CHROMATOGRAPHIC AND ELECTROPHORETIC HETEROGENEITY OF CYTOCHROMES P-450 SOLUBILIZED FROM UNTREATED RAT-LIVER

  • 1 January 1978
    • journal article
    • research article
    • Vol. 6  (4) , 353-362
Abstract
When the hepatic microsomes prepared from 1 or 2 untreated male rats were incubated for 3 h 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 were resolved by anion-exchange chromatography into 4 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 hemoglobin. Each of the 4 fractions of cytochrome P-450 were further resolved by electrofocusing in polyacrylamide into numerous protein bands, more than 8 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 should provide a basis for subsequent chemical, physical, catalytic and immunological investigation of the heterogeneity, and its significance.