Purification and Characterisation of the Fourth Component of Bovine Complement

Abstract

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 α, β and γ 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 α chain. The presence of dual α chains was confirmed by radiolabelling their reactive thiol ester moiety with 14C methylamine. The difference in size of the two bovine α chains is ~ 1,800 daltons. On activation of bovine C4 both α chains were cleaved into α' chains (87,000 and 85,000 daltons) characteristic of C4b.

Analysis of C3 and C4 in Ovine Plasma

“…The y chain of C4 is not usually visible in SDS-PAGE gels run under the con ditions described, since it migrates close to the solvent front. The presence of a y chain in reduced immunoprecipitated sheep C4 has been demonstrated previously by con ventional SDS-PAGE [8].…”

“…Firstly, the plasma of most sheep tested contained C4 which gave two main a chains, designated aA and aB with Mrs of approximately 108 and 95 kd respec tively. In a previous study using purified ovine C4, the presence of two a chains (Mr 108 and 95 kd), each capable of incorporat ing radiolabelled methylamine, was demon strated [8], It seems likely therefore that these a chains are derived from two sub classes of ovine C4. a chains representative of two subclasses of C4 have also been de tected in cattle [15] although the difference in molecular weight of the two cattle a chains was only ~ 1.8 kd.…”

“…C3 concentrations of test sera are reported as a percentage of this control sera. The control serum had been standardized against a highly purified C4 preparation isolated previously [8] and contained 320 mg/1 of C4 protein.…”

“…Rabbit anti-sheep C4 antiserum was prepared from highly purified sheep C4 as described previously [8], The specificity of this antiserum for sheep C4 was established by both immunoelectrophoresis and SDS-PAGE analysis of the protein precipitated by it from sheep plasma. Rabbit anti-sheep C3 antiserum was purchased from Cappell Worthington (Malvern, Pa., USA).…”

Distribution of complement protein C4 concentrations in bovine plasma

“…Aliquots (5 pl) of test specimen or control serum were added to wells punched in the gel and the diameters of the zones of precipitation determined after diffusion was complete. The bovine control serum contained 430 mg/l of C4 protein and had been standardized with a purified C4 preparation isolated previously (Groth et al, 1985). The monospecific rabbit antiserum was prepared using the same C4 preparation as the immunogen.…”