Reactivity and mechanism of the reactions of 4-methylbenzoquinone with amino acid residues in β-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 β-lactoglobulin (β-LG) and amino acids at neutral pH were investigated. LC-MS analysis revealed that Cys121 was likely the most modified residue in β-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 °C and pH 7.0 by stopped-flow spectrophotometry. The rate of reactions decreased in the order: β-LG > His > Trp > Arg > N^α-acetyl His > N^α-acetyl Arg > N^α-acetyl Trp. The rate constants correlated with the pK_a values of the amino acids, showing that the amount of unprotonated amine is the major factor determining the reactivity.