Fig 6. 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 $k_{on}^B$ 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 4^th site when three HABA molecules are already bound: $k_1^{AV-HABA3}$ = 5.1 ± 0.1 × 10⁵ M^-1s^-1 and the HABA dissociation of saturated AV-HABA₄ complex: $k_{-1}^{AV-HABA4}$ = 6.23 ± 0.11 s^-1. The corresponding K_D ^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.