Bhagwat A S, Blokesch A, Irrgang K D, Salnikow J, Vater J
Technische Universität Berlin, Institut für Biochemie und Molekulare Biologie, Germany.
Arch Biochem Biophys. 1993 Jul;304(1):38-44. doi: 10.1006/abbi.1993.1318.
Chemical modification of a purified spinach cytochrome (cyt) b6f complex was carried out using micromolar concentrations of o-phthalaldehyde (OPA). The o-phthalaldehyde-modified complex exhibited a strong and stable fluorescence characteristic of the thioisoindole derivative. o-Phthalaldehyde also caused crosslinking of the polypeptides and on polyacrylamide gel electrophoresis a new band with a molecular mass of 38-40 kDa exhibiting this characteristic fluorescence was observed as the major crosslinked component; in addition, minor protein bands in the mass range 50-60 kDa appeared. Immunostaining with specific antibodies identified the 38-kDa crosslink as a conjugate of cyt b6 with subunit IV and an additional conjugate of 54 kDa involving cyt f and subunit IV. Crosslinking of the Rieske protein with these components was not observed. In OPA-treated cyt b6f complex, the electron transfer from plastoquinol to cyt f is inhibited as well as proton translocation after incorporation of the complex into liposomes. The EPR spectral analysis of the complex modified at various o-phthalaldehyde concentrations showed that the characteristic EPR signal of the Rieske Fe-S center was lost.
使用微摩尔浓度的邻苯二甲醛（OPA）对纯化的菠菜细胞色素（cyt）b6f复合物进行化学修饰。邻苯二甲醛修饰的复合物表现出硫代异吲哚衍生物强烈且稳定的荧光特性。邻苯二甲醛还导致多肽交联，在聚丙烯酰胺凝胶电泳上观察到一条分子量为38 - 40 kDa的新条带，呈现这种特征荧光，是主要的交联成分；此外，还出现了分子量在50 - 60 kDa范围内的次要蛋白条带。用特异性抗体进行免疫染色鉴定出38 kDa的交联物是细胞色素b6与亚基IV的共轭物，以及一个涉及细胞色素f和亚基IV的54 kDa的额外共轭物。未观察到 Rieske 蛋白与这些成分的交联。在OPA处理的细胞色素b6f复合物中，将复合物掺入脂质体后，从质体醌到细胞色素f的电子传递以及质子转运均受到抑制。对在不同邻苯二甲醛浓度下修饰的复合物进行电子顺磁共振（EPR）光谱分析表明，Rieske铁硫中心的特征EPR信号消失。
