Groth D M, Wetherall J D, Umotong B A, Sparrow P, Lee I R, Carrick M J
Complement. 1987;4(1):1-11. doi: 10.1159/000463002.
A relatively rapid method for the isolation of complement protein C4 from bovine plasma is described. The method consists of DEAE Sephacel anion exchange chromatography of plasminogen-depleted bovine plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration on a TSK G3000 SW column. A yield of approximately 20% was obtained. Conventional SDS-PAGE of purified bovine C4 showed the presence of alpha, beta and gamma polypeptide chains, the molecular weights of which were determined from Ferguson plots to be 95,000 +/- 2,500, 80,500 +/- 2,000 and 30,000 +/- 500 daltons, respectively. SDS-PAGE of C4 immunoprecipitated from the plasma of individual cattle in gels with a reduced proportion of crosslinker showed size polymorphism of the alpha chain. The presence of dual alpha chains was confirmed by radiolabelling their reactive thiol ester moiety with 14C methylamine. The difference in size of the two bovine alpha chains is approximately 1,800 daltons. On activation of bovine C4 both alpha chains were cleaved into alpha' chains (87,000 and 85,000 daltons) characteristic of C4b.
本文描述了一种从牛血浆中分离补体蛋白C4的相对快速的方法。该方法包括对去除纤溶酶原的牛血浆进行DEAE Sephacel阴离子交换色谱，随后在CM Sepharose上进行阳离子交换色谱，最后在TSK G3000 SW柱上进行凝胶过滤。获得了约20%的产率。纯化的牛C4的常规SDS-PAGE显示存在α、β和γ多肽链，通过弗格森图确定其分子量分别为95,000±2,500、80,500±2,000和30,000±500道尔顿。在交联剂比例降低的凝胶中，对从个体牛血浆中免疫沉淀的C4进行SDS-PAGE，显示α链存在大小多态性。用14C甲胺对其反应性硫酯部分进行放射性标记，证实了双α链的存在。两条牛α链的大小差异约为1,800道尔顿。牛C4激活后，两条α链均裂解为C4b特有的α'链（87,000和85,000道尔顿）。
