H2O2 detection from intact mitochondria as a measure for one-electron reduction of dioxygen requires a non-invasive assay system
H2O2 detection from intact mitochondria as a measure for one-electron reduction of dioxygen requires a non-invasive assay system
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DOI：
10.1016/s0005-2728(99)00083-3
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发表时间：
1999-10-06
影响因子：
4.3
通讯作者：
Nohl, H
中科院分区：
文献类型：
--
作者：
Staniek, K;Nohl, H
评估超氧自由基 (O-2(.-)) 的存在、产生位点以及将 1-e(-) 从生物氧化还原系统转移到氧气所需的条件，是了解 O-2 稳态失调导致氧化应激的先决条件。线粒体越来越被认为是多种疾病和衰老过程中主要的 O-2(.-) 来源。关于线粒体 O-2(.-) 释放的相互矛盾的报告促使我们批判性地研究常用的 O-2(.-) 检测方法的适用性。由于线粒体外膜对 O-2(.-) 检测系统的大多数成分不具有渗透性，我们决定采用稳定的歧化产物 H2O2。早期表明这种代谢物很容易渗透到细胞质中。除四甲基联苯胺外，由于溶解度问题或消光系数低，没有任何化学反应物能够通过辣根过氧化物酶催化的吸光度变化表明 H2O2 的存在。四甲基联苯胺依赖性 H2O2 检测通过呼吸链的 e(-) 载体重新还原染料来抵消。虽然荧光染料东莨菪素和高香草酸被发现适合检测线粒体 H2O2 释放，但荧光变化受到线粒体蛋白质成分的强烈影响。本研究通过将检测系统与产生 H2O2 的线粒体分离来解决这个问题。 (C) 1999 Elsevier Science B.V. 保留所有权利。
Evaluation of the existence of superoxide radicals (O-2(.-)), the site of generation and conditions required for one-e(-) transfer to oxygen from biological redox systems is a prerequisite for the understanding of the deregulation of O-2 homeostasis leading to oxidative stress. Mitochondria are increasingly considered the major O-2(.-) source in a great variety of diseases and the aging process. Contradictory reports on mitochondrial O-2(.-) release prompted us to critically investigate frequently used O-2(.-) detection methods for their suitability. Due to the impermeability of the external mitochondrial membrane for most constituents of O-2(.-) detection systems we decided to follow the stable dismutation product H2O2. This metabolite was earlier shown to readily permeate into the cytosol. With the exception of tetramethylbenzidine none of the chemical reactants indicating the presence of H2O2 by horseradish peroxidase-catalyzed absorbance change were suited due to solubility problems or low extinction coefficients. Tetramethylbenzidine-dependent H2O2 detection was counteracted by rereduction of the dye through e(-) carriers of the respiratory chain. Although the fluorescent dyes scopoletin and homovanillic acid were found to be suited for the detection of mitochondrial H2O2 release, fluorescence change was strongly affected by mitochondrial protein constituents. The present study has resolved this problem by separating the detection system from H2O2-producing mitochondria. (C) 1999 Elsevier Science B.V. All rights reserved.