Gasotransmitter Regulation of Phosphatase Activity in Live Cells Studied by Three‐Channel Imaging Correlation

Enzyme activity in live cells is dynamically regulated by small‐molecule transmitters for maintaining normal physiological functions. A few probes have been devised to measure intracellular enzyme activities by fluorescent imaging, but the study of the regulation of enzyme activity via gasotransmitters in situ remains a long‐standing challenge. Herein, we report a three‐channel imaging correlation by a single dual‐reactive fluorescent probe to measure the dependence of phosphatase activity on the H 2 S level in cells. The two sites of the probe reactive to H 2 S and phosphatase individually produce blue and green fluorescent responses, respectively, and resonance energy transfer can be triggered by their coexistence. Fluorescent analysis based on the three‐channel imaging correlation shows that cells have an ideal level of H 2 S to promote phosphatase activity up to its maximum. Significantly, a slight deviation from this H 2 S level leads to a sharp decrease of phosphatase activity. The discovery further strengthens our understanding of the importance of H 2 S in cellular signaling and in various human diseases.