Photooxidation of Amplex Red to resorufin: implications of exposing the Amplex Red assay to light.
Photooxidation of Amplex Red to resorufin: implications of exposing the Amplex Red assay to light.
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DOI：
10.1016/j.freeradbiomed.2012.06.034
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发表时间：
2012-09-01
影响因子：
7.4
通讯作者：
Mason, Ronald P.
中科院分区：
文献类型：
--
作者：
Zhao, Baozhong;Summers, Fiona A.;Mason, Ronald P.
Amplex Red试验是一种检测H2O2的荧光试验，依靠H2O2与无色、无荧光的Amplex Red以1:1的化学计量量反应，在辣根过氧化物酶（HRP）的催化下形成彩色、荧光的再间苯二酚。我们发现，当Amplex Red暴露在光线下时，再间苯甲醚是人为形成的。在没有H2O2和HRP的情况下，Amplex Red在室温光或仪器激发光下的吸收光谱和荧光光谱发生了变化，清楚地表明荧光产物间苯二酚已经形成。这种光化学反应是由存在于Amplex Red原液中的微量再间酚引发的。ESR自旋俘获研究表明，超氧自由基是这一过程的中间体。耗氧量测量进一步证实，超氧化物和H2O2是由Amplex红的光氧化人工产生的。超氧化物歧化酶（HRP）的存在也显著增加了间苯二酚的人工形成。这种光氧化过程将导致在环境光照射下对H2O2的敏感性降低，并且在高能量照射（如UVA照射）下可能无效的测量。一般来说，在用Amplex Red测量氧化应激时，应采取预防措施，尽量减少暴露在光下。
The Amplex Red assay, a fluorescent assay for the detection of H2O2, relies on the reaction of H2O2 and colorless, nonfluorescent Amplex Red with a 1:1 stoichiometry to form colored, fluorescent resorufin, catalyzed by horseradish peroxidase (HRP). We have found that resorufin is artifactually formed when Amplex Red is exposed to light. In the absence of H2O2 and HRP, the absorption and fluorescence spectra of Amplex Red changed during exposure to ambient room light or instrumental excitation light, clearly indicating that the fluorescent product resorufin had formed. This photochemistry was initiated by trace amounts of resorufin that are present in Amplex Red stock solutions. ESR spin-trapping studies demonstrated that superoxide radical was an intermediate in this process. Oxygen consumption measurements further confirmed that superoxide and H2O2 were artifactually produced by the photooxidation of Amplex Red. The artifactual formation of resorufin was also significantly increased by the presence of superoxide dismutase or HRP. This photooxidation process will result in a less sensitive assay for H2O2 under ambient light exposure and potentially invalid measurements under high energy exposure such as UVA irradiation. In general, precautions should be taken to minimize exposure to light during measurement of oxidative stress with Amplex Red.
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