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.