title: Revised stability constant, spectroscopic properties and binding mode of Zn(II) to FluoZin-3, the most common zinc probe in life sciences.
abstract: 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 ((C)Kd) values at pH7.4 are slightly discrepant, (15nM or 8.9nM). In this work we determined the (C)Kd for Zn(II) complexation by FluoZin-3 at pH7.4 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 (C)Kd (9.1±0.4nM; -log (C)Kd=8.04) and of the absolute (pH-independent) stability constant log βZnL (8.16±0.05) were provided by fluorescence spectroscopy experiments performed at 1μM concentrations. Our experiments demonstrated that both of aminocarboxylate moieties of FluoZin-3 bind the Zn(II) ion synergistically.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.
authors.forename: I
authors.initials: I
authors.lastname: Marszałek
authors.affiliation: Laboratory of Chemical Biology, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Krężel
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.
authors.forename: W
authors.initials: W
authors.lastname: Goch
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland; NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614 Poznań, Poland.
authors.forename: I
authors.initials: I
authors.lastname: Zhukov
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.
authors.forename: I
authors.initials: I
authors.lastname: Paczkowska
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland. Electronic address: wbal@ibb.waw.pl.
authors.forename: W
authors.initials: W
authors.lastname: Bal
mesh_terms: D006863:Hydrogen-Ion Concentration; D015335:Molecular Probes / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D011083:Polycyclic Compounds / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry*
chemical_list: C451182:FluoZin-3; D015335:Molecular Probes; D011083:Polycyclic Compounds; D015032:Zinc; D009571:Nitrilotriacetic Acid
keywords: FluoZin-3; Fluorescent probes; Spectroscopy; Stability constants; Zinc