Isolation of Component C4 of Human Complement and Its Polypeptide Chains

Abstract

Component C4 of human complement was purified from fresh frozen plasma with a yield of 25% using an initial batch separation with quaternary diethyl-(2-hydroxypropyl)aminoethyl--Sephadex followed by column chromatography on DEAE-cellulose and gel filtration in Sephadex G-200. The final product was homogenous according to polyacrylamide gel electrophoresis and immunochemical methods. Low-speed sedimentation-equilibrium analyses revealed a molecular weight of 189,000, using a value of 0.736 ml/g for the partial specific volume. The polypeptide chains of reduced and alkylated C4 were separated on DEAE-Sepharose in the presence of 8 M urea. Gel filtration in Sepharose 4B in 6 M guanidine hydrochloride revealed molecular weights of 88,000, 72,000 and 32,000 for the alpha, beta and gamma chain respectively. The amino acid compositions of component C4 and its constitutive chains were also determined.