MATERIALS AND METHODS

Materials-Hen egg-white lysozyme, recrystallized six times, was purchased from Seikagaku Kogyo. Bovine pancreas RNase A (Type IIA) was obtained from Sigma. Both proteins were completely deionized by exhaustive dialysis against distilled water and lyophilized before use. GdnHCl was an ultrapure product from Schwarz/Mann. D-Sorbitol was a reagent grade product from Merck AG. Glycerol and other chemicals were special reagent grade products from Wako Pure Chemicals. Circular Dichroism Measurements-The conformational change of a protein induced by GdnHCl was followed by means of CD measurement with a Jasco J-40A spectro polarimeter. The spectropolarimeter was calibrated with d-10-camphorsulfonic acid. Quartz cells with pathlengths of 1 and 5mm were used for CD measurements in the cases of lysozyme and RNase A, respectively. The temperature of the solution in a cell was kept at 25.0± 0.1 C by circulat ing water in the cell holder.

Protein solutions with different concentrations of Gdn HCl and polyol were prepared as follows. First, concen trated stock protein and polyol solutions were prepared by dissolving them in water adjusted with HCl to pH 2.0 for lysozyme and to pH 2.8 for RNase A. The required amount of solid GdnHCl, which was predetermined from the refractive index data (12), was weighed into a 5 ml flask and then dissolved in a known amount(2.5 ml) of a stock polyol solution. Then a given amount(0.5 ml) of a stock protein solution was added, and, finally, the flask was filled up with a small amount of the same HCl solution as used for preparing the stock protein solutions. Thus, 15-18 sample solutions with different GdnHCl concentrations were made with identical polyol (0-30%, w/v) and protein(0.033% for lysozyme and 0.02% for RNase A) concentrations. The mixtures were incubated at 25• Ž for 24h for complete denaturation equilibrium. Each sample solution was intro duced into a quartz cell and then the ellipticity at 222nm was recorded as a function of the GdnHCl concentration. The CD readings were expressed as the mean residue molar ellipticity,[0], assuming a residue molecular weight of 110 for both proteins.