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