A major emphasis of our studies during the past two years has been the use of mechanism-based inactivators to probe the active site of purified forms of P450. Recently, we have investigated the ability of a series of aryl acetylenes to act as mechanism-based inactivators. The inactivation of P450 2B1 by 9-ethynylphenanthrene (9EPh) has been characterized in detail during the past year. 9EPh inactivated the 7-ethoxycoumarin O-deethylase activity of P450 2B1 in a time-and NADPH-dependent manner. After HPLC separation of peptides derived from CNBr digestion,fractions were analyzed by MALDI-MS and peaks were detected at m/z 2720.9 and 2939.9. The lower mass peak represents the MH+ for the peptide Ile290 to Met314, while the higher mass peak corresponds to the MH+ of the modified peptide. The difference in mass would correspond to the addition of a phenanthrylacetyl group to the peptide. When the radiolabeled fraction containing the modified and unmodified peptides was further digested with pepsin and reanalyzed by MALDI-MS, the site of attachment could be assigned to one of the amino acids contained in the peptide Phe297 to Leu307. We are interested in the mass spectral analysis of covalently labeled peptides isolated from cytochrome P450 inactivated by various compounds including several arylacetylenes and furanocoumarins. We are interested in the MALDI-MS analysis of covalently labeled peptides to aid in the identification of adducts and the subsequent sequencing of these peptides in order to determine the site of labeling. In addition, we are using of GC-MS analysis for product identification from incubations containing microsomes or purified P450 after metabolism of various compounds including 8-methoxypsoralen.
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