Chien L F, Brand M D
Department of Biochemistry, University of Cambridge, U.K.
Biochem J. 1996 Dec 15;320 ( Pt 3)(Pt 3):837-45. doi: 10.1042/bj3200837.
The effect of chloroform on mitochondrial respiration with succinate was investigated by applying the method of Brand, Chien and Diolez [(1994) Biochem. J. 297, 27-29] to examine whether chloroform causes redox slip (fewer protons pumped per electron transferred) during mitochondrial electron transport. N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD), which lowers H+/O (the number of protons pumped to the external medium by the electron transport complexes per oxygen atom consumed) by altering the electron flow pathway, was investigated for comparison. Non-phosphorylating mitochondria that had been treated with 350 microM TMPD or 30 mM chloroform were titrated with malonate in the presence of submaximal concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Linear relations between CCCP-induced extra respiration and protonmotive force were obtained. These results showed that there was no measurable protonmotive force-dependent or rate-dependent slip in mitochondria treated with either TMPD or chloroform. However, both TMPD and chloroform seemed to decrease H+/O in a manner independent of protonmotive force and rate. The relationship between non-phosphorylating respiration and protonmotive force was simulated in mitochondria of which 25% of the total population were assumed to have been broken. The simulation showed that the apparent decrease in H+/O on the addition of TMPD or chloroform to mitochondria could be in principle accounted for by breakage. Assays of mitochondrial breakage (ATP hydrolysis in the presence of atractyloside and oxidation of exogenous NADH) showed that chloroform broke mitochondria but TMPD did not. We conclude that chloroform changes the measured H+/O as an artifact by causing mitochondrial breakage and does not cause measurable redox slip, whereas TMPD genuinely lowers H+/O.
通过应用布兰德、钱和迪奥莱兹的方法[（1994年）《生物化学杂志》297卷，27 - 29页]研究了氯仿对琥珀酸介导的线粒体呼吸的影响，以检验氯仿在线粒体电子传递过程中是否会导致氧化还原滑移（每转移一个电子泵出的质子减少）。作为对比，研究了通过改变电子流动途径来降低H⁺/O（电子传递复合物每消耗一个氧原子泵入外部介质的质子数）的N，N，N'，N'-四甲基对苯二胺（TMPD）。在亚最大浓度的解偶联剂羰基氰化物间氯苯腙（CCCP）存在的情况下，用丙二酸对经350微摩尔TMPD或30毫摩尔氯仿处理的非磷酸化线粒体进行滴定。获得了CCCP诱导的额外呼吸与质子动力之间的线性关系。这些结果表明，在用TMPD或氯仿处理的线粒体中，没有可测量的依赖质子动力或速率的滑移。然而，TMPD和氯仿似乎都以独立于质子动力和速率的方式降低H⁺/O。在假定25%的线粒体总数已被破坏的线粒体中模拟了非磷酸化呼吸与质子动力之间的关系。模拟结果表明，向线粒体中添加TMPD或氯仿时H⁺/O的明显降低原则上可能是由线粒体破裂导致的。线粒体破裂的测定（在存在苍术苷的情况下ATP水解和外源NADH的氧化）表明氯仿会使线粒体破裂，而TMPD不会。我们得出结论，氯仿通过导致线粒体破裂改变了测量到的H⁺/O，这是一种假象，且不会导致可测量的氧化还原滑移，而TMPD确实会降低H⁺/O。
