Original ContributionsCritical evaluation of the use of hydroethidine as a measure of superoxide anion radical
Introduction
Hydroethidine (HE)1 is oxidized to the fluorescent ethidium (E+) by O2− whether added as KO2, or produced by activated leukocytes [1]. Moreover, the oxidation of HE to E+ was shown to be rapid when the oxidant was O2−; but not when O2, H2O2, HOCl or ONOO− were used [2]. Consequently HE has been applied as a detector of intracellular O2− [3].
We have investigated chromogenic and lumigenic detectors of O2−, such as the tetrazoliums [4] and lucigenin [5], and have found them to be prone to artifact in that they can cause O2− production even in systems not producing this radical in the absence of these compounds. We wondered whether in hydroethidine we might finally have a convenient measure of O2−, free from such problems.
In the experiments reported herein we verify earlier reports [1] that the oxidation of HE to E+ is rapidly caused by O2− but not by H2O2 [2]. However we note that HE can be oxidized by cytochrome c and more importantly that HE can catalyze the dismutation of O2−. This latter process interferes with the fluorimetric measurement of O2− with HE.
Section snippets
Materials and methods
HE was purchased from Polysciences Inc. and was dissolved, to 63.5 mM, in either N,N-dimethyl formamide or dimethyl sulfoxide. This stock solution was stored under N2 at −80°C. Working solutions were prepared by diluting this stock into 50 mM potassium phosphate at pH 7.8. This was done immediately prior to use. DNA and E+ were from Sigma. The oxidation of HE to E+ was monitored fluorimetrically by exciting at 470 nm and following emission at 590 nm.
The sodA sodB strains of Escherichia coli
Oxidation of HE to E+ by O2−
Line 1, Fig. 1 presents the rate of oxidation of HE to E+ by the flux of O2− generated by the xanthine oxidase reaction. That this oxidation was due to O2−, and not to H2O2 or to O2, is shown by the dose-dependent inhibition by SOD (lines 2, 3 and 4). Catalase, in contrast was without effect (not shown). These results are in full accord with previous reports [1], [2], [3]. It should be noted that xanthine oxidase plus xanthine did not cause the reduction of E+ to HE, even under anaerobic
Discussion
In accord with previous reports [1], [2] we find HE to be oxidized to E+ by O2−, whereas the rates of oxidation by O2 or by H2O2are much slower. The reduction of E+ to HE, by glucose oxidase plus glucose, does increase cyt c reduction, but that is due to a direct effect of HE and not to HE-mediated O2− production; since this cyt c reduction was not SOD-inhibitable. The oxidations of HE by cyt c consumed more than the theoretical 2 cyt c/HE and the product was not E+. This indicates that cyt c
Acknowledgements
This work was supported by grants from the Council for Tobacco Research, U.SA., Inc. (2871BR1) and the National Institutes of Health (HL56025-03).
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