Determination of hydroxyl rate constants by a high-throughput fluorimetric assay: towards a unified reactivity scale for antioxidants

We describe in this article the development of a new method for the determination of rate constants of reaction of the hydroxyl radical, generated by radiolysis of water, with almost any possible molecule. It has been designed to provide a fast and reliable screening of antioxidant banks using microplates. Our particular approach is based on the use of the coumarin molecule as a competitor against the tested molecules: after a fast pulse of low dose irradiation, the fluorescence of 7-hydroxycoumarin produced by the oxidation of coumarin is measured and is inversely proportional to the scavenging ability of the tested antioxidant. We have validated our protocol using 32 molecules whose rate constants with HO˙ had already been evaluated and found a good agreement between our rate constants and the latter ones. The scopes and limitations of our method, as well as those of other rate constant determination methods, are discussed.