DETERMINING STABILITY OF PROTEINS FROM GUANIDINIUM CHLORIDE TRANSITION CURVES

The guanidinium chloride (GdmCl) denaturation of RNAase A, lysozyme and metmyoglobin was investigated at several pH values by using absorbance measurements at 287, 300 and 409 nm respectively. From these measurements the free-energy change on denaturation, DELTAG(app.), was calculated, assuming a two-state mechanism, and values of DELTAG(app.) at zero concentration of the denaturant were measured. For each protein all DELTAG(app.) values were adjusted to pH 7.00 by using the appropriate relationship between DELTAG(app.) and pH. Dependence of the adjusted DELTAG(app.) value on GdmCl concentration increases for metmyoglobin and decreases for the other two proteins as the denaturant concentration decreases. It has been shown that these are expected results if the presence of the acid-denatured state during the GdmCl denaturation of proteins is considered.