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

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Abstract
High performance liquid chromatography [HPLC] was used to fractionate rat liver microsomes under nondenaturing conditions. Selective detection at 405 nm allowed resolution of microsomal heme proteins into 3 peaks (A, B and C). Cytochromes in the peaks retain their native property of binding CO after HPLC. Peak-A, first eluting, contained cytochrome P-450 and was rich in cytochrome P-420. Peak-B was largely Hb and peak-C was a major cytochrome P-450. The ratio of peak-C to A was increased by treatment of rats with phenobarbitone, .beta.-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 was observed on feeding 3,4,5,3'',4'',5''-hexachlorobiphenyl. NADPH cytochrome c reductase eluted earlier than peak-C but cytochrome b5 was not separated from the major cytochrome P-450 peak. The separations obtained were 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.