Superoxide measurement system by pH-jump method

采用 pH 跳跃法的超氧化物测量系统

• 批准号: 03671040
• 负责人: FUJII Hirotada
• 金额: $ 1.28万
• 依托单位: Tokyo Metropolitan Institute of Medical Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03671040/
• 关键词: Superoxide; Active oxygen; pH-Jump; ESR; Nitroso compound; ス-パ-オキシド

For direct measurement of the ESR signal of superoxide anion (O^-_) produced in biological. O^-_generated at a physiological pH was trapped in alkaline media instead of by a rapid freezing method, and then its signal was measured by ESR spectroscopy at 77K.A reaction mixture for O^-_generation, such as neutrophils and NADPH oxidase in neutrophils, was incubated at a physiological pH (pH7.0-7.5) for a suitable reaction period (30 s), then an aliquot (300 mul) was pipetted out and squirted into 600 mul of 0.5 M NaOH to stabilize O^-_(pH-jump). The alkaline mixture was promptly introduced into an ESR tube and frozen by dipping the tube directly into a cooling liquid. A typical signal of O^-_ was detected by ESR spectroscopy and the amount of trapped O^-_ was measured quantitatively at 77 K.Based on this pH-jump method, we developed the superoxide measuring system which is controlled by the personal computer.This system should be widely used for direct measurement of O^-_ produced in biological system at physiological pH.

用于直接测量生物体中产生的超氧阴离子（O^-_）的ESR信号。在生理pH下产生的O^-_被捕获在碱性介质中而不是通过快速冷冻方法，然后通过ESR光谱在77K测量其信号。O^-_生成的反应混合物，例如中性粒细胞和中性粒细胞中的NADPH氧化酶，在生理pH（pH7.0-7.5）下孵育合适的反应时间（30秒），然后移出等分试样（300μl）并喷射加入 600 μl 0.5 M NaOH 中以稳定 O^-_（pH 跃变）。将碱性混合物迅速引入 ESR 管中，并通过将管直接浸入冷却液中进行冷冻。通过ESR光谱法检测到O^-_的典型信号，并在77 K下定量测量被捕获的O^-_的量。基于这种pH跳变方法，我们开发了由个人计算机控制的超氧化物测量系统.该系统应广泛用于直接测量生物系统在生理pH值下产生的O^-_。

项目成果

Hirotada FUJII,Katsuko KAKINUMA: "Electron paramagnetic resonance studies on cytochrome b-558 and peroxidases of pib blood granulocytes" Biochimica et Biophysica Acta. 1136. 239-246 (1992)

Hirotada FUJII、Katsuko KAKINUMA：“对 pib 血粒细胞的细胞色素 b-558 和过氧化物酶的电子顺磁共振研究”Biochimica et Biophysicala Acta。

Hirotada FUJII,Lawrence J.Berliner: "Some applications of ESR to in vivo animal studies and EPR imaging" Biological Magnetic Resonance. 11. 307-320 (1992)

Hirotada FUJII，Lawrence J.Berliner：“ESR 在体内动物研究和 EPR 成像中的一些应用”生物磁共振。

Toshiaki Miki Hirotada Fujii Katsuko Kakinuma: "EPR signals of cytochrome b-558 purified from porcine neutrophils" Journal of Biological Chemistry. 267. 19673-19675 (1992)

Toshiaki Miki Hirotada Fujii Katsuko Kakinuma：“从猪中性粒细胞中纯化的细胞色素 b-558 的 EPR 信号”《生物化学杂志》。

Hirotada Fujii Katsuko Kakinuma: "Electron paramagnetic resonance studies on cytochrome b-558 and peroxidases of pib blood granulocytes" Biochimica et Biophysica Acta. 1136. 239-246 (1992)

Hirotada Fujii Katsuko Kakinuma：“对 pib 血粒细胞的细胞色素 b-558 和过氧化物酶的电子顺磁共振研究”Biochimica et Biophysica Acta。

Hirotada Fujii and Lawrence J. Berliner: "Some applications of ESR to in vivo animal studies and EPR imaging" Biol. Magn. Reson. 11. 307-320 (1992)

Hirotada Fujii 和 Lawrence J. Berliner：“ESR 在体内动物研究和 EPR 成像中的一些应用”Biol。