Abstract
We have carried out guanidinium chloride (GdmCl) and urea denaturations of bovine β-lactoglobulin A (β-lgA) at pH 2.0 and 25 °C, using far-UV and near-UV circular dichroism, near-UV absorption and tryptophan fluorescence spectroscopies. The stable intermediate state that occurs during GdmCl denaturation has been characterized by the far- and near-UV circular dichroism, tryptophan difference absorption, tryptophan fluorescence and 8-anilino-1-naphthalene sulphonic acid binding measurements. Following conclusions have been reached. (a) Urea-induced denaturation is not a two-state process. (b) GdmCl-induced denaturation is composed of two distinct two-state processes. (c) α-Helical content, burial of tryptophan residues and burial of hydrophobic surface area are more in the GdmCl-induced stable intermediate than those originally present in the native protein.