Analysis of kinetics of dihydroethidium fluorescence with superoxide using xanthine oxidase and hypoxanthine assay.
Analysis of kinetics of dihydroethidium fluorescence with superoxide using xanthine oxidase and hypoxanthine assay.
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DOI：
10.1007/s10439-012-0653-x
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发表时间：
2013-02
影响因子：
3.8
通讯作者：
Kavdia, Mahendra
中科院分区：
文献类型：
--
作者：
Chen, Juan;Rogers, Steven C.;Kavdia, Mahendra
超氧化物（O2 −）是一种重要的活性氧（ROS），在生理和病理生理学中起着重要作用。需要准确检测O2 −，以更好地了解许多血管病变。在这项研究中，我们通过使用黄嘌呤氧化酶（XOD）/次黄嘌呤（HX）测定O2 −生成和O2 −敏感荧光染料二氢乙锭（DHE）测量O2 −进行了机制研究。为了量化O2 −和DHE的相互作用，我们使用酶标仪测量了荧光。我们对DHE-O2 −相互作用进行了详细的反应动力学分析，以了解O2 −自歧化的影响并量化DHE-O2 −反应速率。DHE和2-hydroethidium（EOH）（DHE和O2 −相互作用的产物）的荧光取决于反应条件。动力学分析得出DHE-O2-反应的反应速率常数为2.169 ± 0.059 × 103 M − 1s − 1，比报告值2.6 ± 0.6 × 105 M − 1s − 1慢约100倍。此外，O2 −自歧化对DHE-O2 −相互作用有显著影响。对于O2-测量来说，DHE与O2-的反应速率较慢更合理。在这种方式下，DHE不会与超氧化物歧化酶和NO竞争O2 −。结果表明，由于许多因素的竞争性干扰，O2 −产生速率的准确测量可能很困难;但是O2 −浓度可以量化。
Superoxide (O2−) is an important reactive oxygen species (ROS), and has an essential role in physiology and pathophysiology. An accurate detection of O2− is needed to better understand numerous vascular pathologies. In this study, we performed a mechanistic study by using the xanthine oxidase (XOD)/hypoxanthine (HX) assay for O2− generation and a O2− sensitive fluorescent dye dihydroethidium (DHE) for O2− measurement. To quantify O2− and DHE interactions, we measured fluorescence using a microplate reader. We conducted a detailed reaction kinetic analysis for DHE–O2− interaction to understand the effect of O2− self-dismutation and to quantify DHE–O2− reaction rate. Fluorescence of DHE and 2-hydroethidium (EOH), a product of DHE and O2− interaction, were dependent on reaction conditions. Kinetic analysis resulted in a reaction rate constant of 2.169±0.059×103 M−1s−1 for DHE-O2− reaction that is ~ 100× slower than the reported value of 2.6±0.6×105 M−1s−1. In addition, the O2− self-dismutation has significant effect on DHE-O2− interaction. A slower reaction rate of DHE with O2− is more reasonable for O2− measurements. In this manner, the DHE is not competing with superoxide dismutase and NO for O2−. Results suggest that an accurate measurement of O2− production rate may be difficult due to competitive interference for many factors; however O2− concentration may be quantified.
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影响因子：
2.9
作者：
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