A calorimetric study of solute effects on the kinetic stability of α-amylase

In this study we evaluated the applications of isothermal titration calorimetry (ITC) to study solute effects on the kinetics of irreversible protein denaturation. More specifically, denaturation of Bacillus Halmapalus α-amylase (BHA) was initiated by addition of EDTA to the calorimetric cell, which reduces the thermostability of the protein from marginally stable to unstable at the experimental temperature, by removing bound calcium ions. The calorimetric signal was shown to be proportional to the unfolding rate of the protein, since, ΔHagg was shown to be close to zero. Comparison of ITC with chromatographic size exclusion data (SEC) provided an avenue to study unfolding and aggregation separately, which proved to be useful in analysing the mechanism of solute effects on denaturation kinetics. Solute effects are discussed in line with preferential interactions and Wyman linkage function.