Protein and nonprotein cysteinyl thiol modification by N-acetyl-p-benzoquinone imine via a novel Ipso adduct

N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminoph en (APAP), can arylate and oxidize protein and nonprotein thiols in the pat hogenesis of APAP-induced hepatotoxicity. We report the first direct eviden ce for the formation of a labile ipso adduct between glutathione (GSH) and NAPQI using a combination of techniques including liquid chromatography/tan dem mass spectrometry and liquid chromatography/NMR spectroscopy. Decomposi tion kinetics of the GSH-NAPQI ipso adduct and product ratios suggested tha t the ipso adduct was readily reversible back to NAPQI under neutral and ba sic conditions. The significance of the ipso adduct is that it may migrate from its site of formation to other cell compartments where it can either o xidize protein thiols or covalently modify them. Ipso adduct formation with protein thiols was demonstrated with a cysteine protease, papain, whose ca talytic activity relies on the presence of an active site cysteinyl thiol. The formation and reactions of cysteinyl thiol ipso adducts of NAPQI provid es significant new insights into possible reactions of quinone imines with cellular peptides and proteins.