The unfolding curve can be divided into three regions:

1. Pre-transition region : it shows how y for the folded protein i.e. y_F responds to the denaturant.
2. Transition region : it shows how y varies as the unfolding takes place.
3. Post-transition region : it shows how y for the unfolded protein i.e. y_U responds to the denaturant.

The equilibrium constant, K_eq for the reaction can be calculated as follows:

8.4

The free energy change, Δ∆G for the reaction can be calculated from equation 8.5:

8.5

The values of K_eq are most accurately measured near the midpoint of the denaturation curve and the errors become substantial for values outside the range 0.1 – 10. This corresponds to the ΔG values between –5.7 to +5.7 kJ/mole (~ ±1.36 kcal/mol ). The data for this range can be tabulated as shown in table 8.2.

Table 8.2: Analysis of the urea denaturation curve