Fluorescent detection of hydrogen sulfide (H(2)S) through the formation of pyrene excimers enhances H(2)S quantification in biochemical systems

Hydrogen sulfide (H(2)S) is produced endogenously by several enzymatic pathways and modulates physiological functions in mammals. Quantification of H(2)S in biochemical systems remains challenging because of the presence of interferents with similar reactivity, particularly thiols. Herein, we present a new quantification method based on the formation of pyrene excimers in solution. We synthesized the probe 2-(maleimido)ethyl 4-pyrenylbutanoate (MEPB) and determined that MEPB reacted with H(2)S in a two-step reaction to yield the thioether-linked dimer (MEPB)(2)S, which formed excimers upon excitation, with a broad peak of fluorescence emission centered at 480 nm. In contrast, we found that the products formed with thiols showed peaks at 378 and 398 nm. The difference in emission between the products prevented the interference. Furthermore, we showed that the excimer fluorescence signal yielded a linear response to H(2)S, with a limit of detection of 54 nM in a fluorometer. Our quantification method with MEPB was successfully applied to follow the reaction of H(2)S with glutathione disulfide and to quantify the production of H(2)S from cysteine by Escherichia coli. In conclusion, this method represents an addition to the toolkit of biochemists to quantify H(2)S specifically and sensitively in biochemical systems.