Isolation and characteristics of coumarin-specific cytochrome P-450 (P-450Cho) from liver microsomes of DBA/2N mice, induced with pyrazole

Coumarin-specific cytochrome P-450 (P-450Coh) has been isolated from liver microsomes of DBA/2N mice induced with pyrazole. The induction effect was accompanied by a 5.8-5.9-fold increase in the P-450Coh content which made up to 14.4-17% of the total cytochrome P-450 pool in the microsomes. At the final step of P-450Coh purification, variously substituted Sepharoses (hydroxyphenyl-, cholate-, aminooctyl- and t-cytochrome-b5-) were used. The optimal scheme involved solubilization of microsomes with sodium cholate, hydrophobic chromatography on octyl-Sepharose, adsorption on calcium-tartrate gel and hydrophobic ion-exchange chromatography on aminooctyl-Sepharose. According to SDS gel electrophoresis data, the purity of P-450Coh was 95% and Mr was 50,000 Da. The amino acid composition of the protein includes 445 residues. At saturating concentrations of coumarin, more than 90% of P-450Coh are represented by the high spin form. The catalytic activity of P-450Coh was studied in reactions of xenobiotics oxidation.