STUDIES BY UV SPECTROSCOPY OF THERMAL-DENATURATION OF BETA-LACTOGLOBULIN IN UREA AND ALKYLUREA SOLUTIONS

The thermal denaturation of beta-lactoglobulin in aqueous solutions at three different pH's and in aqueous solutions of urea, methyl-, N,N'-dimethyl- and ethylurea was studied by UV spectroscopy. The UV-melting curves,were analyzed on the basis of two-state approximation to obtain the apparent equilibrium constant, K(app), and the apparent standard enthalpy of transition, DELTAH(app)0, for protein unfolding as a function of temperature. From K(app), calculations of DELTAG(app)0, as functions of temperature around transition temperature, T1/2, in urea and alkylurea solutions and different buffer solutions have been carried out. An increase in the observed transition temperature, T1/2, and the corresponding transition enthalpies, DELTAH(app)0, with decreasing pH or denaturant concentration indicate increased stability of protein in these conditions. However, comparison of DELTAH(app)0 with DELTAH(cal) shows that the thermal transition of beta-lactoglobulin in aqueous urea and alkylurea solutions is not a two-state process. It has also been observed that urea and all alkylureas cause a red shift in the absorbance spectrum of beta-lactoglobulin which increases with increasing denaturant concentration and decreasing pH. A similar increase in red shift of beta-lactoglobulin absorbance spectrum has also been observed with increasing temperature.