Human Factor D̄ of the Alternative Complement Pathway: Purification and Characterization

Abstract

D̄ was purified to homogeneity from outdated human plasma by successive chromatography on Bio Rex 70, Sephadex G-200, Bio Rex 70, and Sephadex G-75. Column fractions were monitored for D̄ activity by a hemolytic diffusion plate assay. The overall yield was approximately 4% by activity. A m.w. of 22,900 daltons was established by sedimentation equilibrium. Amino acid analyses have been obtained and Isoleucine has been determined as the NH2-terminus. Incubation of D̄ with purified B and CoVF in the presence of Mg++ resulted in cleavage of B, as judged by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis. D̄ hydrolyzed certain synthetic amino acid esters of arginine, lysine, and tyrosine. Benzoyl-L-arginine methyl ester (BAME) was the most sensitive substrate for D̄ among those tested. The substrate profile of D̄ was distinct when compared to that of C1̄s, C1̄r, plasmin, urokinase, and trypsin. Both the enzymatic and hemolytic activity of D̄ were irreversibly inhibited by treatment with 10 mM DFP as well as by reduction and alkylation.