Pierre Y, Breyton C, Kramer D, Popot J L
Institut de Biologie Physico-Chimique, CNRS URA 1187, Paris, France.
J Biol Chem. 1995 Dec 8;270(49):29342-9. doi: 10.1074/jbc.270.49.29342.
A protocol has been developed for the purification of the cytochrome b6 f complex from the unicellular alga Chlamydomonas reinhardtii. It is based on the use of the neutral detergent Hecameg (6-O-(N-heptylcarbamoyl)-methyl-alpha-D-glycopyranoside) and comprises only three steps: selective solubilization from thylakoid membranes, sucrose gradient sedimentation, and hydroxylapatite chromatography. The purified complex contains two b hemes (alpha bands, 564 nm; Em,8 = -84 and -158 mV) and one chlorophyll alpha (lambda max = 667-668 nm) per cytochrome f (alpha band, 554 nm; Em,8 = +330 mV). It is highly active in transferring electrons from decylplastoquinol to oxidized plastocyanin (turnover number 250-300 s-1). The purified complex contains seven subunits, whose identity has been established by N-terminal sequencing and/or peptide-specific immunolabeling, namely four high molecular weight subunits (cytochrome f, Rieske iron-sulfur protein, cytochrome b6, and subunit IV) and three approximately 4-kDa miniproteins (PetG, PetL, and PetX). Stoichiometry measurements are consistent with every subunit being present as two copies per b6 f dimer.
已开发出一种从单细胞绿藻莱茵衣藻中纯化细胞色素b6 f复合体的方案。该方案基于使用中性去污剂Hecameg（6 - O -（N - 庚基氨基甲酰基）- 甲基 - α - D - 吡喃葡萄糖苷），仅包括三个步骤：从类囊体膜中选择性溶解、蔗糖梯度沉降和羟基磷灰石层析。纯化后的复合体每个细胞色素f（α带，554 nm；Em,8 = +330 mV）含有两个b型血红素（α带，564 nm；Em,8 = -84和 - 158 mV）和一个叶绿素α（λmax = 667 - 668 nm）。它在将电子从癸基质体醌转移到氧化型质体蓝素方面具有高活性（周转数250 - 300 s-1）。纯化后的复合体包含七个亚基，通过N端测序和/或肽特异性免疫标记确定了它们的身份，即四个高分子量亚基（细胞色素f、 Rieske铁硫蛋白、细胞色素b6和亚基IV）和三个约4 kDa的小蛋白（PetG、PetL和PetX）。化学计量测量结果与每个亚基在每个b6 f二聚体中以两个拷贝存在一致。
