| Immobilized ligand/soluble analyte . | PCNA . | |||||
|---|---|---|---|---|---|---|
| ka1 (M−1 s−1) . | kd1 (s−1) . | kforward (s−1) . | kbackward (s−1) . | KD (M) . | ||
| p47phox (two-state model) | 8.7 × 103 | 8.4 × 10−3 | 7.5 × 10−3 | 6.5 × 10−4 | 7.7 × 10−8 | |
| p47-PX domain (1:1 model) | 85.2 | 2.8 × 10−3 | NA | NA | 3.3 × 10−5 | |
| p47 peptide 106–127 | 1.0 × 104 | 0.35 | NA | NA | 3.4 × 10−5 (KD1) | |
| (Heterogeneous ligand model) | 160 | 0.0111 | NA | NA | 6.9 × 10−5 (KD2) | |
| p47 peptides | ||||||
| 83–110 | ND | ND | ND | ND | ND | |
| 63–90 | ||||||
| 46–67 | ||||||
| Rac2 (two-state model) | 541 | 17 × 10−3 | 5.7 × 10−3 | 6.6 × 10−4 | 3.3 × 10−6 | |
| Immobilized ligand/soluble analyte . | PCNA . | |||||
|---|---|---|---|---|---|---|
| ka1 (M−1 s−1) . | kd1 (s−1) . | kforward (s−1) . | kbackward (s−1) . | KD (M) . | ||
| p47phox (two-state model) | 8.7 × 103 | 8.4 × 10−3 | 7.5 × 10−3 | 6.5 × 10−4 | 7.7 × 10−8 | |
| p47-PX domain (1:1 model) | 85.2 | 2.8 × 10−3 | NA | NA | 3.3 × 10−5 | |
| p47 peptide 106–127 | 1.0 × 104 | 0.35 | NA | NA | 3.4 × 10−5 (KD1) | |
| (Heterogeneous ligand model) | 160 | 0.0111 | NA | NA | 6.9 × 10−5 (KD2) | |
| p47 peptides | ||||||
| 83–110 | ND | ND | ND | ND | ND | |
| 63–90 | ||||||
| 46–67 | ||||||
| Rac2 (two-state model) | 541 | 17 × 10−3 | 5.7 × 10−3 | 6.6 × 10−4 | 3.3 × 10−6 | |
No significant binding was detected for p40phox and p67phox for concentrations ≤1 µM. The binding of PCNA with p47phox (25–400 nM), p47-PX domain (1.83–29.5 µM), or p47peptide 106–127 (0.31–40 µM) was investigated by SPR as described in Materials and methods. Kinetics parameters were calculated using either two-state, 1:1 Langmuir, or heterogeneous ligand reactions models. For the two-state binding model (A + B ↔ AB ↔ AB*) that fit the best for full-length p47phox, ka1, kd1, kforward, and kbackward constants were determined by global fitting. The dissociation constant KD was determined from the (kd1/ka1)/(1 + kforward/kbackward) ratio. The heterogeneous ligand model accounts for two different binding sites on the immobilized PCNA, and the corresponding two kinetic values were calculated. For 1:1 Langmuir and heterogeneous ligand models, the equilibrium dissociation constant is calculated by KD = kd/ka. The data presented were obtained with a statistic χ2 value <2. Rac2, p40phox, and p67phox were also tested for their interaction with PCNA. Binding of Rac2 (0.51–8, 2 µM) to PCNA was measured and evaluated as reported below (χ2 value 0.6). NA, not applicable; ND, the very low binding signal detected for p47 83–110, 63–90, and 46–67 peptides did not allow evaluation of kinetic data.