Photophysics and fluorimetric titrations of fluorescent ion

Abstract

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.