INCREASED THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF AMINO-ACIDS

This study is a systematic attempt to understand the roles of osmolytes in protecting proteins against denaturing stress. Thermal denaturation of cytochrome c has been studied in the presence of various concentrations of all L-amino acids that are more hydrophobic than glycine and have a solubility of 0.1 M or higher in water at 25 degrees C. The basic observations are as follows. (1) Arginine and histidine destabilize the native protein; both T-m (the midpoint of thermal transition) and Delta G(D)(H2O) (25 degrees C) (the Gibbs energy of stabilization) decrease with increasing amino acid concentration. (2) Isoleucine, leucine and phenylalanine have no effect on T-m and Delta G(D)(H2O) (25 degrees C). (3) Valine and less hydrophobic amino acids stabilize the protein in terms of T-m but Delta G(D)(H2O) (25 degrees C) is unchanged. This observation was confirmed by the study of isothermal denaturation of cytochrome c by guanidinium chloride which suggested that Delta G(D)(H2O) is independent of osmolyte concentration, but C-m (the midpoint of transition) is increased in their presence. (4) In the case of stabilizers, change in T-m/mol of amino acid decreases with increasing hydrophobicity of these osmolytes.