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.

The distributions of plasma concentrations of complement proteins C3 and C4 were studied in sample populations of merino and Suffolk sheep. No differences between the breeds or the sexes were observed. The distribution for ovine C4 was polymodal and very disperse relative to that for C3. It was found, however, that C3 concentrations were elevated in specimens from 20 merino sheep bred as high responders to a Trichostrongylus vaccine. Significantly decreased plasma C4 concentrations were observed in representatives of both merino and merino X Border Leicester cross-bred sheep affected with congenital progressive ovine muscular dystrophy. Agarose gel electrophoretic variants of ovine C3 were not detected. Evidence for electrophoretic variants of ovine C4 in agarose gels was found although individual allotypes could not be reliably identified. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) did not reveal size heterogeneity for the α and β chains of immunoprecipitated ovine C3. Analysis of reduced immunoprecipitated ovine C4 by SDS-PAGE revealed considerable size heterogeneity in the α chain consistent with isotypic and/or allotypic variability. The data presented strongly suggest the presence of two C4 loci in sheep, each of which exhibits polymorphism.