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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Widespread sulfenic acid formation in tissues in response to hydrogen peroxide.
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Widespread sulfenic acid formation in tissues in response to hydrogen peroxide.

机译：响应过氧化氢而在组织中形成广泛的亚磺酸。

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A principal product of the reaction between a protein cysteinyl thiol and hydrogen peroxide is a protein sulfenic acid. Because protein sulfenic acid formation is reversible, it provides a mechanism whereby changes in cellular hydrogen peroxide concentration may directly control protein function. We have developed methods for the detection and purification of proteins oxidized in this way. The methodology is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin-maleimide. Arsenite-dependent signal generation was fully blocked by pretreatment with dimedone, consistent with its reactivity with sulfenic acids to form a covalent adduct that is nonreducible by thiols. The biotin tag facilitates the detection of protein sulfenic acids on Western blots probed with streptavidin-horseradish peroxidase and also their purification by streptavidin-agarose. We have characterized protein sulfenic acid formation in isolated hearts subjected to hydrogen peroxide treatment. We have also purified and identified a number of the proteins that are oxidized in this way by using a proteomic approach. Using Western immunoblotting we demonstrated that a highly significant proportion of some individual proteins (68% of total in one case) form the sulfenic derivative. We conclude that protein sulfenic acids are widespread physiologically relevant posttranslational oxidative modifications that can be detected at basal levels in healthy tissue, and are elevated in response to hydrogen peroxide. These approaches may find widespread utility in the study of oxidative stress, particularly because hydrogen peroxide is used extensively in models of disease or redox signaling.
机译：蛋白半胱氨酰硫醇和过氧化氢之间反应的主要产物是蛋白亚磺酸。由于蛋白质亚磺酸的形成是可逆的，因此它提供了一种机制，细胞中过氧化氢浓度的变化可以直接控制蛋白质的功能。我们已经开发了检测和纯化以此方式氧化的蛋白质的方法。该方法基于变性条件下蛋白质亚磺酸的亚砷酸盐特异性还原以及随后用生物素-马来酰亚胺标记。通过用二甲酮预处理完全阻断了砷依赖性信号的产生，这与其与亚磺酸的反应性形成了无法被硫醇还原的共价加合物相一致。生物素标签有助于在用链霉亲和素-辣根过氧化物酶探测的蛋白质印迹上检测蛋白质亚磺酸，以及通过链霉亲和素-琼脂糖对其进行纯化。我们已经表征了过氧化氢处理过的离体心脏中蛋白质亚磺酸的形成。我们还使用蛋白质组学方法纯化并鉴定了许多以这种方式氧化的蛋白质。使用Western免疫印迹，我们证明了某些蛋白质（在一种情况下占总蛋白质的68％）中相当高的比例形成了亚硫衍生物。我们得出结论，蛋白质亚磺酸是广泛存在的生理相关的翻译后氧化修饰，可以在健康组织的基础水平上检测到，并且在对过氧化氢的响应中升高。这些方法可能在氧化应激的研究中找到了广泛的用途，特别是因为过氧化氢被广泛用于疾病或氧化还原信号的模型中。

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