Photophysics and Fluorimetric Titrations of Fluorescent Ion Indicators

A test based on time-resolved fluorescence experiments (Anal. Biochem.245, 28–37, 1997) allows one to assess the interference of the excited-state association with the fluorimetric determination of the ground-state dissociation constant K_d of fluorescent ion:indicator complexes. If an inflection point occurs in the plot of the fluorescence signal vs – log[ion] in the ion concentration range where both decay times are invariant, this inflection point can be associated with the correct K_d. Here we apply this test to the fluorescent ion indicators SBFO (for Na⁺), Mag-fura-2 (for Mg²⁺), and APTRA-BTC (for Ca²⁺). In all three cases the decay times are invariant in the concentration ranges of the respective ions where the fluorescence titrations show unique inflection points, indicating that the fluorimetrically determined K_d values are the true K_d values.