Hydrogen sulfide deactivates common nitrobenzofurazan-based fluorescent thiol labeling reagents.
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 incompatibility of nitrobenzofurazan-based thiol labeling reagents in the presence of H2S. Taken together, these studies highlight the differential reactivity of thiols and H2S toward common thiol-labeling reagents and suggest that sufficient care must be taken when labeling or measuring thiols in cellular environments that produce H2S due to the potential for both false-positive and eroded responses.
Department of Chemistry and Biochemistry, Institute of Molecular Biology, Materials Science Institute, 1253 University of Oregon , Eugene, Oregon 97403, United States.
Leticia A
LA
Montoya
Michael D
MD
Pluth
D001572:Benzofurans / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006862:Hydrogen Sulfide / Q000737:chemistry*; D007202:Indicators and Reagents / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D001572:Benzofurans; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D013438:Sulfhydryl Compounds; C105430:benzofuran; D006862:Hydrogen Sulfide