Revised stability constant, spectroscopic properties and binding mode of Zn(II) to FluoZin-3, the most common zinc probe in life sciences

2-[2-[2-[2-[bis(carboxylatomethyl)amino]-5-methoxyphenoxylethoxy]-4-(2,7-difluoro-3-oxido-6-oxo-4a,9a-dihydroxanthen-9-yl)anilino]acetate (FluoZin-3) is used very broadly in life sciences as intra- and extracellular Zn(II) sensor selective for Zn(II) over Co(II), Ca(II) and Mg(II) ions at their physiological concentrations. It has been used for determination of relative and absolute levels of exchangeable Zn(II) in cells and extracellular fluids. Despite its popularity, the knowledge of its acid/base and Zn(II) coordination abilities and of its spectroscopic properties remained very limited. Also the published conditional dissociation constant (K-C(d)) values at pH 7.4 are slightly discrepant, (15 nM or 8.9 nM). In this work we determined the K-C(d) for Zn(II) complexation by FluoZin-3 at pH 7.4 with nitrilotriacetic acid (NTA) as competitor using two independent methods: fluorimetry and UV-Vis spectroscopy. For the first time, we investigated FluoZin-3 alone and complexed with Zn(II) in the wide range of pH, determining the total of eight plc values from fluorescence spectra and from various regions of UV-Vis spectra. The validated values of K-C(d) (9.1 +/- 0.4 nM; -log(C)K(d) = 8.04) and of the absolute (pH-independent) stability constant log beta(znL) (8.16 +/- 0.05) were provided by fluorescence spectroscopy experiments performed at 1 mu M concentrations. Our experiments demonstrated that both of aminocarboxylate moieties of FluoZin-3 bind the Zn(II) ion synergistically. (C) 2016 Elsevier Inc. All rights reserved.