Calorimetric studies on renaturation by CaCl2 addition of metal-free α-amylase from Bacillus Licheniformis (BLA)

In our previous work we characterized the Ca binding properties of BLA by analysis of Ca-induced renaturation in the presence of urea. In this study we focused on the renaturing capacity of CaCl2 in the absence of urea and analysed the apparent thermodynamic and kinetic properties of renatured BLA by DSC, CD and dynamic light scattering (DLS) measurements. Ca-free protein did not return fully to the native state even after extensive dialysis against high concentrations of calcium. Thus Ca removal provokes changes in protein structure that can not be reversed completely even by addition of an excess of calcium. However, activity studies performed simultaneously with the DSC experiments showed a significant return of enzymatic activity despite the failure of complete return of native stability. This conclusion is in excellent agreement with findings of Machius et al. who suggested on the basis of their X-ray studies that it is not possible to remove CaI and CaII without introducing significant structural changes. It is important to note that all unfolding reactions of metal-free, partially renatured and native protein were found to be irreversible. Therefore the DSC transition curves were fitted using irreversible transition models. It turned out that the apparent heat capacity profiles of partially renatured BLA could be well represented by superposition of two irreversible processes each following the two-state irreversible model.