Thermodynamics of the Binding of Chymotrypsin with the Black-eyed Pea Trypsin and Chymotrypsin Inhibitor (BTCI) |
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Authors: | Sonia M de Freitas Hiroaki Ikemoto and Manuel M Ventura |
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Institution: | (1) Departamento de Biologia Celular, Universidade de Brasília, 70910-900 Brasília, DF, Brazil |
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Abstract: | The binding of -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: H
vh
°
= 10.8 kcal mol-1 (from inhibitory assays) and 11.1 kcal mol–1 (from fluorescence polarization); S° = 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 H
cal
°
= 4.9 kcal mol-1. These results indicate that the binding of chymotrypsin to BTCI is an entropically driven process. |
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Keywords: | Bowman-Birk inhibitor microcalorimetry enthalpy fluorescence polarization |
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