Brunmark P, Harriman S, Skipper P L, Wishnok J S, Amin S, Tannenbaum S R
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139, USA.
Chem Res Toxicol. 1997 Aug;10(8):880-6. doi: 10.1021/tx9700782.
Covalent adducts between serum albumin and low molecular weight organic electrophiles are formed with a high degree of regioselectivity mostly 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 different subdomain. The regioselectivity of PAH epoxide and diol epoxide binding was examined in this study by reaction of human serum albumin in vitro with the racemic trans,anti-isomers of 7,8-dihydrobenzo[a]pyrene-7,8-diol 9,10-epoxide (1), 2,3-dihydrofluoranthene-2,3-diol 1,10b-epoxide (2), 1,2-dihydrochrysene-1,2-diol 3,4-epoxide (5), 6-methyl-1,2-dihydrochrysene-1,2-diol 3,4-epoxide (6), 5-methyl-1,2-dihydrochrysene-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-epoxide (9), and 11,12-dihydrodibenzo[a,l]pyrene-11,12-diol 13,14-epoxide (10) and the racemic epoxides cyclopenta[cd]pyrene 3,4-epoxide (3) and benzo[a]pyrene 4,5-epoxide (4) followed by determination of the linkage site. Adducted albumin was digested enzymatically, and digests were chromatographed by reversed-phase HPLC to purify peptide adducts, which were analyzed by electrospray ionization collision-induced dissociation (CID) tandem mass spectrometry. Product ion spectra revealed that adducts fragmented predominantly by cleavage of the peptide-PAH bond with retention of charge by the peptide as well as by the hydrocarbon. Peptide sequences were determined by MS/MS analysis of the peptide ions formed by in-source CID to cleave the adduct bond. Longer peptide sequences established site selectivity by virtue of their uniqueness, while shorter sequences revealed the reactant amino acid within the site. Epoxide 4 and diol epoxides 1, 2, 5, and 6 reacted predominantly with His146; epoxide 3 and diol epoxides 7-9 reacted predominantly with Lys137. Both residues are situated in subdomain IB. The binding site for 10 could not be determined uniquely, but one of the several possibilities was Lys159, which is also located in subdomain IB. The results, taken together with previous findings, demonstrate that the reaction of polycyclic aromatic hydrocarbon epoxides with human serum albumin is highly selective for a small number of residues in subdomain IB.
血清白蛋白与低分子量有机亲电试剂之间形成的共价加合物具有高度的区域选择性，主要针对位于亚结构域IIA和IIIA中的亲核氨基酸残基。先前的研究表明，多环芳烃（PAH）的二醇环氧化物代谢物可能靶向不同亚结构域中的残基。本研究通过人血清白蛋白在体外与7,8-二氢苯并[a]芘-7,8-二醇9,10-环氧化物（1）、2,3-二氢荧蒽-2,3-二醇1,10b-环氧化物（2）、1,2-二氢 Chrysene-1,2-二醇3,4-环氧化物（5）、6-甲基-1,2-二氢 Chrysene-1,2-二醇3,4-环氧化物（6）、5-甲基-1,2-二氢 Chrysene-1,2-二醇3,4-环氧化物（7）、3,4-二氢苯并[c]菲-3,4-二醇1,2-环氧化物（8）、11,12-二氢苯并[g] Chrysene-11,12-二醇13,14-环氧化物（9）和11,12-二氢二苯并[a,l]芘-11,12-二醇13,14-环氧化物（10）的外消旋反式、反式异构体以及外消旋环氧化物环戊[cd]芘3,4-环氧化物（3）和苯并[a]芘4,5-环氧化物（4）反应，随后测定连接位点。加合的白蛋白经酶消化，消化物通过反相HPLC进行色谱分离以纯化肽加合物，通过电喷雾电离碰撞诱导解离（CID）串联质谱对其进行分析。产物离子光谱显示，加合物主要通过肽-PAH键的断裂而碎片化，肽以及烃保留电荷。通过对源内CID形成的肽离子进行MS/MS分析以裂解加合键来确定肽序列。较长的肽序列因其独特性确定了位点选择性，而较短的序列则揭示了该位点内的反应性氨基酸残基。环氧化物4和二醇环氧化物1、2、5和6主要与His146反应；环氧化物3和二醇环氧化物7-9主要与Lys137反应。这两个残基都位于亚结构域IB中。10的结合位点无法唯一确定，但几种可能性之一是Lys159，其也位于亚结构域IB中。这些结果与先前的发现一起表明，多环芳烃环氧化物与人血清白蛋白的反应对亚结构域IB中的少数残基具有高度选择性。
