Hydrogen Sulfide Deactivates Common Nitrobenzofurazan-BasedFluorescent Thiol Labeling Reagents

摘要

Sulfhydryl-containing compounds, including thiols and hydrogen sulfide (H2S), play important but differential roles in biological structure and function. One major challenge in separating the biological roles of thiols and H2S 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 H2S. Although H2S 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 H2S. By contrast, nitrobenzofurazan-based labeling reagents were deactivated by H2S addition. Furthermore, the addition of H2S to thiol-activated nitrobenzofurazan-based reagents reduced the fluorescence signal, thus establishing the incompatibilityof nitrobenzofurazan-based thiol labeling reagents in the presenceof H2S. Taken together, these studies highlight the differentialreactivity of thiols and H2S toward common thiol-labelingreagents and suggest that sufficient care must be taken when labelingor measuring thiols in cellular environments that produce H2S due to the potential for both false-positive and eroded responses.