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Toward Understanding the Reaction Kinetics of a Coumarin‐Fluorescein FRET based Dithio Probe for Quantitation of Cellular Thiols

Thio containing compounds like glutathione and protein cysteines are essential for the proper function of a cell. The levels of thiols and their oxidation state determine the cellular redox‐potential, which is associated with oxidative stress, and disease state of a cell. We recently reported synthesis and preliminary characterization of a coumarin‐fluorescein‐ reagent (Donor‐S‐S‐Acceptor, DSSA), a FRET‐based fluorescent dithio probe with unusually low reduction potential (‐ 0.6 V) that can detect changes in intracellular thiols and cross membranes of bacterial and mammalian cells. Here we report in vitro reaction kinetics of this probe with glutathione, a major cellular redox potential regulator. The dithio probe was reacted with reduced and oxidized glutathione and their ratiometric mixtures under physiologically relevant conditions. We have demonstrated that this probe is reduced by reduced glutathione (GSH) in a two step process via an intermediate. The first step involves an initial burst, with an intermediate which is more fluorescent than the initial probe, followed by stead state increase in fluorescence in the second step. Reactions with oxidized dithios showed a rapid one step process due to disulfide exchange,while ratiometric mixtures of oxidized and reduced thiols show predictable trends relating to their concentration ratios. Understanding of these mechanisms is critical in utilizing this probe to quantify cellular thiols in real time. This research was supported by a Biomedical Technology Alliance grant (01‐KMS LEG FY06‐12368) to D.S.S.