Potential Misevaluation of the Ground-State Dissociation Constant from Fluorimetric Titrations: Application to the Ion Indicators SBFI, PBFI, and Fura-2

Fluorescence Lifetime Microscopy of the Sodium Indicator Sodium-Binding Benzofuran Isophthalate in HeLa Cells

“…The analysis of the decay curves revealed that the decays are biexponential, with decay times virtually independent of [Na ϩ ] in the concentration range 0 -145 mM. This means that the complex formation in the excited-state is negligible for the investigated concentration range (4). Consequently, the decays were analyzed simultaneously with a biexponential expression, with the decay times being linked over [Na ϩ ].…”

“…The measurements were performed in the absence of Na ϩ (ᮀ) and in the presence of 145 mM Na ϩ (s). 4 shows the emission spectra of SBFI in buffer solutions containing various concentrations of BSA, both in the absence of Na ϩ and in the presence of a saturating Na ϩ concentration (145 mM). Although such a high [Na ϩ ] is not reached inside the cytoplasm, the in vitro measurements at this [Na ϩ ] allow one to investigate the effects of proteins on the Na ϩ -bound form of the indicator.…”

“…When the emission is recorded at 510 nm, SBFI in pure buffer has an isoexcitation point at Ϸ360 nm (6), although a value of 375 nm has also been reported (3). The relative emission of free SBFI at 510 nm to that of the Na ϩ ʚSBFI complex is 0.18 (4). The photophysics of SBFI in simple buffer solution was recently described (4).…”

“…Na ϩ binds to SBFI in a 1:1 stoichiometry (1, 4). The ground-state dissociation constant K d is in the range 4.2-7.4 mM (1,4). The selectivity of Na ϩ over K ϩ is about 20 (1) and the effective K d in the presence of K ϩ is about 17 mM (1).…”

“…The relative emission of free SBFI at 510 nm to that of the Na ϩ ʚSBFI complex is 0.18 (4). The photophysics of SBFI in simple buffer solution was recently described (4). The fluorescence intensity decays of the indicator were analyzed in terms of an intermolecular two-state excited-state process.…”