Dynamic equilibria between subcomponents of C1, the first component of human complement.
C1r and C1s, the serine protease components of activated C1, form a tetramer in the presence of Ca2+. The stability of this tetramer is sufficient that its association with the third component, C1q, 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 C1s (s*) to monitor assembly and subcomponent exchange in 0.15 mol/l NaCl, 0.001 mol/l Ca2+ 0.02 mol/l Tris, pH 7.4. Addition of q to r2s*2 causes a small but measurable delta A of 0.01-0.02. The response is too fast to measure at 37 degrees but can be readily followed at 4 degrees where t 1/2 = 0.6 min when [q] = [r2s*2] = 0.5 mumol/l. The increase in A can be readily reversed by dilution or by addition of unlabeled C1s. Slow incremental addition of q to a solution of r2s*2 produces a dose-dependent delta A from which stoichiometry and dissociation constants can be derived. Measurements of Kd as a function of temperature establish an inverse temperature dependence with delta H = -15 kcal/mol and a value of Kd = 0.031 mumol/l at 37 degrees (delta G = + 11, T delta 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.
Ingham
K C
KC
American Red Cross Biomedical Research and Development Laboratory, Rockville, MD 20855.
Milasincic
D J
DJ
Busby
T F
TF
Strickland
D K
DK
D015923:Complement C1r / Q000737:chemistry / Q000378:metabolism*; D003173:Complement C1s / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D009994:Osmolar Concentration; D011485:Protein Binding; D013696:Temperature; D013816:Thermodynamics
D046911:Macromolecular Substances; D015923:Complement C1r; D003173:Complement C1s