Some problems in Michaelis-Menten kinetic analysis of benzpyrene hydroxylase in hepatic microsomes from polycyclic hydrocarbon-pretreated animals

Several difficulties which severely compromised the interpretation of hepatic microsomal benzpyrene hydroxylase activity according to Michaelis-Menten kinetics were encountered. With hepatic microsomes from control, benzpyrene-treated or 3-methylcholanthrene-treated rats or mice, the apparent K m of benzpyrene hydroxylase was dependent upon the microsomal protein concentration in the incubation mixture. When hepatic microsomes from benzpyrene- or 3-methylcholanthrene-treated rats were used, accurate estimations of initial reaction velocities were difficult to make due to rapid metabolic depletion of substrate and subsequent disappearance of fluorescent product(s) during even short incubation periods. The extremely low solubility of benzpyrene in aqueous solutions, together with the propensity of this polycyclic hydrocarbon to bind to non-hydroxylating sites on proteins, suggested that the kinetics of substrate availability might significantly contribute to the observed non-Michaelis-Menten kinetics of hepatic microsomal benzpyrene hydroxylase from benzpyrene-treated rats.