Quantitative detection of nitroxyl upon trapping with glutathione and labeling with a specific fluorogenic reagent

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

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

Donors of nitroxyl (HNO) have shown promise for treatment of stroke, heart failure, alcoholism and cancer. However, comparing the pharmacological capacities of various donors is difficult without first quantifying the amount of HNO released from each donor. Detection and quantitation of HNO has been complicated by the rapid self-consumption of HNO through irreversible dimerization, poor selectivity of trapping agents against other nitrogen oxides, and/or low sensitivity towards HNO. Here, an assay is described for the trapping of HNO by glutathione (GSH) followed by labeling of GSH with the fluorogenic agent, naphthalene-2,3-dicarboxaldehyde (NDA), and subsequent quantitation by fluorescence difference. The newly developed assay was used to validate the pH-dependence of HNO release from isopropylamine NONOate (IPA/NO), which is a dual donor of HNO and NO at physiological pH. Furthermore, varied assay conditions were utilized to suggest the ratios of the products of the reaction of GSH with HNO. At intracellular concentrations of GSH, the disulfide (GSSG) was the major product, but significant concentrations of glutathione sulfinamide (GS(O)NH2) were also detected. This suggests that GS(O)NH 2, which is a selective biomarker of HNO, may be produced in concentrations that are amenable to in vivo analysis.

Original languageEnglish (US)
Pages (from-to)476-484
Number of pages9
JournalFree Radical Biology and Medicine
Volume63
DOIs
StatePublished - 2013

Bibliographical note

Funding Information:
We are grateful for financial support from NSF ( CHE0645818 ). We thank Prof. Craig Aspinwall for helpful discussions. We also thank Dr. Debashree Basudhar and Gaurav Bharadwaj for synthesis of the NONOates.

Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.

Keywords

  • Angeli's salt
  • Free radicals
  • Glutathione
  • Isopropylamine NONOate
  • Naphthalene-2,3-dicarboxaldehyde
  • Nitric oxide
  • Nitroxyl
  • Sulfinamide

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