T'sai A L, Palmer G
Biochim Biophys Acta. 1982 Sep 15;681(3):484-95. doi: 10.1016/0005-2728(82)90191-8.
A simple, high-yield purification procedure for cytochrome b from yeast Complex III has been developed. This procedure involves solubilization using chemical modification of the lysine residues with 3,4,5,6-tetrahydrophthalic anhydride followed by hydroxyapatite column chromatography. This purified cytochrome b has a heme content of 37.0 nmol cytochrome b/mg and a molecular weight on SDS gels of 25000-26000. Amino acid analysis indicates high hydrophobicity and is very comparable to the composition deduced from the gene sequence (Nobrega, F.G. and Tzagoloff, A. (1980) J. Biol. Chem. 255, 9828-9837). The latter data indicate a molecular weight of 42000 for the polypeptide; our heme analyses thus imply the presence of two hemes per polypeptide chain. Optical and MCD spectra are typical of a low-spin b-type cytochrome. MCD-potentiometric titration indicates a one-electron carrier with a single midpoint potential of -44 mV at pH 7.4 and 25 degrees C. The EPR spectrum of isolated cytochrome b has only one gz signal at 3.70, indicating that the 'strained' heme structure (Carter, K., T'sai, A. and Palmer, G. (1981) FEBS Lett. 132, 243--246) is still maintained. No indication of antimycin binding was demonstrated either by the direct-fluorescence method or binding-precipitation method although stoichiometric binding to the parent Complex III was readily demonstrated.
已开发出一种从酵母复合物III中纯化细胞色素b的简单、高产率方法。该方法包括用3,4,5,6 - 四氢邻苯二甲酸酐对赖氨酸残基进行化学修饰以实现溶解，随后进行羟基磷灰石柱色谱分离。这种纯化的细胞色素b的血红素含量为37.0 nmol细胞色素b/mg，在SDS凝胶上的分子量为25000 - 26000。氨基酸分析表明其具有高疏水性，并且与从基因序列推导的组成非常相似（诺布雷加，F.G.和察戈洛夫，A.（1980年）《生物化学杂志》255卷，9828 - 9837页）。后者的数据表明该多肽的分子量为42000；因此我们的血红素分析表明每条多肽链存在两个血红素。光学和MCD光谱是低自旋b型细胞色素的典型光谱。MCD电位滴定表明在pH 7.4和25℃下，它是一个单电子载体，具有-44 mV的单一中点电位。分离出的细胞色素b的EPR光谱在3.70处只有一个gz信号，表明“应变”血红素结构（卡特，K.、蔡，A.和帕尔默，G.（1981年）《欧洲生物化学学会联合会快报》132卷，243 - 246页）仍然得以维持。尽管已很容易证明与亲本复合物III存在化学计量结合，但无论是直接荧光法还是结合沉淀法都未显示抗霉素结合的迹象。
