Potential misevaluation of the ground-state dissociation constant from fluorimetric titrations: Application to the ion indicators SBFI, PBFI, and Fura-2

A test based on time-resolved fluorescence experiments is proposed to assess the interference of an excited-state reaction with the fluorimetric determination of the ground-state dissociation constant K-d of ion subset of fluorescent indicator complexes. If an inflection point occurs in the plot of the fluorescence signal vs -log [ion] in the concentration range of the ion where both decay times are invariant, this inflection point can be associated With the correct K-d. In contrast, the inflection point(s) in the concentration range where the decay times vary cannot be attributed to K-d. The test is applied to the fluorescent ion indicators SBFI (for Na+), PBFI (for K+), and Fura-2 (for Ca2+). In all three cases the decay times are invariant in the physiological concentration ranges of the respective ions, indicating that the fluorimetrically determined K-d values are actually the true K-d values. (C) 1997 Academic Press.