KINETIC CHARACTERIZATION OF AFFINITY-CHROMATOGRAPHY PURIFIED CLOSTRIPAIN

The cysteine protease clostripain, purified by affinity chromatography on a large scale, shows very high activity against BAEE using the tit rimetric standard assay. Furthermore, titration of the active site wit h the irreversible inhibitor tosyl-lysyl-chloromethane resulted in a m ore than two times higher specific activity compared with literature d ata (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 the N-alpha-prot ected dipeptide ester Mal-Tyr-Arg-OEt. It was demonstrated from the ki netic data that the highly purified clostripain is the most active enz yme preparation available up to now. In contrast to the standard subst rate, Mal-Tyr-Arg-OEt shows a threefold lower specificity constant.