Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach
Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach
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DOI：
10.1016/j.aca.2017.03.027
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发表时间：
2017-05-29
影响因子：
6.2
通讯作者：
Maruyama, Toru
中科院分区：
文献类型：
--
作者：
Ikeda, Mayumi;Ishima, Yu;Maruyama, Toru
硫化氢（H2S）信号传导涉及多种蛋白质上的多硫化物（RSSnSR '）形成。然而，目前缺乏敏感的多硫化物检测分析提出了理解硫烷硫稳态和信号的方法学挑战。我们开发了一种新的组合测定，通过修改硫化物抗氧化剂缓冲液（SAOB），以产生一种“消除方法的硫化物从多硫化物”（EMSP）的处理溶液，释放硫化物，随后与亚甲蓝（MB）硫化物检测测定。对低分子量硫物质进行的EMSP-MB硫化物联合检测试验表明，硫化物由三硫化物化合物（如谷胱甘肽三硫化物和二烯丙基三硫化物）产生，但不是由巯基化合物（如半胱氨酸、胱氨酸和谷胱甘肽）产生。就血浆蛋白而言，这种新型联合检测方法显示约为14.7。1.7. 3.9. 3.7分别从人血清白蛋白、α（1）-抗胰蛋白酶、α（1）-酸性糖蛋白和卵清蛋白中释放硫化物mol/mol，表明血清白蛋白是人体血液循环中多硫化物的主要库。结合不同物种白蛋白的结果，释放的硫化物与半胱氨酸而不是蛋氨酸具有良好的相关性，表明多硫化物的掺入位点是半胱氨酸。用这种新的硫化物滞留测定法，在人健康血浆、唾液和泪液中分别定量了约8，000、120和1100 μ M的多硫化物浓度。我们有前途的多硫化物特异性检测方法可以是一个非常重要的工具，因为多硫化物的定量测定揭示了蛋白质结合的半胱氨酸多硫化物的功能后果，并扩大了活性氧的研究领域，活性多硫化物物种。(C)2017 Elsevier B. V.版权所有。
Hydrogen sulfide (H2S) signaling involves polysulfide (RSSnSR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7. 1.7. 3.9. 3.7 sulfide mol/mol released from human serum albumin, alpha(1)-anti-trypsin, alpha(1)-acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 mu M of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species. (C) 2017 Elsevier B.V. All rights reserved.