Ding A, Zia-Amirhosseini P, McDonagh A F, Burlingame A L, Benet L Z
Department of Pharmacy, University of California, San Francisco, USA.
Drug Metab Dispos. 1995 Mar;23(3):369-76.
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.
通过质谱法确定了药物（托美汀）葡萄糖醛酸苷（TG）与人血清白蛋白（HSA）体外孵育形成的加合物结构，以及该蛋白上的优先结合位点。此外，研究了在三种不同的TG与HSA浓度比下，TG对HSA共价修饰的浓度依赖性。对蛋白质加合物进行酶解，肽片段通过高效液相色谱法分离。含托美汀的肽（通过313nm处的吸光度指示）通过液体二次离子质谱法、连续流快原子轰击质谱法以及使用四扇区串联质谱仪的碰撞诱导解离、基质辅助激光解吸电离飞行时间质谱法进行分析，在某些情况下还通过埃德曼测序法进行分析。鉴定出的肽包含通过葡萄糖醛酸共价连接到蛋白质赖氨酸基团（赖氨酸199，在较小程度上还有赖氨酸195和525）的托美汀，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）的托美汀。此外，有间接证据表明TG与赖氨酸541结合，以及托美汀与精氨酸521结合。我们的结果表明，这些反应性代谢物与蛋白质亲核位点的结合通过两种不同机制发生：一种涉及亚胺（席夫碱）形成，另一种涉及葡萄糖醛酸的亲核取代。然而，我们的数据表明，在较低（更接近生理）代谢物浓度下，前者（其中酰基葡萄糖醛酸的葡萄糖醛酸部分保留在加合物中）更受青睐。
