Mobile phase | Immobilized ligand | ka [M−1 s− 1] | kd [s− 1] | KD [nM] |
|---|
MucB | MucA’ | 6.39 × 104 | 1.27 × 10− 4 | 1.99 |
MucB | β | 3.87 × 104 | 1.2 × 10− 5 | 0.31 |
- In SPR, a ligand, i.e., MucA’ or β, is immobilized on a sensor chip, and an analyte, in this case MucB, is injected over the chip surface in a mobile phase. If the ligand and analyte interact, the resulting increase in mass on the chip surface is detected and plotted as an increase in response units (RU) over time, allowing calculation of the apparent association rate constant (ka) value. After the injection of protein in the mobile phase is complete, buffer is passed over the chip, and the dissociation of the proteins is observed as a loss in mass over time from which the apparent dissociation rate constant (kd) value can be calculated. From these data, the equilibrium dissociation constant (KD) is obtained (KD = kd/ka). In the calculation, we used the first flow cell with immobilized BSA as a reference cell because MucB protein did not show any binding to it similar to the intact CM surface. To determine the kinetic constants with highest accuracy we calculated the values at several different densities of immobilized ligands and selected the values with the best fits according to the residual and lag plots
