*3.7. Analysis of Denaturation Spectral Measurements*

The spectral property (*F*344) plotted against the concentration of urea/GdmCl generated the transition curve. The molar concentration of urea/GdmCl were used to calculate thermodynamic properties such as stability parameters (∆*G 0 <sup>D</sup>*), slope (*m*), and Cm (=∆*G 0 <sup>D</sup>/m*) where ∆*G* 0 *<sup>D</sup>* is the Gibbs free energy change without the denaturants, *m* is

*(∂*∆*GD/∂[urea/GdmCl*]), and Cm is the transition midpoint of chemical-induced denaturation curve where ∆*G<sup>D</sup>* = 0. The analysis was done based on the least-square method to fit the denaturation curve using the following Equation (1):

$$y = \frac{yN + yD \times Exp[-(\Delta G^\circ \,\_D - m[\murea/Gdm\text{Cl}])/\text{RT}]}{(1 + Exp[-(\Delta G^\circ \,\_D - m[\murea/Gdm\text{Cl}]/\text{RT}])} \tag{1}$$

where *yN* and *yD* are the estimated optical properties of the native protein and the denatured protein, respectively, under the similar experimental condition in which y has been measured, *T* is the temperature in Kelvin, and *R* is the universal gas constant.
