Assessment of nitric oxide signals by triiodide chemiluminescence

Alfred Hausladen, Ruslan Rafikov, Michael Angelo, David J. Singel, Evgeny Nudler, Jonathan S. Stamler

Research output: Contribution to journalArticlepeer-review

71 Scopus citations

Abstract

Nitric oxide (NO) bioactivity is mainly conveyed through reactions with iron and thiols, furnishing iron nitrosyls and S-nitrosothiols with wide-ranging stabilities and reactivities. Triiodide chemiluminescence methodology has been popularized as uniquely capable of quantifying these species together with NO byproducts, such as nitrite and nitrosamines. Studies with triiodide, however, have challenged basic ideas of NO biochemistry. The assay, which involves addition of multiple reagents whose chemistry is not fully understood, thus requires extensive validation: Few protein standards have in fact been characterized; NO mass balance in biological mixtures has not been verified; and recovery of species that span the range of NO-group reactivities has not been assessed. Here we report on the performance of the triiodide assay vs. photolysis chemiluminescence in side-by-side assays of multiple nitrosylated standards of varied reactivities and in assays of endogenous Fe- and 5-nitrosylated hemoglobin. Although the photolysis method consistently gives quantitative recoveries, the yields by triiodide are variable and generally low (approaching zero with some standards and endogenous samples). Moreover, in triiodide, added chemical reagents, changes in sample pH, and altered ionic composition result in decreased recoveries and misidentification of NO species. We further show that triiodide, rather than directly and exclusively producing NO, also produces the highly potent nitrosating agent, nitrosyliodide. Overall, we find that the triiodide assay is strongly influenced by sample composition and reactivity and does not reliably identify, quantify, or differentiate NO species in complex biological mixtures.

Original languageEnglish (US)
Pages (from-to)2157-2162
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number7
DOIs
StatePublished - Feb 13 2007
Externally publishedYes

Keywords

  • Red blood cell vasodilation
  • S-nitrosohemoglobin
  • S-nitrosylation

ASJC Scopus subject areas

  • General

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