Kinetics of factor IXa and EGR-factor IXa binding to LRP and recombinant cluster II and IV fragments
| Injected protein . | Immobilized protein . | koff(s−1) . | kon (s−1M−1) . | Kd (nmol/L) . |
|---|---|---|---|---|
| Factor IXa | LRP | (1) 4.6 (± 0.6) × 10−3 | (1) 1.7 (± 0.2) × 105 | (1) 27.1 ± 4.8 |
| (2) 8.3 (± 1.4) × 10−2 | (2) 1.2 (± 0.2) × 106 | (2) 69.2 ± 16.4 | ||
| EGR-factor IXa | LRP | (1) 3.7 (± 0.3) × 10−3 | (1) 1.4 (± 0.2) × 105 | (1) 26.4 ± 4.3 |
| (2) 9.9 (± 1.0) × 10−2 | (2) 1.4 (± 0.2) × 106 | (2) 70.7 ± 12.4 | ||
| Cluster II | Factor IXa | 5.7 (± 1.2) × 10−3 | 2.5 (± 0.3) × 104 | 227.4 ± 55.1 |
| Cluster IV | Factor IXa | 9.6 (± 0.5) × 10−3 | 1.8 (± 0.2) × 105 | 53.3 ± 6.5 |
| Injected protein . | Immobilized protein . | koff(s−1) . | kon (s−1M−1) . | Kd (nmol/L) . |
|---|---|---|---|---|
| Factor IXa | LRP | (1) 4.6 (± 0.6) × 10−3 | (1) 1.7 (± 0.2) × 105 | (1) 27.1 ± 4.8 |
| (2) 8.3 (± 1.4) × 10−2 | (2) 1.2 (± 0.2) × 106 | (2) 69.2 ± 16.4 | ||
| EGR-factor IXa | LRP | (1) 3.7 (± 0.3) × 10−3 | (1) 1.4 (± 0.2) × 105 | (1) 26.4 ± 4.3 |
| (2) 9.9 (± 1.0) × 10−2 | (2) 1.4 (± 0.2) × 106 | (2) 70.7 ± 12.4 | ||
| Cluster II | Factor IXa | 5.7 (± 1.2) × 10−3 | 2.5 (± 0.3) × 104 | 227.4 ± 55.1 |
| Cluster IV | Factor IXa | 9.6 (± 0.5) × 10−3 | 1.8 (± 0.2) × 105 | 53.3 ± 6.5 |
Association and dissociation between either factor IXa or EGR-factor IXa and LRP was determined by passing 6 different concentrations (5-150 nmol/L) over 3 separate channels with immobilized LRP (16 fmol/mm2) and 1 control channel (no immobilized LRP) as described in the legend to Figure 1B. Association and dissociation between cluster II or cluster IV of LRP and factor IXa was assessed in a similar manner except that 5 different concentrations of cluster II or cluster IV (50-150 nmol/L) were passed over immobilized factor IXa. To this end, 122 fmol/mm2 or 32 fmol/mm2 of factor IXa was immobilized to determine cluster II or cluster IV binding constants, respectively. The data, obtained from duplicate experiments, were analyzed by performing nonlinear global fitting of data corrected for bulk refractive index changes to calculate association rate constants (kon) and dissociation rate constants (koff).32 Data represent average values ± SD.