HUMAN FACTOR-D OF ALTERNATIVE COMPLEMENT PATHWAY - PURIFICATION AND CHARACTERIZATION

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  • 1 January 1977
    • journal article
    • research article
    • Vol. 119  (1) , 337-342
Abstract
.hivin.D [activated factor D] was purified to homogeneity from outdated human plasma by successive chromatography on Bio Rex 70, Sephadex G-200, Bio Rex 70 and Sephadex G-75.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 .hivin.D activity by a hemolytic diffusion plate assay. The overall yield was approximately 4% by activity. A MW of 22,900 daltons was established by sedimentation equilibrium. Amino acid analyses were obtained and isoleucine was determined as the NH2-terminus. Incubation of .hivin.D with purified Factor B[B] and CoVF [cobra venom factor] in the presence of Mg++ resulted in cleavage of B, as judged by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis. .hivin.D hydrolyzed certain synthetic amino acid esters of arginine, lysine and tyrosine. Benzoyl-L-arginine methyl ester (BAME) was the most sensitive substrate for .hivin.D among those tested. The substrate profile of .hivin.D was distinct when compared to that of C.hivin.1s [S fragment from activated complement component 1], C.hivin.1r, plasmin, urokinase, and trypsin. The enzymatic and hemolytic activity of .hivin.D were irreversibly inhibited by treatment with 10 mM DFP [diisopropylfluorophosphate] and by reduction and alkylation.

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