Quinone-induced protein modifications: Kinetic preference for reaction of 1,2-benzoquinones with thiol groups in proteins

Oxidation of polyphenols to quinones serves as an antioxidative mechanism, but the resulting quinones may induce damage to proteins as they react through a Michael addition with nucleophilic groups, such as thiols and amines to give protein adducts. In this study, rate constants for the reaction of 4-methylbenzoquinone (4MBQ) with proteins, thiol and amine compounds were determined under pseudo first order conditions by UV-vis stopped-flow spectrophotometry. The chemical structures of the adducts were identified by LC-ESI-MS/MS. Proteins with free thiols were rapidly modified by 4MBQ with apparent second order rate constants, k(2) of (3.1 +/- 0.2) x 10(4) M-1 s(-1) for bovine serum albumin (BSA) and (4.8 +/- 0.2) x 10(2) M-1 s(-1) for human serum albumin at pH 7.0. These values are at least 12-fold greater than that for alpha-lactalbumin (4.0 +/- 0.2) x 10(2) M-1 s(-1), which does not contain any free thiols. Reaction of Cys-34 of BSA with N-ethylmaleimide reduced the thiol concentration by similar to 59%, which resulted in a decrease in k(2) by a similar percentage, consistent with rapid adduction at Cys-34. Reaction of 4MBQ with amines (Gly, N alpha-acetyl-L-Lys, N epsilon-acetyl-L-Lys and L-Lys) and the guanidine group of N alpha-acetyl-L-Arg was at least 5 x 10(5) slower than with low-molecular-mass thiols (L-Cys, N alpha-acetyl-L-Cys, glutathione). The thiol-quinone interactions formed colorless thiol-phenol products via an intermediate adduct, while the amine-quinone interactions generated colored amine-quinone products that require oxygen involvement. These data provide strong evidence for rapid modification of protein thiols by quinone species which may be of considerable significance for biological and food systems. (C) 2016 Elsevier Inc. All rights reserved.