An amphiphilic structure of the ninth component of human complement. Evidence from analysis of fragments produced by alpha-thrombin.

Purified human C9 was treated separately with three proteolytic enzymes: trypsin, plasmin, and alpha-thrombin, and the digestion products were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Trypsin initially cleaved the Mr = 71,000 C9 to produce a Mr = 47,000 fragment plus numerous smaller fragments and prolonged digestion reduced the molecule to small polypeptides. Plasmin produced a Mr = 37,000 fragment which was stable to further digestion, plus fragments smaller than Mr = 10,000. Human alpha-thrombin cleaved C9 (7.8% carbohydrate) at a single internal site to produce a Mr = 37,000 fragment (11.3% carbohydrate) and a Mr = 34,000 fragment (3.9% carbohydrate). Statistical analysis of the amino acid compositions of the fragments and alkaline polyacrylamide gel electrophoresis showed that C9 is highly amphiphilic; the Mr = 34,000 fragment contains a majority of the acidic amino acids and migrates rapidly on alkaline gels; the Mr = 37,000 fragment is hydrophobic with a slow electrophoretic mobility. The two fragments remain noncovalently associated, but were separated by sodium dodecyl sulfate-hydroxylapatite chromatography. The NH2-terminal sequence analysis of native C9, of alpha-thrombin-cleaved C9, and for the isolated fragments showed that the acidic Mr = 34,000 fragment is the NH2-terminal C9a domain and the more hydrophobic Mr = 37,000 fragment is the carboxyl-terminal C9b domain. Hemolytic activity of C9 was unaffected by alpha-thrombin cleavage.