DIFFERENCES IN THE PROCESSES OF BETA-LACTOGLOBULIN COLD AND HEAT DENATURATIONS

The changes in P-lactoglobulin upon cold and heat denaturation were st udied by scanning calorimetry, CD, and NMR spectroscopy. It is shown t hat, in the presence of urea, these processes of beta-lactoglobulin de naturation below and above 308 K are accompanied by different structur al and thermodynamic changes. Analysis of the NOE spectra of beta-lact oglobulin shows that changes in the spin diffusion of beta-lactoglobul in after disruption of the unique tertiary structure upon cold denatur ation are much more substantial than those upon heat denaturation. In cold denatured P-lactoglobulin, the network of residual interactions i n hydrophobic and hydrophilic regions of the molecule is more extensiv e than after heat denaturation. This suggests that upon cold- and heat -induced unfolding, the molecule undergoes different structural rearra ngements, passing through different denaturation intermediates. From t his point of view, cold denaturation can be considered to be a two sta ge process with a stable intermediate. A similar equilibrium intermedi ate can be obtained at 35 degrees C in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of the intermediate. However , these processes differ in cooperativity, showing noncommensurate sig moidal-like changes in efficiency of spin diffusion, ellipticity at 22 2 nm, and partial heat capacity. The disruption with cooling is accomp anied by cooperative changes in heat capacity, whereas with heating th e heat capacity changes only gradually. Considering the sigmoidal shap e of the heat capacity change an extended heat absorption peak, we pro pose that the intermediate state is stabilized by enthalpic interactio ns.