Calorimetric study of the reduction of the disulfide bonds in insulin

Calorimetric measurement was made on the reduction of the three disulfide bonds of insulin by dithiothreitol (DTT). The reaction was performed at 298 K in three different buffer solutions of pH 9.6. The observed heat changes were corrected for the enthalpy of proton release from the buffer components and the net heat of reaction of insulin with DTT was determined to be delta H0 = 51.5 +/- 1.4 kJ(mol insulin)-1. By subtracting the enthalpy of DTT oxidation (Fukada, H. & Takahashi, K. (1980) J. Biochem. 87, 1105--1110), the standard enthalpy of reduction of insulin was found to be delta H = 78.8 +/- 7.9 kJ(mol insulin)-1. Using the enthalpy change for reduction of random-coil proteins by dithioerythritol (Johnson, R.E., Adams, P., & Rupley, J.A. (1978) Biochemistry 17, 1479-1484), the enthalpy change associated with the conformational change of insulin alone was evaluated to be delta H = 74 +/- 2 kJ(mol insulin)-1 (or 13 J(g protein)-1, a value very similar to that observed for the denaturation of other globular proteins.