Analysis of fluorometric titration curves

The fluorometric determination of the ground-state dissociation constant Kd of a complex between ligand and titrant with 1:n stoichiometry in the presence and absence of excited-state association is discussed. This report extends the results of a previous study (J. Phys. Chem. 1994, 98, 8585) where a 1:1 complex between ligand and titrant was assumed. For that case, it was demonstrated that adequate experimental design, i.e., by monitoring the fluorescence at the isoemissive point, could eliminate the interference of the excited-state association. In this report we use the direct parametric fit of the fluorometric titration to recover reliable estimates for Kd. It is shown that in the presence of the excited-state association this approach yields the unique value of Kd if a priori knowledge of the excited-state rate constants is available. This method also offers an effective way for discriminating between competitive models (presence and absence of the excited-state association reaction with non 1:1 stoi...