Groth D M, Wetherall J D, Taylor L, Sparrow P R, Lee I R
School of Medical Technology, Curtin University of Technology, Bentley, Western Australia.
Mol Immunol. 1988 Jun;25(6):577-84. doi: 10.1016/0161-5890(88)90080-6.
A relatively rapid procedure is described for the isolation of the fourth component of complement (C4) from ovine plasma. The method, which recovers approximately 30% C4, is based upon DEAE Sephacel anion exchange chromatography of PEG precipitated plasminogen depleted plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration. SDS-PAGE of purified ovine C4 under reducing conditions revealed a complex pattern of bands which was interpreted on the basis of a three polypeptide chain structure for each of two distinct species, or isotypes, of C4 molecule herein termed C4A and C4B. Each isotype differs in the mol. wt of the alpha chain--108 and 95 K respectively. Nucleophilic substitution of immunoprecipitated ovine C4 with radiolabelled methylamine revealed that both C4 species contained a reactive thiol ester site and that each could be cleaved into an activated form (presumably C4b) characterised by a truncated alpha' chain some 8 K lower in mol. wt. A comparison of the isotype composition of purified C4 with that of immunoprecipitated C4 from the same animal indicated that the purification procedure favoured isolation of the C4B isotype. The mol. wts of both the alpha and beta chains were lowered following digestion of ovine C4 with neuraminidase.
本文描述了一种从绵羊血浆中分离补体第四成分（C4）的相对快速的方法。该方法回收率约为30%的C4，其基于聚乙二醇沉淀去除纤溶酶原的血浆在DEAE Sephacel阴离子交换柱上进行层析，随后在CM Sepharose上进行阳离子交换层析，最后进行凝胶过滤。还原条件下纯化的绵羊C4的SDS-PAGE显示出复杂的条带模式，基于本文中称为C4A和C4B的两种不同类型或同种型的C4分子中每一种的三链多肽结构进行解释。每种同种型的α链分子量不同，分别为108k和95k。用放射性标记的甲胺对免疫沉淀的绵羊C4进行亲核取代，结果显示两种C4类型均含有一个反应性硫酯位点，且每种均可裂解为一种活化形式（推测为C4b），其特征为α'链截短，分子量低约8k。将纯化的C4的同种型组成与同一动物免疫沉淀的C4的同种型组成进行比较，结果表明纯化过程有利于C4B同种型的分离。用神经氨酸酶消化绵羊C4后，α链和β链的分子量均降低。
