Calorimetry of some trypsin-trypsin inhibitor reactions.

The heats of reaction of trypsin with soybean (Kunitz), ovomucoid, and lima bean inhibitors have been measured at pH 5.0 and 10’ and 25”. All the reactions were endothermic, with AH ranging from 8.6 Cal per mole for the lima bean inhibitor to 15.3 Cal per mole for the soybean inhibitor. Equilibrium constants for the association were calculated from the dependence of residual tryptic activity upon added inhibitor. Free energy changes ranged from -9.8 to -12.7 Cal per mole, and AS varied from 48 to 79 cal per mole degree. The change in heat capacity, calculated from (AHZg8 AH2&/15, varied from -263 to -442 cal per mole degree. The reactions are in general characterized by large negative AG”, moderate positive AH, large positive AS, and moderately large -ACp. The thermodynamic quantities for conversion of native soybean inhibitor to its state in which the arg 64-ile 65 peptide bond is cleaved were calculated as AGZ,, = -1400 cal per mole, AH 298 = -4040 cal per mole, AS298 = -8.8 cal per mole degree, and ACp = -55 cal per mole degree. The results for complex formation and for conversion of native soybean inhibitor to its hydrolyzed form are thought to indicate that interactions other than those directly involved in changes in covalent bonding are thermodynamically significant.