Identification of a second binding site for C5 at the CUB domain of C3b. (A) Binding competition between C3/C5 and CRIg on C3b. A total of 1500 RUs of biotinylated C3b molecules was immobilized on a streptavidin-coated SAP sensor chip. C3 (0.3 µM) and C5 (0.15 µM) were flowed over the chip alone (brown and red, respectively) or in a mixture with CRIg (30 µM) (purple and blue). The individual injection of CRIg is shown in green. Duplicates of each binding event were performed to show reproducibility. (B-C) Determination of dissociation constants for C3/C5 binding to iC3b. An iC3b immobilization signal of 1100 RUs was reached. C3 was flowed over in concentrations ranging from 0.046 to 6 µM, C5 in concentrations from 0.023 to 3 µM. The equilibrium dissociation constant K_D was estimated by plotting the steady-state response over the corresponding concentration and using a 1:1 steady-state affinity model. Note that the estimated K_D for the interaction of C5 with iC3b is higher than the highest concentration assayed and thus is less precise when compared with the other K_D estimations. (D) As in panel A, but on iC3b. A total of 1100 RUs of biotinylated iC3b was immobilized on a SAP sensor chip. The number of independent SPR assays performed were: 1 typical assay of 2 independent assays (A,C-D [right side, C5 as analyte]); 1 assay was performed (B,D [left side, C3 as analyte]).