Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic Biol Med 2003 Jun 01;34(11):1359-68
Date
05/22/2003Pubmed ID
12757846DOI
10.1016/s0891-5849(03)00142-4Scopus ID
2-s2.0-0038368985 (requires institutional sign-in at Scopus site) 681 CitationsAbstract
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.
Author List
Zhao H, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vásquez-Vivar J, Kalyanaraman BAuthors
Balaraman Kalyanaraman PhD Professor in the Biophysics department at Medical College of WisconsinJeannette M. Vasquez-Vivar PhD Professor in the Biophysics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Chromatography, High Pressure LiquidDNA
Ethidium
Fluorescence
Fluorescent Dyes
Hydrazines
Mass Spectrometry
Nitric Oxide
Nitric Oxide Donors
Nitric Oxide Synthase
Nitric Oxide Synthase Type III
Nitrogen Oxides
Oxidation-Reduction
Phenanthridines
Superoxide Dismutase
Superoxides
Xanthine
Xanthine Oxidase