EVIDENCE FOR COVALENT BINDING OF ACYL GLUCURONIDES TO SERUM-ALBUMIN VIA AN IMINE MECHANISM AS REVEALED BY TANDEM MASS-SPECTROMETRY

Acyl glucuronide metabolites of bilirubin and many drugs can react wit h serum albumin in vivo to form covalent adducts. Such adducts may be responsible for some toxic effects of carboxylic nonsteroidal antiinfl ammatory agents. The mechanism of formation of the adducts and their c hemical structures are unknown. In this paper we describe the use of t andem mass spectrometry to locate binding sites and elucidate the bind ing mechanism involved in the formation of covalent adducts from tolme tin glucuronide and albumin in vitro. Human serum albumin and excess t olmetin glucuronide were coincubated in the presence of sodium cyanobo rohydride to trap imine intermediates. The total protein product was r educed, carboxymethylated, and digested with trypsin. Six tolmetin-con taining peptides (indicated by absorbance at 313 nm) were isolated by high-pressure liquid chromatography and analyzed by liquid secondary-i on mass spectrometry and collision-induced dissociation, using a four- sector tandem mass spectrometer. All six peptides contained tolmetin l inked covalently via a glucuronic acid to protein lysine groups. Major attachment sites on the protein were Lys-195, -199, and -525; minor s ites were identified as Lys-137, -351, and -541. Our results show unam biguously that the glucuronic acid moiety of acyl glucuronides can be retained within the structure when these reactive metabolites bind cov alently to proteins, and they suggest that acyl migration followed by Schiff base (imine) formation is a credible mechanism for the generati on of covalent adducts in vivo.