title: Nitroxidative chemistry interferes with fluorescent probe chemistry: implications for nitric oxide detection using 2,3-diaminonaphthalene.
abstract: Simultaneous production of nitric oxide (NO) and superoxide generates peroxynitrite and causes nitroxidative stress. The fluorometric method for NO detection is based on the formation of a fluorescent product from the reaction of a nonfluorescent probe molecule with NO-derived nitrosating species. Here, we present an example of how nitroxidative chemistry could interact with fluorescent probe chemistry. 2,3-Naphthotriazole (NAT) is the NO-derived fluorescent product of 2,3-diaminonaphthalene (DAN), a commonly used NO-detecting molecule. We show that NO/superoxide cogeneration, and particularly peroxynitrite, mediates the chemical decomposition of NAT. Moreover, the extent of NAT decomposition depends on the relative fluxes of NO and superoxide; the maximum effect being reached at almost equivalent generation rates for both radicals. The rate constant for the reaction of NAT with peroxynitrite was determined to be 2.2×10(3)M(-1)s(-1). Further, various peroxynitrite scavengers were shown to effectively inhibit NO/superoxide- and peroxynitrite-mediated decomposition of NAT. Taken together, the present study suggests that the interference of a fluorometric NO assay can be originated from the interaction between the final fluorescent product and the formed reactive nitrogen and oxygen species.
authors: School of Pharmacy, National Defense Medical Center, Taipei, Taiwan, ROC. Electronic address: tmhu@ndmctsgh.edu.tw.
authors: Teh-Min
authors: TM
authors: Hu
authors: College of Pharmacy, Taipei Medical University, Taipei, Taiwan, ROC.
authors: Shih-Jiuan
authors: SJ
authors: Chiu
authors: School of Pharmacy, National Defense Medical Center, Taipei, Taiwan, ROC.
authors: Yu-Ming
authors: YM
authors: Hsu
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry*; D016166:Free Radical Scavengers / Q000737:chemistry; D007700:Kinetics; D009569:Nitric Oxide / Q000032:analysis*; D015538:Nitrosation; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000737:chemistry; D014230:Triazoles / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D016166:Free Radical Scavengers; D014230:Triazoles; D013481:Superoxides; D030421:Peroxynitrous Acid; C026373:2,3-diaminonaphthalene; D009569:Nitric Oxide; D015081:2-Naphthylamine
keywords: Fluorescent probe; Naphthotriazole; Nitric oxide; Peroxynitrite; Superoxide