Abstract

When the hepatic microsomes prepared from one or two untreated male rats were incubated for 3 hr at room temperature in buffered Emulgen 911, cholate, glycerol, and EDTA, almost complete recovery of the cytochromes P-450 in solubilized form was obtained. The intact cytochromes P-450 in this preparation were retained by Sephadex G-200. They could be resolved by anion-exchange chromatography into four fractions with 70--80% recovery of cytochrome P-450 and little or no conversion to P-420. In the process, the p-450 hemoproteins were separated from cytochrome b5, NADPH-cytochrome c reductase, and hemoglobulin. Each of the four fractions of cytochrome P-450 could be further resolved by electrofocusing in polyacrylamide into numerous protein bands, more than eight of which stained for heme. Extracts from focused gels retained P-450 spectral character. Mixtures of the various fractions electrofocused additively. Added hematin focused only in the acidic region of the gels. Although these results indicate extensive heterogeneity in the cytochromes P-450, the complexities of electrofocusing in the presence of detergent warrants cautious interpretation. These methods, however, should provide a basis for subsequent chemical, physical, catalytic, and immunological investigations of the heterogeneity, and its significance.