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Assessment of the Maximal Activity of Complex IV in the Inner Mitochondrial Membrane by Tandem Electrochemistry and Respirometry
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-12-15 , DOI: 10.1021/acs.analchem.0c02910 Nathan L Frantz 1 , Gabrielle Brakoniecki 1 , Dawei Chen 1 , Denis A Proshlyakov 1, 2
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-12-15 , DOI: 10.1021/acs.analchem.0c02910 Nathan L Frantz 1 , Gabrielle Brakoniecki 1 , Dawei Chen 1 , Denis A Proshlyakov 1, 2
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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.
中文翻译：
通过串联电化学和呼吸测定法评估线粒体内膜中复合物 IV 的最大活性
评估线粒体电子传递酶的活性对于理解代谢疾病的机制很重要，但线粒体的结构组织和低样本可用性给原位功能研究带来了独特的挑战。我们报告了串联微流体呼吸计的开发，该呼吸计可同时跟踪电极上介质的减少以及随后线粒体内膜中复合物 IV 导致的 O 2减少。对于 150 μm 厚的样品，O 2消耗对多个交替电势阶跃的响应时间约为 10 s。稳定的 O 2消耗与所提供的电荷显示出良好的定量相关性，Pearson's r = 0.994。用碳墨或气相二氧化硅修饰的生物相容性金电极上介体的还原可以与线粒体对介体的氧化竞争，产生与抗坏血酸化学还原相当的总体呼吸活性。 O 2消耗对介体和线粒体悬浮液浓度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。通过 NaN 3部分抑制复合物 IV 时内在线粒体活性的模拟变化的辐射测量来验证介导的电化学方法。这种方法使得能够开发不依赖于O 2 的仿生电化学测定，其狭隘地针对其原生环境中的电子传输链的组分。
更新日期：2021-01-26
中文翻译：
通过串联电化学和呼吸测定法评估线粒体内膜中复合物 IV 的最大活性
评估线粒体电子传递酶的活性对于理解代谢疾病的机制很重要，但线粒体的结构组织和低样本可用性给原位功能研究带来了独特的挑战。我们报告了串联微流体呼吸计的开发，该呼吸计可同时跟踪电极上介质的减少以及随后线粒体内膜中复合物 IV 导致的 O 2减少。对于 150 μm 厚的样品，O 2消耗对多个交替电势阶跃的响应时间约为 10 s。稳定的 O 2消耗与所提供的电荷显示出良好的定量相关性，Pearson's r = 0.994。用碳墨或气相二氧化硅修饰的生物相容性金电极上介体的还原可以与线粒体对介体的氧化竞争，产生与抗坏血酸化学还原相当的总体呼吸活性。 O 2消耗对介体和线粒体悬浮液浓度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。通过 NaN 3部分抑制复合物 IV 时内在线粒体活性的模拟变化的辐射测量来验证介导的电化学方法。这种方法使得能够开发不依赖于O 2 的仿生电化学测定，其狭隘地针对其原生环境中的电子传输链的组分。
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