线粒体电子传递链电子漏的化学发光测定  

Measuring mitochondrial reactive oxygen species by a chemiluminescence method

作　　者：周智波[1] 钟丽君[1] 程时[1] 

机构地区：[1]北京大学基础医学院生物物理学系,北京100083

出　　处：《动物学报》2004年第1期120-125,共6页ACTA ZOOLOGICA SINICA

基　　金：生物膜与膜生物工程国家重点实验室开放课题基金支持~~

摘　　要：本实验用差速离心法分离正常大鼠肝脏和心肌线粒体 ,以lucigenin (探测超氧阴离子 )与luminol (探测过氧化氢 )为探剂 ,用化学发光法测定METC电子漏的生成。在反应体系中加入外源底物 ,其发光强度明显高于空白对照 (体系中无线粒体 )。在肝线粒体体系中 ,无论是lucigenin还是luminol诱发的发光 ,琥珀酸底物引起的发光强均要高于丙酮酸 /苹果酸引起的发光强度。在心肌线粒体 luminol体系中也有与肝线粒体相似的结果 ,在心肌线粒体 lucigenin体系中 ,加入外源底物丙酮酸We employed differential centrifugation to isolate the liver mitochond ria and the heart mitochondria.The mitochondrial protein was measured with Bradford using bovine serum albumin as the standard. For detecting and measuring the mitochondria-derived reactive o xygen species, both lucigenin and luminol were used as chemilumigenic probes. In liver mitochondria, by adding exogenous substrates, succinate or pyruvate/malat e, after incubation with lucigenin (10 μmol/L) at 37℃, lucigenin-derived che mi luminescence (CL) was produced and its intensity was markedly above the backgrou nd of the luminometer. The CL response with the succinate-driven liver mitochon d ria was higher than that of the pyruvate/malate-supported liver mitochondria, a n d the lucigenin-derived CL elicited with succinate-driven mitochondria was pos it ively correlated with the concentration of lucigenin added. To measure the H 2O 2 production, luminol was used. In the presence of horseradish peroxidase, luminol -derived CL was elicited with substrate-supplemented mitochondria. The intensi ty of luminol-derived CL was related with the concentration of H 2O 2, which is the product of superoxide detoxification by Mn SOD, and the luminol-derived CL resp o nse was similar to that of elicited by lucigenin, namely, the CL with succinate -supported mitochondria was higher than that of pyruvate/malate-supplemented m it o chondria. In heart mitochondria, the luminol-derived CL elicited with succinate -or pyruvate/malate-supported mitochondria was higher than instrument backgro un d . Luminol-derived CL’s intensity of succinate-supported mitochondria w as higher than that of pyruvate/malate-supported mitochondria. But in measuring heart mi t ochondrial O 2 ·- , the CL response with the succinate-supported mitocho ndria was lower than that of pyruvate/malate-supported mitochondria, which was different from the CL response with liver mitochondria

关 键 词：化学发光 线粒体电子传递链 电子漏 活性氧 大鼠肝脏 心肌线粒体 

分 类 号：Q62[生物学—生物物理学]