Thermal aggregation of α-chymotrypsin: Role of hydrophobic and electrostatic interactions

We have recently reported that electrostatic interactions may play a critical role in alcohol-induced aggregation of α-chymotrypsin (CT). In the present study, we have investigated the heat-induced aggregation of this protein. Thermal aggregation of CT obeyed a characteristic pattern, with a clear lag phase followed by a sharp rise in turbidity. Intrinsic and ANS fluorescence studies, together with fluorescence quenching by acrylamide, suggested that the hydrophobic patches are more exposed in the denatured conformation. Typical chaperone-like proteins, including α- and β-caseins and α-crystalline could inhibit thermal aggregation of CT, and their inhibitory effect was nearly pH-independent (within the pH range of 7-9). This was partially counteracted by α-, β- and especially γ-cyclodextrins, suggesting that hydrophobic interactions may play a major role. Loss of thermal aggregation at extreme acidic and basic conditions, combined with changes in net charge/pH profile of aggregation upon chemical modification of lysine residues are taken to support concomitant involvement of electrostatic interactions.