE were compared with those for theNα-protected dipeptide ester Mal-Tyr-Arg-OEt. It was demonstrated from the kinetic data that the highly purified clostripain is the most active enzyme preparation available up to now. In contrast to the standard substrate, Mal-Tyr-Arg-OEt shows a threefold lower specificity constant.

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