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

FEBS Letters

Joseph, Joy¹  Kalyanaraman, B¹  Vasquez-Vivar, Jeannette⁴  Nsanzumuhire, Cline³  Martásek, Pavel²  Zhang, Hao¹  Tordo, Paul³  Karoui, Hakim³ 

[1] Biophysics Research Institute, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA;University of Texas Health Sciences Center, San Antonio, TX, USA;Laboratoire ‘Structure et Réactivité des Espèces Paramagnétiques’, Université de Provence, Marseille, France;Department of Pathology and Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI, USA

关键词: Electron spin resonance;    Spin trap;    Nitric oxide synthase;    Vascular biology;    Tetrahydrobiopterin;    ESR;    electron spin resonance;    DMPO;    5;    5-dimethyl-1-pyrroline N-oxide;    DEPMPO;    5-(dimethoxyphosphoryl)-5′-methyl-1-pyrroline N-oxide;    EMPO;    5-ethoxycarbonyl-5-methyl-pyrroline N-oxide;    nNOS;    neuronal nitric oxide synthase;    eNOS;    endothelial nitric oxide synthase;    DMPO–OOH;    DMPO superoxide adduct;    DEPMPO–OOH;    DEPMPO superoxide adduct;    EMPO–OOH;    EMPO superoxide adduct;    EMPO–OH;    EMPO hydroxyl adduct;   

DOI  :  10.1016/S0014-5793(00)01498-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We describe the synthesis and biological applications of a novel nitrogen-15-labeled nitrone spin trap, 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide ([¹⁵N]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 [¹⁴N]EMPO–OOH and [¹⁵N]EMPO–OOH are less complex and easier to interpret. Potential uses of [¹⁵N]EMPO in elucidating the mechanism of superoxide formation from nitric oxide synthases, and in ischemia/reperfusion injury are discussed.

【 授权许可】

Unknown