As a first step in the investigation of papain catalysis using subzero temperatures to detect, accumulate, and characterize enzyme-substrate intermediates, we have studied some potential cryosolvents and carried out preliminary intermediate trapping experiments. The effects of subzero temperatures and aqueous dimethyl sulfoxide solutions on the papain-catalyzed hydrolysis of Nalpha-carbobenzoxy-L-lysine p-nitrophenyl ester have been investigated in detail. At 0 degrees C, the value of kcat decreases with increasing dimethyl sulfoxide concentration, decreasing in proportion to the decreased water concentration; however, the value of Km increases exponentially. The effect on Km can be accounted for by a combination of both dielectric and competitive inhibition effects. The Arrhenius plot for the deacylation reaction in 7.65 M (60% v/v) dimethyl sulfoxide is linear over the temperature range 0 to -45 degrees C and extrapolates to a calculated value of kcat at 25 degrees C in excellent agreement with that obtained in the absence of organic solvent. The pH-rate profile is not substantially perturbed by the presence of 7.65 M dimethyl sulfoxide. At -45 degrees C and below, turnover occurs extremely slowly, and is essentially negligible, although acylation is still quite rapid. Consequently, the acyl enzyme, Na-carbobenzoxy-L-lysyl-papain, can be readily accumulated and trapped at temperatures below -50 degrees C. At these low temperatures, under conditions of excess substrate, the amount of p-nitrophenol liberated in the acylation reaction is equivalent to the active-site normality of the enzyme, indicating a 1:1 stoichiometry in formation of the acyl enzyme. The effect of dimethyl sulfoxide up to 7.65 M, on the intrinsic ultraviolet, fluorescence, and circular dichroic properties of the enzyme shows no evidence of any solvent-induced structural changes. All experimental observations are consistent with the conclusion that 7.65 M dimethyl sulfoxide and subzero temperatures have no deleterious effects on papain-catalyzed reactions. A related series of experiments indicate that aqueous ethanol cryosolvents up to 13.7 M (80% v/v) are also suitable. Preliminary experiments at subzero temperatures using Na-carbobenzoxy-L-lysine methyl ester suggest the existence of three enzyme-substrate intermediates which can be detected and accumulated.