Fig. 3.
SPR analysis of activated convertase formation by native fB. (A) The variant fB proteins were purified from the plasma of donors known to be homozygous for fB32R or fB32Q. FB was flowed over the surface of the C3b-coated chip in the presence of 1 μg/mL fD at concentrations between 460 and 7 nM in Biacore buffer (10 mM Hepes, pH 7.4/50 mM NaCl/1 mM MgCl2/0.005% surfactant P20). Sensorgrams from fB32R are solid lines and fB32Q are dotted lines; identical concentrations are illustrated for the 2 variants. Arrows indicate sensorgram generated by either variant at 115 nM for comparison. Kinetic information from 2 independent experiments (different surface and preparation of fB each time) were analyzed by using 2-state reaction model, and data were identical for both experiments. KD: fB32R, 2.9 nM; and fB32Q, 7.1 nM (χ2 in replicate experiments was between 6 and 13). Data were also analyzed by using a 1:1 Langmuir model; although the fit was not as good, kinetic analysis revealed similar affinities (3.2 nM, fB32R; 7.7 nM, fB32Q) (χ2 in replicates was between 7 and 32). (B) All 3 variants were purified from plasma of homozygous donors, and flowed over a C3b surface at 2 concentrations (156, 39 nM). Sensorgrams from fB32R are solid lines, fB32Q are dotted lines, and fB32W are in gray. Data were normalized in the y axis, and sensorgrams overlaid.