Equilibrium and kinetics of the unfolding of α-lactalbumin by guanidine hydrochloride

Abstract

The reversible unfolding of α-lactalbumin by guanidine hydrochloride has been studied at 25.0°C by means of difference spectra and optical rotation. At about pH 5.50, two tryptophanyl residues buried in the interior of the native protein were considered to be exposed on its surface in the denaturated state. The dependence of the equilibrium constant of the unfolding reaction in aqueous solutions of guanidine hydrochloride on pH can be described in terms of abnormal histidyl residues. The dependence on guanidine hydrochloride concentration results in a smaller intrinsic binding constant of the denaturant with the protein, a smaller difference between the numbers of the sites on the denaturated and the native protein molecules, and a more unstable structure in the native state, than for lysozyme. On the other hand, from kinetic measurements, the unfolding was considered to be an apparent two-state transition. Apparent rate constants and half-times of the transition suggest a faster transition than in the case of lysozyme.