Effects of storage on hepatic microsomal cytochromes and substrate-induced difference spectra

Abstract

Microsomal cytochrome P450, the ethyl isocyanide difference spectrum of dithionite-reduced microsomes (measured at the pH giving equal absorbance of 430 and 455 mμ peaks), and the aniline type II difference spectrum were affected similarly after phenobarbital induction and during the subsequent aging in vitro (about 90 hr at 1° under nitrogen) of hepatic microsomes from rats pretreated with phenobarbital. In these same microsomal preparations from phenobarbital-pretreated rats, type I spectra (caused by adding hexobarbital or (+) benzphetamine to microsomes) were increased to a greater extent and were less stable to aging in vitro than the previously mentioned components. During aging, decreases in the type II spectra paralleled the loss of total heme, but large decreases in the type I spectra were observed with little or no loss of heme. Also, aging did not change the pH giving equal absorbance of the 430 and 455 mμ peaks in the ethyl isocyanide difference spectrum, nor was there any shift in the wavelength of the maximum (450 mμ) in the carbon monoxide difference spectrum of dithionite-reduced microsomes. The binding associated with type I or II spectral changes did not appear to conform to simple Michaelis-Menten kinetics. Plots of ΔA vs. ΔA/S (where ΔA is the difference in absorbance between the trough and the peak in the 350 to 450 mμ region of the type I or II substrate-induced microsomal difference spectrum, and S is the corresponding substrate concentration) exhibited curvature (concave upwards). During aging, the curvature associated with plots of ΔA vs. ΔA/S using ( + ) benzphetamine increased, while the curvature associated with plots of ΔA vs. ΔA/S using aniline decreased. Some correlation was observed between increases in the rate of N-demethylation of (+) benzphetamine or p-hydroxylation of aniline and the increases in the respective type I or II difference spectra, after phenobarbital treatment of rats. Also it appeared that the rate of p-hydroxylation of aniline decreased to a lesser extent on aging than the rate of N-demethylation of (+) benzphetamine.