Relaxation kinetics of the AV-HABA complex and unlabeled biotin.

The [HABA]/[B7] is the concentration ratio of these two ligands and the relaxation rate is in s-1 (Eq 3). The data points were fitted to a linear regression model yielding a slope and intercept of 0.0156 ± 0.0012 and 0.161 ± 0.005, respectively, resulting in a of 5.3 ± 0.9 × 106 M-1s-1 at 23°C. The additional values required for this calculation were HABA association constant to the 4th site when three HABA molecules are already bound: = 5.1 ± 0.1 × 105 M-1s-1 and the HABA dissociation of saturated AV-HABA4 complex: = 6.23 ± 0.11 s-1. The corresponding KD AV-HABA = 12.2 ± 0.3 × 10−6 M and is in excellent agreement with the 12 × 10−6 M reported by Green [60] at pH 8.