Evidence for free radical formation during the oxidation of 2′-7′-dichlorofluorescin to the fluorescent dye 2′-7′-dichlorofluorescein by horseradish peroxidase:: Possible implications for oxidative stress measurements

Abstract

The oxidation of 2′-7′-dichlorofluorescin (DCFH) to the fluorescent 2′-7′-dichlorofluorescein (DCF) by horseradish peroxidase (HRP) was investigated by fluorescence, absorption, and electron spin resonance spectroscopy (ESR). As has been previously reported, HRP/H 2O 2 oxidized DCFH to the highly fluorescent DCF. However, DCF fluorescence was still observed when H 2O 2 was omitted, although its intensity was reduced by 50%. Surprisingly, the fluorescence increase, in the absence of exogenous H 2O 2, was still strongly inhibited by catalase, demonstrating that H 2O 2 was present and necessary for DCF formation. H 2O 2was apparently formed during either chemical or enzymatic deacetylation of 2′-7′-dichlorofluorescin diacetate (DCFH-DA), probably by auto-oxidation. Spectrophotometric measurements clearly showed that DCFH could be oxidized either by HRP-compound I or HRP-compound II with the obligate generation of the DCF semiquinone free radical (DCF •−). Oxidation of DCF •− to DCF by oxygen would yield superoxide (O 2 •−). ESR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the presence of both superoxide and hydroxyl radicals in the DCFH/H 2O 2/HRP system. Both radicals were also detected in the absence of added H 2O 2, although the intensities of the resultant adducts were decreased. This work demonstrates that DCF fluorescence cannot be used reliably to measure O 2 •− in cells because O 2 •− itself is formed during the conversion of DCFH to DCF by peroxidases. The disproportionation of superoxide forms H 2O 2 which, in the presence of peroxidase activity, will oxidize more DCFH to DCF with self-amplification of the fluorescence. Because the deacetylation of DCFH-DA, even by esterases, can produce H 2O 2, the use of this probe to measure H 2O 2 production in cells is problematic.