Thermal stability of proteins analyzed by temperature scanning pH-stat titration

Based on the fact that pH changes occur during the thermal unfolding of a protein, a pH-stat titrimetric procedure is described for the analysis of thermal stability. In all cases the agreement with other stability measurements was good, including a correlation with activity loss in enzymes. A model for the titration curves, assuming first-order denaturation kinetics, linear temperature increase, and validity of the Arrhenius equation, has been proposed and analyzed. Thus, thermodynamic constants can be calculated from tritration curves, or transition temperatures estimated if the Arrhenius constants are known. The equipment consists of a pH-stat, a programmable heating unit, and a temperature measuring/recording system. Analysis can be done quickly and on partially purified solutions, provided the buffer capacity is low, using about 20 mg protein/10 ml sample. The effects of pH, Ca²⁺ ions, substrate, chemical modification, etc., on thermal stability are conveniently analyzed up to about 90°C.