Purification and Molecular Characterization of β-Naphthoflavone–Inducible Cytochrome P-450 from Rat Epidermis

This study was designed to characterize epidermal cytochrome P-450 (P-450) induced by skin application of β-naphthoflavone (β-NP). Topical application of β-NF (40 mg/kg) to rats resulted in a 2.6-times increase in epidermal P-450 content and a 3-14-times increase in epidermal monooxygenase activities. The purified epidermal P-450 showed a major band at 54kDa on SDS-PAGE, which co-migrated with hepatic P-4501A1 and cross-reacted with monoclonal and polyclonal antibodies specific to P-4501A1. The specific content of purified epidermal P-450 was 1.53 nmol/mg protein, representing 42-times purification. HPLC analysis of the purified epidermal P-450 showed similar elution profile and retention time as that of hepatic P-4501A1. The purified preparation efficiently catalyzedbenzo(a)pyrene hydroxylation when reconstituted with purified NADPH-P-450 reductase and phospholipid. Peptide fingerprint analysis of the purified epidermal P-450 and hepatic P-4501A1 showed similar monoclonal antibody 1-7-1 reacting epitopes. Partial N-terminal amino acid sequence analysis of purified epidermal P-450 showed complete homology with the known sequence of P-4501A1. Similarly, HPLC analysis of tryptic digest of purified epidermal P-450 and hepatic P-4501A1 showed identical peptide peaks with comparable retention times. N-terminal amino acid sequence analysis of three randomly selected tryptic peptides showed complete homology with the known sequence of P-4501A1. These results indicate that rat epidermal P-450 induced by β-NF is similar to hepatic P-4501A1