Bioluminescent detection of peroxynitrite with a boronic acid-caged luciferin

Nathan A. Sieracki, Benjamin N. Gantner, Mao Mao, John H. Horner, Richard D. Ye, Asrar B. Malik, Martin E. Newcomb, Marcelo G. Bonini*

*Corresponding author for this work

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

Peroxynitrite, a highly reactive biological oxidant, is formed under pathophysiologic conditions from the diffusion-limited reaction of nitric oxide and superoxide radical anion. Peroxynitrite has been implicated as the mediator of nitric oxide toxicity in many diseases and as an important signaling disrupting molecule (L. Liaudet et al., Front. Biosci. 14, 4809-4814, 2009) [1]. Biosensors effective at capturing peroxynitrite in a specific and fast enough manner for detection, along with readouts compatible with in vivo studies, are lacking. Here we report that the boronic acid-based bioluminescent system PCL-1 (peroxy-caged luciferin-1), previously reported as a chemoselective sensor for hydrogen peroxide (G.C. Van de Bittner et al., Proc. Natl. Acad. Sci. USA 107, 21316-21321, 2010) [2], reacts with peroxynitrite stoichiometrically with a rate constant of 9.8±0.3×105 M-1 s-1 and a bioluminescence detection limit of 16 nM, compared to values of 1.2±0.3 M-1 s-1 and 231 nM for hydrogen peroxide. Further, we demonstrate bioluminescent detection of peroxynitrite in the presence of physiological competitors: carbon dioxide, glutathione, albumin, and catalase. We also demonstrate the utility of this method to assess peroxynitrite formation in mammalian cells by measuring peroxynitrite generated under normal culture conditions after stimulation of macrophages with bacterial endotoxin lipopolysaccharide. Thus, the PCL-1 method for measuring peroxynitrite generation shows superior selectivity over other oxidants under in vivo conditions.

Original languageEnglish (US)
Pages (from-to)40-50
Number of pages11
JournalFree Radical Biology and Medicine
Volume61
DOIs
StatePublished - 2013

Keywords

  • Boronate
  • Free radicals
  • Luciferin
  • PCL-1
  • Peroxynitrite
  • Sensor

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

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