Figure 2

Surface plasmon resonance analysis of S100A12-RAGE interaction. (A and B). Sensorgrams on Ca²⁺- and Zn²⁺-dependent binding of S100A12 to immobilized RAGE. Sensorgrams show association and dissociation with varying conditions in which either Ca²⁺ was kept stable with varying Zn²⁺ concentrations or vice versa. One representative out of three independent experiments is shown as response units (RU; y-axis). Time is shown in seconds. (C). Kinetic and affinity analysis of RAGE binding to immobilized S100A12. Recombinant human RAGE/Fc (125 nM) was injected over a surface where recombinant S100A12 was immobilized at a density of 2,933 RU on a CM5 chip using amine coupling chemistry. Samples were injected for 3 min at a flow rate of 30 μl/min. HBS-P buffer (Biacore) containing 1 mM Ca²⁺ and 20 μM Zn²⁺ was used as sample and running buffer. The sensorgram obtained after injection of 125 nM RAGE (straight line) was perfectly fit to a 1:1 Langmuir model (dotted line; BIAevaluation software; Biacore) for calculation of affinity and on-/off-rates. The K_D, kon, koff and Chi² values were calculated to: 4.6 × 10-9 M; 6.8 × 104 1/Ms; 3.2 × 10-4 1/s; and 0.47, respectively.