Instantaneous monitoring of hydroxyl radical-mediated protein alterations by green fluorescent protein

Abstract

In the present study, green fluorescent protein (GFP) is successfully applied for instantaneous monitoring of hydroxyl radical-mediated protein alterations. Hydroxyl radical generated from metal-mediated Fenton’s reaction (in the presence of 50 μM copper ions, 10 mM ascorbic acid and 1.05 % hydrogen peroxide) rapidly suppressed the fluorescent emission of 60 % in a few seconds followed by a gradual decrease up to 75 % maximum inactivation was reached. The production of hydroxyl radical was experimentally proven to be specifically derived from copper-catalyzed Fenton’s reaction in which other divalent cations (e. g. Zn2+, Cd2+, Mn2+, Co2+ and Ni2+) exerted no inhibitory interaction. Supplementation of oxidative scavengers and metal chelators into the assay reaction provided protective effects on the fluorescent intensity. The degree of protection was in the order of EDTA > histidine >>> glutathione ~ sodium azide > thiourea ~ mannitol. The findings herein gain insights not only into the deleterious effect of reactive oxygen species on biological macromolecules but also the potential applicability as a versatile antioxidant screening assay.