Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species
Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species
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DOI：
10.1074/jbc.m209264200
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发表时间：
2003-01-31
影响因子：
4.8
通讯作者：
Nagano, T
中科院分区：
文献类型：
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作者：
Setsukinai, K;Urano, Y;Nagano, T
我们设计并合成了2-[6-(4‘-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic酸(HPF)和2[6-(4’-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic酸(APF)作为新型荧光探针，用于选择性地检测羟基自由基((OH)-O-)等高活性氧物种(HROS)。和过氧化物酶的活性中间体。虽然HPF和APF本身几乎不发光，但APF与HROS和次氯酸盐(-OCL)反应时，选择性地和剂量依赖地提供了一种强荧光化合物荧光素，而不是其他活性氧物种(ROS)。类似地，HPF仅在与hROS反应时提供荧光素。因此，单独使用HPF或APF不仅可以区分HRO与过氧化氢(H_2O_2)、一氧化氮(NO)和超氧化物(O-2(.-))，而且联合使用HPF和APF也可以特异性地检测到-OCL。此外，我们将HPF和APF应用于活细胞，发现HPF和APF不同于2‘，7’-二氯二氢荧光素，它们对光诱导的自氧化反应具有抵抗力，并且我们首次可以观察到在刺激的中性粒细胞中产生-OCL。HPF和APF可以作为研究HROS和-OCL在许多生物和化学应用中的作用的有用工具。
We designed and synthesized 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) as novel fluorescence probes to detect selectively highly reactive oxygen species (hROS) such as hydroxyl radical ((OH)-O-.) and reactive intermediates of peroxidase. Although HPF and APF themselves scarcely fluoresced, APF selectively and dose-dependently afforded a strongly fluorescent compound, fluorescein, upon reaction with hROS and hypochlorite (-OCl), but not other reactive oxygen species (ROS). HPF similarly afforded fluorescein upon reaction with hROS only. Therefore, not only can hROS be differentiated from hydrogen peroxide (H2O2), nitric oxide (NO), and superoxide (O-2(.-)) by using HPF or APF alone, but -OCl can also be specifically detected by using HPF and APF together. Furthermore, we applied HPF and APF to living cells and found that HPF and APF were resistant to light-induced autoxidation, unlike 2',7'-dichlorodihydrofluoreseein, and for the first time we could visualize -OCl generated in stimulated neutrophils. HPF and APF should be useful as tools to study the roles of hROS and -OCl in many biological and chemical applications.