Affinity of the C9 molecule for the C5b-8 complex compared with that for the complex containing C9 molecules

Gram-negative bacterial cells exposed to a complement source may carry membrane attack complexes containing variable numbers of C9 molecules per C5b-8 site. In order to investigate the assembly of this complex, the ability of C9 molecules to bind to C5b-8 complexes was compared with the binding characteristics of C9 for C5b-8 complexes containing variable numbers of bound C9 molecules. The apparent dissociation constant (Kd) of the C9 molecule for the C5b-8 site on a complement-sensitive strain of Escherichia coli was 1.2 (+/- 0.15) nM at 0 degree C. These conditions allow the binding of one C9 molecule per C5b-8 site. The C5b-8 site containing one C9 molecule bound a second C9 molecule at 0 degree C only after incubation at 37 degrees C. The binding of C9 to a C5b-8 site containing one C9 molecule was found to be 1.3 (+/- 0.2) nM. Therefore, the presence of a C9 molecule did not significantly alter the binding capacity of the C5b-8 site for additional C9 molecules. A similar result was obtained by using rabbit erythrocytes bearing either C5b-8 sites or C5b-8 sites containing one molecule of C9 per complex at 0 degree C. The similarity of binding characteristics for the first and second C9 molecules argues that the initial C9 molecule in the complex does not affect the binding of subsequent C9 molecules. This suggests that a unique C9 binding site that does not involve previously bound C9 molecules may exist on the forming membrane attack complex.