The novel red-fluorescent probe DAR-4M measures reactive nitrogen species rather than NO
The novel red-fluorescent probe DAR-4M measures reactive nitrogen species rather than NO
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DOI：
10.1016/j.vascn.2005.06.004
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发表时间：
2005-11-01
影响因子：
1.9
通讯作者：
Busija, David W.
中科院分区：
文献类型：
--
作者：
Lacza, Zsombor;Horvath, Eszter M.;Busija, David W.
简介：几种荧光探针被设计用于测量一氧化氮（NO），然而，在生物样品中它们的特异性和灵敏度方面出现了问题。在本研究中，我们测试了一种新的罗丹明为基础的发色团二氨基罗丹明-4M（DAR-4 M）与NO和其他活性氮和氧物种的反应。方法：我们在96孔板中进行荧光测定，在无细胞缓冲液中具有与细胞质相似的离子浓度。使用各种NO供体和活性氮和氧物种产生剂量响应曲线。比较了红色荧光DAR-4 M及其绿色荧光对应物4-氨基-5-甲氨基-2 '，7'-二氟荧光素（DAF-FM）之间的效果。结果如下：DAR-4 M对NO供体的荧光产率明显高于DAF-FM，而两种探针的灵敏度阈值相当（在0.1 mM硝普钠的范围内）。这两种染料与各种NO供体以剂量依赖性的方式反应，而超氧化物，过氧化氢，过氧硝酸盐，或硝酰基未能改变探针的荧光强度。DAR-4 M在过氧亚硝酸盐的存在下被增强以与DAF-FM类似的方式与低水平的NO供体反应。讨论内容：我们的结论是DAR-4 M是一个合适的红色荧光探针的活性氮物质的生产的定性评估，但不具体的NO。样品之间的定量比较是不合适的，因为荧光产率的影响，在样品中存在的其他氧化剂。(C)2005年爱思唯尔公司All rights reserved.
Introduction: Several fluorescent probes were designed for the measurement of nitric oxide (NO), however, questions arose regarding their specificity and sensitivity in biological samples. In the present study we tested the reaction of a novel rhodamine-based chromophore diaminorhodamine-4M (DAR-4M) with NO and other reactive nitrogen and oxygen species. Methods: We performed fluorometry in 96-well plates in a cell-free buffer with similar ion concentrations as the cytoplasm. Dose-response curves were generated using various NO donors and reactive nitrogen and oxygen species. The effects were compared between the red-fluorescent DAR-4M and its green-fluorescent counterpart 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM). Results: DAR-4M had a markedly higher fluorescence yield to NO donors than DAF-FM, while both probes had a comparable threshold of sensitivity (in the range of 0.1 mM nitroprusside). Both dyes reacted with various NO donors in a dose-dependent manner, while superoxide, hydrogen peroxide, peroxynitrate, or nitroxyl failed to change the fluorescence intensity of the probes. DAR-4M was potentiated in the presence of peroxynitrite to react with low levels of NO donors in a similar manner to DAF-FM. Discussion: We conclude that DAR-4M is a suitable red-fluorescent probe for the qualitative assessment of reactive nitrogen species production, but not specific for NO. Quantitative comparisons among samples is inappropriate since the fluorescent yield is affected by the presence of other oxidants in the sample. (C) 2005 Elsevier Inc. All rights reserved.