Expanded Dynamic Range of Free Zinc Ion Determination by Fluorescence Anisotropy

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Abstract
We demonstrate that by use of a biosensor approach employing wild type human apocarbonic anhydrase II and a newly synthesized fluorescent ligand, ABD-M, free Zn(II) may be determined in solution at concentrations in the picomolar range with good accuracy by fluorescence anisotropy. Fluorescence anisotropy enjoys the same freedom from artifact as wavelength ratiometric approaches widely used for determining metal ions in solution such as Ca(II). In addition, we demonstrate that anisotropy-based determinations exhibit an important advantage, a broad dynamic range, which has not been demonstrated for wavelength ratiometric approaches. In particular, by judicious choice of excitation and emission wavelengths, the concentration range over which Zn(II) may be determined accurately can be increased by approximately 2 orders of magnitude. As ABD-M also exhibits significant changes in excitation and emission spectra as well as lifetime upon binding to the active-site Zn(II) in holocarbonic anhydrase, it should also be useful for wavelength ratiometric and lifetime-based determinations.