Separation and Function of the Fourth Component of Complement

Various methods of purification of C′4 have been reported; however, hemolytic activity of C′4 was occasionally lost during purification and it was difficult to separate C′4 from the C′1 inactivator. The following new method of purification of C′4 was then developed: By salting out serum with sodium sulfate, C′4 activity was separated from the C′1 inactivator; loss of C′4 activity was prevented by blocking the action of C′4 inactivating substances with di-isopropyl fluorophosphate. Further purification was achieved by chromatography on carboxymethyl (CM)- and diethylaminoethyl (DEAE)-cellulose columns, and by gel filtration on Sephadex G-200. Guinea pig, human and rat C′4 were separated by this new process and the effects of C′4 sites on the formation of C′3 sites and C′5 sites were investigated. Human C′3 was more reactive with EAC′1gp4hu2gp than with EAC′1gp4gp2gp as detected by immune adherence; guinea pig C′3 was equally reactive with the two kinds of cells.