Differential scanning calorimetry (DSC) of the thermal denaturation of wild-type phytase and the disulfide variants.

A) Representative DSC profiles at a protein concentration of 0.5 mg/mL and a scan rate of 200 degrees/hour. B) Plot of transition temperature (derived from the results in panel A) versus number of engineered bridges. C) Plot of degree of reversibility of the thermal denaturation process versus the number of engineered bridges. The degrees of reversibility shown were derived from DSC experiments performed with a protein concentration of 0.25 mg/mL, at a scanning rate of 200°C /hour. In all cases, the first scan was stopped at 100°C and a re-heating run was performed after letting the sample cool in the calorimetric cell. The degree of reversibility was calculated as the ratio between the maximum heat capacity values for the reheating and the first transitions after suitable chemical baseline correction. D) Degree of reversibility for the variant with three engineered bridges versus protein concentration. E) Scan-rate effect on the transition temperatures (circles) derived from DSC experiments. Extrapolation to 1/v = 0 (i.e., to infinite scan-rate) should remove kinetic distortions associated to partial irreversibility (the squares at 1/v = 0 actually represent the denaturation temperatures for equilibrium unfolding determined from model fitting to rate data). In panels B–E the colors refer to the different variant studied as specified in panel A.