THE SEPARATION OF FUNCTIONALLY DISTINCT FORMS OF THE 3RD COMPONENT OF HUMAN-COMPLEMENT (C-3)

Complement component C3 contained the following trace contaminants: C3b, hemolytically inactive C3 with intact .alpha.- and .beta.-chains (C3u) and degraded C3 (apparent MW 140,000) with an intact .beta.-chain but with a fragmented .alpha.-chain. The proportion of C3u in the C3 is increased on standing and by freezing and thawing. These contaminants could be separated from each other and from native C3 by chromatography on sulfated Sepharose. They were characterized by their susceptibility to C3b inactivator in the presence of .beta.1H, their ability to be cleaved by C3 convertase and their ability to form alternative-pathway C3 convertase in solution. C3b or C3u incubation with .beta.1H and C3b inactivator resulted in C3 species cleavage; the .alpha.''-chain of C3b was cleaved to fragments of apparent MW 67,000 and 43,000, the .alpha.-chain of C3u was cleaved to fragments of apparent MW 75,000 and 43,000. Native C3 and degraded C3 were unaffected by incubation with .beta.1H and C3b inactivator. C3u, unlike C3, was not cleaved to C3b by the classical- or alternative-pathway C3 convertase in solution. When C3b or C3 was incubated with factors B and D, forming C3 convertase, the initial factor-B cleavage rate was several orders of magnitude lower in the presence of C3 than in the presence of C3b. The slow rate observed for C3 could be decreased by preincubation with .beta.1H and C3b inactivator or by C3 rechromatography. The degraded C3 did not support factor-B cleavage by factor .hivin.D.