期刊
FREE RADICAL BIOLOGY AND MEDICINE
卷 49, 期 1, 页码 61-66出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.freeradbiomed.2010.03.014
关键词
Superoxide detection; Hydrogen peroxide; Superoxide reductase; Amplex red; Horseradish peroxidase; Free radicals
资金
- FCT/MCTES (Portugal) [PTDC/SAU-GMG/70033/2006, PTDC/QUI/70101/2006, SFRH/BPD/34763/2007]
- Fundação para a Ciência e a Tecnologia [PTDC/SAU-GMG/70033/2006, PTDC/QUI/70101/2006, SFRH/BPD/34763/2007] Funding Source: FCT
Superoxide reductase (SOR) is an enzyme that converts superoxide into hydrogen peroxide at a twofold higher yield than canonical superoxide dismutases (SOD). Superoxide radical detection was investigated using the Amplex red (AR)/peroxidase system to measure the difference in hydrogen peroxide production yield in the presence of SOR or SOD. We found that reduced SOR reacts with the AR oxidation intermediate, a one-electron reduced AR radical, by reducing this intermediate back to the initial AR leuco compound. Ascorbate also quenched this radical in a concentration-dependent manner and could be used to compete efficiently with SOR; at concentrations of ascorbate higher than 5 mu M, SOR no longer interfered with the detection of H2O2. By using xanthine/xanthine oxidase as a superoxide-generating system, it was possible to successfully quantify superoxide and hydrogen peroxide in vitro using the AR/peroxidase/SOR system, either by visible absorption or by fluorescence emission, with a considerable low detection limit of 10 nM/min. The use of enzymes with diffusion-limited reactivity toward superoxide substantially increases specificity and detection threshold for superoxide and turns this approach into a powerful system to detect ROS in biological systems. (C) 2010 Elsevier Inc. All rights reserved.
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