Detection of nitric oxide from pig trachea by a fluorescence method

Evgenia M. Lozinsky, Lioubov V. Martina, Alexander I. Shames, Natalya Uzlaner, Alexandra Masarwa, Gertz I. Likhtenshtein, Dan Meyerstein, Vladimir V. Martin, Zvi Priel

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

A nitronyl nitroxide radical covalently linked to an organic fluorophore, pyrene, was used to detect nitric oxide (NO) from freshly excited tissues. This approach is based on the phenomenon of the intramolecular fluorescence quenching of the fluorophore fragment by the nitroxide. The pyrene-nitronyl (PN) reacts with NO to yield a pyrene-imino nitroxide radical (PI) and NO 2. Conversion of PN to PI is accompanied by changes in the electron paramagnetic resonance (EPR) spectrum from a five-line pattern (two equivalent N nuclei) into a seven-line pattern (two nonequivalent N nuclei). The transformation of the EPR signal is accompanied by an increase in the fluorescence intensity since the imino nitroxide radical is a weaker quencher than the nitronyl one. The results indicate that the fluorescence measurements enable detection of nanomolar concentrations of NO compared to a sensitivity threshold of only several micromolar for the EPR technique. The method was applied to the determination of NO and S-nitroso compounds in tissue from pig trachea epithelia. The measured basal flux of S-nitroso compounds obtained from the tissues was about 1.2nmol/g×min, and NO-synthase stimulated by extracellular adenosine 5′-triphosphate produced NO flux of 0.9nmol/g×min.

Original languageEnglish
Pages (from-to)139-145
Number of pages7
JournalAnalytical Biochemistry
Volume326
Issue number2
DOIs
StatePublished - 15 Mar 2004

Keywords

  • Cilia
  • Electron paramagnetic resonance (EPR)
  • Fluorescence
  • Fluorophore-nitronyl-nitroxide
  • Nitric oxide
  • Trachea

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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