Giulivi C, Boveris A, Cadenas E
Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles 90033.
Arch Biochem Biophys. 1995 Feb 1;316(2):909-16. doi: 10.1006/abbi.1995.1122.
Production of hydroxyl radicals (HO.) by substrate-supplemented beef heart submitochondrial particles was studied by electron paramagnetic resonance in conjunction with the spin trap 5,5'-dimethyl-1-pirroline-N-oxide (DMPO). Supplementation of submitochondrial particles with NADH or succinate in the presence of antimycin resulted in the formation hydroxyl-, alpha-hydroxyethyl-, and methyl radical adducts. The latter two adducts were derived from HO. attack of ethanol or dimethyl sulfoxide (DMSO), respectively, the solvents used for the inhibitors of the respiratory chain. These ESR signals were slightly increased by superoxide dismutase and abolished by catalase. Further support for the production of HO. during mitochondrial electron transfer was furnished by kinetic competition experiments with DMSO as the HO. scavenger. This approach yielded a kappa SCAVENGER/kappa DMPO value of 1.7, in agreement with a competitive spin trapping of free HO. using DMSO as a scavenger. The scission of H2O2 to HO. requires consideration of a Fenton chemistry, i.e., the participation of metals or redox active metal pools in mitochondria to drive this reaction. The effect of several metal chelators on the formation of both HO. and H2O2 was examined. Bathophenantroline, bathocuproine, and desferrioxamine decreased the DMPO-HO. signal and increased accumulation of H2O2. Conversely, EDTA or diethylenetriaminepentaacetic acid substantially increased the DMPO-HO. signal intensity and decreased H2O2 accumulation. These different results were rationalized in terms of the reduction potential of the redox couples involved, i.e., that of the ligated metal and those encompassed in the one-electron reduction of superoxide radical and of hydrogen peroxide. The formation of 8-hydroxydesoxyguanosine in mitochondrial DNA was examined under experimental conditions in which H2O2 production by isolated mitochondria was enhanced. The formation of 8-hydroxydesoxyguanosine increased with increasing rates of H2O2 formation. The biological significance of H2O2 and HO. formation during mitochondrial electron transfer is discussed in terms of oxidative damage of mitochondrial DNA and the implications for mitochondrial functions and aging.
通过电子顺磁共振结合自旋捕获剂5,5'-二甲基-1-吡咯啉-N-氧化物（DMPO），研究了底物补充的牛心亚线粒体颗粒产生羟基自由基（HO·）的情况。在抗霉素存在下，用NADH或琥珀酸补充亚线粒体颗粒会导致羟基、α-羟乙基和甲基自由基加合物的形成。后两种加合物分别源自HO·对乙醇或二甲基亚砜（DMSO）的攻击，乙醇和二甲基亚砜是呼吸链抑制剂所使用的溶剂。超氧化物歧化酶会使这些电子顺磁共振信号略有增加，而过氧化氢酶则会使其消失。用DMSO作为HO·清除剂的动力学竞争实验进一步支持了线粒体电子传递过程中HO·的产生。这种方法得到的κ清除剂/κDMPO值为1.7，这与使用DMSO作为清除剂对游离HO·进行竞争性自旋捕获的结果一致。H2O2裂解为HO·需要考虑芬顿化学，即线粒体中金属或氧化还原活性金属池参与驱动此反应。研究了几种金属螯合剂对HO·和H2O2形成的影响。红菲绕啉、铜试剂和去铁胺降低了DMPO-HO·信号，并增加了H2O2的积累。相反，EDTA或二乙烯三胺五乙酸显著增加了DMPO-HO·信号强度，并减少了H2O2的积累。根据所涉及的氧化还原对的还原电位，即连接金属的还原电位以及超氧自由基和过氧化氢单电子还原中所包含的还原电位，对这些不同结果进行了合理化解释。在增强分离线粒体产生H2O2的实验条件下，检测了线粒体DNA中8-羟基脱氧鸟苷的形成。8-羟基脱氧鸟苷的形成随着H2O2形成速率的增加而增加。从线粒体DNA的氧化损伤以及对线粒体功能和衰老的影响方面，讨论了线粒体电子传递过程中H2O2和HO·形成的生物学意义。
