Kinetic and equilibrium constants for binding of WT and mutant FVIII-C2 proteins to BO2C11.

Based on the good agreement between the theoretical Rmax (determined using the GE Biacore T100 software) and the measured Rmax values, a 1:1 binding model was determined to be appropriate for these experiments. Occasionally, the sensorgram corresponding to the highest concentration was omitted from analysis due to apparent non-specific binding, but in these cases the binding curves at lower FVIII-C2 concentrations were adequate to calculate reliable rate constants. The entries in bold are the 16 substitutions at 15 positions at the BO2C11 binding interface (“contacts” defined as FVIII-C2-BO2C11 interatomic d < 3.8 Å). Residence time = 1/kd. Dissociation half-life = ln 2 /kd. *SE = Standard Error of the curve fit calculated by the Biacore analysis software. †These substitutions resulted in a kd >4X that of WT-FVIII-C2 for BO2C11 or abrogated binding to BO2C11. ‡These substitutions decreased the residence time for the FVIII-C2-mutein/BO2C11 complex at least 10X. The kinetic range for the Biacore T100 is: ka constants from ~103–107 M-1s-1; kd constants from ~10-5–0.5 s-1. For fast associations with fitted ka ≥ 3.2E+06, the evaluation software warns that the value approaches the limit that can be measured by the instrument. All ka values between 1.0E+07 and 3.0E+07 and kinetic data calculated using these ka values are underlined. All fitted ka values >3.0E+07 are indicated as “>3.0E+07” and the corresponding KD values were not calculated. For slow dissociations with fitted kd ≤ 3.0E-05, the evaluation software warns that the value approaches the limit that can be measured by the instrument. All kd values between 1.0E-05 and 3.0E-05 and kinetic data calculated using these kd values are underlined.
Kinetic and equilibrium constants for binding of WT and mutant FVIII-C2 proteins to BO2C11.