Isolation of Late Complement Components by Affinity Chromatography: I. Purification of the Human Complement Component C9 and Production of a C9-Defective Human Serum

Abstract

A new procedure for the isolation of the human complement component C9 is described. This procedure offers the possibility to prepare functionally pure C9 in a one-step procedure with a high recovery of 10–22% of the biological activity. The ¹²⁵iodinated C9 had a molecular weight of 78,000 daltons and was only contaminated in traces with other proteins. Further purification by absorption with an «anti-impurity» column lead to a C9 preparation which behaved as a homogenous single polypeptide chain in SDS polyacrylamide gel electrophoresis after reduction with mercaptoethanol. It formed a single bell-shaped precipitate in crossed immunoelectrophoresis with antibodies against human serum in the gel of the second dimension. The recovery of the biological activity after the second purification step was in the order of 6–10%. Both preparative steps could be performed within a few hours. 450 μg C9 protein were isolated from 135 ml human serum. A human serum completely defective in C9 was prepared by the extensive absorption of a smaller volume of human serum proteins with the anti-C9 column.