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2′,7′-dichlorofluorescin-based analysis of Fenton chemistry reveals auto-amplification of probe fluorescence and albumin as catalyst for the detection of hydrogen peroxide
Biochemical Journal ( IF 4.1 ) Pub Date : 2020-12-23 , DOI: 10.1042/bcj20200602
Teresa Gonzalez 1 , Franck Peiretti 1 , Catherine Defoort 1 , Patrick Borel 1 , Roland Govers 1
Affiliation  

Fluorophore 2′,7′-dichlorofluorescin (DCF) is the most frequently used probe for measuring oxidative stress in cells, but many aspects of DCF remain to be revealed. Here, DCF was used to study the Fenton reaction in detail, which confirmed that in a cell-free system, the hydroxyl radical was easily measured by DCF, accompanied by the consumption of H2O2 and the conversion of ferrous iron into ferric iron. DCF fluorescence was more specific for hydroxyl radicals than the measurement of thiobarbituric acid (TBA)-reactive 2-deoxy-D-ribose degradation products, which also detected H2O2. As expected, hydroxyl radical-induced DCF fluorescence was inhibited by iron chelation, anti-oxidants, and hydroxyl radical scavengers and enhanced by low concentrations of ascorbate. Remarkably, due to DCF fluorescence auto-amplification, Fenton reaction-induced DCF fluorescence steadily increased in time even when all ferrous iron was oxidized. Surprisingly, the addition of bovine serum albumin rendered DCF sensitive to H2O2 as well. Within cells, DCF appeared not to react directly with H2O2 but indirect via the formation of hydroxyl radicals, since H2O2-induced cellular DCF fluorescence was fully abolished by iron chelation and hydroxyl radical scavenging. Iron chelation in H2O2-stimulated cells in which DCF fluorescence was already increasing did not abrogate further increases in fluorescence, suggesting DCF fluorescence auto-amplification in cells. Collectively, these data demonstrate that DCF is a very useful probe to detect hydroxyl radicals and hydrogen peroxide and to study Fenton chemistry, both in test tubes as well as in intact cells, and that fluorescence auto-amplification is an intrinsic property of DCF.

中文翻译:

基于2',7'-二氯荧光素的Fenton化学分析显示自动扩增探针荧光和白蛋白作为检测过氧化氢的催化剂

荧光团2',7'-二氯荧光素(DCF)是最常用于测量细胞氧化应激的探针,但是DCF的许多方面仍有待揭示。在这里,DCF被用来详细研究Fenton反应,这证实了在无细胞系统中,DCF可以很容易地测量羟基自由基,伴随着H2O2的消耗和亚铁转化为三价铁。DCF荧光对羟基自由基的特异性比对硫代巴比妥酸(TBA)反应性的2-脱氧-D-核糖降解产物的测量要好,后者也可以检测到H2O2。不出所料,铁螯合剂,抗氧化剂和羟自由基清除剂可抑制羟自由基诱导的DCF荧光,而低浓度的抗坏血酸盐会增强羟自由基诱导的DCF荧光。值得注意的是,由于DCF荧光自动扩增,即使所有亚铁都被氧化,Fenton反应诱导的DCF荧光也随时间稳定增加。令人惊讶的是,添加牛血清白蛋白也使DCF对H2O2敏感。在细胞内,DCF似乎不直接与H2O2反应,而是通过形成羟基自由基间接发生反应,因为H2O2诱导的细胞DCF荧光被铁螯合和羟基自由基清除完全消除了。DCF荧光已经增加的H2O2刺激的细胞中的铁螯合并不能消除荧光的进一步增加,这表明DCF荧光会在细胞中自动扩增。总的来说,这些数据表明DCF是检测试管和完整细胞中羟基自由基和过氧化氢以及研究Fenton化学的非常有用的探针,
更新日期:2020-12-18
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