Purification and properties of cytochrome P-450 generally acting as a catalyst on benzo[a]pyrene hydroxylation from liver microsomes of untreated rats

A form of cytochrome P-450 generally catalyzing benzoa]pyrene (Ba]P) hydroxylation was purified from liver microsomes of untreated rats on the basis of the catalytic activity. The purification procedures consisted of cholate solubilization and chromatography in 3 steps, on DEAE-Toyopearl (at room temperature), hydroxylapatite, and CM-Toyopearl columns. Cytochrome P-450 purified in this way (named P-450/Ba]P) was homogeneous on SDS-polyacrylamide gel electrophoresis, and the molecular weight was estimated to be 51,000. The absorption spectra of the oxidized form of P-450/Ba]P showed a Soret peak at 417 nm, characteristic of low-spin hemoprotein, and the Soret peak of the reduced cytochrome P-450-CO complex was at 451 nm. Immunochemical analysis of P-450/Ba]P indicated that P-450/Ba]P is immunologically distinct from P-450b (a major phenobarbital-inducible form of P-450) and P-450c (a major 3-methylcholanthrene-inducible form of P-450, which highly catalyzes the hydroxylation of Ba]P). Ba]P hydroxylase activity in liver microsomes of untreated rats was inhibited to about 20% by the P-450/Ba]P antibody. These results demonstrate that P-450/Ba]P is a different form of P-450 from P-450b and P-450c, and generally catalyzes Ba]P hydroxylation in liver microsomes of untreated rats.