Fig 9. Dissociation kinetics of SAV-BcO and SAV-BFl complexes.

(A) Concentration dependence of the displacement reaction of SAV-BcO complex (AB₁ model) by unlabeled B₇ at 20°C. The concentration of challenging B₇ was 740 nM (blue), 1240 nM (pink) and 1740 nM (red) after the remaining free binding sites were filled. The half-times were 56.6 s, 33.9 s and 24.2 s, respectively; with a release amplitude of 79 ± 1%. (B) Temperature dependence of the displacement reaction of SAV-BcO by unlabeled B₇ for the AB₁ filling model (at 20°C and 27°C) and for the AB₄ model (at 20°C). The corresponding $k_{off}^{displacement}$ (calculated from the slope) were 1.6 ± 0.4 × 10⁵ M^-1s^-1, 4.6 ± 0.3 × 10⁵ M^-1s^-1 and 1.2 ± 0.3 × 10⁵ M^-1s^-1, respective. (C) Displacement reaction of unlabeled B₇ and SAV-BFl complex (AB₁ filling model) at 30°C. The concentration of challenging B₇ was 1400 nM which produced a release of only 5% of the bound probe. The green curve is the observed data and black curve is the fitted curve for which only 6.5% displacement was observed for SAV-BFl complex in contrast with 79% in case of the complex formed with the longer linker BcO.