Molecular Pharmacology, Vol 12, 483-493, Copyright © 1976 by the American Society for Pharmacology and Experimental Therapeutics

Separation and Purification of Two Forms of Hepatic Cytochrome P-450 from Untreated Rabbits

¹ Pharmacology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Two forms of hepatic cytochrome P-450 have been separated and partially purified from untreated rabbits. The carbon monoxide-reduced minus reduced difference spectrum of one form (A) had a peak of absorption at 450.2 nm while the same spectrum of the other form (B) gave a peak at 448 nm. Differences in the spectral properties of the two forms of the cytochrome were also seen in the Soret region of the spectra of oxidized preparations, in the Soret and visible regions of the spectra of reduced preparations complexed with carbon monoxide, and in the difference spectra formed by the addition of type I and type II compounds. The cytochromes were purified to 6.5 and 14.3 nmoles/mg of protein for forms A and B, respectively. The partially purified preparations were free of cytochrome b₅ and had barely detectable concentrations of NADPH-cytochrome c reductase, epoxide hydrase, and cytochrome P-420. The cytochrome in fraction A was about twice as active as the cytochrome in fraction B in support of benzpyrene hydroxylase, benzphetamine N-demethylase, and 7-ethoxycoumarin O-deethylase activities when combined with NADPH-cytochrome c reductase and lipid fractions. Monomeric molecular weights for both forms of the cytochrome were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be near 51,500.

Note:
ACKNOWLEDGMENTS The authors are indebted to Dr. J. R. Fouts, Chief, Pharmacology Branch, National Institute of Environmental Health Sciences, for his support and encouragement, and to Mr. Gary Foureman for determining epoxide hydrase activities.