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

Abstract

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.

A New Mechanism for Hydroxyl Radical Production in Irradiated Nanoparticle Solutions

“…32.5 nm nanoparticles were put in the presence of coumarin to measure the hydroxyl radical production after irradiation as it was suggested to be the main radical responsible for target degradation when nanoparticles are irradiated by ionizing radiation . Among the numerous ways of quantifying hydroxyl radical production, the coumarin HO • trapping assay was chosen because it is fully compatible with nanoparticles as it doesn' induce any aggregation and because fluorescence detection that can be run is a very sensitive technique allowing the detection of quantities down to 30 n m of hydroxyl radical …”

“…[ 14 ] Among the numerous ways of quantifying hydroxyl radical production, the coumarin HO • trapping assay [18][19][20] was chosen because it is fully compatible with nanoparticles as it doesn't induce any aggregation and because fl uorescence detection that can be run is a very sensitive technique allowing the detection of quantities down to 30 n m of hydroxyl radical. [ 21 ] Louit et al performed HO • quantifi cation by titration of 7-hydroxycoumarin that was shown to be the main fl uorescent product of coumarin oxidation. This titration was validated both by liquid chromatography coupled to fl uorescence detection and by fl uorescence titration without preliminary separation of the oxidized coumarin derivatives.…”

Small Molecule Signaling Agents: The Integrated Chemistry and Biochemistry of Nitrogen Oxides, Oxides of Carbon, Dioxygen, Hydrogen Sulfide, and Their Derived Species

“…The second order rate constants for the reaction of HO· with a variety of biologically relevant molecules are typically near the diffusion-controlled limit (>10 9 M −1 s −1 ), indicating that very few collisions are required for a reaction to occur. 28 Thus, it will be expected that biological damage by HO· will be localized to its site of generation, which in many cases means the site where a catalytic metal is present (reaction 13). In fact, evidence suggests that under certain conditions “free” HO· is not generated in the Fenton reaction but that rather a metal-bound oxidant is made, and this is the ultimate oxidant.…”

Reaching new levels of realism in modeling biological macromolecules in cellular environments

“…That is in principle also possible using computational methods [232, 233], but it would be rather tedious and expensive to carry out such calculations for a large number of different enzymes and reactions. There is hope, however, that this may not be necessary given recent efforts to obtain kinetic rate constants in a high-throughput and consistent manner that should result in more reliable data [234, 235]. …”