Ratiometric Measurement of Hydrogen Sulfide and Cysteine/Homocysteine Ratios Using a Dual-Fluorophore Fragmentation Strategy

Abstract

Hydrogen sulfide (H2S) is an integral signaling molecule in biology with complex generation, translocation, and metabolism processes that are intertwined with cellular thiols. Differentiating the complex interplay between H2S and biological thiols, however, remains challenging due to the difficulty of monitoring H2S and thiol levels simultaneously in complex redox environments. As a step toward unraveling the complexities of H2S and thiols in sulfur redox homeostasis, we present a dual-fluorophore fragmentation strategy that allows for the ratiometric determination of relative H2S and cysteine (Cys) or homocysteine (Hcy) concentrations, two important metabolites in H2S biosynthesis. The key design principle is based on a nitrobenzofurazan-coumarin (NBD-Coum) construct, which fragments into spectroscopically differentiable products upon nucleophilic aromatic substitution with either H2S or Cys/Hcy. Measurement of the ratio of fluorescence intensities from coumarin and the NBD-Cys or NBD-Hcy adducts generates a sigmoidal response with a dynamic range of 3 orders of magnitude. The developed scaffold displays a rapid response (<1 min) and is selective for sulfhydryl-containing nucleophiles over other reactive sulfur, oxygen, and nitrogen species, including alcohol- and amine-functionalized amino acids, polyatomic anionic sulfur species, NO, and HNO. Additionally, NBD-Coum is demonstrated to differentiate and report on different oxidative stress stimuli in simulated sulfur pools containing H2S, Cys, and cystine.

A simple and new fluorescent and colorimetric probe based on NBD–maleimide for detecting thiols in living cells

“…2 Abnormal levels of thiols in biological systems can cause several health problems, such as liver damage, slow growth, loss of muscle and fat and Alzheimer's disease. 18 Maleimide is a typical Michael receptor in organic reactions and it has been used in designing new uorescent sensors for detection of biothiols. Recently, several detection methods for thiols have been developed, such as mass spectrometry, 4 high performance liquid chromatography (HPLC), 5 capillary electrophoresis, 6 UV-Vis spectroscopy, 7 and uorimetric sensing.…”

A multi-emissive fluorescent probe for the discrimination of glutathione and cysteine

“…Therefore, we introduced a covalently linked coumarin as the internal standard fluorophore (Hammers et al, 2014;Chen et al, 2016;Hu et al, 2016). We anticipated that, upon addition of Cys, free coumarin would be released, and the internal standard signal from coumarin would be observed other than the weakly fluorescent nitrogen-BODIPY.…”

Chemiluminescent Detection of Enzymatically Produced H2S

“…Such reaction-based systems make use of the unique chemical properties of H 2 S to attack activated electrophiles (Chen, Zhu, et al, 2013; Liu et al, 2011; Montoya, Pearce, Hansen, Zakharov, & Pluth, 2013; Montoya & Pluth, 2014; Qian et al, 2011; Shen et al, 2011), precipitate metal salts (Sasakura et al, 2011), or to reduce azide or nitro groups on masked fluorophores (Lippert, New, & Chang, 2011; Montoya & Pluth, 2012; Peng et al, 2011; Thorson, Majtan, Kraus, & Barrios, 2013). More recent advances allow for the simultaneous measurement of both thiols and H 2 S (Hammers & Pluth, 2014) and offer probes functionalized with targeting groups to report on H 2 S in specific organelles (Bae et al, 2013; Liu, Xu, Spring, & Cui, 2013), proteins (Chen, Chen, Ren, & Ai, 2012), or cellular environments (Lin, Lippert, & Chang, 2013). The advent and elaboration of reaction-based H 2 S detection methods has expanded the arsenal of tools available to further advance our understanding of biological H 2 S.…”