title: Covalent binding of zomepirac glucuronide to proteins: evidence for a Schiff base mechanism.
abstract: Carboxylic acids can undergo irreversible binding to proteins via their acyl glucuronide metabolites. Such binding occurs in humans and in vitro for bilirubin and the anti-inflammatory drugs zomepirac and tolmetin and may have toxicological significance. The mechanism of the binding is uncertain, but is thought to involve nucleophilic displacement of glucuronic acid by free cysteine thiols, tyrosine, or lysine residues of the protein. We now present evidence, using zomepirac glucuronide, for an alternative mechanism where irreversible binding occurs by isomerization of the glucuronide and formation of an imine linkage between the glucuronic acid moiety of the conjugate and a lysine or terminal amino group of the protein. Involvement of a protein thiol in the binding mechanism was ruled out by experiments with the thiol blocking agent, p-hydroxymercuribenzoate. Moreover, significant irreversible binding occurred when the cysteine free protein, alpha s1-casein, was incubated in solution with zomepirac glucuronide in vitro. When zomepirac glucuronide was added to solutions with albumin in the presence of the imine trapping reagent, sodium cyanide, or the reducing agent, sodium cyanoborohydride, irreversible binding of zomepirac increased 8.3- and 5.5-fold relative to controls, respectively, after 6 hr at 37 degrees C. The product formed after treatment of solutions of zomepirac glucuronide and albumin with sodium cyanoborohydride was stable in dilute acid, whereas similar acid treatment of zomepirac-albumin adduct from control incubations, obtained after sodium cyanide exposure, released 30% of the total adduct. Only isomeric conjugates of zomepirac glucuronide were released from zomepirac-albumin adduct by mild acid; no beta 1-isomer was detected.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Liver Center, School of Medicine, University of California, San Francisco 94143.
authors.forename: P C
authors.initials: PC
authors.lastname: Smith
authors.forename: L Z
authors.initials: LZ
authors.lastname: Benet
authors.forename: A F
authors.initials: AF
authors.lastname: McDonagh
mesh_terms: D000818:Animals; D001241:Aspirin / Q000494:pharmacology; D002364:Caseins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D066298:In Vitro Techniques; D007536:Isomerism; D008862:Microsomes, Liver / Q000378:metabolism; D011485:Protein Binding; D012459:Salicylates / Q000494:pharmacology; D020156:Salicylic Acid; D012545:Schiff Bases / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism; D012756:Sheep; D014046:Tolmetin / Q000031:analogs & derivatives* / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism
chemical_list: D002364:Caseins; D006902:Hydroxymercuribenzoates; D012459:Salicylates; D012545:Schiff Bases; D012709:Serum Albumin; C030601:4-hydroxymercuribenzoate; D014535:Uridine Diphosphate Glucuronic Acid; C027368:zomepirac glucuronide; D014046:Tolmetin; D020156:Salicylic Acid; D001241:Aspirin