Assaying for hydroxyl radicals: Hydroxylated terephthalate is a superior fluorescence marker than hydroxylated benzoate

Abstract

Generation of hydroxyl radicals in terephthalate (benzene-1,4-dicarboxylic acid) solution yields fluorescent 2-hydroxy-terephthalate. The reaction product is stable for hours and can readily be assessed using standard fluorimeters. The efficiency, i.e. the relative increase of fluorescence per ·OH radical, is about three times higher than that of the formation of salicylate (2-hydroxy-benzoate) from benzoic acid and approximately hundred-fold higher than that of the hydroxylation of phenylalanine. As the terephthalate molecule is symmetric with respect to ring-hydroxylation, only one isomer is formed; hence, mechanistic interpretation of the hydroxylation reaction is facilitated.The scavenging rate constant of terephthalate for ·OH yielding the hydroxycyclohexadienyl adduct as first intermediate is close to the diffusion controlled limit (k = 3.3 × 109 M-1s-1). Therefore, competition of the detector molecule with biomolecules being present under physiological conditions is expected to be efficient. The assay can be used to detect ‘free’ ·OH radicals produced by the radiolysis of water as well as ‘hydroxyl analogous species’ that have been suggested to arise from the interaction of complex-bound reduced metal with either oxygen or hydrogen peroxide, e.g. from Fenton reactions.Based on calibration with radiolytically generated hydroxyl radicals the detection limit of the method is estimated to be around 50 nmol/dm3. Terephthalate is classified non-toxic and hence may also prove useful for microdialysis and continuous flow experiments as observation of fluorescence is ‘non-destructive’ and the reporter substance does not necessarily have to be subjected to HPLC.