1. Eight different forms of cytochrome P-450 have been purified to electrophoretic homogeneity. Electrophoretic, spectral and catalytic properties of these cytochrome P-450s are presented and comparison is made with preparations presented elsewhere in the literature.2. The levels of these forms of cytochrome P-450 present in liver microsomes of rats treated with various compounds have now been quantified. Several forms of cytochrome P-450 are induced, in a more or less coordinate manner, while levels of other cytochrome P-450s are lowered, during administration of commonly used inducing agents.3. The role of cytochrome P-450 purification and characterization studies in the understanding of the total field is discussed, along with directions in which future research is needed.

Eight different forms of cytochrome P-450 (P-450) were purified to electrophoretic homogeneity by a common procedure from liver microsomes of rats treated with phenobarbital or beta-naphthoflavone. Antibodies were prepared to seven of these forms in rabbits. The eight P-450s were distinguished by spectral properties of the ferric, ferrous, and ferrous carbonyl forms, apparent monomeric molecular weights, peptide mapping, immunological reactivity as discerned by double-diffusion immunoprecipitin analysis and crossed immunoelectrophoresis, and catalytic activities toward the substrates acetanilide, aminopyrine, aniline, benzo[a]-pyrene, d-benzphetamine, N,N-dimethylnitrosamine, 7-ethoxycoumarin, 7-ethoxyresorufin, ethylmorphine, p-nitroanisole, testosterone, and (R)- and (S)-warfarin. Crossed sodium dodecyl sulfate-polyacrylamide gel immunoelectrophoresis was used to estimate the levels of each of the eight forms of P-450 present in the liver microsomes of untreated rats and rats treated with phenobarbital, 5,6-benzoflavone, pregnenolone-16 alpha-carbonitrile, isosafrole, or the polychlorinated biphenyl mixture Aroclor 1254. In each situation, the sum of the levels of these eight P-450s was at least as high as the spectrally determined P-450 content. The results clearly demonstrate that individual forms of P-450 can be induced by different compounds and that a single compound can lower the level of one form of P-450 while inducing one or more other forms of P-450. Catalytic activities toward each of the substrates observed with microsomal preparations are compared to rates predicted on the basis of the content of each of the eight P-450s. These studies provide a basis for further studies on the regulation of individual P-450s, the physical properties of the different P-450s, and the metabolic consequences of changes in the forms of P-450 in rat liver models.

Highly-purified rat liver microsomal cytochrome P-450 converted cyclohexane to cyclohexanol in the presence of iodosobenzene. Oxygen from 18O-iodosobenzene was not incorporated into cyclohexanol but oxygen from H218O was readily incorporated. Cytochrome P-450 catalyzed the facile exchange of oxygen between iodosobenzene and water but neither cytochrome P-420 nor the apoenzyme did. Under these conditions cytochrome P-450 readily incorporated oxygen from 18O2 into cyclohexanol in the presence of NADPH-cytochrome P-450 reductase and NADPH. The results are interpreted in a mechanism in which cytochrome P-450 forms a common hydroxylating species in the presence of iodosobenzene or O2 plus NADPH.