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

• FluoZin-3 is the most popular fluorescent probe selective for Zn(II) ions.
• Protonation and stability constants of FluoZin-3 and its Zn(II) complex were determined.
• The affinity of FluoZin-3 for Zn(II) in a broad pH range was established.
• Both aminocarboxylate moieties of FluoZin-3 participate in Zn(II) binding synergistically.

2-[2-[2-[2-[bis(carboxylatomethyl)amino]-5-methoxyphenoxy]ethoxy]-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 (CKd) values at pH 7.4 are slightly discrepant, (15 nM or 8.9 nM). In this work we determined the CKd 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 pKa values from fluorescence spectra and from various regions of UV–Vis spectra. The validated values of CKd (9.1 ± 0.4 nM; − log CKd = 8.04) and of the absolute (pH-independent) stability constant log βZnL (8.16 ± 0.05) were provided by fluorescence spectroscopy experiments performed at 1 μM concentrations. Our experiments demonstrated that both of aminocarboxylate moieties of FluoZin-3 bind the Zn(II) ion synergistically.

Spectroscopic characterization of acid/base and zinc binding properties of the Zn(II) selective probe FluoZin-3 yielded the revised affinity constant and its pH dependence, as well as the information about the zinc binding mode.Figure optionsDownload as PowerPoint slide