BENZO[A]PYRENE ANTI-DIOL EPOXIDE COVALENTLY MODIFIES HUMAN SERUM-ALBUMIN CARBOXYLATE SIDE-CHAINS AND IMIDAZOLE SIDE-CHAIN OF HISTIDINE(146)

Human serum albumin was reacted with (+/-)-r-7,t-8-dihydroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BaPDE) in vitro and extracted to remove byproducts not covalently bound to the protein. Enzymatic digestion of the adducted protein in (HO)-H-2-O-18 at pH 8.2 and pH 10.0, followed by analysis of the released 7,8,9,10-tetrahydrotetrols by positive chemical ionization mass spectrometry for O-18 incorporation, revealed that carboxylic esters are formed by the epoxide. Analysis by HPLC/UV of the protein digest indicated that esters are the major product formed. Two additional stable products were also observed, accounting for 22 and 8% of chromatographed material. These were identical in UV absorption spectral characteristics with synthetic N(im)-histidine-anti-BaPDE adducts. Amino acid analysis of the peptide portion of the major product, in combination with its FAB mass spectrum, was consistent with a composition of histidine, proline, and tyrosine, while like analysis of the minor adducted peptide was consistent with a composition of histidine and proline. The first combination of amino acids occurs only once within the sequence of human albumin as His(146)-Pro(147)-Tyr(148). The second could be a subsequence of the first or correspond to His(338)-Pro(339) or His(440)-Pro(441). When synthetic His-Pro-Tyr was reacted with anti-BaPDE, a product which was chromatographically and spectrally (UV, FAB-MS) identical with the material isolated from alkylated albumin was formed in low yield. Reaction with fluorescamine followed by acid-catalyzed rearrangement of the products and analysis of the fluorescence spectra from the resulting materials revealed that the adducts in the protein resulted from alkylation of the imidazole tau-nitrogen of histidine. These results indicate that, in addition to the unknown amino acids esterified, His(146) and possibly His(338) or His(440), the former two of which are in a previously recognized binding site for certain covalent and noncovalent bulky aromatic ligands, are alkylated by anti-BaPDE to form enzymatically stable adducts.