Kim C H, King T E
J Biol Chem. 1983 Nov 25;258(22):13543-51.
A new mitochondrial protein was isolated to pure form. This protein was indispensable for the formation of the cytochrome c1-c complex; hence, it was provisionally named the hinge protein for formation of the cytochrome c1-c complex, or for simplicity, merely called the hinge protein. The simplest method for the preparation of the pure protein involved essentially pH 5.5 treatment of high purity of "two-band" cytochrome c1 prepared from an improved method. The use of two band cytochrome c1 prepared by an improved method was preferred because the improved method apparently yielded less tight bonding between the heme-containing and colorless protein entities than that from the original methods (King, T. E. (1978) Methods Enzymol. 53, 181-191). The c1-c complex comprised 1 molar equivalent each of the hinge protein, "one-band" cytochrome c1 and cytochrome c. It was demonstrated by gel filtration chromatography that in the absence of the hinge protein, there was no complex formation between cytochromes c and one-band c1. In titration of the complex formed between one-band cytochrome c1 and cytochrome c with the hinge protein present by using the increase of the Soret-Cotton effect as a criterion (Chiang, Y. L., Kaminsky, L. S., and King, T. E. (1976) J. Biol. Chem. 251, 29-36), a sharp break was observed which showed the three species to be present in equivalent amounts. The hinge protein showed low extinction in the 280 nm region and exhibited poor color value and diffuse character of the band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after staining with Coomassie brilliant blue. The molecular weight was found to be (i) 9,800 from sedimentation equilibrium, (ii) 11,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iii) 23,000 with a Stokes radius of 22.4 A from gel filtration chromatography estimated from a standard curve with proteins of known molecular parameters. The disparities in these data from the actual value of 9,175 from calculations based on amino acid sequence, as previously reported (Wakabayashi, S., Takeda, H., Matsubara, H., Kim, C. H., and King, T. E. (1982) J. Biochem. (Tokyo) 91, 2077-2085), have been discussed.
一种新的线粒体蛋白被分离纯化。这种蛋白对于细胞色素c1-c复合物的形成是不可或缺的；因此，它被暂时命名为细胞色素c1-c复合物形成的铰链蛋白，或者简单地称为铰链蛋白。制备纯蛋白的最简单方法主要涉及用pH 5.5处理由改进方法制备的高纯度“双带”细胞色素c1。使用改进方法制备的双带细胞色素c1是优选的，因为改进方法产生的含血红素和无色蛋白实体之间的结合似乎比原始方法产生的结合更松散（金，T.E.（1978年）《酶学方法》53卷，181 - 191页）。c1-c复合物由1摩尔当量的铰链蛋白、“单带”细胞色素c1和细胞色素c组成。凝胶过滤色谱法表明，在没有铰链蛋白的情况下，细胞色素c和单带c1之间不会形成复合物。在用铰链蛋白滴定单带细胞色素c1和细胞色素c之间形成的复合物时，以Soret - Cotton效应的增加为标准（蒋，Y.L.，卡明斯基，L.S.，和金，T.E.（1976年）《生物化学杂志》251卷，29 - 36页），观察到一个明显的转折点，表明这三种物质以等量存在。铰链蛋白在280nm区域的消光值较低，在用考马斯亮蓝染色后，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中显示出较差的色值和条带的弥散特征。分子量通过以下方法测定：（i）沉降平衡法测得为9800；（ii）十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳法测得为11000；（iii）凝胶过滤色谱法根据已知分子参数的蛋白质标准曲线估计，分子量为23000，斯托克斯半径为22.4 Å。如先前报道（若林，S.，武田，H.，松原，H.，金，C.H.，和金，T.E.（1982年）《生物化学杂志》（东京）91卷，2077 - 2085页），基于氨基酸序列计算得到的实际值为9175，对这些数据与实际值之间的差异进行了讨论。
