2.5. Detection of methanethiol using Ellman's reagent (DTNB)

To assess to what extent volatile methanethiol is available as MTO substrate when generated in an adjacent well of the 384-well plate, methanethiol-induced reduction of DTNB (5,5′-Dithiobis-2-nitrobenzoic acid; Carl Roth) to 2-nitro-5-thiobenzoate (TNB) was followed. The assay was prepared as described above, with 40 μl of DTNB solution (4 mg/ml DTNB in 0.1 M potassium phosphate buffer; 1 mM EDTA; pH 8.0) added instead of MTO samples in wells next to the MGL reaction well. In negative controls, the MGL reaction well contained methionine solution only, without added MGL. After starting the reaction (at 37 °C), aliquots were taken at different times, diluted 1:50 in potassium phosphate buffer (equilibrated to room temperature) and absorption was measured at 412 nm. Using the TNB extinction coefficient of 14,150 M⁻¹ cm⁻¹ [19], available methanethiol concentrations were assessed.