Direct chemiluminescence detection of nitric oxide in aqueous solutions using the natural nitric oxide target soluble guanylyl cyclase

Yakov Y. Woldman, Jian Sun, Jay L. Zweier, Valery V. Khramtsov

Research output: Contribution to journalArticle

Abstract

Nitric oxide (NO) is a free radical involved in many physiological processes including regulation of blood pressure, immune response, and neurotransmission. However, the measurement of extremely low, in some cases subnanomolar, physiological concentrations of nitric oxide presents an analytical challenge. The purpose of this methods article is to introduce a new highly sensitive chemiluminescence approach to direct NO detection in aqueous solutions using a natural nitric oxide target, soluble guanylyl cyclase (sGC), which catalyzes the conversion of guanosine triphosphate to guanosine 3′,5′-cyclic monophosphate and inorganic pyrophosphate. The suggested enzymatic assay uses the fact that the rate of the reaction increases by about 200 times when NO binds with sGC and, in so doing, provides a sensor for nitric oxide. Luminescence detection of the above reaction is accomplished by converting inorganic pyrophosphate into ATP with the help of ATP sulfurylase followed by light emission from the ATP-dependent luciferin-luciferase reaction. Detailed protocols for NO quantification in aqueous samples are provided. The examples of applications include measurement of NO generated by a nitric oxide donor (PAPA-NONOate), nitric oxide synthase, and NO gas dissolved in buffer. The method allows for the measurement of NO concentrations in the nanomolar range and NO generation rates as low as 100 pM/min.

Original languageEnglish (US)
Pages (from-to)1339-1345
Number of pages7
JournalFree Radical Biology and Medicine
Volume47
Issue number10
DOIs
StatePublished - Nov 15 2009
Externally publishedYes

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Keywords

  • Chemiluminescence
  • Free radicals
  • Guanylyl cyclase
  • Luciferase
  • Nitric oxide
  • Nitric oxide synthase
  • NO donors
  • Pyrophosphate

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

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