5TH COMPONENT OF HUMAN-COMPLEMENT - PURIFICATION FROM PLASMA AND POLYPEPTIDE-CHAIN STRUCTURE

Human C5 has been isolated from fresh plasma utilizing an approach which also provides for the copurification of human C3 [Tack, B. F., & Prahl, J. W. (1976) Biochemistry 15, 4513-4521], Initial fractionation of plasma with poly(ethylene glycol) followed by depletion of plasminogen on Sepharose-L-lysine, chromatography on diethylaminoethylcellulose, gel filtration on Sepharose 6B, chromatography on hydroxylapatite, and depletion of IgG subclasses 1, 2, and 4 on protein A-Sepharose proved satisfactory for obtaining C5 in high yield and biochemical purity. Final recoveries were 32% of the initial C5 protein and 29% of the initial C5 hemolytic activity as quantitated by specific immunoprecipitant and hemolytic titration assays. Apparent homogeneity was indicated by monodisperse behavior on immunoelectrophoresis and polyacrylamide gel electrophoresis. Total reduction and alkylation of disulfide bonds revealed a two-polypeptide chain structure: an α chain and a β chain with respective molecular weights of 115 000±12 000 and 75 000±8000. A preparative separation of the chains was obtained by gel filtration on a Sepharose CL-4B column equilibrated with 0.2% sodium dodecyl sulfate in 0.1 M sodium bicarbonate (pH 7.9). Automated Edman degradation of C5 gave a single N-terminal sequence of Thr-Leu-Gln-Lys-Lys-Ile-Glx-Glx-Ile-Ala. An identical N-terminal sequence was observed with the isolated α chain. Several attempts to sequence the N-terminal region of the β chain have been unsuccessful. Digestion of each chain with carboxypeptidases A and Y has indicated a common carboxyl-terminal sequence of (Ala, Val)-Ala-Gly-Ser. The identity in N-terminal structure between the C5 α chain and the C5a anaphylatoxin (Fernandez & Hugli, 1976) provides direct evidence that this potent anaphylactic and chemotactic polypeptide is released from the amino-terminal region of this chain. © 1979, American Chemical Society. All rights reserved.