Thermal unfolding of bovine β-lactoglobulin studied by UV spectroscopy and fluorescence quenching

In spite of numerous physical and biochemical studies on thermal denaturation of bovine β-lactoglobulin (β-LG), the definition of the N- and U-states, and the extent of reversibility assigned to the process are not clearly determined. The application of two-state model and dissociation coupled unfolding model to interpret the thermal unfolding profiles obtained by different authors using UV-difference absorbance at 293 nm, was analyzed and discussed. We conclude that both models fail to describe correctly the β-LG thermal unfolding process in the conditions assayed in these previous works (at protein concentrations below 4 mg mL−1, at low ionic strength and near neutral pH). The probable causes of this failure are: (1) below 55 °C, β-LG solutions are a mixture of monomers and dimers, (2) between 55 and 90 °C, β-LG heating induces irreversible changes in the monomeric protein structure and promotes the formation of aggregates of low molecular weight, and (3) above 90 °C, β-LG aggregates of high molecular weight are formed irreversibly. In this work, the presence of irreversible changes in β-LG heating was demonstrated by quenching the intrinsic fluorescence of the protein by added acrylamide.