Heme interaction with C3 favors the formation of the AP C3/C5 convertases. C3 convertase was formed on an SPR chip coated with (A) C3 and (B) C3b. MgCl2 (1 mM final) was added in HEPES running buffer for interactions involving FB. C3 or C3b on the chip was exposed to increasing concentrations of heme as indicated. Subsequently, a series of injections of C3, FB + FD, C3, and C5 allowed formation of a C3/C5 convertase on the chip. One representative experiment of 4 is shown. The influence of heme on the capacity of FB and FD to bind to (C) C3 and (D) C3b was measured by SPR. C3 and C3b were immobilized on the chip and FB and FD, either native or exposed to five- and 10-fold molar excess of heme, were injected. One representative sensorogram set of 3 experiments is shown. Each interaction was tested on at least two different chips at least twice per chip, and the data were double referenced and analyzed by using ProteOn manager software.
