Superoxide-driven NAD(P)H oxidation induced by EDTA-manganese complex and mercaptoethanol

Abstract

A purely chemical system for NAD(P)H oxidation to biologically active NAD(P) + has been developed and characterized. Suitable amounts of EDTA, manganous ions and mercaptoethanol, combined at physiological pH, induce nucleotide oxidation through a chain length also involving molecular oxygen, which eventually undergoes quantitative reduction to hydrogen peroxide. Mn 2+ is specifically required for activity, while both EDTA and mercaptoethanol can be replaced by analogs. Optimal molar ratios of chelator/metal ion (2 : 1) yield an active coordination compound which catalyzes thiol autoxidation to thiyl radical. The latter is further oxidized to disulfide by molecular oxygen whose one-electron reduction generates superoxide radical. Superoxide dismutase (SOD) inhibits both thiol oxidation and oxygen consumption as well as oxidation of NAD(P)H if present in the mixture. A tentative scheme for the chain length occurring in the system is proposed according to stoichiometry of reactions involved. Two steps appear of special importance in nucleotide oxidation: (a) the supposed transient formation of NAD(P) • from the reaction between NAD(P)H and thiyl radicals; (b) the oxidation of the reduced complex by superoxide to keep thiol oxidation cycling.