INFLUENCE OF QUINONE METHIDE REACTIVITY ON THE ALKYLATION OF THIOL AND AMINO-GROUPS IN PROTEINS - STUDIES UTILIZING AMINO-ACID AND PEPTIDE MODELS

Quinone methides (QMs) are electrophiles formed in several biological processes including direct oxidations of 4-alkylphenols by cytochromes P450. These species may be responsible for the adverse effects of cer tain phenolic compounds through protein alkylation, but little informa tion is available concerning specific targets or the resulting mechani sms of cell injury. The present goal was to determine the most likely sites of adduct formation among competing protein nucleophiles utilizi ng QMs of varying electrophilicity. Reactions of poorly reactive, mode rately reactive, and highly reactive QMs, 2,6-di-tert-butyl-4-methylen e-2,5-cyclohexadienone (BHT-QM), 1'-dimethylethyl)-4-methylene-2,5-cyc lohexadienone (BHTOH-QM), and rt-butyl-6-methyl-4-methylene-2,5-cycloh exadienone (BDMP-QM), respectively, were investigated in aqueous solut ions with nucleophilic amino acids. Each QM rapidly formed a thioether derivative of cysteine with little or no competition from the additio n of water (hydration). The a-amino groups were the primary sites of a lkylation for all other amino acids examined including lysine, histidi ne, tyrosine, and serine, and the pseudo-first order rates were 5 to 8 -fold greater than the rates of hydration. Alkylation of the side chai n nitrogens of lysine and histidine occurred at about one-fourth the r ate of hydration for BDMP-QM, but no reaction was detectable for BHT-Q M and no reactions occurred between QMs and amino acid hydroxyl groups . The results indicate that, based on chemical reactivity, peptide alk ylation should occur in the order cysteine thiol > N-terminal amino > N epsilon-lysine = NIm-histidine, with side chain modifications occurr ing only with the more electrophilic QMs. Reactions of QMs with the tr ipeptide Gly-His-lys confirmed the results with amino acids as N alpha -glycine alkylation predominated, but side chain adducts also formed w ith BHTOH-QM and BDMP-QM. Human hemoglobin was treated with QMs, hydro lyzed, and assayed by HPLC-thermospray mass spectrometry. This work re vealed that NE-lysine was the main alkylation site, emphasizing the im portance of factors, in addition to chemical reactivity, which influen ce protein modification by electrophiles. (C) 1997 Elsevier Science Ir eland Ltd.