Pou S, Huang Y I, Bhan A, Bhadti V S, Hosmane R S, Wu S Y, Cao G L, Rosen G M
Department of Pharmacology and Toxicology, University of Maryland School of Pharmacy, Baltimore 21201.
Anal Biochem. 1993 Jul;212(1):85-90. doi: 10.1006/abio.1993.1295.
Toward the development of a fluorescence assay in combination with confocal microscopy to image free radicals generated by cells, we synthesized a fluorophore-nitroxide, 5-((2-carboxy)phenyl)-5-hydroxy-1-((2,2,5,5-tetramethyl-1-oxypyrrolid in-3- yl)methyl)-3-phenyl-2-pyrrolin-4-one sodium salt, and tested the applicability of this probe to detect oxygen-centered free radicals. The reaction of the fluorophore-nitroxide with superoxide (10 microM/min) generated either by the reaction of xanthine oxidase on xanthine or by PMA-activated neutrophils in the presence of cysteine (200 microM) resulted in a loss of electron spin resonance (ESR) signal intensity concurrent with an increase in fluorescence emission. The decrease in ESR signal and the augmentation in fluorescence emission were inhibited by the addition of superoxide dismutase. This fluorophore-nitroxide also reacted with methyl radical generated by the reaction of hydroxyl radical with DMSO (0.14 M). In this case a loss in ESR signal intensity concomitant with an increase in fluorescence emission which were inhibited by catalase (300 U/ml), was recorded. These results clearly demonstrated the feasibility of using fluorescence methodology in conjunction with a fluorophore-nitroxide to detect oxygen-centered free radicals in biological systems.
为了开发一种结合共聚焦显微镜的荧光测定法来对细胞产生的自由基进行成像，我们合成了一种荧光团 - 氮氧化物，即5 - （（2 - 羧基）苯基） - 5 - 羟基 - 1 - （（2,2,5,5 - 四甲基 - 1 - 氧化吡咯烷 - 3 - 基）甲基） - 3 - 苯基 - 2 - 吡咯啉 - 4 - 酮钠盐，并测试了该探针检测以氧为中心的自由基的适用性。荧光团 - 氮氧化物与黄嘌呤氧化酶作用于黄嘌呤产生的超氧化物（10微摩尔/分钟）或在半胱氨酸（200微摩尔）存在下PMA激活的中性粒细胞产生的超氧化物反应，导致电子自旋共振（ESR）信号强度降低，同时荧光发射增加。加入超氧化物歧化酶可抑制ESR信号的降低和荧光发射的增强。这种荧光团 - 氮氧化物还与羟基自由基与DMSO（0.14 M）反应产生的甲基自由基反应。在这种情况下，记录到ESR信号强度降低，同时荧光发射增加，而过氧化氢酶（300 U/ml）可抑制这种现象。这些结果清楚地证明了使用荧光方法结合荧光团 - 氮氧化物检测生物系统中以氧为中心的自由基的可行性。
