Purification of the third and fifth components of human complement: Application of hydrophobic chromatography

Abstract

By application of both hydrophobic and salt-mediated hydrophobic chromatography the third (C3) and fifth (C5) components of human serum complement were purified. Serum was initially adsorbed onto the hydrophobic resin (omega-amino hexyl agarose). This first step yields greater than 95% of the starting C3 and C5. Subsequent fractionation of the C3/C5 pool on hydroxylapatite was used to separate C5 and C3. Salt-mediated hydrophobic chromatography, and either gel filtration (C3) or calcium phosphate (C5) yielded homogeneous samples of C3 and C5 as assessed by SDS polyacrylamide gel electrophoresis. Quantitative radial immunodiffusion and hemolytic assays were used to measure recoveries at each step in the purification scheme. The final recovery yields of C3 were 34% of the initial C3 protein while that for C5 was 46%. Polyacrylamide gel electrophoresis of either reduced C5 or reduced C3 resulted in the identification of two polypeptide chains with molecular weights of 115,000 and 75,000 daltons. Non-reduced C3 and C5 showed migration patterns indicative of different molecular weights (possibly due to the carbohydrate content of C5). The molecular weight of both C3 and C5 was shown to be approximately 200,000 daltons by gel filtration. Trypsinization of pure C5 was shown to produce extremely potent biologically active material as assessed by the ability to cause enzyme release from neutrophils. This procedure allows for the rapid isolation of pure and hemolytically active C3 and C5 from human serum.