Intracellular calcium measurements by 19F NMR of fluorine-labeled chelators.

Symmetrically substituted difluoro derivatives of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (nFBAPTA) show large 19F NMR chemical shifts on chelating divalent cations. The complexes of Ca2+ with 4FBAPTA and 5FBAPTA show fast and slow exchange behavior, respectively, and the chemical shift or the areas of the resonances from the free and complexed forms can be used to determine the free Ca2+ concentration. The measurement of the free Ca2+ concentration by either ligand is unaffected by free Mg2+ concentrations less than 10 mM, by pH 6-8, or by contaminating divalent ions of high affinity (Zn2+, Fe2+, Mn2+). The tetraacetoxymethyl ester derivative of 5FBAPTA was used to load mouse thymocytes with 5FBAPTA to intracellular concentrations of 1 mM, and the 19F spectrum indicated a free intracellular Ca2+ concentration [( Ca]i) of 250 nM. The [Ca]i was increased to 350 nM by addition of succinylated concanavalin A at mitogenic concentrations, and the addition of A23187 saturated the intracellular chelator with Ca2+ from the external medium. The method provides a measurement of [Ca]i and other divalent cation concentrations with direct identification of the ionic species chelated.