Thermodynamics of the Binding of Chymotrypsin with the Black-eyed Pea Trypsin and Chymotrypsin Inhibitor (BTCI)

Abstract

The binding of α-chymotrypsin to black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) has been studied using the inhibitory activity against the enzyme and the formation of the complex enzyme/inhibitor followed by measurements of fluorescence polarization. Apparent equilibrium constants were estimated for several temperatures and the values obtained range from 0.32 × 10⁷ to 1.36 × 10⁷ M⁻¹. The following values were found from van't Hoff plots: Δ H ^°_vh = 10.8 kcal mol^-1 (from inhibitory assays) and 11.1 kcal mol⁻¹ (from fluorescence polarization); ΔS° = 67.9 and = 67.8 kcal K⁻¹ mol⁻¹, respectively. Calorimetric binding enthalpy was determined (corrected for the ionization heat of the buffer) and the resulting value was ΔH ^°_cal = 4.9 kcal mol^-1. These results indicate that the binding of chymotrypsin to BTCI is an entropically driven process.