The substituent effect on complex formation between alpha-trypsin and para-substituted benzamidinium ions: a...

1. Dissociation constants, Ki, were determined spectrophotometrically by measuring the absorbance at 410 nm, using N alpha-benzoyl-D,L-arginine-para-nitroanilide (Bz-D,L-Arg-Nan) as substrate. The Ki values for the complexes of alpha-trypsin with each of the para-derivatives of the benzamidinium ion -NH2, -CH3, -H, -F, -Cl, -Br, -COOEt, and -NO2 were measured at six temperatures (8, 15, 20, 25, 29 and 33 degrees C), in order to determine the thermodynamic parameters delta G0, delta H0, delta S0, and delta C0P for complex formation. 2. The standard enthalpy change (delta H0) was constant (-12.45 kcal/mol) and all other parameters were also negative. The large negative values obtained for the standard heat capacity change (delta C0P) suggest that the process occurs with a conformational adaptation in the enzyme structure. 3. The apparent partial specific volumes of free alpha-trypsin and alpha-trypsin bound to benzamidinium ion indicated that there is a decrease of approximately 0.10 cm3/g in the enzyme volume when the inhibitor binds. This contraction is consistent with the release of about 130 water molecules per enzyme molecule.