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 (Cmax), the time required to reach half of Cmax (t1/2), maximum releasing rate of H2S (Rmax), and time at H2S (tr-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.