REACTIVITY OF TOLMETIN GLUCURONIDE WITH HUMAN SERUM-ALBUMIN - IDENTIFICATION OF BINDING-SITES AND MECHANISMS OF REACTION BY TANDEM MASS-SPECTROMETRY

The structures of adducts formed from in vitro incubation of a drug (t olmetin) glucuronide (TG) and human serum albumin (HSA), and the prefe rred binding sites on this protein were determined by mass spectrometr y, 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 fragm ents were separated by HPLC, Tolmetin-containing peptides (indicated b y absorbance at 313 nm) were analyzed by liquid secondary-ion mass spe ctrometry, continuous flow-fast atom bombardment mass spectrometry, an d collision-induced dissociation using a four-sector tandem mass spect rometer, matrix-assisted laser desorption ionization-time-of-flight-ma ss spectrometry, and in selected cases by Edman sequencing, The identi fied peptides contained tolmetin linked covalently via a glucuronic ac id to a protein lysine group (lysine 199 and to a lesser extent lysine s 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 lysin e 541, and binding of tolmetin to arginine 521. Our results establish that the binding of these reactive metabolites to nucleophilic sites o f proteins occur via two different mechanisms: one involving imine (Sc hiff base) formation and the other involving nucleophilic displacement of glucuronic acid, Our data suggest, however, that the former, in wh ich the glucuronic acid moiety of the acyl glucuronide is retained wit hin the adducts, is favored at lower (closer to physiological) metabol ite concentrations.