PP17 - Kinetic investigation of the reaction of 4-methylbenzoquinone with thiol and amine compounds: consequences for protein modification

Abstract

Plant extracts rich in polyphenols are added as antioxidants to various foods. 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. The reaction of quinones with amino acids is involved in many physiological and pathological phenomena, such as skin inflammation, cataract and bone marrow toxicity. In this project we have determined rate constants for reaction of 4-methylbenzoquinone (4MBQ) (the oxidized form of 4-methylcatechol; generated by electrolysis) with thiols and amines (L-cysteine, N-α-acetyl-L-cysteine, glutathione (GSH), L-glycine, N-α-acetyl-L-lysine, N-α-acetyl-L-arginine) under pseudo first-order conditions. The rate constants were determined by following the loss of 4MBQ at 401 nm, and adduct formation at 294 nm by stopped-flow spectrophotometry over the pH range 4.5-6.5. Autoreduction of 4MBQ has k ~0.073 s -1 at pH 6.5. With L-glycine k is 0.102 s -1 at pH 6.5 so this is a slow reaction. No reaction was observed with side chain amine groups. Reaction of thiols with quinones occurs via reversible formation of an intermediate (with equilibrium constant K) and subsequent irreversible conversion to the final phenol adduct, with rate constant, k . With the thiols examined, the highest rate constant was obtained for L-cysteine ( k 2150 s -1 ). Lower values were obtained for N-α-acetyl-L-cysteine, where the amino group of is blocked by acetylation, and for GSH, suggesting that a free amine increases the reaction rate. These data shows that thiol groups are major targets for quinones such as 4MBQ, with rate constants at least 10,000-fold greater than for reaction with the side chain amine group of Lys, the free amino acid of amino acids, and the guanidine group of Arg residues. Thiols are therefore the major kinetic targets for protein modifications by quinones.