Evidence for free radical formation during the oxidation of 2′-7′-dichlorofluorescin to the fluorescent dye 2′-7′-dichlorofluorescein by horseradish peroxidase:: Possible implications for oxidative stress measurements
Evidence for free radical formation during the oxidation of 2′-7′-dichlorofluorescin to the fluorescent dye 2′-7′-dichlorofluorescein by horseradish peroxidase:: Possible implications for oxidative stress measurements
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DOI：
10.1016/s0891-5849(99)00137-9
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发表时间：
1999-10-01
影响因子：
7.4
通讯作者：
Mason, RP
中科院分区：
文献类型：
--
作者：
Rota, C;Chignell, CF;Mason, RP
采用荧光、吸收和电子自旋共振光谱（ESR）研究了辣根过氧化物酶（HRP）氧化2'-7'-二氯荧光素（DCFH）生成2'-7'-二氯荧光素（DCF）的过程。如前所述，HRP/H2O2氧化DCFH生成高荧光DCF。而忽略H2O2时，DCF荧光仍然存在，但强度降低了50%。令人惊讶的是，在没有外源H2O2的情况下，荧光增强仍然被过氧化氢酶强烈抑制，这表明H2O2存在并且是DCF形成所必需的。H2O2显然是在2'-7'-双乙酸二氯荧光素（DCFH-DA）的化学或酶脱乙酰过程中形成的，可能是通过自氧化。分光光度测定清楚地表明，DCFH可以被酶标化合物I或酶标化合物II氧化，并产生DCF半醌自由基（DCF.-）。DCF氧化。-被氧转化成DCF会生成超氧化物（O-2(.-)）。ESR光谱结合自旋阱5,5-二甲基-1-吡咯啉n -氧化物（DMPO）发现DCFH/H2O2/HRP系统中存在超氧自由基和羟基自由基。在不添加H2O2的情况下，这两种自由基也被检测到，尽管结果加合物的强度降低了。这项工作表明，DCF荧光不能可靠地用于测量O-2（）。-)，因为O-2(。-)本身是在过氧化物酶将DCFH转化为DCF时形成的。超氧化物歧化形成H2O2，在过氧化物酶活性的作用下，将更多的DCFH氧化为DCF，荧光自扩增。因为DCFH-DA的去乙酰化，即使是通过酯酶，也会产生H2O2，所以使用这种探针来测量细胞中H2O2的产生是有问题的。(C) 1999 Elsevier Science Inc.；
The oxidation of 2'-7'-dichlorofluorescin (DCFH) to the fluorescent 2'-7'-dichlorofluorescein (DCF) by horseradish peroxidase (HRP) was investigated by fluorescence, absorption, and electron spin resonance spectroscopy (ESR). As has been previously reported, HRP/H2O2 oxidized DCFH to the highly fluorescent DCF. However, DCF fluorescence was still observed when H2O2 was omitted, although its intensity was reduced by 50%. Surprisingly, the fluorescence increase, in the absence of exogenous H2O2, was still strongly inhibited by catalase, demonstrating that H2O2 was present and necessary for DCF formation. H2O2 was apparently formed during either chemical or enzymatic deacetylation of 2'-7'-dichlorofluorescin diacetate (DCFH-DA), probably by auto-oxidation. Spectrophotometric measurements clearly showed that DCFH could be oxidized either by HRP-compound I or HRP-compound II with the obligate generation of the DCF semiquinone free radical (DCF.-). Oxidation of DCF.- to DCF by oxygen would yield superoxide (O-2(.-)). ESR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the presence of both superoxide and hydroxyl radicals in the DCFH/H2O2/HRP system. Both radicals were also detected in the absence of added H2O2, although the intensities of the resultant adducts were decreased. This work demonstrates that DCF fluorescence cannot be used reliably to measure O-2(.-) in cells because O-2(.-) itself is formed during the conversion of DCFH to DCF by peroxidases. The disproportionation of superoxide forms H2O2 which, in the presence of peroxidase activity, will oxidize more DCFH to DCF with self-amplification of the fluorescence. Because the deacetylation of DCFH-DA, even by esterases, can produce H2O2, the use of this probe to measure H2O2 production in cells is problematic. (C) 1999 Elsevier Science Inc.