Involvement of the Superoxide Anion Radical in the Autoxidation of Pyrogallol and a Convenient Assay for Superoxide Dismutase

Abstract

The autoxidation of pyrogallol was investigated in the presence ofEDTA in the pH range7.9-10.6. The rate of autoxidation increases with increasing pH. At pH 7.9 the reaction is inhibited to 99 by superoxide dismutase, indicating an almost total dependence on the participation of the superoxide anion radical, 02.-, in the reaction. Up to pH 9.1 the reaction is still inhibited to over 90% by superoxide dismutase, but at higher alkalinity, O,.--independent mechanisms rapidly become dominant.Catalase has no effect on the autoxidation but decreases the oxygen consumption by half, showing that H202 is the stable product of oxygen and that H20, is not involved in the autoxidation mechanism.A simple and rapid method for the assay of superoxide dismutase is described, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol.A plausible explanation is given for the non-competitive part of the inhibition of catechol 0-methyltransferase brought about by pyrogallol.Pyrogallol (1,2,3-benzenetriol) has long been known to autoxidize rapidly, especially in alkaline solution and the reaction has been employed for the removal of oxygen from gases.Molecular oxygen, carrying two unpaired electrons with parallel spins, has a preference for univalent reduction because spin restrictions arise when reduction with electron pairs is attempted [l]. The recently discovered enzyme superoxide dismutase [2] extremely rapidly dismutases univalently reduced oxygen 02.-, the superoxide anion radical (2 02.-+ 2 H + -+ 0, no saturation kinetics at the O,.-concentrations which have been feasible to produce [7] and hence induces a pseudo-first order dismutation of O,.-.The present paper describes studies of the autoxidation of pyrogallol under various conditions. The role of 02.-in the reactions was investigated with the aid of superoxide dismutase. The data obtained allotted suitable conditions for a convenient assay of superoxide dismutase. MATERIALS AND METHODS+ H,O,). The enzyme has proven to be a useful probe for studying the participation of the radical in reac-

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