Reactivity of tolmetin glucuronide with human serum albumin. Identification of binding sites and mechanisms of reaction by tandem mass spectrometry.
Reactivity of tolmetin glucuronide with human serum albumin. Identification of binding sites and mechanisms of reaction by tandem mass spectrometry.
复制标题
DOI：
--
复制
发表时间：
1995-03
期刊：
影响因子：
--
通讯作者：
A. Ding;P. Zia‐Amirhosseini;Antony F. McDonagh;A. L. Burlingame;Leslie Z. Benet
中科院分区：
文献类型：
--
作者：
A. Ding;P. Zia‐Amirhosseini;Antony F. McDonagh;A. L. Burlingame;Leslie Z. Benet
用质谱法测定了托美汀（tolmetin）、葡萄糖醛酸酯（TG）和人血清白蛋白（HSA）体外培养形成的加合物的结构及其在该蛋白上的首选结合位点。此外，在三种不同的TG与HSA的浓度比下，研究了TG对HSA共价修饰的浓度依赖性。酶解蛋白质加合物，高效液相色谱分离肽段。含tolmetin的肽（以313 nm的吸光度表示）通过液体二次离子质谱、连续流快速原子轰击质谱、碰撞诱导解离（使用四扇区串联质谱、基质辅助激光解吸电离飞行时间质谱）和Edman测序进行分析。所鉴定的多肽含有通过葡萄糖醛酸共价连接到蛋白质赖氨酸基团（赖氨酸199和赖氨酸195和525）或直接连接到赖氨酸（赖氨酸199和541）、丝氨酸（丝氨酸220、232和480）或精氨酸（精氨酸222）的tolmetin。此外，有间接证据表明TG与赖氨酸541结合，tolmetin与精氨酸521结合。我们的研究结果表明，这些活性代谢物与蛋白质亲核位点的结合通过两种不同的机制发生：一种涉及亚胺（希夫碱）的形成，另一种涉及葡萄糖醛酸的亲核位移。然而，我们的数据表明，前者，其中酰基葡萄糖醛酸部分保留在加合物中，有利于较低（更接近生理）代谢物浓度。
The structures of adducts formed from in vitro incubation of a drug (tolmetin) glucuronide (TG) and human serum albumin (HSA), and the preferred binding sites on this protein were determined by mass spectrometry. In addition, the concentration dependence of covalent modification of HSA by TG was studied at three different concentration ratios of TG to HSA. Protein adducts were enzymatically digested and peptide fragments were separated by HPLC. Tolmetin-containing peptides (indicated by absorbance at 313 nm) were analyzed by liquid secondary-ion mass spectrometry, continuous flow-fast atom bombardment mass spectrometry, and collision-induced dissociation using a four-sector tandem mass spectrometer, matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry, and in selected cases by Edman sequencing. The identified peptides contained tolmetin linked covalently via a glucuronic acid to a protein lysine group (lysine 199 and to a lesser extent lysines 195 and 525) or tolmetin directly linked to lysines (lysines 199 and 541), serines (serines 220, 232, and 480), or arginines (arginine 222). In addition, there was indirect evidence for binding of TG to lysine 541, and binding of tolmetin to arginine 521. Our results establish that the binding of these reactive metabolites to nucleophilic sites of proteins occur via two different mechanisms: one involving imine (Schiff base) formation and the other involving nucleophilic displacement of glucuronic acid. Our data suggest, however, that the former, in which the glucuronic acid moiety of the acyl glucuronide is retained within the adducts, is favored at lower (closer to physiological) metabolite concentrations.