INCREASED THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF NATURALLY-OCCURRING OSMOLYTES

Organisms and cellular systems which have adapted to stresses such as high temperature, desiccation, and urea-concentrating environments have responded by concentrating particular organic solutes known as osmolytes. These osmolytes are believed to confer protection to enzyme and other macromolecular systems against such denaturing stresses. Differential scanning calorimetric (DSC) experiments were performed on ribonuclease A and hen egg white lysozyme in the presence of varying concentrations of the osmolytes glycine, sarcosine, N,N-dimethylglycine, and betaine. Solutions containing up to several molar concentrations of these solutes were found to result in considerable increases in the thermal unfolding transition temperature (T(m)) for these proteins. DSC scans of ribonuclease A in the presence of up to 8.2 M sarcosine resulted in reversible two-state unfolding transitions with T(m) increases of up to 22-degrees-C and unfolding enthalpy changes which were independent of T(m). On the basis of the thermodynamic parameters observed, 8.2 M sarcosine results in a stabilization free energy increase of 7.2 kcal/mol for ribonuclease A at 65-degrees-C. This translates into more than a 45 000-fold increase in stability of the native form of ribonuclease A over that in the absence of sarcosine at this temperature. Catalytic activity measurements in the presence of 4 M sarcosine give k(cat) and K(m) values that are largely unchanged from those in the absence of sarcosine. DSC of lysozyme unfolding in the presence of these osmolytes also results in T(m) increases of up to 23-degrees-C; however, significant irreversibly occurs with this protein. Naturally occurring glycine-based osmolytes appear to provide a general method of stabilizing proteins against thermal unfolding even well beyond the physiological concentration range for osmolyte, and the degree of stabilization can be extraordinary.