Our current understanding of the intracellular thermodynamics and kinetics of Zn(II) ions is largely based\ud on the application of fluorescent sensor molecules, used to study and visualize the concentration,\ud distribution and transport of Zn(II) ions in real time. Such agents are designed for high selectivity for zinc\ud in respect to other biological metal ions. However, the issue of their sensitivity to physiological levels of\ud 10 low molecular weight Zn(II) ligands (LMWLs) has not been addressed. We followed the effects of eight\ud such compounds on the fluorescence of ZnAF-1 and ZnAF-2F, two representatives of ZnAF family of\ud fluorescein-based zinc sensors containing the N,N-bis(2-pyridylmethyl)ethylenediamine chelating unit.\ud Fluorescence titrations of equimolar Zn(II)/ZnAF-1 and Zn(II)/ZnAF-2F solutions with acetate,\ud phosphate, citrate, glycine, glutamic acid, histidine, ATP and GSH demonstrated strong fluorescence\ud 15 quenching. These results are interpreted in terms of an interplay of the formation of the [ZnAF-Zn(II)-\ud LMWL] ternary complexes and the competition for Zn(II) between ZnAF and LMWLs. UV-vis\ud spectroscopic titrations revealed the existence of supramolecular interactions between the fluorescein\ud moiety of ZnAF-1 and ATP and His, which, however, did not contribute to fluorescence quenching.\ud Therefore, the obtained results show that the ZnAF sensors, other currently used zinc sensors containing\ud 20 the N,N-bis(2-pyridylmethyl)ethylenediamine unit, and, in general, all sensors that do not saturate the\ud Zn(II) coordination sphere, may co-report cellular metabolites and Zn(II) ions, leading to\ud misrepresentations of the concentrations and fluxes of biological zinc
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