Smith G A, Hesketh R T, Metcalfe J C, Feeney J, Morris P G
Proc Natl Acad Sci U S A. 1983 Dec;80(23):7178-82. doi: 10.1073/pnas.80.23.7178.
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.
1,2 - 双（邻氨基苯氧基）乙烷 - N，N，N′，N′ - 四乙酸（nFBAPTA）的对称取代二氟衍生物在螯合二价阳离子时会出现较大的¹⁹F核磁共振化学位移。Ca²⁺与4FBAPTA和5FBAPTA形成的配合物分别表现出快速和慢速交换行为，游离态和配合态的化学位移或共振峰面积可用于测定游离Ca²⁺浓度。使用这两种配体测定游离Ca²⁺浓度不受游离Mg²⁺浓度低于10 mM、pH值在6 - 8范围内或高亲和力污染二价离子（Zn²⁺、Fe²⁺、Mn²⁺）的影响。5FBAPTA的四乙酰氧基甲酯衍生物用于将5FBAPTA载入小鼠胸腺细胞，使其细胞内浓度达到1 mM，¹⁹F光谱表明细胞内游离Ca²⁺浓度[Ca]i为250 nM。在促有丝分裂浓度下添加琥珀酰化伴刀豆球蛋白A可使[Ca]i增加到350 nM，添加A23187可使细胞内螯合剂与细胞外介质中的Ca²⁺饱和。该方法可测量[Ca]i和其他二价阳离子浓度，并能直接鉴定螯合的离子种类。
