A Convenient Method for Determination of Dissociation Constants of Enzyme-Ligand Complexes Based on Difference Absorption Spectra. : A Model Case of Ribonuclease F1-Guanosine 2'-Monophosphate

A convenient method has been developed for investigating interaction of an enzyme with a specific ligand on the basis of the difference absorption spectrum. The experimental procedure has been described in detail. Briefly, to a solution of the enzyme, a concentrated solution of the ligand was added successively in small volume aliquots and the increase in absorbance after each addition was measured at two wavelengths which corresponded to a maximum and a crossover point of the difference absorption spectrum. Processing the obtained data gave the difference at the maximum wavelength as a function of the total ligand concentration. The data set thus generated was fitted to the equilibrium equation and the best fit values for the dissociation constant, K_d and the molar difference absorption coefficient at the maximum wavelength, Aemax, were determined. The method was successfully applied to ribonuclease F1-guanosine 2'-phosphate system and gave the following parameters: K_d=2.5 μM and Δε_<290>=3890 M^<-1>cm^<-1>.