The oxidation of 2′,7′-dichlorofluorescin to reactive oxygen species:: A self-fulfilling prophesy?
The oxidation of 2′,7′-dichlorofluorescin to reactive oxygen species:: A self-fulfilling prophesy?
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DOI：
10.1016/j.freeradbiomed.2005.10.042
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发表时间：
2006-03-15
影响因子：
7.4
通讯作者：
Mason, RP
中科院分区：
文献类型：
--
作者：
Bonini, MG;Rota, C;Mason, RP
本文研究了HRP/过氧亚硝基阴离子体系对2 ′，7 ′-二氯荧光素（DCFH）及其二乙酸盐（DCFHDA）的氧化作用。DCFH和DCFHDA都被氧化成荧光产物。然而，一个主要的异常，是观察到荧光继续建立唇长后过氧亚硝酸盐总分解和初始HRP化合物I还原，这表明生产的氧化剂的系统。事实上，预先形成的HRP化合物I立即被DCFH和DCFHDA还原为化合物II，并专门形成DCF中心点-半醌和DCFHDA衍生的自由基。过氧化氢酶强烈抑制荧光和EPR信号，这表明H2 O2的中间体形成。综上所述，数据表明过氧亚硝酸盐将HRP快速氧化成HRP化合物I，其被DCFH及其二乙酸盐形式还原，同时形成DCF中心点-半醌和DCFH DA-衍生的自由基。它们被O-2氧化，产生O-2（中心点-）-（如EPR和氧消耗实验所示），其歧化产生H2 O2，其用于通过HRP催化进一步促进DCFH/DCFHDA氧化。此外，DCFHDA显示出比其水解产物DCFH更显著地抗氧化，大概是因为不存在容易氧化的苯酚部分。DCFHDA/DCFH已被用于研究各种系统中的自由基产生。我们的研究结果表明，这种测定是受到一个严重的伪影，因为它产生了它的目的是测量，因此，它在生物系统中的使用应谨慎对待。爱思唯尔公司出版
The oxidation of 2',7'-dichlorofluorescin (DCFH) and its diacetate form (DCFHDA) by the HRP/peroxynitrite system was investigated. Both DCFH and DCFHDA were oxidized to fluorescent products. A major anomaly, however, was the observation that fluorescence continued to build Lip long after peroxynitrite total decomposition and the initial HRP compound I reduction, suggesting the production of oxidants by the system. Indeed, preformed HRP compound I was instantly reduced by DCFH and DCFHDA to compound II with the obligate formation of DCF center dot- semiquinone and DCFHDA-derived radicals. Catalase strongly inhibited fluorescence and EPR signals, suggesting the intermediate formation of H2O2. Taken together the data indicate that peroxynitrite rapidly oxidizes HRP to HRP compound I, which is reduced by DCFH and its diacetate form with the concomitant formation of DCF center dot- semiquinone and DCFH DA-derived radicals. These are oxidized by O-2, producing O-2(center dot-) - (as demonstrated by EPR and oxygen consumption experiments), which dismutates to produce H2O2, which serves to fuel further DCFH/DCFHDA oxidation via HRP catalysis. Also DCFHDA was shown to be considerably more resistant to oxidation than its hydrolyzed product DCFH, presumably because of the absence of the easily oxidizable phenol moieties. DCFHDA/DCFH have been used to study free radical production in a variety of systems. Our findings demonstrate that this assay is subject to a serious artifact in that it produces what it is purported to measure; therefore, its use in biological systems should be approached with caution. Published by Elsevier Inc.