The αPROX assay: fluorescence screening of the inhibitory effects of hydrophilic antioxidants on protein oxidation

Abstract

We report on a new and convenient high-throughput fluorescence technique for determining antioxidant capacities of hydrophilic food samples. The new method is called alphaPROX (anti protein oxidation) and is based on an equimolar complex of diphenylhexatriene propionic acid (DPHPA) and bovine serum albumin (BSA) in aqueous buffer at pH 7.4. DPHPA is a reporter fluorophore that becomes nonfluorescent upon free radical-induced oxidation. In a typical assay, the DPHPA/BSA complex is challenged with peroxyl radicals and shows almost the same susceptibility to oxidation as unlabeled BSA. The progress of protein oxidation and its inhibition by antioxidants at physiological pH is determined from the time-dependent decrease in DPHPA fluorescence intensity. The alphaPROX method was compared to other techniques frequently used to measure antioxidant capacities. In this article, representative results are provided for the inhibitory effects of pure food components, fruit juices, wines, and various polar plant extracts on protein oxidation.