Pyridoindole antioxidant stobadine protected bovine serum albumin against the hydroxyl radical mediated cross-linking in vitro

On exposure to free radicals generated by the Fenton reaction system of Fe2+/EDTA/H2O2/ ascorbate, bovine serum albumin (BSA), used as a model of water-soluble protein, was losing its water solubility depending on the concentration of the chelated iron. The precipitate was found irreversibly insoluble even in concentrated urea. In the soluble fraction, SDS-PAGE analysis proved the presence of dimers and trimers of BSA, accompanied by enhanced bityrosine fluorescence. The pyridoindole antioxidant stobadine inhibited the process of albumin insolubilization in a concentration-dependent manner, the protective effect being more efficient than that of 2-keto-4-methiolbutyric acid (KMBA). Stobadine was, however, less effective than trolox. The inhibitory effect of the antioxidants, expressed as IC50, correlated well with the reciprocal values of corresponding second order rate constants for scavenging OH (.) radicals. The results indicated that the insolubilization of BSA induced by the Fenton system of Fe2+/EDTA/H2O2/ascorbate was caused by OH. radical mediated cross-linking of the albumin. The model system proved to be suitable for convenient testing of OH. radical scavenging ability of new antioxidants in a non-lipid environment. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.