Environmental conditions that might be of importance for the polymerization of the 9th component (C9) of human complement were investigated. In disagreement with earlier reports summarized by Tschopp et al. [Tschopp, J. Muller-Eberhard, H. J., and Podack, E.R. (1982)] no evidence for significant aggregation or loss of hemolytic activity of C9 when incubated at 37.degree. C even after 12 days of incubation was found. Higher temperatures cause denaturation of the protein and formation of stringlike aggregates. In contrast, short-term proteolysis with 1% (w/w) trypsin at room temperature causes rapid polymerization of part of the C9 into tubular structures (poly-C9), and the remainder of the monomeric C9 is digested. This polymerization reaction is inhibitable by trypsin inhibitor: .alpha.-thrombin and proteinase K are ineffective in creating polymers. A 2nd discrepancy to the earlier reports is the finding that monomeric C9 immediately interacts with small unilamellar lipid vesicles (SUV) without a required heating step. As a result of this interaction about half of the C9 aggregates to form strings and tubules, and these aggregates cause agglutination of vesicles. The other half of the C9 associates with a 2nd population of SUV without causing a change in Stokes'' radius of these vesicles and no proteinaceous structures are detectable on the vesicle surface by EM. When these 2 vesicle populations are tested for their membrane integrity, no release of an encapsulated fluorescent marker can be detected, nor is there leakage of K+ across the bilayer membrane since a membrane diffusion potential can be developed. The earlier postulate that C9 is a cytotoxic molecule was not confirmed but it is concluded that neither monomeric nor polymerized C9 by itself is membranolytic in general. Monomeric C9 interacts with SUV above or below the lipid phase transition, but it does not associate with large multilamellar vesicles, indicating that the lipid packing density may be of importance for protein penetration. The relevance of this polymerization reaction to the function of C9 is uncertain. The intermediate C5b-8 complex may cause C9 aggregation in situ either because of an intrinsic proteolytic activity or because it changes the lipid packing in its vicinity, thereby allowing C9 to penetrate into the bilayer and subsequently to aggregate. The polymerization process may be a side reaction that functions to deactivate active C9 molecules.
