title: Bioluminescent detection of peroxynitrite with a boronic acid-caged luciferin.
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. USA107, 21316-21321, 2010) [2], reacts with peroxynitrite stoichiometrically with a rate constant of 9.8±0.3×10(5)M(-1)s(-1) and a bioluminescence detection limit of 16nM, compared to values of 1.2±0.3M(-1)s(-1) and 231nM 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.
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Sieracki
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Benjamin N
authors.initials: BN
authors.lastname: Gantner
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612, USA; Section of Cardiology, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Mao
authors.initials: M
authors.lastname: Mao
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: John H
authors.initials: JH
authors.lastname: Horner
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Ye
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Martin E
authors.initials: ME
authors.lastname: Newcomb
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612, USA; Section of Cardiology, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA. Electronic address: mbonini@uic.edu.
authors.forename: Marcelo G
authors.initials: MG
authors.lastname: Bonini
mesh_terms: D000818:Animals; D052160:Benzothiazoles / Q000378:metabolism*; D001897:Boronic Acids; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D052247:Nitric Oxide Synthase Type II / Q000502:physiology; D030421:Peroxynitrous Acid / Q000032:analysis* / Q000097:blood
chemical_list: D052160:Benzothiazoles; D001897:Boronic Acids; C532924:D-luciferin; D030421:Peroxynitrous Acid; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse
keywords: Boronate; Free radicals; Luciferin; PCL-1; Peroxynitrite; Sensor