Apps D K, Boisclair M D, Gavine F S, Pettigrew G W
Biochim Biophys Acta. 1984 Jan 30;764(1):8-16. doi: 10.1016/0005-2728(84)90134-8.
Redox titrations of cytochrome b-561 have been performed with the purified cytochrome and with intact and detergent-solubilized chromaffin-granule membranes. The midpoint redox potential of the cytochrome is 100-130 mV; this depends upon the composition of the buffer, but is independent of pH in the range 5.5-7.5; partial proteolysis of the cytochrome raises the midpoint potential to 160 mV. The Nernst plots of titration data have slopes of 75-115 mV, and are in some cases sigmoid in shape. This may be explained by negative cooperativity during redox transitions in oligomeric cytochrome b-561. Measurements of the haem and cytochrome content of chromaffin granule membrane suggest a haem content of 1 mol/mol protein. Chemical crosslinking of cytochrome b-561 suggests that it may exist as an oligomer of 4-6 polypeptide chains within the chromaffin granule membrane. Aggregation of purified cytochrome b-561 was shown by gel filtration studies and by immunological methods in SDS-polyacrylamide gels. Studies of the molecular weight of the aggregates suggest that the monomer has a molecular weight close to 22 000, but migrates anomalously slowly during electrophoresis.
已使用纯化的细胞色素b - 561以及完整的和经去污剂增溶的嗜铬颗粒膜进行了细胞色素b - 561的氧化还原滴定。细胞色素的中点氧化还原电位为100 - 130 mV；这取决于缓冲液的组成，但在5.5 - 7.5的pH范围内与pH无关；细胞色素的部分蛋白酶解将中点电位提高到160 mV。滴定数据的能斯特图斜率为75 - 115 mV，在某些情况下呈S形。这可能是由于寡聚细胞色素b - 561氧化还原转变过程中的负协同作用所致。嗜铬颗粒膜血红素和细胞色素含量的测量表明血红素含量为1摩尔/摩尔蛋白质。细胞色素b - 561的化学交联表明它可能以4 - 6条多肽链的寡聚体形式存在于嗜铬颗粒膜内。凝胶过滤研究和SDS - 聚丙烯酰胺凝胶中的免疫学方法显示了纯化的细胞色素b - 561的聚集。对聚集体分子量的研究表明单体的分子量接近22000，但在电泳过程中迁移异常缓慢。
