Commercially lyophilized preparation of Ribonuclease-A (type III-A) from bovine pancreas (RNase-A) was purchased from Sigma Chemical Co. Trimethylamine N-oxide (TMAO), betaine, sarcosine, proline, glycine, β-alanine and cytidine 2′–3′ cyclic monophosphate (C>p) were also purchased from Sigma Chemical Co. Guanidinium chloride (GdmCl) and urea were obtained from MP Biomedicals. These and other chemicals, which were of analytical grade, were used without further purification.
RNase-A solution was dialyzed extensively against 0.1 M KCl solution at pH 7.0 and 4°C. Protein stock solution was filtered using 0.22 µm millipore syringe filter. The protein gave a single band during polyacrylamide gel electrophoresis. Concentration of the protein solution was determined experimentally using molar absorption coefficient, ε (M−1 cm−1) value of 9800 at 277.5 nm [21] (link). The concentrations of GdmCl and urea stock solutions were determined by refractive index measurements [22] (link). All solutions for optical measurements were prepared in the degassed 0.05 M cacodylic acid buffer containing 0.1 M KCl. Since pH of the protein solution may change upon addition of the osmolytes, pH of each solution was measured after the denaturation and refolding experiments. It was observed that the change in pH was not significant (∼0.02–0.04).
RNase-A solution was dialyzed extensively against 0.1 M KCl solution at pH 7.0 and 4°C. Protein stock solution was filtered using 0.22 µm millipore syringe filter. The protein gave a single band during polyacrylamide gel electrophoresis. Concentration of the protein solution was determined experimentally using molar absorption coefficient, ε (M−1 cm−1) value of 9800 at 277.5 nm [21] (link). The concentrations of GdmCl and urea stock solutions were determined by refractive index measurements [22] (link). All solutions for optical measurements were prepared in the degassed 0.05 M cacodylic acid buffer containing 0.1 M KCl. Since pH of the protein solution may change upon addition of the osmolytes, pH of each solution was measured after the denaturation and refolding experiments. It was observed that the change in pH was not significant (∼0.02–0.04).