CALORIMETRIC INVESTIGATIONS OF BINDING OF INHIBITORS TO ALPHA-CHYMOTRYPSIN .I. ENTHALPY OF DILUTION OF ALPHA-CHYMOTRYPSIN AND OF PROFLAVIN, AND ENTHALPY OF BINDING OF INDOLE . N-ACETYL-D-TRYPTOPHAN, AND PROFLAVIN TO ALPHA-CHYMOTRYPSIN

A flow microcalorimeter has been employed to measure the enthalpies of binding of indole, N-acetyl-D-tryptophan, and proflavin to α-chymotrypsin at pH 7.8 and also the heats of dilution of α-chymotrypsin and proflavin at the same pH. The following points are discussed. (1) The studies of the heats of dilution of α-chymotrypsin as a function of enzyme concentration lead to the suggestion that monomer-dimer equilibrium of this enzyme exists at pH 7.8. The calculated thermodynamic functions for this equilibrium are consistent with the view that the dimeric forms of the enzyme can be identified as the enzyme-substrate intermediates preceding the autolysis reaction. (2) The heat effects due to the dilution of proflavin are shown to be consistent with the assumption that the self-association of this compound can be adequately represented by a series of equilibria involving dimer, trimer, and other higher polymers. The large negative enthalpy of polymerization (-4.0 kcal/mole) shows that the self-association of proflavin is accompanied by an unfavorable entropy change. (3) The apparent heats of binding of inhibitors to α-chymotrypsin were observed to be strongly dependent on enzyme concentration. These observations lead to the suggestion that the dimeric forms of the enzyme are incapable of binding inhibitors. (4) The enthalpies of binding of indole, N-acetyl-D-tryptophan, and proflavin to α-chymotrypsin were observed to be -15.2, -19.0, and -11.3 kcal/mole, respectively, at pH 7.8. On the basis of these numbers it is concluded that conformational changes in the enzyme are induced by the binding of inhibitors. © 1969, American Chemical Society. All rights reserved.