The Effect of Stabilizers and Denaturants on the Cold Denaturation Temperatures of Proteins and Implications for Freeze-Drying

Purpose. The aim of the study is to investigate the effects of stabilizers and denaturants on the thermal and cold denaturation temperatures of selected proteins in systems of interest to freeze-drying. Methods. β-Lactoglobulin and phosphoglycerate kinase (PGK) were chosen as model proteins. Protein thermal and cold denaturation temperatures were determined by both conventional and modulated differential scanning calorimetry and verified by tryptophan emission spectroscopy in selected systems. Results. The cold denaturation of p-lactoglobulin was reversible, whereas the thermal denaturation was only reversible at high scanning rate (10°C/min). The cold denaturation temperatures of p-lactoglobulin decreased with an increase in protein concentration (self-stabilization). The cold denaturation temperature increased with increases in pH (from pH 2 to 7) with about 4.6°C increase per unit pH change. All stabilizers studied (i.e., sucrose, trehalose and glycerol) increased the thermal denaturation temperature of the proteins studied and decreased the cold denaturation temperature. The effect of sucrose in decreasing the PGK cold denaturation temperature [40°C per molar concentration increase (40°C/M)] was of the same magnitude as for p-lactoglobulin (36°C/M). The effect of stabilizers on cold denaturation temperatures is much greater than the effect on thermal denaturation temperatures. With sucrose, the p-lactoglobulin thermal denaturation temperature increases only about 5°C from 0 to 2.7 M, whereas the decrease in cold denaturation temperature was more than 35°C even at sucrose concentrations as low as 0.9 M. Denaturants (urea and guanidine hydrochloride) increased the cold denaturation temperatures of proteins and thereby destabilized protein; the magnitudes were 9°C/M (urea on T c d of p-lactoglobulin) and 65°C/M (guanidine hydrochloride on PGK) compared with literature data of 16°C/ M (guanidine hydrochloride on p-lactoglobulin). The cold denaturation temperatures of p-lactoglobulin and PGK extrapolated to zero concentration of denaturants were -14 and -26°C, respectively. Conclusions. The protein cold denaturation temperature was pH-, protein concentration-, and additive-dependent. Stabilizers, such as sugars and/or polyols, can stabilize both protein thermal and cold denaturation, whereas the denaturants destabilize protein cold denaturation. The stabilization effect on protein cold denaturation is much larger than on thermal denaturation, a result of great importance in protein freeze-drying.