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The C5b-6 Complex: Reaction with C7, C8, C9¹

From the Department of Molecular Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037

Abstract

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^-12 M, K_D (C8) = 0.9 to 8.6 x 10^-12 M, and K_D (C9) = 0.1 to 0.5 x 10^-12 M. The free energy for C5b-9 formation from C5b-6, C7, C8, and C9 is estimated to be -50 kcal/mole.

Inactivation of C5b-6 by C7 is a rapid, time and temperature dependent process following second order kinetics. Complex formation at 37°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 membrance binding site, C5b-7*, 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 C5b-7* 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: G = -17 kcal/mole, H = +8.6 kcal/mole and S = 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.

Footnotes

¹ This work was supported by United States Public Health Service Grants AI 07007 and HL 16411. This is Publication No. 1487 from the Research Institute of Scripps Clinic.

² Recipient of Investigatorship. PO 167-1 and PO 167-2 from the Deutsche Forschungsgemeinschaft.

³ Supported by United States Public Health Service Training Grant HI 07195.

⁴ Recipient of Research Career Development Award 5 KO4 CA 70841 from the United States Public Health Service. Present address: Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284.

⁵ Cecil H. and Ida M. Green Investigator in Medical Research, Research Institute of Scripps Clinic.

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