High stability to irreversible inactivation at elevated temperatures of enzymes covalently modified by hydrophilic reagents: alpha-Chymotrypsin.

Based on the idea that proteins can be stabilized by a decrease in the thermodynamically unfavorable contact of the hydrophobic surface clusters with water, alpha-chymotrypsin (CT) was acylated with carboxylic acid anhydrides or reductively alkylated with aliphatic aldehydes. Modification of CT with hydrophilic reagents leads to 100-1000-fold increase in stability against the irreversible thermoinactivation. The correlation holds: the greater the hydrophilization increment brought about by the modification, the higher is the protein thermostability. After some limiting value, however, a further increase in hydrophilicity does not change thermostability.It follows from the dependence of the thermoinactivation rate constants on temperature that for hydrophilized CT there is the conformational transition at 55-65 degrees C into an unfolded state in which inactivation is much slower than that of the low-temperature conformation. The thermodynamic analysis and fluorescent spectral data confirm that the slow inactivation of hydrophilized CT at high temperatures proceeds via a chemical mechanism rather than Incorrect refolding operative for both the native and low-temperature form of the modified enzyme. Hence, the hydrophilization stabilizes the unfolded high-temperature conformation by eliminating the incorrect refolding.