Generation of the Thiyl Radical in Bovine Serum Albumin by the Superoxide Anion Radical

Abstract

The mechanism for the beneficial effects of vitamin C (ascorbic acid, AsA) on the quality of the heatinduced fish gel, kamaboko, has been proposed by Nishimura et al. to involve the production of the thiyl radical (S •E ). However, the detection of S •E in myosin is very difficult, because its large molecular weight (about 500,000) produces a strained electron spin resonance (ESR) signal. The generation of S •E in myosin has therefore not yet been elucidated. We conducted an ESR analysis with bovine serum albumin (BSA) which is a lower-molecular-weight protein (about 60, 000). Although the signal with four main peaks that was observed under illumination at 650,000 lx in a mixture containing riboflavin, a spintrapping reagent, and reduced BSA by dithiothreitol, non-reduced BSA produced only a small signal. The intensity of the signal obtained by using reduced BSA, however, decreased in response to superoxide dismutase (SOD; 200 units/ml), while inactivated SOD had little effect. These results suggest that S •E could be produced in protein by the action of O2-. (Received November 2, 2004; Accepted in revised form May 30, 2005)