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Logo of jbcThe Journal of Biological Chemistry
J Biol Chem. 2010 May 7; 285(19): e99938.
PMCID: PMC2863233

The Peroxynitrite Plateau♦

Peroxynitrite Is the Major Species Formed from Different Flux Ratios of Co-generated Nitric Oxide and Superoxide. Direct Reaction with Boronate-based Fluorescent Probe

♦ See referenced article, J. Biol. Chem. 2010, 285, 14210–14216

Peroxynitrite (ONOO) is a potent, unstable intermediate formed from the reaction between nitric oxide (NO) and superoxide (O2[bar dot]). Due to its extremely short life-span, ONOO is very difficult to monitor in cells, leading to some controversy regarding this reaction. For example, indirect measurements have suggested that ONOO yield follows a bell-shaped curve with a maximum at an NO:O2[bar dot] molar ratio of 1:1; however, these results might be based off of secondary radical reactions and thus may be erroneous. In this Paper of the Week, Jacek Zielonka and colleagues used a novel fluorogenic probe, coumarin-7-boronic acid (CBA), to assay ONOO formation; CBA reacts rapidly and stoichiometrically with ONOO to produce the fluorescent molecule 7-hydroxycoumarin. They found that in conditions using constant O2[bar dot], ONOO formation increased linearly with increased NO amounts until the ratio reached 1:1, at which point yields plateaued, but did not decrease as in previous work. Zielonka and colleagues performed these assays further in the presence of superoxide dismutase and catalase, and the lack of inhibition confirmed that ONOO, and not other nitrogen species, was reacting with the CBA. This elegantly simple study lends crisp new insight into redox-related cellular reactions and also highlights the utility of boronate derivatives for detecting such molecules.

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onoo formation (measured as rate of 7-hydroxycoumarin fluorescence increase) under a constant superoxide flux of 2 μm/min peaks at a 1:1 molar ratio with nitric oxide and then plateaus.

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