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

Abstract In this study, we characterized the potential H 2 S-releasing properties of seven different H 2 S donors, including sodium sulfide (Na 2 S), sodium hydrosulfide (NaHS), diallyl disulfide (DADS), diallyl trisulfide (DATS), sodium thiosulfate (Na 2 S 2 O 3 ), 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 H 2 S-releasing characteristics of the various donors, we evaluated four parameters, maximum concentration of H 2 S at the steady state (C max ), the time required to reach half of C max (t 1/2 ), maximum releasing rate of H 2 S (R max ), and time at H 2 S (t r-max ). The results showed that the H 2 S-releasing kinetics of each H 2 S 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 H 2 S among the four H 2 S donors in the following order: DATS > DADS > Na 2 S ~ NaHS. The H 2 S-releasing characteristics of the H 2 S 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 H 2 S-releasing dynamics.