Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: Potential implications in intracellular fluorescence detection of superoxide
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.
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The neutrophil respiratory burst is usually measured spectrophotometrically by reduction of ferricytochrome C to ferrocytochrome C with absorbance at 550 nm, or histologically by reduction of nitro blue tetrazolium to insoluble formazan.
“The neutrophil respiratory burst is usually measured spectrophotometrically: (a) following the reduction of ferricytochrome C Fe3+ which is reduced by superoxide, producing oxygen and bright-red ferrocytochrome C Fe2+ with a detectable spectrophotometric absorbance at 550 nm [152,153]; and (b) histologically using the tetrazolium salt, nitro blue tetrazolium, which is reduced intracellularly to an”
From Superoxide Anion Chemistry—Its Role at the Core of the Innate Immunity (2023) · cites this paper as [152]
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- Authors
- Zhao, H., Kalivendi, S., Zhang, H., Joseph, J., Nithipatikom, K., Vásquez-Vivar, J., & Kalyanaraman, B.
- Affiliations
- Medical College of Wisconsin (US)
- Corresponding author
- Kalyanaraman, B.
- Venue
- Free Radical Biology and Medicine
- Publisher
- Elsevier BV
- Published
- 2003-06
- Volume
- 34
- Issue
- 11
- Pages
- 1359-1368
- ISSN
- 0891-5849 (ISSN-L)
- OpenAlex
- W2020600203
- Cited by
- 803
- References
- 30
- Citations by year
- 2026: 23 · 2025: 20 · 2024: 18 · 2023: 21 · 2022: 21 · 2021: 29 · 2020: 35 · 2019: 27 · 2018: 23 · 2017: 33 · 2016: 31 · 2015: 46 · 2014: 36 · 2013: 33 · 2012: 45
- FWCI
- 14.14
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Free Radical Biology and Medicine (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Heart, Lung, and Blood Institute (1P1 HL 68769-01, P01HL068769, R01HL067244, HL067244)
- National Cancer Institute (R01CA077822, CA-77822)
- National Institute of Neurological Disorders and Stroke (R01NS039958, NS 39958, NS40494, R01NS040494)
- National Center for Research Resources (P41RR001008, RR 01008)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
- Physical Sciences › Chemistry › Spectroscopy › Molecular Sensors and Ion Detection
- Health Sciences › Medicine › Biochemistry › Antioxidant Activity and Oxidative Stress
- Keywords
- hydroethidine, dihydroethidium, superoxide, ethidium, superoxide detection, reaction product identification, DNA binding, xanthine/xanthine oxidase, endothelial nitric oxide synthase, potassium superoxide
- MeSH terms
- Chromatography, High Pressure Liquid, DNA, Ethidium, Fluorescence, Fluorescent Dyes, Hydrazines, Nitric Oxide, Nitrogen Oxides, Oxidation-Reduction, Phenanthridines, Mass Spectrometry, Superoxides, Superoxide Dismutase, Xanthine Oxidase, Nitric Oxide Synthase, Xanthine, Nitric Oxide Donors, Nitric Oxide Synthase Type III
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