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Detection of superoxide anion using an isotopically labeled nitrone spin trap: potential biological applications
Author(s) -
Zhang Hao,
Joseph Joy,
Vasquez-Vivar Jeannette,
Karoui Hakim,
Nsanzumuhire Cline,
Martásek Pavel,
Tordo Paul,
Kalyanaraman B
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01498-8
Subject(s) - superoxide , nitrone , chemistry , adduct , xanthine oxidase , xanthine , nitric oxide , photochemistry , biochemistry , enzyme , organic chemistry , cycloaddition , catalysis
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.