Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium-HCl, urea, and pH.

The stability of bovine lactoperoxidase to denaturation by guanidinium-HCl, urea, or high temperature was examined by differential scanning calorimetry (DSC) and tryptophan fluorescence. The calorimetric scans were observed to be dependent on the heating scan rate, indicating that lactoperoxidase stability at temperatures near Tm is controlled by kinetics. The values for the thermal transition, Tm, at slow heating scan rate were 66.8, 61.1, and 47.2 degrees C in the presence of 0.5, 1, and 2 M guanidinium-HCl, respectively. The extrapolated value for Tm in the absence of guanidinium-HCl is 73.7 degrees C, compared with 70.2 degrees C obtained by experiment; a lower experimental value without a denaturant is consistent with distortion of the thermal profile due to aggregation or other irreversible phenomenon. Values for the heat capacity, Cp, at Tm and Ea for the thermal transition decrease under conditions where Tm is lowered. At a given concentration, urea is less effective than guanidinium-HCl in reducing Tm, but urea reduces Cp relatively more. Both fluorescence and DSC indicate that thermally denatured protein is not random coil. A change in fluorescence around 35 degrees C, which was previously reported for EPR and CD measurements (Boscolo et al. Biochim. Biophys. Acta 1774 (2007) 1164-1172), is not seen by calorimetry, suggesting that a local and not a global change in protein conformation produces this fluorescence change. Copyright (c) 2010 Elsevier B.V. All rights reserved.