Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium:: Potential implications in intracellular fluorescence detection of superoxide
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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DOI：
10.1016/s0891-5849(03)00142-4
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发表时间：
2003-06-01
影响因子：
7.4
通讯作者：
Kalyanaraman, B
中科院分区：
文献类型：
--
作者：
Zhao, HT;Kalivendi, S;Kalyanaraman, B
氢乙啶 (HE) 或二氢乙锭 (DHE) 是一种氧化还原敏感探针，已广泛用于检测细胞内超氧阴离子。人们普遍认为，超氧化物和 HE 之间的反应会形成双电子氧化产物乙锭 (E+)，乙锭 (E+) 与 DNA 结合，导致荧光增强（激发，500-530 nm；发射，590-620 nm）。然而，超氧阴离子氧化HE的机制仍不清楚。在本研究中，我们表明在几种酶或化学系统（例如黄嘌呤/黄嘌呤氧化酶、内皮一氧化氮合酶或超氧化钾）中产生的超氧化物将HE氧化成与E+完全不同的荧光产物（激发，480 nm；发射，567 nm）。 HPLC 测量表明 HE/超氧化物反应产物的洗脱方式与 E+ 不同。这种新产品在 DNA 存在下表现出荧光增强。质谱数据表明HE/超氧化物反应产物的分子量为330，而乙锭的分子量为314。我们得出结论，超氧化物和HE之间的反应形成了与乙锭不同的荧光标记产物。讨论了这一发现对细胞内超氧化物检测和成像的潜在影响。 (C) 2003 爱思唯尔公司。
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. (C) 2003 Elsevier Inc.