Resolution by high-pressure liquid chromatography and partial characterization of multiple forms of cytochrome P-450 from hepatic microsomes of phenobarbital-treated rats.
Seven cytochromes P-450 (A, B, C, D, E1, E2 and F) were isolated from hepatic microsomes of phenobarbital-induced rats by a modification of the procedure of Guengerich and Martin [Arch. Biochem. Biophys. (1980) 205, 365-379]. The modification consisted of replacing DEAE-cellulose column by two DEAE-Sepharose CL-6B columns connected in tandem, changing the elution scheme and monitoring the resulting fractions by high-pressure liquid chromatography (HPLC). Cytochrome P-450 forms D, E1, E2 and F having molecular masses of 52.5 kDa, 52.5 kDa, 53.3 kDa and 53.2 kDa, respectively were resolved from the major form of cytochrome P-450 'peak B2' of Guengerich and Martin (above reference). These four cytochromes P-450 were immunologically identical by Ouchterlony double-diffusion analysis. Slight but significant differences were evident in the partial peptide digest maps of these four cytochromes P-450 and catalytic properties of these four forms, though qualitatively similar, demonstrated distinct quantitative differences. Furthermore, HPLC retention times of these four cytochrome P-450s were quite different. Cytochrome P-450 forms A, B and C were distinctly different from each other and from the forms D, E1, E2 and F in the following respects: partial peptide digest maps, catalytic activities, and HPLC retention times. The present results show that cytochromes P-450 considered homogeneous by sodium dodecyl sulfate/polyacrylamide gel electrophoresis may be heterogeneous and contain multiple forms of cytochromes P-450 with different net charges but similar molecular-masses. These studies also demonstrate the capability of HPLC in providing a simple and effective tool for monitoring the separation of cytochromes P-450 showing charge heterogeneity.
D016604:Aflatoxin B1; D000348:Aflatoxins / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013057:Spectrum Analysis
D000348:Aflatoxins; D010446:Peptide Fragments; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D010634:Phenobarbital