Fluorescent probes are useful tools for the detection of sulfane sulfurs in biological systems. In this work, we report the development of SSP4, a widely used probe generated in our laboratory. We describe its evolution, preparation, and physical/chemical properties. Fluorescence analyses of SSP4 determined its high selectivity and sensitivity to sulfane sulfurs, even with the interfering presence of other species, such as amino acids and metal ions. Protocols for using SSP4 in a relatively quick and simple manner for the detection of persulfidated proteins, including papain, BSA, and GAPDH were developed. The method was then applied to human protein disulfide isomerase (PDI), leading to the discovery that persulfidation can occur at PDI's non-active site cysteines, and that PDI reductase activity is affected by sulfane sulfur treatment. Protocols for using SSP4 for the bioimaging of exogenous and endogenous sulfane sulfurs in different -cell lines were also established. These results should guide further applications of SSP4.

荧光探针是检测生物系统中硫烷硫的有用工具。在这项工作中，我们报告了 SSP4 的开发，SSP4 是我们实验室生成的一种广泛使用的探针。我们描述了它的演变、制备和物理/化学性质。SSP4 的荧光分析确定了它对硫烷硫的高选择性和敏感性，即使存在其他物质的干扰，如氨基酸和金属离子。开发了以相对快速和简单的方式使用 SSP4 检测过硫化蛋白（包括木瓜蛋白酶、BSA 和 GAPDH）的协议。然后将该方法应用于人蛋白质二硫键异构酶 (PDI)，导致发现 PDI 的非活性位点半胱氨酸可能发生过硫化，并且 PDI 还原酶活性受硫烷硫处理的影响。还建立了使用 SSP4 对不同细胞系中的外源性和内源性硫烷硫进行生物成像的方案。这些结果应该指导 SSP4 的进一步应用。