Phenoxyl Free Radical Formation during the Oxidation of the Fluorescent Dye 2′,7′-Dichlorofluorescein by Horseradish Peroxidase

Abstract

The oxidation of the fluorescent dye 2,7-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF ⅐ ). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF ⅐ oxidizes GSH, generating the glutathione thiyl radical (GS ⅐ ), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS ⅐ radical. Similarly, DCF ⅐ oxidized NADH, generating the NAD ⅐ radical that reduced oxygen to superoxide (O 2 . ), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H 2 O 2 , which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H 2 O 2 production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.2Ј,7Ј-Dichlorofluorescin (DCFH) 1 is widely used to measure oxidative stress in cells. The diacetate form of DCFH enters the cell and is hydrolyzed by intracellular esterases to liberate DCFH. Upon reaction with oxidizing species, the highly fluorescent compound 2Ј,7Ј-dichlorofluorescein (DCF) is formed. The fluorescence intensity can be measured and is the basis of the popular cellular assay for oxidative stress (1).Although this assay is commonly used, there are many controversies regarding its real validity. It is not clear which oxidative species is responsible for the oxidation of DCFH. Furthermore, there are some disagreements in the literature concerning the effect of superoxide dismutase on oxidative stress in cells monitored by the DCF fluorometric assay. Some studies report an inhibitory effect by superoxide dismutase (2-15), while almost the same number of studies report no effect (16 -26). A recent paper by LeBel et al. (27) mentioned that the interpretation of specific reactive oxygen species involved in the oxidation of DCFH to DCF in biological systems should be approached with caution. It has also been demonstrated that the photoreduction of DCF results in the formation of the DCF semiquinone free radical (DCF . ), which, under aerobic conditions, is oxidized by oxygen to its parent dye, DCF, concomitantly forming superoxide radical (28). Moreover, it was reported that DCFH, upon reacting with horseradish peroxidase-compound I or -compound II, was oxidized to DCF . , which was subsequently air-ox...

Imaging Dynamic Redox Changes in Mammalian Cells with Green Fluorescent Protein Indicators

“…Targeting the probe to the plasma membrane or mutating the roGFPs to enhance sensitivity to H 2 O 2 failed to uncover sensitivity to EGF in NR6 cells or by LPA in HeLa cells, even though the modified probes still reacted to exogenous oxidants. Meanwhile, we verified in parallel experiments that the classic ROS probe 2Ј,7Ј-dichlorodihydrofluorescein diacetate in NR6 cells responded to EGF, although 2Ј,7Ј-dichlorodihydrofluorescein diacetate has been reported to be vulnerable to autocatalytic oxidation or other artifacts (35,36). Therefore, we doubt that the above growth factors cause significant acute global perturbations in thioldisulfide redox potential.…”

Reactive oxygen species and cellular oxygen sensing

“…For instance, DCFH has been shown to react with agents other than H 2 O 2 making it more of a measure of total redox status rather than cellular H 2 O 2 [54,55]. Moreover, both lucigenin and DCFH have been shown to yield a fluorescent signal in the absence of ROS, giving rise to the potential for high background and false positive readings [56][57][58]. In addition to these limitations, one must also assume that cellular uptake of DCFH-DA is equivalent under different conditions if a direct comparison is to be made.…”

Characterization of Wintertime Reactive Oxygen Species Concentrations in Flushing, New York

“…A calibration curve (shown in supplementary material, Figure S1) was drawn from the measured fluorescence of standard H 2 O 2 solutions, and the fluorescence intensity data from the analysis of the samples was converted into equivalent H 2 O 2 concentrations using the obtained calibration curve. In prior biological studies, it has been documented that removal of the diacetate groups in DCFH-DA may result in trace H 2 O 2 production (Rota et al 1999). However, we suggest that these are quantitatively insignificant sources of error, based on the observed background levels of DCF fluorescence.…”