FIGURE 4.

Influence of the concentration of ligand B present in the calorimetric cell. Titrations at different total concentrations of ligand B in the case of negative cooperativity have been simulated. The concentration of ligand A in the syringe is 300 μM, and the concentration of macromolecule in the calorimetric cell is 20 μM. The concentrations of ligand B in the calorimetric cell are 0 μM (solid squares), 10 μM (open squares), 20 μM (solid circles), 50 μM (open circles), 100 μM (solid triangles), 200 μM (open triangles), and 500 μM (solid upside-down triangles). The binding parameters are K_A = 10⁸ M⁻¹, ΔH_A = 10 kcal/mol, K_B = 10⁵ M⁻¹, and ΔH_B = 5 kcal/mol. The cooperativity parameters are α = 0.01 and Δh = 3 kcal/mol. (Inset) Apparent binding parameters for ligand A estimated by nonlinear regression of each titration represented as a function of free ligand B: apparent association constant (solid squares) and apparent binding enthalpy (open squares). Due to interparameter dependency and correlation, both interaction parameters could not be estimated by nonlinear regression analysis according to the approximate method (Eqs. 10 and 12). If α is given a fixed value of 0.01, the estimated value for Δh is 3 ± 2 kcal/mol; if Δh is given a fixed value of 3 kcal/mol, the estimated value for α is 0.011 ± 0.005 kcal/mol. Again, using the total concentration of ligand B or the difference between the total concentration of ligand B and macromolecule at the beginning of the experiment slightly improved the estimations. The interaction parameters estimated by nonlinear regression analysis of only one experiment ([B]_T = 200 μM) according to the exact method (Eq. 17) are α = 0.010 ± 0.001 and Δh = 3.01 ± 0.02 kcal/mol.