Binding of alkylamines to trypsin as measured by difference spectroscopy

The effect of n-propylamine, n-butylamine and n-pentylamine on trypsin at pH 5.4, at 25 degrees and 7 degrees C was investigated by the difference spectroscopic technique. The difference spectra were found to be similar for all three alkylamines tested. The difference between the absorbance minimum near 290 nm and the absorbance maximum near 297 nm taken as the measure of perturbation, the following values were obtained for the dissociation constant of the enzyme-ligand complex as calculated from the dependence of this perturbation upon alkylamine concentration at 25 degrees: propylamine, 6.0 X 10(-3) M; butylamine, 3.0 X 10(-3) M; pentylamine, 4.3 X 10(-4) M; and at 7 degrees: butylamine: 5.6 X 10(-3) M the chromophores situated in the active centre. It seems probable that the binding of an alkylamine gives rise to a conformational change which results in the rotation of Tyr-228 and Trp-215 residues towards the hydrophobic surroundings. This dislocation is a part of the minute change that leads to the activation of the catalytic site.