Microplate-Based Colorimetric Detection of Free Hydrogen Sulfide

Abstract

Hydrogen sulfide (H2S) has recently been recognized as an important physiologically relevant gasotransmitter. Produced by the enzymes involved in the transsulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), H2S has been implicated to control biological activity in virtually every organ system. In recent years it is being recognized that many commonly used H2S assays do not measure free H2S specifically and may be prone to artifacts. This has led to large variations in the reported H2S biological concentrations. In order to accurately study H2S's functions in biological systems accurate assays which measure free H2S specifically are required. In this work we present a simple microplate-based colorimetric assay for H2S gas. The underside of a 96-well microplate cover was coated with Nafion polymer doped with Ag(+) ions. H2S is a highly volatile gas, and as it is volatilized in the microplate well it reacts with Ag(+) to produce Ag2S nanoparticles, which have a strong absorbance in the low-UV range. By monitoring the absorbance change from formation of Ag2S nanoparticles, H2S production can be monitored in real time. The assay has a limit of detection (LOD) of 2.61 nmol (8.70 μM) and a liner range up to 30 nmol (100 μM). Using the assay, the KM and Vmax of recombinant CSE enzyme were determined to be 11.13 ± 0.57 mM and 0.45 ± 0.01 nmol min(-1), respectively. H2S production from mouse liver homogenate under aerobic conditions in the presence of cysteine was measured and determined to be 4.89 ± 0.19 nmol min(-1) mL(-1) homogenate. The assay is simple, low cost, and specific to free H2S gas.