The effect of thiol compounds on the autolysis of papain

Incubation of papain with 3--17.5 mM dithiothreitol and dithioerythritol at pH 8.5 causes inactivation owing to autolysis. Such inactivation is not observed on incubation with equivalent concentrations of mercaptoethanol, cysteine or 2,3-dimercaptopropanol. The inactivation rate is independent of papain concentration within the range of 0.5--2% and is proportional to dithiothreitol concentration. This is in agreement with a sequence of two reactions: (Formula: see text) the first reaction being rate-limiting. S-Carboxymethyl-papain (I) and S-carboxamidomethyl-papain (II) were incubated with 18 mM dithiothreitol at pH 8.5 and, after stopping the reaction with iodoacetic acid, were subjected to gel electrophoresis. Electrophoretograms of both I and II exhibited a small new band attributable to a species with one disulphide bond reduced and carboxymethylated. The new band on II was more pronounced than that of I. It is argued that (Formula: see text) is a papain species with one reduced disulphide bond, sensitive to proteolytic attack by native papain.