Quantitative Detection of Nitroxyl Upon Trapping with Glutathione and Labeling with a Specific Fluorogenic Reagent

Johnson, Gail M.; Chozinski, Tyler J.; Salmon, Debra J.; Moghaddam, Alan D.; Chen, Hsin Chih; Miranda, Katrina M.

doi:10.1016/j.freeradbiomed.2013.05.011

Cited by 24 publications

(18 citation statements)

References 60 publications

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“…Although these results are consistent with our previous analysis [45], a higher degree of reliability is suggested here since GS(O)NH 2 itself is detected rather than a decrease in fluorescence intensity. Additionally, the CZE-LIF method requires much smaller sample volumes (10 nL) than our high throughput assay (200 μL), while also providing enhanced sensitivity.…”

Section: Resultssupporting

confidence: 94%

“…This assay was used to quantify HNO from primary amine diazeniumdiolates (NONOates), such as IPA/NO. Furthermore, the yield of GS(O)NH 2 and GSSG formed from the reaction of HNO with GSH was confirmed to be dependent on the initial concentration of GSH (Scheme 1; see reference [45] for complete discussion). In particular, when the concentrations of GSH and Angeli’s salt are less than 50 μM, GS(O)NH 2 is the exclusive product.…”

Section: Resultsmentioning

confidence: 94%

“…This is presumably due to quenching of fluorescence from labeling of two amines in close proximity [57, 58], which has been previously observed upon labeling of lysine [51]. Therefore, to detect changes in GSSG concentration, GSSG was reduced first to GSH with TCEP before blocking with NEM and fluorogenic labeling, as previously described [45]. …”

Section: Resultsmentioning

confidence: 99%

“…We have previously used Angeli’s salt to evaluate the performance of a related fluorescence assay that employed a plate reader for high throughput analysis of HNO from donors [45]. Briefly, HNO released from Angeli’s salt was trapped by GSH, followed by labeling unreacted GSH with NDA, and subsequent quantitation of HNO by fluorescence difference.…”

Section: Resultsmentioning

confidence: 99%

“…These donors were stored at −20 °C for several weeks and then on ice while in use. GS(O)NH 2 was synthesized as previously described [45]. Stock solutions of peroxynitrite (ONOO − ) were prepared as previously reported [32, 46] and diluted in 10 mM NaOH to yield a concentration of 5 mM.…”

Section: Methodsmentioning

confidence: 99%

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Glutathione sulfinamide serves as a selective, endogenous biomarker for nitroxyl after exposure to therapeutic levels of donors

Johnson

Chozinski

Gallagher

et al. 2014

Free Radical Biology and Medicine

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Nitroxyl (HNO) donors exhibit promising pharmacological characteristics for treatment of cardiovascular disorders, cancer and alcoholism. However, whether HNO also serves as an endogenous signaling agent is currently unknown, largely due to the inability to selectively and sensitively detect HNO in a cellular environment. Although a number of methods to detect HNO have been developed recently, sensitivity and selectivity against other nitrogen oxides or biological reductants remain problematic. To improve selectivity, the electrophilic nature of HNO has been harnessed to generate modifications of thiols and phosphines that are unique to HNO, especially compared to nitric oxide (NO). Given high bioavailability, glutathione (GSH) is expected to be a major target of HNO. As a result, the putative selective product glutathione sulfinamide (GS(O)NH2) may serve as a high yield biomarker of HNO production. In this work, the formation of GS(O)NH2 following exposure to HNO donors was investigated. Fluorescent labeling followed by separation and detection using capillary zone electrophoresis with laser-induced fluorescence allowed quantitation of GS(O)NH2 with nanomolar sensitivity, even in the presence of GSH and derivatives. Formation of GS(O)NH2 was found to occur exclusively upon exposure of GSH to HNO donors, thus confirming selectively. GS(O)NH2 was detected in the lysate of cells treated with low micromolar concentrations of HNO donors, verifying that this marker has sufficient stability to server as a biomarker of HNO. Additionally, the concentration-dependent formation of GS(O)NH2 in cells treated with an HNO donor suggests that the concentration of GS(O)NH2 can be correlated to intracellular levels of HNO.

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Section: Resultssupporting

confidence: 94%

Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Glutathione sulfinamide serves as a selective, endogenous biomarker for nitroxyl after exposure to therapeutic levels of donors

Johnson

Chozinski

Gallagher

et al. 2014

Free Radical Biology and Medicine

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Mechanistic Origin of Favorable Substituent Effects in Excellent Cu Cyclam Based HNO Sensors

et al. 2022

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HNO has broad chemical and biomedical properties. Metal complexes and derivatives are widely used to make excellent HNO sensors. However, their favorable mechanistic origins are largely unknown. Cu cyclam is a useful platform to make excellent HNO sensors including imaging agents. A quantum chemical study of Cu cyclams with various substitutions was performed, which reproduced diverse experimental reactivities. Structural, electronic, and energetic profiles along reaction pathways show the importance of HNO binding and a proton‐coupled electron transfer mechanism for HNO reaction. Results reveal that steric effect is primary and electronic factor is secondary (if the redox potential is sufficient), but their interwoven effects can lead to unexpected reactivity, which looks mysterious experimentally but can be explained computationally. This work suggests rational substituent design ideas and recommends a theoretical study of a new design to save time and cost due to its subtle effect.

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Mechanistic Origin of Favorable Substituent Effects in Excellent Cu Cyclam Based HNO Sensors

et al. 2022

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Quantitative Detection of Nitroxyl Upon Trapping with Glutathione and Labeling with a Specific Fluorogenic Reagent

Cited by 24 publications

References 60 publications

Glutathione sulfinamide serves as a selective, endogenous biomarker for nitroxyl after exposure to therapeutic levels of donors

Glutathione sulfinamide serves as a selective, endogenous biomarker for nitroxyl after exposure to therapeutic levels of donors

Mechanistic Origin of Favorable Substituent Effects in Excellent Cu Cyclam Based HNO Sensors

Mechanistic Origin of Favorable Substituent Effects in Excellent Cu Cyclam Based HNO Sensors

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