Evidence for covalent binding of acyl glucuronides to serum albumin via an imine mechanism as revealed by tandem mass spectrometry.
Evidence for covalent binding of acyl glucuronides to serum albumin via an imine mechanism as revealed by tandem mass spectrometry.
复制标题
串联质谱法揭示了酰基葡萄糖醛酸苷通过亚胺机制与血清白蛋白共价结合的证据。
DOI：
10.1073/pnas.90.9.3797
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发表时间：
1993
影响因子：
11.1
通讯作者：
Benet,LZ
中科院分区：
文献类型：
--
作者：
Ding,A;Ojingwa,JC;McDonagh,AF;Burlingame,AL;Benet,LZ
胆红素和许多药物的酰基葡糖苷酸代谢产物在体内可与血清白蛋白反应形成共价加合物。这些加合物可能是羧酸类非甾体类抗炎药的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法定位结合位点，并阐明了在体外从托美汀葡糖苷酸和白蛋白形成共价加合物的结合机制。人血清白蛋白和过量托美汀葡糖苷酸在氰基硼氢化钠存在下共孵育，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。六托美汀含有肽（由在313 nm处的吸光度表示），通过高压液相色谱法分离，并通过液体二次离子质谱法和碰撞诱导解离，使用四扇区串联质谱仪进行分析。所有六种肽均含有托美丁，托美丁通过葡萄糖醛酸与蛋白质赖氨酸基团共价连接。蛋白质上的主要附着位点为Lys-195、-199和-525;次要位点被鉴定为Lys-137、-351和-541。我们的研究结果明确表明，酰基葡萄糖醛酸苷的葡萄糖醛酸部分可以保留在结构内，当这些反应性代谢产物共价结合到蛋白质，他们建议，酰基迁移，然后席夫碱（亚胺）形成是一个可靠的机制，在体内产生共价加合物。
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.