Characterization of H2S releasing properties of various H2S donors utilizing microplate cover-based colorimetric assay

In this study, we characterized the potential H₂S-releasing properties of seven different H₂S donors, including sodium sulfide (Na₂S), sodium hydrosulfide (NaHS), diallyl disulfide (DADS), diallyl trisulfide (DATS), sodium thiosulfate (Na₂S₂O₃), morpholin-4-ium 4-methoxyphenyl-morpholino-phosphinodithioate (GYY4137), and Lawesson's reagent, in three assay solutions, phosphate buffered saline (PBS, pH 7.4), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (HBS, pH 7.6), and cell growth media (GM), utilizing our microplate cover-based colorimetric assay. For quantitative analyses of H₂S-releasing characteristics of the various donors, we evaluated four parameters, maximum concentration of H₂S at the steady state (C_max), the time required to reach half of C_max (t_1/2), maximum releasing rate of H₂S (R_max), and time at H₂S (t_r-max). The results showed that the H₂S-releasing kinetics of each H₂S donor were dependent on the type of assay solution. In particular, the addition of GSH to DATS in GM released the fastest and highest amounts of H₂S among the four H₂S donors in the following order: DATS > DADS > Na₂S ~ NaHS. The H₂S-releasing characteristics of the H₂S donors were well-matched with cell viability results of human prostate cancer PC-3 cells. Therefore, the microplate cover-based colorimetric assay will be a useful tool for accurate and efficient measurements of H₂S-releasing dynamics.