IDENTIFICATION OF SUBDOMAIN IB IN HUMAN SERUM-ALBUMIN AS A MAJOR BINDING-SITE FOR POLYCYCLIC AROMATIC HYDROCARBON EPOXIDES

Covalent adducts between serum albumin and low molecular weight organi c electrophiles are formed with a high degree of regioselectivity most ly for nucleophilic amino acid residues located in subdomains IIA and IIIA. Previous studies have indicated that diol epoxide metabolites of polycyclic aromatic hydrocarbons (PAH) may target residues in a diffe rent subdomain. The regioselectivity of PAH epoxide and diol epoxide b inding was examined in this study by reaction of human serum albumin i n vitro with the racemic trans,anti-isomers of 7,8-dihydrobenzo[a]pyre ne-7,8-diol 9,10-epoxide (1), 2,3-dihydrofluoranthene-2,3-diol 1,10b-e poxide (2), 1,2-dihydrachrysene-1,2-diol 3,4-epoxide (5), 6-methyl-1,2 -dihydrochrysene-1,2-diol 3,4-epoxide (6), 5-methyl-1,2-dihydrochrysen e-1,2-diol 3,4-epoxide (7), 3,4-dihydrobenzo-[c]phenanthrene-3,4-diol 1,2-epoxide (8), 11,12-dihydrobenzo[g]chrysene-11,12-diol 13,14-epoxid e (9), and 11,12-dihydrodibenzo[a,l]pyrene-11,12-diol 13,14-epoxide (1 0) and the racemic epoxides cyclopenta[cd]pyrene 3,4-epoxide (3) and b enzo[a]pyrene 4,5-epoxide (4) followed by determination of the linkage site. Adducted albumin was digested enzymatically, and digests were c hromatographed by reversed-phase HPLC to purify peptide adducts, which were analyzed by electrospray ionization collision-induced dissociati on (CID) tandem mass spectrometry. Product ion spectra revealed that a dducts fragmented predominantly by cleavage of the peptide-PAH bond wi th retention of charge by the peptide as well as by the hydrocarbon. P eptide sequences were determined by MS/MS analysis of the peptide ions formed by in-source CID to cleave the adduct bond, Longer peptide seq uences established site selectivity by virtue of their uniqueness, whi le shorter sequences revealed the reactant amino acid within the site. Epoxide 4 and diol epoxides 1, 2, 5, and 6 reacted predominantly with His(146); epoxide 3 and dial epoxides 7-9 reacted predominantly with Lys(137). Both residues are situated in subdomain IB. The binding site for 10 could not be determined uniquely, but one of the several possi bilities was Lys(159), which is also located in subdomain IB. The resu lts, taken together with previous findings, demonstrate that the react ion of polycyclic aromatic hydrocarbon epoxides with human serum album in is highly selective for a small number of residues in subdomain IB.