el-Demerdash M, Salnikow J, Vater J
Technical University Berlin, Institute of Biochemistry and Molecular Biology, West Germany.
Arch Biochem Biophys. 1988 Jan;260(1):408-15. doi: 10.1016/0003-9861(88)90464-x.
The cytochrome b6f complex of spinach chloroplasts was prepared with minor modification according to the method of E. Hurt and G. Hauska (1981) Eur. J. Biochem. 117, 591-599) replacing, however, the final ultracentrifugation step by hydroxyapatite chromatography as suggested by M. F. Doyle and C.-A Yu (1985) Biochem. Biophys. Res. Commun. 131, 700-706). The purified complex was partially dissociated by treatment with 4 M urea or 0.1% sodium dodecyl sulfate (SDS) in the absence of reducing agents. A binary subcomplex consisting of cytochrome f and the Rieske iron-sulfur protein was observed under these conditions by three different methods: (a) hydroxyapatite chromatography; (b) extraction with an isopropanol/water/trifluoroacetic acid mixture; and (c) gel filtration in the presence of low SDS concentrations. The subcomplex dissociated into its components by treatment with mercaptoethanol. These results suggest a close interaction of the cytochrome f with the Rieske protein involving SH groups which under reducing conditions leads to complete dissociation of the subcomplex.
菠菜叶绿体细胞色素b6f复合物的制备方法根据E. Hurt和G. Hauska（1981年，《欧洲生物化学杂志》117卷，591 - 599页）的方法稍作修改，不过，按照M. F. Doyle和C.-A Yu（1985年，《生物化学与生物物理研究通讯》131卷，700 - 706页）的建议，用羟基磷灰石色谱法取代了最后的超速离心步骤。纯化后的复合物在无还原剂的情况下用4 M尿素或0.1%十二烷基硫酸钠（SDS）处理会部分解离。在这些条件下，通过三种不同方法观察到了由细胞色素f和 Rieske铁硫蛋白组成的二元亚复合物：（a）羟基磷灰石色谱法；（b）用异丙醇/水/三氟乙酸混合物萃取；（c）在低SDS浓度下进行凝胶过滤。该亚复合物经巯基乙醇处理后会解离成其组成成分。这些结果表明细胞色素f与 Rieske蛋白之间存在紧密的相互作用，涉及SH基团。在还原条件下，这种相互作用会导致亚复合物完全解离。
