Purification of the subunit C1q from the first component of equine complement

Initial separation and concentration of Clq from fresh, normal equine serum was accomplished by precipitation in 0.02 M acetate buffer, pH 5.5, at 4° for 24 hr. The re-dissolved precipitate was clarified by centrifugation at 80,000 g for 1 hr and then dialysed against Tris-HCl buffer (0.05 M, pH 8.0) containing 10^-3 M EDTA. The clarified dialysate remained biologically active at 5° for at least 4 wk. Biological activity of equine Clq was determined by assay of its ability to agglutinate sensitized sheep erythrocytes (EA). Following ammonium sulphate fractionation, Sepharose 4B gel filtration yielded three major peaks. Two protein bands were demonstrated on analysis of the second Sepharose peak by disc acrylamide electrophoresis, pH 8.3. Elution of the protein bands showed EA-agglutinating activity only in the band which migrated furthest toward the cathode. Equine Clq isolated by this method yielded an approximate forty-fold purification in specific activity. Some properties of equine Clq were characterized. Equine Clq was heat-labile, as shown by loss of its EA-agglutinating activity after heating at 58° for 15 min. Moreover, storage at 4° and freeze-thaw cycles greatly reduced EA agglutination. Preliminary determination of the sedimentation coefficient indicated that it was comparable to that reported for human and rabbit Clq.