Steady state affinity data for the binding of activated rFXIII-A to captured GSTα fragment 1-9 by SPR
| Ligand GSTα fragment . | Molecular mass of GSTα fragments, kDa . | Molecular mass of GST removed α fragments, kDa . | Equilibrium dissociation constant (Kd) μM . |
|---|---|---|---|
| GSTα fragment 1 (233-425) | 50.0 | 20.02 | 2.35 ± 0.09 |
| GSTα fragment 2 (23-403) | 47.4 | 17.43 | 2.64 ± 1.21 |
| GSTα fragment 3 (233-388) | 45.8 | 15.83 | * |
| GSTα fragment 4 (233-375) | 44.6 | 14.57 | * |
| GSTα fragment 5 (233-341) | 41.2 | 11.18 | * |
| GSTα fragment 6 (233-290) | 36.4 | 6.41 | * |
| GSTα fragment 7 (233-266) | 34.1 | 4.1 | * |
| GSTα fragment 8 (289-425) | 43.7 | 13.7 | 1.81 ± 0.45 |
| GSTα fragment 9 (371-425) | 35.7 | 5.7 | 3.19 ± 0.57 |
| Ligand GSTα fragment . | Molecular mass of GSTα fragments, kDa . | Molecular mass of GST removed α fragments, kDa . | Equilibrium dissociation constant (Kd) μM . |
|---|---|---|---|
| GSTα fragment 1 (233-425) | 50.0 | 20.02 | 2.35 ± 0.09 |
| GSTα fragment 2 (23-403) | 47.4 | 17.43 | 2.64 ± 1.21 |
| GSTα fragment 3 (233-388) | 45.8 | 15.83 | * |
| GSTα fragment 4 (233-375) | 44.6 | 14.57 | * |
| GSTα fragment 5 (233-341) | 41.2 | 11.18 | * |
| GSTα fragment 6 (233-290) | 36.4 | 6.41 | * |
| GSTα fragment 7 (233-266) | 34.1 | 4.1 | * |
| GSTα fragment 8 (289-425) | 43.7 | 13.7 | 1.81 ± 0.45 |
| GSTα fragment 9 (371-425) | 35.7 | 5.7 | 3.19 ± 0.57 |
Molecular mass of recombinant fibrinogen αC fragments 1-9 in the presence and absence of the GST tag are also shown. With the use of a CM5 sensor chip anti-GST antibody capture approach on a BIAcore 3000 platform, a 2-fold serial dilution of activated rFXIII-A was injected for 60 seconds over captured GSTα fragments 1-9. The data were evaluated with BIAcore 3000 BIAevaluation 4.1 software fitted to a predefined steady state affinity model with the use of blank reference and buffer subtracted data.
Negligible binding was observed for GSTα fragments 3, 4, 5, 6, and 7, which was too low to undertake formal analysis. (n = 3).