Quantitation of the binding affinities between 4.1R fragments and MESA
Origin and region of 4.1R . | Ka, M-1sec-1 . | Kd, sec-1 . | K(D)kin, μM . | K(D)Scat, μM . |
|---|---|---|---|---|
| RBC | ||||
| 80 kDa | 1.0 ± 0.02 × 105 | 2.7 ± 0.10 × 10-2 | 0.27 | 0.14 |
| 30 kDa | 2.3 ± 0.02 × 104 | 4.4 ± 0.04 × 10-2 | 1.90 | 3.20 |
| GST-fusion | ||||
| 30 kDa | 1.2 ± 0.02 × 104 | 1.5 ± 0.04 × 10-2 | 1.25 | 1.47 |
| 30-kDa F1 | No binding | No binding | No binding | No binding |
| 30-kDa F2 | No binding | No binding | No binding | No binding |
| 30-kDa F3 | No binding | No binding | No binding | No binding |
| 30-kDa F4 | No binding | No binding | No binding | No binding |
| 30-kDa F5 | 3.3 ± 0.09 × 104 | 3.7 ± 0.1 × 10-2 | 1.17 | 1.20 |
| 30-kDa F6 | No binding | No binding | No binding | No binding |
| 30-kDa F7 | No binding | No binding | No binding | No binding |
| 30-kDa F8 | No binding | No binding | No binding | No binding |
| (30 + 16) kDa | 1.0 ± 0.06 × 105 | 1.3 ± 0.06 × 10-2 | 0.13 | 0.24 |
| 16 kDa | No binding | No binding | No binding | No binding |
| 10 kDa | No binding | No binding | No binding | No binding |
| 22/24 kDa | No binding | No binding | No binding | No binding |
| GST | No binding | No binding | No binding | No binding |
| BSA | No binding | No binding | No binding | No binding |
Origin and region of 4.1R . | Ka, M-1sec-1 . | Kd, sec-1 . | K(D)kin, μM . | K(D)Scat, μM . |
|---|---|---|---|---|
| RBC | ||||
| 80 kDa | 1.0 ± 0.02 × 105 | 2.7 ± 0.10 × 10-2 | 0.27 | 0.14 |
| 30 kDa | 2.3 ± 0.02 × 104 | 4.4 ± 0.04 × 10-2 | 1.90 | 3.20 |
| GST-fusion | ||||
| 30 kDa | 1.2 ± 0.02 × 104 | 1.5 ± 0.04 × 10-2 | 1.25 | 1.47 |
| 30-kDa F1 | No binding | No binding | No binding | No binding |
| 30-kDa F2 | No binding | No binding | No binding | No binding |
| 30-kDa F3 | No binding | No binding | No binding | No binding |
| 30-kDa F4 | No binding | No binding | No binding | No binding |
| 30-kDa F5 | 3.3 ± 0.09 × 104 | 3.7 ± 0.1 × 10-2 | 1.17 | 1.20 |
| 30-kDa F6 | No binding | No binding | No binding | No binding |
| 30-kDa F7 | No binding | No binding | No binding | No binding |
| 30-kDa F8 | No binding | No binding | No binding | No binding |
| (30 + 16) kDa | 1.0 ± 0.06 × 105 | 1.3 ± 0.06 × 10-2 | 0.13 | 0.24 |
| 16 kDa | No binding | No binding | No binding | No binding |
| 10 kDa | No binding | No binding | No binding | No binding |
| 22/24 kDa | No binding | No binding | No binding | No binding |
| GST | No binding | No binding | No binding | No binding |
| BSA | No binding | No binding | No binding | No binding |
Purified RBC 4.1R and recombinant 4.1R fragments were used in protein interaction assays using an IAsys resonant mirror biosensor (Affinity Sensors), using previously described methods.5 Briefly, interaction assays were performed using a 19-residue MESA peptide, synthesized as previously described,8 and purified GST-MF3(S)+19, GST-MF3(S)Δ19, and GST-MF4(S) fusion proteins (Figure 1). In these experiments, aminosilane cuvettes were coated with the 19-residue synthetic peptide, GST-MF3(S)+19, GST-MF3(S)Δ19, GST-MF4(S), GST, or bovine serum albumin (BSA) and purified. RBC 4.1R or GST-4.1R or GST-4.1R fusion proteins were added to the cuvettes in aqueous solution. Binding assays were carried out in PBS containing 0.05% (vol/vol) Tween 20. From the binding curves obtained, Ka, Kd, K(D)kin, and K(D)Scat (dissociation constants determined by kinetic and Scatchard analysis, respectively) were determined using FASTfit software (Affinity Sensors).