纯化膜蛋白质复合体Cyt b_6f的新方法

A NEW METHOD FOR PURIFING Cyt b 6f PROTEIN COMPLEX 

建立了纯化光合膜蛋白质复合体Ｃｙｔｂ６ｆ的新方法。与现有方法相比，新方法简单明了，只包括透析离心和用硫酸铵分级沉淀两个步骤，而且适于大批量制备。按此方法从菠菜叶绿体分离纯化的制剂含９．８ｎｍｏｌＣｙｔｆ·ｍｇ－１；ＳＤＳＰＡＧＥ显示４条主要区带及１条低分子量区带，背景干净；Ｃｙｔｂ６（ｂ型血红素）∶Ｃｙｔｆ＝２∶１（ｍｏｌ∶ｍｏｌ），Ｃｈｌａ∶Ｃｙｔｆ＝１∶１（ｍｏｌ∶ｍｏｌ）；酶活性（ＰＱ２Ｈ２→Ｃｙｔｃ）８０μｍｏｌＣｙｔｃ·ｎｍｏｌＣｙｔｆ－１·ｈ－１左右；产率可达３８％。

A new protocol which is much simpler than current procedures, has been developed for purification of the Cyt b 6f protein complex. The protocol contained only two steps——dialysis centrifugation and stepwise precipitation with ammonium sulfate. Moreover, this method is suitable for larger scale preparation. The purified complex from spinach (Spinacia oleracea L.) contained 9.8 nmol Cyt f per milligram protein. 2 Cyt b 6(b hemes) and 1 Chl a per Cyt f. SDS PAGE showed four main bands and one weak band with low molecular weight. Its activity(PQ 2H 2→Cyt c)was around 80 μmol Cyt c·nmol Cyt f -1 ·h -1 .