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: 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 is a papain species with one reduced disulphide bond, sensitive to proteolytic attack by native papain