COMPACTNESS OF THERMALLY AND CHEMICALLY DENATURED RIBONUCLEASE-A AS REVEALED BY VOLUME AND COMPRESSIBILITY

The conformational changes of ribonuclease A due to thermal and guanid ine hydrochloride denaturation were monitored by means of precise dens ity and sound velocity measurements. It was found that the apparent mo lar volume decreased but the adiabatic compressibility increased on th ermal denaturation under acidic conditions (pHs 1.60, 1.90, and 2.08). On the other hand, guanidine hydrochloride denaturation (pH 2.00) bro ught about large decreases in the compressibility and apparent molar v olume. These results indicate that the conformation of the denatured p rotein is greatly different between the two types of denaturation: the thermally denatured state corresponds to the structure with enhanced thermal fluctuation having a residual secondary structure and a high l ocal concentration of nonpolar groups exposed, but the guanidine hydro chloride denaturation leads to exposure of a large amount of amino aci d residues, resulting in an increase in hydration and a decrease in th e internal cavity. The compressibility changes due to both types of de naturation were not correlated to a loss of the secondary structure, a s judged by means of circular dichroism. These findings suggest that t he compactness and thermal fluctuation of the protein cannot be descri bed by a two-state denaturation model and that there are some molten-g lobule-like intermediates in the denaturation processes.