Dynamic equilibria between subcomponents of CĪ, the first component of human complement

Abstract

Clr̄ and Cls̄, the serine protease components of activated Cl, form a tetramer in the presence of Ca 2+. The stability of this tetramer is sufficient that its association with the third component, Clq, has been successfully treated as a reversible bimolecular equilibrium reaction [Siegel and Schumaker, Molec. Immun. 20, 53–66 (1983)]. We have used the fluorescence anisotropy ( A) of fluorescein-labeled Cls̄ (s ∗) to monitor assembly and subcomponent exchange in 0.15 mol/1 NaCl, 0.001 mol/l Ca 2+ 0.02 mol/l Tris, pH 7.4. Addition of q to r 2s 2 ∗ causes a small but measurable Δ A of 0.01–0.02. The response is too fast to measure at 37° but can be readily followed at 4° where t 1 2 = 0.6 min when [q] = [r̄ 2s̄ 2 ∗] = 0.5 μmol/l. The increase in A can be readily reversed by dilution or by addition of unlabeled Cls̄. Slow incremental addition of q to a solution of r̄ 2s̄ 2 ∗ produces a dose-dependent Δ A from which stoichiometry and dissociation constants can be derived. Measurements of K d as a function of temperature establish an inverse temperature dependence with Δ H = − 15 kcal/mol and a value of K d = 0.031 μmol/l at 37° († G = + 11, T† S = − 26 kcal/mol). Thus, the assembly process appears to be entropy-driven presumably due to the exclusion of structured water from protein-protein interfaces in the complex.