2.9. Detection of Sulfane Sulfurs Using SSP4

SSP4 (Sulfane Sulfur Probe 4) is a fluorescent probe to detect sulfane sulfurs specifically. SSP4 itself is non-fluorescent, but it emits strong green fluorescence after it reacts with sulfane sulfurs. Reactions of thiosulfate with SSP4 were conducted by mixing 10 μM SSP4 with 50 mM, 100 mM, or 300 mM thiosulfate in 200 μL HEPES buffer (100 mM, pH 3.0). The mixture was incubated at room temperature for 30 min, and then the fluorescence was detected by using the Synergy H1 microplate reader. The excitation wavelength was set to 482 nm and the emission wavelength was set to 515 nm.

For analysis of thiosulfate decomposition, 1 M thiosulfate was diluted into 200 μL HEPES buffer (100 mM, pH 4.0) to a final concentration of 300 mM. 100 mM Arg, Thr, or Gly was added individually. At the same time, 10 μM of SSP4 was added to incubate with thiosulfate for 30 min, and fluorescence was detected every 30 s by using the Synergy H1 microplate reader.