Hydrogen Sulfide Deactivates Common Nitrobenzofurazan-Based Fluorescent Thiol Labeling Reagents

[Image: see text] Sulfhydryl-containing compounds, including thiols and hydrogen sulfide (H(2)S), play important but differential roles in biological structure and function. One major challenge in separating the biological roles of thiols and H(2)S is developing tools to effectively separate the reactivity of these sulfhydryl-containing compounds. To address this challenge, we report the differential responses of common electrophilic fluorescent thiol labeling reagents, including nitrobenzofurazan-based scaffolds, maleimides, alkylating agents, and electrophilic aldehydes, toward cysteine and H(2)S. Although H(2)S reacted with all of the investigated scaffolds, the photophysical response to each scaffold was significantly different. Maleimide-based, alkylating, and aldehydic thiol labeling reagents provided a diminished fluorescence response when treated with H(2)S. By contrast, nitrobenzofurazan-based labeling reagents were deactivated by H(2)S addition. Furthermore, the addition of H(2)S to thiol-activated nitrobenzofurazan-based reagents reduced the fluorescence signal, thus establishing the incompatibility of nitrobenzofurazan-based thiol labeling reagents in the presence of H(2)S. Taken together, these studies highlight the differential reactivity of thiols and H(2)S toward common thiol-labeling reagents and suggest that sufficient care must be taken when labeling or measuring thiols in cellular environments that produce H(2)S due to the potential for both false-positive and eroded responses.