Use of fluorescence methods to monitor unfolding transitions in β-lactoglobulin

The degree of exposure of tryptophanyl residues (Trp) in beta-lactoglobutin (beta-LG) molecules can be evaluated by following the external quenching of the intrinsic protein fluorescence by added acrylamide. Based on this technique, we propose a method to monitor beta-LG equilibrium denaturation profile by urea. The results were analyzed by the dissociation coupled unfolding (DCU) model. This model takes into account the impact of dimerization on beta-LG stability. The values of free energy change for denaturing beta-LG (DeltaGdegrees(DCU)) obtained in this work were 63.3 (+/-0.5) kJ mol(-1) at pH 6.8 and 73.4 (+/-2.3) kJ mol(-1) at pH 2.5. These results are in good agreement with previous results reported by other authors, that monitored the denaturation process by ultraviolet difference spectrophotometry. In addition, the protein dependence of denaturation equilibrium profiles by urea followed by fluorescence polarization measurements suggests that beta-LG denatures by 3-state DCU process at both pH 6.8 and pH 2.5, with the dissociation of dimers preceding the unfolding of the monomers. (C) 2002 Elsevier Science Ltd. All rights reserved.