Sensitivity evaluation of NBD-SCN towards cysteine/homocysteine and its bioimaging applications

Chen, Yen‐Hao; Tsai, Jia-Chun; Cheng, Tsan-Hwang; Yuan, Shyng‐Shiou F.; Wang, Yun-Ming

doi:10.1016/j.bios.2014.01.009

Cited by 45 publications

(10 citation statements)

References 66 publications

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“…Although NBD-SH and NBD-SR are both nonfluorescent, NBD-adducts of Cys and Hcy undergo subsequent intermolecular rearrangement with adjacent amine functionalities to form fluorescent amino-bound NBD-NHR compounds (Scheme 2a, b). 59−61 The second fluorophore appended to the NBD scaffold through an ether linkage provides an additional fluorescent reporter that is liberated upon nucleophilic substitution. On the basis of this design, the coumarin fluorescence is directly proportional to the combined H 2 S and Cys/Hcy concentrations in solution upon fragmentation of the probe, whereas the NBD component is proportional to the Cys/Hcy concentration exclusively, given that NBD-SH is nonfluorescent.…”

Section: Resultsmentioning

confidence: 99%

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

2014

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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.

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Section: Resultsmentioning

confidence: 99%

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

2014

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“…The images were obtained by the fluorescence microscope ( λ ex 460 nm, λ em 524 nm; Axiovert 40 CFL, Zeiss). As for 4-nitro-7-thiocyanatobenz-2-oxa-1,3-diazole (NBD-SCN) that was used for detecting H 2 S, the cells were incubated with 5 μ M NBD-SCN for 30 min and then subjected to fluorescence microscope ( λ ex 460 nm, λ em 550 nm) [22]. For confocal fluorescence images study, ECs were seeded at a density of 2 × 10 5 cells/well on cover glasses (24 × 24 mm 2 ) and grown for 24 h. The cells with 10 μ M of FA-OMe incubation were fixed with 4% formaldehyde solution for 20 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Hydrogen Sulfide Increases Nitric Oxide Production and Subsequent S-Nitrosylation in Endothelial Cells

Chen

Wang

et al. 2014

The Scientific World Journal

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Hydrogen sulfide (H2S) and nitric oxide (NO), two endogenous gaseous molecules in endothelial cells, got increased attention with respect to their protective roles in the cardiovascular system. However, the details of the signaling pathways between H2S and NO in endothelia cells remain unclear. In this study, a treatment with NaHS profoundly increased the expression and the activity of endothelial nitric oxide synthase. Elevated gaseous NO levels were observed by a novel and specific fluorescent probe, 5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid methyl ester (FA-OMe), and quantified by flow cytometry. Further study indicated an increase of upstream regulator for eNOS activation, AMP-activated protein kinase (AMPK), and protein kinase B (Akt). By using a biotin switch, the level of NO-mediated protein S-nitrosylation was also enhanced. However, with the addition of the NO donor, NOC-18, the expressions of cystathionine-γ-lyase, cystathionine-β-synthase, and 3-mercaptopyruvate sulfurtransferase were not changed. The level of H2S was also monitored by a new designed fluorescent probe, 4-nitro-7-thiocyanatobenz-2-oxa-1,3-diazole (NBD-SCN) with high specificity. Therefore, NO did not reciprocally increase the expression of H2S-generating enzymes and the H2S level. The present study provides an integrated insight of cellular responses to H2S and NO from protein expression to gaseous molecule generation, which indicates the upstream role of H2S in modulating NO production and protein S-nitrosylation.

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“…Axiovert 40 CFL, Zeiss, G€ ottingen, Germany) [25]. After fluorescence microscopic observations, the ECs were washed twice with PBS buffer and detached by tryptic reaction.…”

Section: Applications Of Fluorescent Probe and Flow Cytometrymentioning

confidence: 99%

“…Even though several approaches were designed to measure H 2 S, the accuracy and a long operation time were unsatisfying [23,24]. Hence, a fluorescence probe was applied, using the reaction of 4-nitro-7-thiocyanatobenz-2-oxa-1,3-diazole (NBD-SCN) with cysteine and homocysteine [22,25]. The cysteine/homocysteine then can be converted to H 2 S via CSE, CBS and 3-MST [14].…”

Section: Introductionmentioning

confidence: 99%

Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells

Huang

Chen

et al. 2015

Biochemical and Biophysical Research Communications

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Sensitivity evaluation of NBD-SCN towards cysteine/homocysteine and its bioimaging applications

Cited by 45 publications

References 66 publications

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

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

Hydrogen Sulfide Increases Nitric Oxide Production and Subsequent S-Nitrosylation in Endothelial Cells

Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells

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