Global analysis of fluorometric titration curves in the presence of excited-state association and quenching.
The fluorometric determination of the ground-state dissociation constant K(d) of a complex between ligand and titrant with 1:1 stoichiometry in the presence of excited-state association and quenching is discussed. This report extends the results of a previous study (Novikov, E.; Stobiecka, A.; Boens, N. J. Phys. Chem. A 2000, 104, 5388), where the direct parametric fit of the fluorometric titration was used to recover reliable estimates for K(d). Here, we show that in the presence of excited-state association and quenching the unique value of K(d) can be obtained from global analysis of four fluorometric titration curves measured at two emission wavelengths and two excitation wavelengths. The same identifiability criterion is applicable for systems where quenching can be neglected. The linked parameters in the global analysis are rational functions of the rate constants, independent of the excitation and emission wavelengths. The developed algorithms for the global parametric fit of fluorometric titration curves are explored using simulations.
Novikov
Eugene
E
Service Bioinformatique, Institut Curie, 26 Rue d'Ulm, Paris Cedex 05, 75248 France.
Boens
Noël
N