Sinapinic acid can replace ascorbate in the biotin switch assay
Sinapinic acid can replace ascorbate in the biotin switch assay
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DOI：
10.1016/j.bbagen.2009.10.004
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发表时间：
2010-01-01
影响因子：
3
通讯作者：
Mutus, Bulent
中科院分区：
文献类型：
--
作者：
Kallakunta, Vasantha Madhuri;Staruch, Andrea;Mutus, Bulent
背景：蛋白质S-亚硝化是一种重要的蛋白质翻译后修饰，改变蛋白质的功能。一氧化氮与硫醇的相互作用是一个活跃的研究领域，是NO发挥其生物学效应的机制之一。生物素开关分析是目前发展起来的一种鉴定S-亚硝化蛋白质的方法。生物素转换试验的主要问题包括还原二硫化物，这可能导致假阳性。我们报告的生物素开关检测芥子酸是利用，而不是抗坏血酸，以消除潜在的文物中的检测S-亚硝化protein.Methods：芥子酸的脱亚硝化能力进行了评估，通过监测无论是NO或NO2-释放荧光NO检测或由格里斯测定，分别。DTNB测定用于比较抗坏血酸和芥子酸的二硫键还原。芥子酸和抗坏血酸在RAW 264.7细胞+/-S-亚硝基半胱氨酸（CysNO）exposure.Results中的S-亚硝基蛋白的生物素开关检测中进行了比较：我们表明芥子酸具有在pH 7.0下脱亚硝基S-亚硝基硫醇的能力，并且在pH 8和8.5下具有反硝化加脱亚硝基的能力。与抗坏血酸，芥子酸降解S-亚硝基硫醇，但它不减少二硫bridge.Conclusions：芥子酸denitrosate RSNO和不减少二硫化物。因此，可以很容易地取代抗坏血酸在检测S-亚硝化蛋白质的生物素开关assay.General意义：所描述的工作是重要的，鉴于蛋白质S-亚硝化。在这项研究中，我们提供了一个重要的修改，消除文物广泛使用的技术检测S-亚硝基蛋白质组，生物素开关测定。(C)2009 Elsevier B. V.保留所有权利。
Background: Protein S-nitrosation is an important post-translational modification altering protein function. Interaction of nitric oxide with thiols is an active area of research, and is one of the mechanisms by which NO exerts its biological effects. Biotin switch assay is the method, which has been developed to identify S-nitrosated proteins. The major concern with biotin switch assay includes reducing disulfide which may lead to false positives. We report a modification of the biotin switch assay where sinapinic acid is utilized instead of ascorbate to eliminate potential artifacts in the detection of S-nitrosated proteins.Methods: The denitrosation ability of sinapinic acid was assessed by monitoring either the NO or NO2- released by chemiluminescent NO detection or by the griess assay, respectively. DTNB assay was used to compare disulfide reduction by ascorbate and sinapinic acid. Sinapinic acid and ascorbate were compared in the biotin switch detection of S-nitrosoproteins in RAW 264.7 cells +/- S-nitrosocysteine (CysNO) exposure.Results: We show that sinapinic acid has the ability to denitrosate S-nitrosothiols at pH 7.0 and denitrate plus denitrosate at pHs 8 and 8.5. Unlike ascorbate, sinapinic acid degrades S-nitrosothiols, but it does not reduce disulfide bridges.Conclusions: Sinapinic acid denitrosate RSNO and does not reduce disulfides. Thus can readily replace ascorbate in detection of S-nitrosated proteins in biotin switch assay.General significance: The work described is important in view of protein S-nitrosation. In this study we provide an important modification that eliminates artifacts in widely used technique for detecting the S-nitrosoproteome, the biotin switch assay. (C) 2009 Elsevier B.V. All rights reserved.