Optimization of a Method for Detecting Intracellular Sulfane Sulfur Levels and Evaluation of Reagents That Affect the Levels in <i>Escherichia coli</i>

Sulfane sulfur is a class of compounds containing zero-valent sulfur. Most sulfane sulfur compounds are reactive and play important signaling roles. Key enzymes involved in the production and metabolism of sulfane sulfur have been characterized; however, little is known about how to change intracellular sulfane sulfur (iSS) levels. To accurately measure iSS, we optimized a previously reported method, in which reactive iSS reacts with sulfite to produce thiosulfate, a stable sulfane sulfur compound, before detection. With the improved method, several factors were tested to influence iSS in <i>Escherichia coli</i>. Temperature, pH, and osmotic pressure showed little effect. At commonly used concentrations, most tested oxidants, including hydrogen peroxide, tert-butyl hydroperoxide, hypochlorous acid, and diamide, did not affect iSS, but carbonyl cyanide m-chlorophenyl hydrazone increased iSS. For reductants, 10 mM dithiothreitol significantly decreased iSS, but <i>tris</i>(2-carboxyethyl)phosphine did not. Among different sulfur-bearing compounds, NaHS, cysteine, S₂O₃²⁻ and diallyl disulfide increased iSS, of which only S₂O₃²⁻ did not inhibit <i>E. coli</i> growth at 10 mM or less. Thus, with the improved method, we have identified reagents that may be used to change iSS in <i>E. coli</i> and other organisms, providing tools to further study the physiological functions of iSS.