Purification and characterization of a macromolecular weight cofactor for C3b-inactivator,C4bC3bINA-cofactor,of human plasma

The macromolecular weight cofactor for C3b-inactivator, C4bC3bINA-cofactor, was purified by an improved method and its protein nature was investigated. The purification procedures were composed of the removal of cold insoluble globulin with gelatin-Sepharose, polyethylene glycol fractionation, gel filtration on Bio-Gel A-15m, heparin-Sepharose chromatography, and the removal of IgM with anti-IgM-Sepharose. The molecular weight of C4bC3bINA-cofactor was estimated to be 450,000 by SDS-polyacrylamide gel electrophoresis.¹ The reduced cofactor gave a single band of 75,000 daltons upon SDS-polyacrylamide gel electrophoresis, suggesting that the cofactor is a disulfide-linked polymer (probably a hexamer) of similar or identical polypeptide chains, each with a molecular weight of 75,000. The cofactor is a glycoprotein and the isoelectric point of the cofactor was estimated to be 6.7. No N-terminal amino acid was detected by the dansylation technique, suggesting that the N-terminal of the cofactor would be blocked. Upon immunoelectrophoresis, the cofactor migrates as a slow β-globulin, and its mobility was shifted towards the anodal side when C4b was added to the cofactor. This result suggested that the cofactor was functionally the same as the C4-binding protein.