Ding A, Ojingwa J C, McDonagh A F, Burlingame A L, Benet L Z
Department of Pharmacy, University of California, San Francisco 94143-0446.
Proc Natl Acad Sci U S A. 1993 May 1;90(9):3797-801. doi: 10.1073/pnas.90.9.3797.
Acyl glucuronide metabolites of bilirubin and many drugs can react with serum albumin in vivo to form covalent adducts. Such adducts may be responsible for some toxic effects of carboxylic nonsteroidal antiinflammatory agents. The mechanism of formation of the adducts and their chemical structures are unknown. In this paper we describe the use of tandem mass spectrometry to locate binding sites and elucidate the binding mechanism involved in the formation of covalent adducts from tolmetin glucuronide and albumin in vitro. Human serum albumin and excess tolmetin glucuronide were coincubated in the presence of sodium cyanoborohydride to trap imine intermediates. The total protein product was reduced, carboxymethylated, and digested with trypsin. Six tolmetin-containing peptides (indicated by absorbance at 313 nm) were isolated by high-pressure liquid chromatography and analyzed by liquid secondary-ion mass spectrometry and collision-induced dissociation, using a four-sector tandem mass spectrometer. All six peptides contained tolmetin linked covalently via a glucuronic acid to protein lysine groups. Major attachment sites on the protein were Lys-195, -199, and -525; minor sites were identified as Lys-137, -351, and -541. Our results show unambiguously that the glucuronic acid moiety of acyl glucuronides can be retained within the structure when these reactive metabolites bind covalently to proteins, and they suggest that acyl migration followed by Schiff base (imine) formation is a credible mechanism for the generation of covalent adducts in vivo.
胆红素和许多药物的酰基葡萄糖醛酸代谢产物可在体内与血清白蛋白反应形成共价加合物。此类加合物可能是羧酸类非甾体抗炎药某些毒性作用的原因。加合物的形成机制及其化学结构尚不清楚。在本文中，我们描述了使用串联质谱来定位结合位点，并阐明体外托美丁葡萄糖醛酸与白蛋白形成共价加合物所涉及的结合机制。在氰基硼氢化钠存在下，将人血清白蛋白与过量的托美丁葡萄糖醛酸共同孵育以捕获亚胺中间体。将总蛋白产物还原、羧甲基化并用胰蛋白酶消化。通过高压液相色谱分离出六个含托美丁的肽段（通过313nm处的吸光度指示），并使用四扇区串联质谱仪通过液相二次离子质谱和碰撞诱导解离进行分析。所有六个肽段均含有通过葡萄糖醛酸与蛋白质赖氨酸基团共价连接的托美丁。蛋白质上的主要结合位点是Lys-195、-199和-525；次要位点鉴定为Lys-137、-351和-541。我们的结果明确表明，当这些反应性代谢产物与蛋白质共价结合时，酰基葡萄糖醛酸的葡萄糖醛酸部分可保留在结构内，并且它们表明酰基迁移继之以席夫碱（亚胺）形成是体内产生共价加合物的可靠机制。
