Reactivity and mechanism of the reactions of 4-methylbenzoquinone with amino acid residues in S-lactoglobulin: A kinetic and product investigation

Quinones, produced by the oxidation of phenolic compounds, covalently bind to nucleophilic groups on amino acids or proteins. In this study, the reactions of 4-methylbenzoquinone (4MBQ) with S-lactoglobulin (S-LG) and amino acids at neutral pH were investigated. LC-MS analysis revealed that Cys121 was likely the most modified residue in S-LG. Identification of reaction products by LC-MS/MS showed that Michael addition occurred in all reactions with amino acids tested. The formation of Schiff base and a di-adduct was found in His and Trp samples. Apparent second-order rate constants (k2) were determined at 25 degrees C and pH 7.0 by stopped-flow spectrophotometry. The rate of reactions decreased in the order: S-LG > His > Trp > Arg > N degrees-acetyl His > N degrees-acetyl Arg > N degrees-acetyl Trp. The rate constants correlated with the pKa values of the amino acids, showing that the amount of unprotonated amine is the major factor determining the reactivity.

Automated summary

This study investigated the reactions of 4-methylbenzoquinone with S-lactoglobulin and amino acids at neutral pH. The results showed that Cys121 was the most modified residue in S-LG, and Michael addition occurred in all reactions with amino acids. The rate constants correlated with the pKa values of the amino acids, indicating that the amount of unprotonated amine is the major factor determining reactivity.