Measurement of the association constants of the complexes formed between intact C1q or pepsin-treated C1q stalks and the unactivated or activated C1r2C1s2 tetramers.
The association constants between C1q and C1r2C1s2 and between C1q and C1r2C1s2 were measured in solution using a new technique which employs sucrose gradient ultracentrifugation to estimate thermodynamic association constants. In this technique, zones of dilute, radioiodine-labeled C1q were sedimented through uniform concentrations of either C1r2C1s2 or C1r2C1s2. The zones remained intact, indicating that the dynamic equilibrium was rapid compared with the time of centrifugation. The observed increases in the sedimentation coefficients of the C1q zones were assumed to be directly proportional to the fraction of C1q bound in the dynamic equilibrium. Binding curves were constructed by performing the measurements at many C1r2C1s2 and C1r2C1s2 concentrations. The association constants were estimated from the midpoints of the binding curves and found to be 6.7 X 10(7)M-1 for C1r2C1s2 binding to 125I-C1q. After activation of the C1r2C1s2 the association constant decreased 10-fold to 7.1 X 10(6)M-1. These association constants refer to solvent conditions of pH 7.35, 1 mM Tris, 5 mM Ca2+ and 150 mM NaC1, pH 7.35. Similar measurements were performed with the collagenous peptic fragment of C1q and both 125I-C1r2C1s2 and 125I-C1r2C1s2. The association constants were independent of the state of activation and both found to be about 2 X 10(7) M-1, suggesting that most if not all of the interactions between C1q and C1r2C1s2 were confined to the collagenous portion of C1q.
D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003166:Complement Activating Enzymes / Q000378:metabolism*; D003167:Complement Activation; D015922:Complement C1q; D015923:Complement C1r; D003173:Complement C1s; D004591:Electrophoresis, Polyacrylamide Gel; D006461:Hemolysis; D006801:Humans; D008722:Methods; D010434:Pepsin A; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism
D010446:Peptide Fragments; D015922:Complement C1q; D003166:Complement Activating Enzymes; D015923:Complement C1r; D003173:Complement C1s; D010434:Pepsin A