Thermodynamics of the unfolding of α-lactalbumin by guanidine thiocyanate

Abstract

The reversible unfolding of α-lactalbumin by guanidine thiocyanate has been studied at 25.0 °C by means of difference spectra and optical rotation measurements. At pH 7.35, two tryptophanyl residues buried in the interior of the protein in the native state were considered to be partly exposed on its surface in the unfolded state. The dependence of the equilibrium constant of the unfolding reaction in aqueous solutions of guanidine thiocyanate on pH can be described in terms of the abnormal histidyl residues and indicated the protein is in the most stable state at pH 7.30. The dependence on the denaturant activity, which was obtained by the isopiestic method, results in a similar difference between the numbers of the binding sites on the unfolded and the native protein molecules to that in the case of the unfolding by guanidine hydrochloride, a larger intrinsic binding constant of the denaturant with the protein than that of the guanidine hydrochloride, and a more unstable structure in the native state than that of lysozyme. Also the dependence on the denaturant concentration results in the higher degree of exposure of both peptide units and hydrophobic side chains in the native state to solvent than in lysozyme.