Urea and Guanidine Hydrochloride Denaturation of Ribonuclease, Lysozyme, &Zhymotrypsin, and @Lactoglobulin*

The unfolding of ribonuclease, lysozyme, a-chymotrypsin, and goat P-lactoglobulin by urea and guanidine hydrochloride (GmHCl) has been followed with the use of optical rotation measurements. Urea denaturation leads to a more negative rotation for each protein than does GmHCl denaturation, but the concentration dependence is such that the rotations are almost identical in the absence of denaturant. This indicates that both urea and GmHCl denaturation lead to a similar, randomly~ coiled conformations By assuming a two-state mechanism, an apparent free energy of unfolding, AG &Pp, has been calculated as a function of denaturant concentration. AG,,, varies linearly with denaturant concentration. The dependence of AG,,, on concentration ranges from 1.1 to 2.1 Cal per mole per molar concentration for urea and from 1.9 to 4.1 Cal per mole per molar concentration for GmHCl for the four proteins. These values depend on the size and composition of the part of the poly peptide chain which is freshly exposed to denaturant by unfolding. By using model compound data and a procedure developed by Tanford (Adunn. Protein Chem. (1970) 24, l-95), an estimate of the amount of buried polypeptide chain needed to account for the experimental results has been obtained. The estimates based on urea and on GmHCl are in excellent agreement. This offers further support for the idea that the extent of unfolding is the same for the two denaturants and also suggests that the mechanism of unfolding is the same in the presence of the two denaturants. The relative effectiveness of the two denaturants depends on the protein. GmHCl is 2.8 times more effective than urea in unfolding ribonuclease but only 1.7 times more effective for lysozyme. This results because the buried polypeptide chain is more polar for ribonuclease than for lysozyme and the solubilizing ability of GmHCl relative to urea is greater for polar groups than for nonpolar groups. Estimates of AG,,, in the absence of denaturant, AGf$f, were obtained from the analysis with the use of model compound data and also from a direct linear extrapolation of ‘=a,, to zero concentration of denaturant. For urea, the two approaches lead to values of AG$z in reasonably good agreement. For GmHCl, only the values of AGF;: from the direct extrapolation are in good agreement with the