METHOD FOR SIMULTANEOUS QUANTITATIVE ESTIMATION OF CYTOCHROMES A,B,C1, + C IN MITOCHONDRIA
METHOD FOR SIMULTANEOUS QUANTITATIVE ESTIMATION OF CYTOCHROMES A,B,C1, + C IN MITOCHONDRIA
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DOI：
10.1016/0003-9861(64)90313-3
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发表时间：
1964-01-01
影响因子：
3.9
通讯作者：
WILLIAMS, JN
中科院分区：
文献类型：
--
作者：
WILLIAMS, JN
建立了一种同时定量测定单个线粒体样品中4种细胞色素a、B、c1和c的方法。分析仅需0.5 - 1.0 μ mol的每种细胞色素。在存在脱氧胆酸盐的情况下记录线粒体样品的差谱（还原与氧化），以澄清线粒体。已知每个细胞色素的吸光度对每个其他细胞色素的最大值和最小值的贡献，可以导出一组4个未知数的4个联立方程。提出了一种求解这些方程的简化方法。对由此获得的细胞色素浓度值的有效性进行了检查。这些方法包括回收加入的纯化细胞色素，从线粒体和线粒体血色原的差谱中淬灭纯化细胞色素和它们的血色原的差谱，用0.9%KCl耗尽提取细胞色素c，提取细胞色素B的血红素，并随后分析血红素作为其吡啶血色原，以及从线粒体的血色原光谱直接计算细胞色素A浓度。有效性研究中各线粒体细胞色素的回收率或百分比的平均值分别为105%（细胞色素a）、95%（细胞色素B）和101%。细胞色素c1为100%，细胞色素c为100%，总平均值为101%。
A method has been developed for quantitatively estimating the 4 cytochromes a, b, c1, and c simultaneously in a single mitochondrial sample. Only 0.5-1.0 m[mu]mole of each cytochrome is necessary for the analysis. The difference spectrum of the mitochondrial sample (reduced vs. oxidized) is recorded in the presence of desoxycholate to clarify the mitochondria. Knowing the contributions in absorbancy of each cytochrome to the major maxima and minima of each of the other cytochromes, a set of 4 simultaneous equations in 4 unknowns can be derived. A simplified procedure for the solution of these equations is presented. Checks on the validity of the values for the cytochrome concentrations thus obtained have been carried out. These procedures include recovery of added purified cytochromes, quenching of the difference spectra of purified cytochromes and their hemochromogens from the difference spectra of the mitochondria and of the mitochondrial hemochromogens, exhaustive extraction of cytochrome c by 0.9% KC1, extraction of the heme of cytochrome b and subsequent analysis of the heme as its pyridine hemochromogen, and direct calculation of cytochrome a concentration from the hemochromogen spectrum of the mitochondria. The average values for recovery or percentage of each mitochondrial cytochrome accounted for in the validity studies were 105% for cytochrome a, 95% for cytochrome b, 101%. for cytochrome c1, and 100% for cytochrome c, with an over-all average of 101%.