Quantitative measurement of superoxide generation using the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide
Quantitative measurement of superoxide generation using the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide
复制标题
DOI：
10.1006/abio.1997.2067
复制
发表时间：
1997-05-01
影响因子：
2.9
通讯作者：
Zweier, JL
中科院分区：
文献类型：
--
作者：
Roubaud, V;Sankarapandi, S;Zweier, JL
通过电子顺磁共振（EPR）使用自旋阱5，5-二甲基-1-吡咯啉-N-氧化物（DMPO）测量和定量超氧化物受到超氧化物加合物DMPO-OOH的短半衰期（在pH 7下类似于50 s）的限制。最近的β-磷酸化硝酮，5-（diethoxyphosphoryl）-5-甲基-1-吡咯啉-N-氧化物（DEPMPO），被开发和报告，形成一个更稳定的超氧化物加合物的半衰期类似于15分钟。我们评估使用DEPMPO的化学和生化系统中的超氧化物的定量测量。为了估计捕获效率，EPR血氧测定法用于测量氧消耗和DEPMPO-OOH信号的强度以测量超氧化物生成和加合物衰变。与超氧化物生成系统，核黄素/光和黄嘌呤/黄嘌呤氧化酶。DEPMPO捕获类似于65%的超氧化物产生。将DEPMPO捕获超氧化物的效率与常用的细胞色素c还原方法进行比较。当超氧化物产生>20 μ M时，细胞色素c检测到类似于100%的超氧化物产生，而DEPMPO捕获60至70%。然而，用DEPMPO进行EPR检测的灵敏度是细胞色素c的40倍。因此，DEPMPO是一种有效的自旋捕集器，能够特异性和灵敏地检测和定量超氧化物的产生。(C)北京：科学出版社.
Measurement and quantitation of superoxide by electron paramagnetic resonance (EPR) using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) have been limited by the short half-life of the superoxide adduct DMPO-OOH (similar to 50 s at pH 7). Recently a beta-phosphorylated nitrone, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), was developed and reported to form a more stable superoxide adduct with a half-life of similar to 15 min. We evaluated the use of DEPMPO for quantitative measurement of superoxide in chemical and biochemical systems. To estimate the efficiency of trapping, EPR oximetry was used to measure oxygen consumption and the intensity of the DEPMPO-OOH signal to measure superoxide generation and adduct decay. With the superoxide generating systems, riboflavin/light and xanthine/xanthine oxidase. DEPMPO trapped similar to 65% of the superoxide produced. The efficiency of superoxide trapping by DEPMPO was compared to the commonly used cytochrome c reduction method. When superoxide production was >20 mu M, cytochrome c detected similar to 100% of the superoxide produced, while DEPMPO trapped 60 to 70%. However, EPR detection with DEPMPO was 40-fold more sensitive than cytochrome c. Thus, DEPMPO is an efficient spin trap which enables specific and sensitive detection and quantitation of superoxide generation. (C) 1997 Academic Press.