A fluorophore-containing nitroxide as a probe to detect superoxide and hydroxyl radical generated by stimulated neutrophils.
A fluorophore-containing nitroxide as a probe to detect superoxide and hydroxyl radical generated by stimulated neutrophils.
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DOI：
10.1006/abio.1993.1295
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发表时间：
1993-07
影响因子：
2.9
通讯作者：
S. Pou;Y. I. Huang;A. Bhan;V. Bhadti;R. Hosmane;Sing-yung Wu;G. Cao;G. Rosen
中科院分区：
文献类型：
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作者：
S. Pou;Y. I. Huang;A. Bhan;V. Bhadti;R. Hosmane;Sing-yung Wu;G. Cao;G. Rosen
为了开发与共聚焦显微镜相结合的荧光测定以成像细胞产生的自由基，我们合成了荧光团-氮氧自由基，5-（（2-羧基）苯基）-5-羟基-1-（（2，2，5，5-四甲基-1-氧代吡咯烷-3-基）甲基）-3-苯基-2-吡咯啉-4-酮钠盐，并测试了该探针检测氧中心自由基的适用性。荧光团-氮氧自由基与超氧化物（10 μ M/min）的反应产生的黄嘌呤氧化酶对黄嘌呤的反应或PMA激活的中性粒细胞在半胱氨酸（200 μ M）的存在下，导致电子自旋共振（ESR）信号强度的损失，同时增加荧光发射。超氧化物歧化酶的加入抑制了ESR信号的降低和荧光发射的增强。该荧光团-氮氧还与通过羟基自由基与DMSO（0.14 M）反应产生的甲基自由基反应。在这种情况下，记录了ESR信号强度的损失，伴随着被过氧化氢酶（300 U/ml）抑制的荧光发射的增加。这些结果清楚地证明了使用荧光方法结合荧光团-氮氧化物来检测生物系统中以氧为中心的自由基的可行性。
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.