Detection of superoxide anion using an isotopically labeled nitrone spin trap: potential biological applications.
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.
Zhang
H
H
Biophysics Research Institute, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Joseph
J
J
Vasquez-Vivar
J
J
Karoui
H
H
Nsanzumuhire
C
C
Martásek
P
P
Tordo
P
P
Kalyanaraman
B
B
D002147:Calmodulin / Q000378:metabolism; D002956:Citrulline / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000138:chemical synthesis / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006207:Half-Life; D017665:Hydroxyl Radical / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009587:Nitrogen Isotopes; D009589:Nitrogen Oxides / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism; D012680:Sensitivity and Specificity; D013113:Spin Labels / Q000138:chemical synthesis; D019097:Spin Trapping / Q000379:methods; D013481:Superoxides / Q000032:analysis / Q000378:metabolism*; D019820:Xanthine / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism
C408629:5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide; D002147:Calmodulin; D003497:Cyclic N-Oxides; D009587:Nitrogen Isotopes; D009589:Nitrogen Oxides; D013113:Spin Labels; C015388:nitrones; D013481:Superoxides; D019820:Xanthine; D002956:Citrulline; D017665:Hydroxyl Radical; D009249:NADP; D019001:Nitric Oxide Synthase; D014969:Xanthine Oxidase