The reaction of purified C5b-6 with purified C7, C8, and C9 was studied in cellfree solution. C5b-6 binds C7, C8, and C9 in a sequential manner and thereby forms hemolytically inactive complexes. Calculations of molar ratios indicate the binding of multiple C9 molecules per C5b-8 complex, which contains equimolar amounts of C5b, C6, C7, and C8. The dissociation constants for the terminal components were calculated from Scatchard plots: K(D)(C7)= 0.2 to 2.9 x 10-12M, K(D)(C8)= 0.9 to 8.6 x 10-12M, and K(D)(C9)= 0.1 to 0.5 x 10-12M. The free energy for C5b-9 formation from C5b-6, C7, C8, and C9 is estimated to be -50 kcal/mole. Inactivation of C5-6 by C7 is a rapid, time and temperature dependent process following second order kinetics. Complex formation at 37degr. C is diffusion controlled; at 4°C it is, in addition, controlled by activation energy requirement since the activation energy for complex formation was found to be 33.7 kcal/mole. Association of C5b-6 and C7 results in the formation of a labile membrane binding site, C5b-7(hemolytically active), which decays rapidly to yield C5b-7(i). C5b-7 formation from its precursors C5b-6 and C7 is the rate limiting step, decay of (5b-7(hemolytically active) to C5b-7(i) is rapid compared to the association reaction. The following thermodynamic parameters were obtained at 30° C for the C5b-7(i) formation from C5b-6 and C7: DeltaG = -17 kcal/mole, DeltaH = +8.6 kcal/mole and DeltaS = 77 e.u. The data are compatible with the interpretation that the protein-protein interactions occurring upon complex formation are accompanied by release of protein bound water or by conformational changes or both. C5b-7 formation results in acquisition of a labile binding site, a markedly anodal electrophoretic mobility, expression of a second neoantigen, and in aggregation in the absence of cells or the S-protein.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Immunology|
|State||Published - Dec 1 1978|
ASJC Scopus subject areas
- Immunology and Allergy