Fluorescein isothiocyanate, a platform for the selective and sensitive detection of S-Nitrosothiols and hydrogen...

Here we show that the fluorescence of fluorescein isothiocyanate (FITC) is not altered by its reaction with primary amines. However, the fluorescence is rapidly quenched upon reaction with small molecular weight thiols including cysteine, glutathione, homocysteine, dithiothreitol, and sulfide. We have taken advantage of the thiol-dependent quenching of FITC to devise a sulfide specific assay by utilizing polydimethylsiloxane (PDMS) membranes that are permeable to hydrogen sulfide but not to larger charged thiols. In addition, we have discovered that the fluorescein dithiocarbamate (FDTC) formed by the reaction with sulfide can specifically react with S-nitrosothiols (RSNO) to regenerate FITC, thus serving as a specific, fluorogenic reagent to detect picomol levels of RSNO. FDTC was tested as an intracellular RSNO-sensor in germinating tomato seedlings (Solanum lycopersicum) via epifluorescence microscopy. Control plant roots exposed to FDTC showed low intracellular fluorescence which increased ∼3-fold upon exposure to extracellular S-nitrosoglutathione and ∼4-fold in the presence of N6022, a S-nitrosoglutathione reductase (GSNOR) inhibitor, demonstrating that FDTC can be used to visualize intracellular RSNO levels.