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Detection of superoxide anion using an isotopically labeled nitrone spin trap: potential biological applications

FEBS Letters, ISSN: 0014-5793, Vol: 473, Issue: 1, Page: 58-62
2000
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Article Description

We describe the synthesis and biological applications of a novel nitrogen-15-labeled nitrone spin trap, 5-ethoxycarbonyl-5-methyl-1-pyrroline N -oxide ([ 15 N]EMPO) for detecting superoxide anion. Superoxide anion generated in xanthine/xanthine oxidase (100 nM min −1 ) and NADPH/calcium-calmodulin/nitric oxide synthase systems was readily detected using EMPO, a nitrone analog of 5,5′-dimethyl-1-pyrroline N -oxide (DMPO). Unlike DMPO-superoxide adduct (DMPO–OOH), the superoxide adduct of EMPO (EMPO–OOH) does not spontaneously decay to the corresponding hydroxyl adduct, making spectral interpretation less confounding. Although the superoxide adduct of 5-(diethoxyphosphoryl)-5-methyl-pyrroline N -oxide is more persistent than EMPO–OOH, the electron spin resonance spectra of [ 14 N]EMPO–OOH and [ 15 N]EMPO–OOH are less complex and easier to interpret. Potential uses of [ 15 N]EMPO in elucidating the mechanism of superoxide formation from nitric oxide synthases, and in ischemia/reperfusion injury are discussed.

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