Fractionation by High Performance Liquid Chromatography of Microsomal Cytochrome P-450 Induced by Hexachlorobiphenyl Isomers

Abstract

High performance liquid chromatography has been employed to fractionate rat liver microsomes under nondenaturing conditions. Selective detection at 405 nm allowed resolution of microsomal heme proteins into three peaks (A, B, and C). Cytochromes in the peaks retain their native property of binding CO after HPLC. Peak-A, first eluting, contains P-450 and is rich in cytochrome P-420. Peak-B is largely hemoglobin and peak-C is a major cytochrome P-450. The ratio of peak-C to A is increased by treatment of rats with phenobarbitone, β-naphthoflavone, 2,3,5,2′,3′,5′-hexachlorobiphenyl and 3,4,5,3′,4′,5′-hexachlorobiphenyl as compared to controls. The highest increment in the ratio is observed on feeding 3,4,5,3′,4′,5′-hexachlorobiphenyl. NADPH cytochrome c reductase elutes earlier than peak-C but cytochrome b₅ is not separated from the major cytochrome P-450 peak. The separations obtained are highly reproducible and considerably faster than conventional gel permeation chromatography. The data presented here are very promising in establishing the role of HPLC in the studies of insoluble proteins and enzymes in general and cytochrome P-450 in particular.