Assessment of the Maximal Activity of Complex IV in the Inner Mitochondrial Membrane by Tandem Electrochemistry and Respirometry.
Assessment of the Maximal Activity of Complex IV in the Inner Mitochondrial Membrane by Tandem Electrochemistry and Respirometry.
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DOI：
10.1021/acs.analchem.0c02910
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发表时间：
2021-01-26
影响因子：
7.4
通讯作者：
Proshlyakov DA
中科院分区：
文献类型：
--
作者：
Frantz NL;Brakoniecki G;Chen D;Proshlyakov DA
线粒体电子传递酶活性的评估对于理解代谢性疾病的机制是重要的，但是线粒体的结构组织和低样品可用性对原位功能研究提出了独特的挑战。我们报告了一个串联的微流控呼吸计的发展，同时跟踪减少介质的电极和随后的减少O2复合物IV在线粒体内膜。对于150 μ m厚的样品，O2消耗对多个交变电位阶跃的响应时间约为10 s。稳定的O2消耗显示出与所提供的电荷的良好的定量相关性，皮尔逊相关系数r = 0.994。在用碳墨或气相二氧化硅修饰的生物相容性金电极上介质的还原可以与线粒体对介质的氧化竞争，产生与抗坏血酸盐化学还原后相当的总体呼吸活性。介体和线粒体悬浮液浓度上的O2消耗的依赖性表明，电极和线粒体之间的传质并不限制后者的生物活性。介导的电化学方法通过放射性测量模拟的线粒体固有活性变化来验证，该变化是由NaN3部分抑制复合物IV后发生的。这种方法能够开发出不依赖于O2的仿生电化学分析，其目标是在其天然环境中的电子传递链的组分。
Assessment of activities of mitochondrial electron transport enzymes is important for understanding mechanisms of metabolic diseases, but structural organization of mitochondria and low sample availability pose distinctive challenges for in situ functional studies. We report the development of a tandem microfluidic respirometer that simultaneously tracks both the reduction of mediators on the electrode and the ensuing reduction of O2 by complex IV in the inner mitochondrial membrane. The response time of O2 consumption to multiple alternating potential steps is of approximately 10 s for a 150 μm-thick sample. Steady O2 depletion shows good quantitative correlation with the supplied electric charge, Pearson’s r = 0.994. Reduction of mediators on biocompatible gold electrodes modified with carbon ink or fumed silica can compete with the oxidation of mediators by mitochondria, yielding an overall respiratory activity comparable to that upon chemical reduction by ascorbate. The dependence of O2 consumption on mediator and mitochondrial suspension concentrations shows that mass transport between the electrode and mitochondria does not limit biological activity of the latter. The mediated electrochemical approach is validated by the radiometric measurements of simulated changes in the intrinsic mitochondrial activity upon partial inhibition of complex IV by NaN3. This approach enables the development of O2-independent, biomimetic electrochemical assays narrowly targeting components of the electron transport chains in their native environments.
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影响因子：
4.7
作者：
Lin GG;Scott JG
通讯作者：
Scott JG
影响因子：
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作者：
Levitsky, Yan;Hammer, Sandra S.;Proshlyakov, Denis A.
通讯作者：
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作者：
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通讯作者：
Proshlyakov, Denis A.