Protocol for using organic persulfides to measure the chemical reactivity of persulfide sensors.
Protocol for using organic persulfides to measure the chemical reactivity of persulfide sensors.
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DOI：
10.1016/j.xpro.2022.101424
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发表时间：
2022-06-17
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硫化氢（H2S）和下游活性硫物质（RSS），包括有机过硫化物，保护细菌细胞免受各种氧化应激。专门的基于二硫醇的转录抑制子直接感知过硫化物以控制细胞H2S/RSS并避免毒性。在这里，我们提出了一个协议，以量化半胱氨酸的化学反应动力学在两个细菌过硫化物传感器对半胱氨酸过硫化物和谷胱甘肽过硫化物，与LC-ESI-MS分析，结果在动力学模型。该方案具有潜在的应用到其他含半胱氨酸的蛋白质和氧化剂。有关本方案使用和执行的完整详细信息，请参见和。通过氧化半胱氨酸和谷胱甘肽的硫化物还原产生过硫化物半胱氨酸反应性分析策略，针对响应RSS的目标蛋白质进行LC-ESI-MS分析，以研究RSS半胱氨酸的产物分布和动力学出版商注：进行任何实验方案都需要遵守当地机构的实验室安全和伦理指南。硫化氢（H2S）和下游活性硫物质（RSS），包括有机过硫化物，保护细菌细胞免受各种氧化应激。专门的基于二硫醇的转录抑制子直接感知过硫化物以控制细胞H2S/RSS并避免毒性。在这里，我们提出了一个协议，以量化半胱氨酸的化学反应动力学在两个细菌过硫化物传感器对半胱氨酸过硫化物和谷胱甘肽过硫化物，与LC-ESI-MS分析，结果在动力学模型。该方案具有潜在的应用到其他含半胱氨酸的蛋白质和氧化剂。
Hydrogen sulfide (H2S) and downstream reactive sulfur species (RSS), including organic persulfides, protect bacterial cells against diverse oxidative stressors. Specialized dithiol-based transcriptional repressors sense persulfides directly to control cellular H2S/RSS and avoid toxicity. Here, we present a protocol to quantify the kinetics of chemical reactivity of cysteines in two bacterial persulfide sensors toward cysteine persulfide and glutathione persulfide, with a LC-ESI-MS analysis that results in a kinetic model. This protocol has potential applications to other cysteine-containing proteins and oxidants. For complete details on the use and execution of this protocol, please refer to and. Persulfide generation from reduction with sulfide of oxidized cysteine and glutathione Cysteine reactivity profiling strategy for proteins of interest that respond to RSS LC-ESI-MS analysis to study product distribution and kinetics of cysteines with RSS Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Hydrogen sulfide (H2S) and downstream reactive sulfur species (RSS), including organic persulfides, protect bacterial cells against diverse oxidative stressors. Specialized dithiol-based transcriptional repressors sense persulfides directly to control cellular H2S/RSS and avoid toxicity. Here, we present a protocol to quantify the kinetics of chemical reactivity of cysteines in two bacterial persulfide sensors toward cysteine persulfide and glutathione persulfide, with a LC-ESI-MS analysis that results in a kinetic model. This protocol has potential applications to other cysteine-containing proteins and oxidants.