FIGURE 5.

Consequences of ignoring either diffusion or local equilibrium behavior. (A) To investigate the impact of improperly neglecting diffusion, we used the Axial and Radial Binding Model and generated FRAP curves using binding parameters that would produce recoveries that depended on diffusion (either from the full model or the local equilibrium domains). These diffusion-dependent recoveries were then fit using the simplified solution of the Axial and Radial Binding Model that ignored diffusion (the reaction-dominant solution). We found we could obtain decent fits with this approach, but estimates of the binding parameters were often in error by almost two orders of magnitude (true parameters in black text, estimated parameters in red text). This improper fit could in fact be rejected by applying the reaction-dominant consistency check in Table 2, namely that For the reaction-dominant fit here, μm²/s and μm. Therefore, the estimated value for must satisfy s⁻¹, which is violated by the estimate of 6.3 s⁻¹ (red text). (B) To investigate the consequences of ignoring the constraints imposed by local-equilibrium behavior, we generated FRAP data within this domain using the Axial and Radial Binding Model. We were able to achieve excellent fits to these data using the same model and a range of other binding parameters. Shown here is a case where the binding rates differ by an order of magnitude (purple versus orange text), but even larger differences were possible as long as the ratio k*_2on/k_2off was kept constant. (C) A constant ratio k*_2on/k_2off = 100 yields the equation log(k*_2on) − log(k_2off) = 2, which produces a line of slope +1 (dotted black line) in the log-log plot of rate constants. All points along this line yield virtually identical FRAP curves demonstrating that independent estimates of the rate constants cannot be obtained in the local equilibrium domain.