Identification of subdomain IB in human serum albumin as a major binding site for polycyclic aromatic hydrocarbon epoxides
Identification of subdomain IB in human serum albumin as a major binding site for polycyclic aromatic hydrocarbon epoxides
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DOI：
10.1021/tx9700782
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发表时间：
1997-08-01
影响因子：
4.1
通讯作者：
Tannenbaum, SR
中科院分区：
文献类型：
--
作者：
Brunmark, P;Harriman, S;Tannenbaum, SR
血清白蛋白和低分子量有机亲电子试剂之间形成的共价加合物具有高度区域选择性，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以不同子域中的残基为目标。本研究通过体外人血清白蛋白与 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) 的外消旋反式反式异构体的反应，检查了 PAH 环氧化物和二醇环氧化物结合的区域选择性。 1,2-二氢-1,2-二醇3,4-环氧化物(5)、6-甲基-1,2-二氢-1,2-二醇3,4-环氧化物(6)、5-甲基-1,2-二氢-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]屈-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)和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)，然后确定连接位点。加合物白蛋白经酶消化，消化物通过反相 HPLC 进行色谱纯化，以纯化肽加合物，并通过电喷雾电离碰撞诱导解离 (CID) 串联质谱法进行分析。产物离子谱表明，加合物主要通过肽-PAH 键的断裂而碎裂，并通过肽和碳氢化合物保留电荷。通过对源内 CID 形成的肽离子进行 MS/MS 分析来确定肽序列，以裂解加合物键。较长的肽序列凭借其独特性建立了位点选择性，而较短的序列则揭示了位点内的反应物氨基酸。环氧化物4和二醇环氧化物1、2、5和6主要与His(146)反应；环氧化物3和二醇环氧化物7-9主要与Lys(137)反应。两个残基均位于子结构域IB中。 10 的结合位点无法唯一确定，但几种可能性之一是 Lys(159)，它也位于子结构域 IB 中。该结果与之前的发现相结合，表明多环芳烃环氧化物与人血清白蛋白的反应对于亚结构域IB中的少量残基具有高度选择性。
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-dihydrachrysene-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 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 possibilities was Lys(159), 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.