The cytochrome P450-mixed function oxidase system plays a crucial role in the metabolism of many endogenous and exogenous compounds, catalyzing the detoxification of chemical carcinogens and toxins as well as the activation of certain compounds to highly reactive intermediates. We are interested in identifying the specific peptide and amino acid residues in the P450 2B1 active site that are involved in substrate binding and catalysis. Conventional enzyme digestion, HPLC separation and gas-phase N-terminal sequencing are usually not successful because P450s and peptides derived from P450s are very hydrophobic. MALDI-MS analysis makes it possible for use to analyze the entire cyanogen bromide digest of the P450 2B1 and to detect and identify the uniquely labeled peptide. In addition, sequence information on the modified peptide(s) can be obtained by PSD analysis of MALDI ions. We have subjected P450 2B1 inactivated with benzyl-aminobenzotriazole (BBT) as well as control protein to cleavage with cyanogen bromide. Preliminary results suggested that peptide #10 (AA 347-376) might be modified by a BBT metabolite. We are now in the process of synthesizing radiolabeled BBT to facilitate the identification of the modified peptide(s) in the reverse-phase fractions. The isolated, labeled peptide will be subjected to MALDI-MS and PSD analysis. We also plan to identify the products produced during P450 2B1 catalysis with the different mechanism-based inactivators in order to obtain a better understanding of the mechanism of action by P450s. Because P450 2B1 can generate low levels of a complex mixture of products we require GC-MS analysis to identify all the different metabolites. Initial studies with BBT have confirmed the identity of three of the five products that were generated during BBT metabolism by P450 2B1.
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