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Mechanistic investigations reveal that dibromobimane extrudes sulfur from biological sulfhydryl sources other than hydrogen sulfide†Electronic supplementary information (ESI) available: Experimental details, pH stability data for BTE, NMR spectra. See DOI: 10.1039/c4sc01875cClick here for additional data file.

Montoya LA, Shen X, McDermott JJ, Kevil CG, Pluth MD - Chem Sci (2014)

Bottom Line: During the growth of this field, significant controversy has arisen centered on the physiological concentrations of H2S.Upon use of the dBB method in wild-type and CSE(-/-) mice, however, dBB reports significantly higher sulfide levels than those measured using mBB.Taken together, the dBB method is a highly sensitive method for H2S but is not compatible for use in studies in which other thiols are present.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry , Institute of Molecular Biology , Materials Science Institute , University of Oregon , Eugene , OR 97403 , USA . Email: pluth@uoregon.edu.

ABSTRACT

Hydrogen sulfide (H2S) has emerged as an important biological signaling molecule in the last decade. During the growth of this field, significant controversy has arisen centered on the physiological concentrations of H2S. Recently, a monobromobimane (mBB) method has been developed for the quantification of different biologically-relevant sulfide pools. Based on the prevalence of the mBB method for sulfide quantification, we expand on this method to report the use of dibromobimane (dBB) for sulfide quantification. Reaction of H2S with dBB results in formation of highly-fluorescent bimane thioether (BTE), which is readily quantifiable by HPLC. Additionally, the reaction of sulfide with dBB to form BTE is significantly faster than the reaction of sulfide with mBB to form sulfide dibimane. Using the dBB method, BTE levels as low as 0.6 pM can be detected. Upon use of the dBB method in wild-type and CSE(-/-) mice, however, dBB reports significantly higher sulfide levels than those measured using mBB. Further investigation revealed that dBB is able to extract sulfur from other sulfhydryl sources including thiols. Based on mechanistic studies, we demonstrate that dBB extracts sulfur from thiols with α- or β-hydrogens, thus leading to higher BTE formation than from sulfide alone. Taken together, the dBB method is a highly sensitive method for H2S but is not compatible for use in studies in which other thiols are present.

No MeSH data available.


Quantification of sulfur extrusion from GSH by (a) dBB to form BTE and by (b) mBB to form SdB. BTE and SdB concentrations were quantified by HPLC. GSH concentrations were confirmed by reaction with 4-fluoro-7-sulfobenzofurazan (SBD-F) followed by HPLC quantification. Treatment of dBB with 5 mM GSH resulted in detector saturation (#).
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fig5: Quantification of sulfur extrusion from GSH by (a) dBB to form BTE and by (b) mBB to form SdB. BTE and SdB concentrations were quantified by HPLC. GSH concentrations were confirmed by reaction with 4-fluoro-7-sulfobenzofurazan (SBD-F) followed by HPLC quantification. Treatment of dBB with 5 mM GSH resulted in detector saturation (#).

Mentions: To quantify the amount of sulfur extracted from common thiols by dBB, we next investigated and quantified the amount of BTE formed after treatment with reduced glutathione (GSH) and measured the BTE product by HPLC. Consistent with the 1H NMR studies, BTE formation was observed by HPLC. To further determine the amount of sulfur extruded from GSH, different concentrations of GSH were added to dBB and the BTE product was quantified by HPLC (Fig. 5). Treatment of mBB with increasing concentrations of GSH ranging from 5 μM to 5 mM only generated low nM concentrations of SdB. By contrast, treatment of dBB with identical GSH concentrations results in generation of micromolar concentrations of BTE. Based on the data, after a 30 minute incubation, dBB extracts approximately 7.0% of the sulfur from GSH to form BTE. By comparison, under identical conditions the mBB method extruded less than 0.01% sulfur from GSH. These extraction efficiencies not only explain the higher levels of biological sulfide detected from dBB but also highlight that mBB does not extract appreciable sulfide from endogenous thiol sources.


Mechanistic investigations reveal that dibromobimane extrudes sulfur from biological sulfhydryl sources other than hydrogen sulfide†Electronic supplementary information (ESI) available: Experimental details, pH stability data for BTE, NMR spectra. See DOI: 10.1039/c4sc01875cClick here for additional data file.

Montoya LA, Shen X, McDermott JJ, Kevil CG, Pluth MD - Chem Sci (2014)

Quantification of sulfur extrusion from GSH by (a) dBB to form BTE and by (b) mBB to form SdB. BTE and SdB concentrations were quantified by HPLC. GSH concentrations were confirmed by reaction with 4-fluoro-7-sulfobenzofurazan (SBD-F) followed by HPLC quantification. Treatment of dBB with 5 mM GSH resulted in detector saturation (#).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4302847&req=5

fig5: Quantification of sulfur extrusion from GSH by (a) dBB to form BTE and by (b) mBB to form SdB. BTE and SdB concentrations were quantified by HPLC. GSH concentrations were confirmed by reaction with 4-fluoro-7-sulfobenzofurazan (SBD-F) followed by HPLC quantification. Treatment of dBB with 5 mM GSH resulted in detector saturation (#).
Mentions: To quantify the amount of sulfur extracted from common thiols by dBB, we next investigated and quantified the amount of BTE formed after treatment with reduced glutathione (GSH) and measured the BTE product by HPLC. Consistent with the 1H NMR studies, BTE formation was observed by HPLC. To further determine the amount of sulfur extruded from GSH, different concentrations of GSH were added to dBB and the BTE product was quantified by HPLC (Fig. 5). Treatment of mBB with increasing concentrations of GSH ranging from 5 μM to 5 mM only generated low nM concentrations of SdB. By contrast, treatment of dBB with identical GSH concentrations results in generation of micromolar concentrations of BTE. Based on the data, after a 30 minute incubation, dBB extracts approximately 7.0% of the sulfur from GSH to form BTE. By comparison, under identical conditions the mBB method extruded less than 0.01% sulfur from GSH. These extraction efficiencies not only explain the higher levels of biological sulfide detected from dBB but also highlight that mBB does not extract appreciable sulfide from endogenous thiol sources.

Bottom Line: During the growth of this field, significant controversy has arisen centered on the physiological concentrations of H2S.Upon use of the dBB method in wild-type and CSE(-/-) mice, however, dBB reports significantly higher sulfide levels than those measured using mBB.Taken together, the dBB method is a highly sensitive method for H2S but is not compatible for use in studies in which other thiols are present.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry , Institute of Molecular Biology , Materials Science Institute , University of Oregon , Eugene , OR 97403 , USA . Email: pluth@uoregon.edu.

ABSTRACT

Hydrogen sulfide (H2S) has emerged as an important biological signaling molecule in the last decade. During the growth of this field, significant controversy has arisen centered on the physiological concentrations of H2S. Recently, a monobromobimane (mBB) method has been developed for the quantification of different biologically-relevant sulfide pools. Based on the prevalence of the mBB method for sulfide quantification, we expand on this method to report the use of dibromobimane (dBB) for sulfide quantification. Reaction of H2S with dBB results in formation of highly-fluorescent bimane thioether (BTE), which is readily quantifiable by HPLC. Additionally, the reaction of sulfide with dBB to form BTE is significantly faster than the reaction of sulfide with mBB to form sulfide dibimane. Using the dBB method, BTE levels as low as 0.6 pM can be detected. Upon use of the dBB method in wild-type and CSE(-/-) mice, however, dBB reports significantly higher sulfide levels than those measured using mBB. Further investigation revealed that dBB is able to extract sulfur from other sulfhydryl sources including thiols. Based on mechanistic studies, we demonstrate that dBB extracts sulfur from thiols with α- or β-hydrogens, thus leading to higher BTE formation than from sulfide alone. Taken together, the dBB method is a highly sensitive method for H2S but is not compatible for use in studies in which other thiols are present.

No MeSH data available.