The Effects of Sulphydryl Reagents on the Binding and Mixed Function Oxidation of Hexobarbital in Rat Hepatic Microsomes

Abstract

1. The effects of the sulphydryl reagents p-chloromercuribenzoate, N-ethylmaleimide and iodoacetamide on the binding spectrum, oxygen consumption and formation of a suspected substrate-cytochrome P-450-oxygen complex for hexobarbital in rat liver microsomes were investigated.

2. The oxygen consumption caused by hexobarbital oxidation was inhibited non-competitively by all three agents, with 50% inhibition at 4 × 10^-5 M for p-chloromercuribenzoate, 3·7x10^-4 M for N-ethylmaleimide and 1·9x10^-3 M for iodoacetamide. Cysteamine protected and at least partially reversed this inhibition.

3. p-Chloromercuribenzoate inhibited the formation of the cytochrome P-450-substrate-oxygen complex, while N-ethylmaleimide and iodoacetamide also inhibited the formation of this complex but to a lesser extent. The p-chloromercuribenzoate inhibition was protected against and reversed by cysteamine.

4. p-Chloromercuribenzoate and N-ethylmaleimide caused a 50% reduction in the magnitude of the hexobarbital-induced binding spectrum, and this was paralleled by the conversion of cytochrome P-450 to cytochrome P-420. Cysteamine protected against this effect but could not reverse it. Iodoacetamide had no effect on the binding spectrum of hexobarbital and failed to convert cytochrome P-450 to cytochrome P-420.

5. Points of attack within the reaction sequence of drug oxidation are tentatively ascribed to the sulphydryl reagents used in this study.