Interactions of C1-INH and C1s with heparin and polyP. (A) Analytical affinity chromatography indicates binding of heparin to C1s and C1-INH. C1-INH (gray line), plasma-derived C1s (black line), a 1:1 mixture of both (pre-incubated for 60 minutes at 37°C) (dotted black line), or the C1s A1 mutant (orange solid line), were separately applied to a HiTrap-Heparin column and eluted with a NaCl gradient as described in “Materials and methods.” Conductivity measurements are shown on the right-hand axis. Eluted fractions were analyzed by SDS-PAGE to confirm the identity of the proteins. (B) SPR was used to quantify binding of C1s (CCP12SP) to biotinylated polyP₂₅₀ attached to streptavidin immobilized to the chip. A representative experiment (n = 3) shows curves for 0 to 625 nM C1s flowed over the immobilized polyP₂₅₀. Inset: The response units obtained at equilibrium for each concentration of C1s were plotted and fitted using a one-site binding model on GraphPad Prism (regression coefficient = 0.99). Similar results were obtained in 2 additional independent experiments.