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Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide

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Abstract

Hydroethidine (HE) or dihydroethidium (DHE), a redox-sensitive probe, has been widely used to detect intracellular superoxide anion. It is a common assumption that the reaction between superoxide and HE results in the formation of a two-electron oxidized product, ethidium (E+), which binds to DNA and leads to the enhancement of fluorescence (excitation, 500–530 nm; emission, 590–620 nm). However, the mechanism of oxidation of HE by the superoxide anion still remains unclear. In the present study, we show that superoxide generated in several enzymatic or chemical systems (e.g., xanthine/xanthine oxidase, endothelial nitric oxide synthase, or potassium superoxide) oxidizes HE to a fluorescent product (excitation, 480 nm; emission, 567 nm) that is totally different from E+. HPLC measurements revealed that the HE/superoxide reaction product elutes differently from E+. This new product exhibited an increase in fluorescence in the presence of DNA. Mass spectral data indicated that the molecular weight of the HE/superoxide reaction product is 330, while ethidium has a molecular weight of 314. We conclude that the reaction between superoxide and HE forms a fluorescent marker product that is different from ethidium. Potential implications of this finding in intracellular detection and imaging of superoxide are discussed.

Introduction

One of the most popular assays for detecting superoxide in cells and tissues involves the use of fluorescence-based techniques 1, 2, 3, 4, 5, 6. Generally, the red fluorescence arising from oxidation of hydroethidine (HE) (also dihydroethidium [DHE]) is detected 7, 8, 9, 10, 11, 12, 13, 14 (Fig. 1). This red fluorescence, often referred to as the “ethidium fluorescence,” is inhibited by intracellular superoxide dismutase and other superoxide scavengers 7, 8, 9, 10, 11, 12, 13, 14. HE is synthesized from sodium borohydride reduction of ethidium, a two-electron oxidation product [15]. Most previous fluorescence measurements have been performed using a kinetic mode and typically acquired at a single wavelength corresponding to that of ethidium 16, 17, 18, 19, 20. Alternatively, the red fluorescence due to oxidized HE was visualized in cells and tissues using fluorescence or confocal microscopy. To our knowledge, the question of whether superoxide-dependent oxidation of HE actually generates ethidium as a product has never been considered.

Like many other investigators in this field, we were under the impression that HE is selectively oxidized by superoxide to ethidium 1, 2, 3, 21. Recently, we began a systematic analysis of the chemistry of oxidant-sensitive fluorescent dyes, including dichlorodihydrofluorescein and HE 22, 23. We discovered that superoxide reacts with HE to form a fluorescent product that is distinctly different from ethidium. In the present study, we provide fluorescence, HPLC, and mass spectral evidence in support of this new finding. The potential implications of this finding with respect to intracellular detection, quantitation, and imaging of superoxide using HE as a fluorescent probe, are discussed.

Section snippets

Materials

Hydroethidine (dihydroethidium) was purchased from Molecular Probes Inc. (Eugene, OR, USA). Ethidium bromide, calf thymus, DNA, ferricytochrome c, NADPH, l-arginine, xanthine, calcium chloride, potassium superoxide, and diethylenetriamine pentaacetic acid (DTPA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The potassium superoxide in dimethyl sulfoxide (DMSO) was prepared immediately prior to use [24]. Bovine brain calmodulin was obtained from Calbiochem (San Diego, CA, USA).

Fluorescence spectra of the product formed from superoxide-dependent oxidation of HE

Figure 2A shows the fluorescence spectra for HE (emission maximum = 595 nm) (Table 1), the oxidation product of HE by X/XO (emission maximum = 586 nm) and for ethidium (emission maximum = 605 nm) in phosphate buffer. Superoxide generation was measured to be 8 μM/min. Figure 2B shows the enhancement in the fluorescence intensity of the three compounds in the presence of DNA. DNA did not have any effect on the rate of oxidation of HE in this system (data not shown). Addition of superoxide

Discussion

Our findings demonstrate that superoxide reacts with HE to form a characteristic fluorescent product that is different from E+. In the presence of other reactive oxygen and nitrogen species (e.g., hydrogen peroxide, hydroxyl radical, or peroxynitrite), HE was not oxidized to form the same fluorescent product. The present findings point to a potentially new and improved fluorescence marker for detecting superoxide in cells.

Abbreviations

  • BH4—6R-5, 6, 7, 8-tetrahydrobiopterin

  • BMPO—5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide

  • DEA-NONOate—2-(N, N-diethylamino)-diazenolate-2-oxide

  • DHE—dihydroethidium

  • E+—ethidium

  • HE—hydroethidine

  • NOS—nitric oxide synthase

  • X—xanthine

  • XO—xanthine oxidase

Acknowledgements

This work was supported by grants 1PIHL68769-01, RR01008, NS40494, HL067244, NS39958, and CA77822 from the National Institutes of Health. The authors thank Mr. Hanbing Lu for his help in obtaining the fluorescence photograph of the DNA gel.

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