Huang D, Everly R M, Cheng R H, Heymann J B, Schägger H, Sled V, Ohnishi T, Baker T S, Cramer W A
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Biochemistry. 1994 Apr 12;33(14):4401-9. doi: 10.1021/bi00180a038.
Size analysis of the cytochrome b6f complex by FPLC Superose-12 chromatography and Blue Native PAGE indicated a predominantly dimeric component with M(r) = (1.9-2.5) x 10(5). The true dimer molecular weight including bound lipid, but not detergent, was estimated to be 2.3 x 10(5). Size and shape analysis by negative-stain single-particle electron microscopy indicated that the preparation of dimeric complexes contains a major population that has a protein cross section 40% larger than the monomer, binds more negative stain, and has a geometry with a distinct 2-fold axis of symmetry compared to the monomeric complex. The dimeric species is more stable at higher ionic strength with respect to conversion to the monomeric species. SDS-PAGE of monomer and dimer preparations indicated that both contain the four major polypeptides in approximately equal stoichiometry and also contain the petG M(r) 4000 subunit. One bound chlorophyll a per monomer, part of the bound lipid, is present in monomer and dimer. The in vitro electron-transport activity (decyl-PQH2-->PC-ferricyanide) of the separated dimer was comparable to that of the isolated b6f complex and was 4-5-fold greater than that of the monomer preparation, whose activity could be attributed to residual dimer. No difference in the properties of the dimer and monomer was detected by SDS-PAGE or redox difference spectrophotometry that could account for the difference in activities. However, the concentration of the Rieske [2Fe-2S] center was found by EPR analysis of the gy = 1.90 signal to be lower in the monomer fraction by a factor of 3.5 relative to the dimer.(ABSTRACT TRUNCATED AT 250 WORDS)
通过快速蛋白质液相色谱法（FPLC）Superose - 12色谱和蓝色天然聚丙烯酰胺凝胶电泳（Blue Native PAGE）对细胞色素b6f复合物进行大小分析，结果表明其主要成分是分子量为（1.9 - 2.5）× 10⁵的二聚体。包括结合脂质但不包括去污剂的真实二聚体分子量估计为2.3 × 10⁵。通过负染单颗粒电子显微镜进行的大小和形状分析表明，二聚体复合物制剂中主要群体的蛋白质横截面积比单体大40%，结合更多负染，并且与单体复合物相比具有明显的二重对称轴几何结构。相对于转化为单体形式，二聚体在较高离子强度下更稳定。单体和二聚体制剂的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）表明，两者都含有四种主要多肽，其化学计量比大致相等，并且还含有分子量为4000的petG亚基。单体和二聚体中均存在每个单体一个结合的叶绿素a，它是结合脂质的一部分。分离出的二聚体的体外电子传递活性（癸基 - PQH₂→质体蓝素 - 铁氰化物）与分离出的b6f复合物相当，比单体制剂高4 - 5倍，单体制剂的活性可归因于残留的二聚体。通过SDS - PAGE或氧化还原差示分光光度法未检测到二聚体和单体性质上的差异可以解释活性差异。然而，通过对gy = 1.90信号的电子顺磁共振（EPR）分析发现，单体组分中 Rieske [2Fe - 2S]中心的浓度相对于二聚体低3.5倍。（摘要截短于250字）
