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Design, Validation, and Application of an Enzyme-Coupled Hydrogen Sulfide Detection Assay

Lynch, Michael J., Crane, Brian R.
Biochemistry 2018 v.58 no.6 pp. 474-483
Escherichia coli, Treponema denticola, anions, beta-mercaptoethanol, biosynthesis, crosslinking, cysteine, cysteine synthase, derivatization, dithiothreitol, fluorescent dyes, glutathione, high performance liquid chromatography, hydrogen sulfide, metabolites, methylene blue, thiols
Hydrogen sulfide (H₂S) is a key metabolite in biosynthesis and is increasingly being recognized as an essential gasotransmitter. Owing to its diffusible and reactive nature, H₂S can be difficult to quantify, particularly in situ. Although several detection schemes are available, they have drawbacks. In efforts to quantify sulfide release in the cross-linking reaction of the flagellar protein FlgE, we developed an enzyme-coupled sulfide detection assay using the Escherichia coli O-acetylserine sulfhydrylase enzyme CysM. Conversion of HS– to l-cysteine via CysM followed by derivatization with the thiol-specific fluorescent dye 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin enables for facile detection and quantification of H₂S by fluorescent HPLC. The assay was validated by comparison to the well-established methylene blue sulfide detection assay and the robustness demonstrated by interference assays in the presence of common thiols such as glutathione, 2-mercaptoethanol, dithiothreitol, and l-methionine, as well as a range of anions. We then applied the assay to the aforementioned lysinoalanine cross-linking by the Treponema denticola flagellar hook protein FlgE. Overall, unlike previously reported H₂S detection methods, the assay provides a biologically compatible platform to accurately and specifically measure hydrogen sulfide in situ, even when it is produced on long time scales.