Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109, USA.
ACS Chem Biol. 2010 Apr 16;5(4):405-14. doi: 10.1021/cb900302u.
S-Nitrosothiols (RSNOs) represent an important class of post-translational modifications that preserve and amplify the actions of nitric oxide and regulate enzyme activity. Several regulatory proteins are now verified targets of cellular S-nitrosation, and the direct detection of S-nitrosated residues in proteins has become essential to better understand RSNO-mediated signaling. Current RSNO detection depends on indirect assays that limit their overall specificity and reliability. Herein, we report the reaction of S-nitrosated cysteine, glutathione, and a mutated C165S alkyl hydroperoxide reductase with the water-soluble phosphine tris(4,6-dimethyl-3-sulfonatophenyl)phosphine trisodium salt hydrate (TXPTS). A combination of NMR and MS techniques reveals that these reactions produce covalent S-alkylphosphonium ion adducts (with S-P(+) connectivity), TXPTS oxide, and a TXPTS-derived aza-ylide. Mechanistically, this reaction may proceed through an S-substituted aza-ylide or the direct displacement of nitroxyl from the RSNO group. This work provides a new means for detecting and quantifying S-nitrosated species in solution and suggests that phosphines may be useful tools for understanding the complex physiological roles of S-nitrosation and its implications in cell signaling and homeostasis.
S-亚硝基硫醇（RSNOs）代表了一类重要的翻译后修饰，可保存和放大一氧化氮的作用，并调节酶活性。现在已经有几个调节蛋白被证实是细胞 S-亚硝化的靶标，而直接检测蛋白质中的 S-亚硝基化残基对于更好地理解 RSNO 介导的信号转导至关重要。目前的 RSNO 检测依赖于间接检测方法，这限制了它们的整体特异性和可靠性。在此，我们报告了 S-亚硝基化半胱氨酸、谷胱甘肽和突变的 C165S 烷氧自由基还原酶与水溶性膦三（4,6-二甲基-3-磺酸钠苯基）膦三钠盐水合物（TXPTS）的反应。NMR 和 MS 技术的组合表明，这些反应产生了共价 S-烷基膦离子加合物（具有 S-P(+)连接性）、TXPTS 氧化物和 TXPTS 衍生的氮杂叶立德。从机制上讲，该反应可能通过 S-取代的氮杂叶立德或从 RSNO 基团直接取代硝酰基进行。这项工作为在溶液中检测和定量 S-亚硝基化物质提供了一种新方法，并表明膦可能是理解 S-亚硝化的复杂生理作用及其在细胞信号转导和动态平衡中的意义的有用工具。
