Ligand binding activities of fH and fH/CFHR3. (A) C3b was thiol-coupled to the surface of a CM5 Biacore chip at a high density (1200 RU). Thiol-coupling results in clustering of C3b on a surface, promoting cross-linking and avidity effects resulting from multisite binding. When fH (bottom) or fH/CFHR3 (top) flowed across this surface at 500 ng/mL (gray line) or 125 ng/mL (black line), enhanced binding of fH/CFHR3 was evident. This cannot be accounted for by increased mass (155 kDa vs 186 kDa); it is probably the result of binding through the CFHR3 domain in addition to the fH SCRs. The “tailing” effect illustrates avidity effects in both traces; kinetics cannot be accurately measured under these conditions. Inset: Coomassie-stained SDS-PAGE gel of fH and fH/CFHR3 purified on allele-specific affinity columns. (B) Purified proteins (402Y) were bound to a HiTrap heparin column and eluted with NaCl. The fH/CFHR3 mutant (gray) eluted at 371mM and fH (black) at 350mM, indicating enhanced binding through the C-terminus to heparin. Dashed line indicates salt gradient.