Independent heat stabilization of proteases associated with multiheaded inhibitors. Complexes of chymotrypsin, subtilisin and trypsin with chicken ovoinhibitor and with lima bean protease inhibitor

Abstract

The heat stabilization resulting from specific association of serine proteases with either of two multiheaded protease inhibitors, chicken ovoinhibitor or lima bean protease inhibitor, was determined at pH 6.7 in a differential scanning calorimeter. The 2 : 1 complex of either bovine α-chymotrypsin or subtilisin BPN with ovoinhibitor showed two major denaturation endotherms; each 1 : 1 complex showed one major endotherm. Association with ovoinhibitor increased the kinetic thermal stabilities over those of the free chymotrypsin or subtilisin. Association with lima bean protease inhibitor stabilized bovine β-trypsin > porcine β-trypsin > bovine α-chymotrypsin. Complexes having different proteases bound to the same inhibitor, such as chymotrypsin · ovoinhibitor · subtilisin (1 : 1 : 1) or trypsin · inhibitor · chymotrypsin (1 : 1 : 1), denatured like mixtures of the 1 : 1 complexes. These results show more clearly that 2 : 1 association with multiheaded inhibitors stabilizes the two bound protease molecules independently. Each bound protease and the domain(s) of the inhibitor influenced by specific binding of this protease are denatured as a unit. Thus, 2 : 1 complexes comprise at least two new denaturing units. The extent of heat stabilization appears roughly proportional to the K assoc determined by other methods. The results are consistent with other evidence that binding sites for proteases on multi-headed inhibitors are relatively independent in structure and function.