Enthalpy and heat capacity changes for the reduction of insulin

The enthalpy changes for the reduction of three disulfide bonds of insulin by dithiothreitol (DTT) were calorimetrically measured at various temperatures ranging from 289 to 308 K. The reduction was performed in three different buffer solutions of pH 9.6, and the observed heat changes were corrected for the ionization heats of the buffer components to obtain the net heats of reduction of insulin with DTT. By subtracting the enthalpy of DTT oxidation reported in the previous paper [Fukada, H., & Takahashi, K. (1980a) J. Biochem. (Tokyo) 87, 1105-1110], we determined the standard enthalpy of reduction of insulin to be delta Hr = 93.4 +/- 7.8 kJ (mol of insulin-1 at 298 K. The heat capacity change with delta Cp,r = 3.2 +/- 0.3 kJ mol-1 K-1. Using the heat of oxidation of the cysteine residue, we estimated the enthalpy change for the conformational transition of insulin induced by the cleavage of three disulfide bonds to be delta H conf = 91 kJ mol-1 at 298 K. The heat capacity change was 2.1 kJ mol-1 K-1. These results imply that the conformational transition taking place during the reduction of three disulfide bonds is thermodynamically of the same nature as the thermal denaturation observed for other globular proteins.