EXPANDED DYNAMIC-RANGE OF FREE ZINC ION DETERMINATION BY FLUORESCENCEANISOTROPY

We demonstrate that by use of a biosensor approach employing wild type human apocarbonic anhydrase II and a newly synthesized fluorescent li gand, ABD-M, free Zn(II) may be determined in solution at concentratio ns in the picomolar range with good accuracy by fluorescence anisotrop y. Fluorescence anisotropy enjoys the same freedom from artifact as wa velength ratiometric approaches widely used for determining metal ions in solution such as Ca(II). In addition, we demonstrate that anisotro py-based determinations exhibit an important advantage, a broad dynami c range, which has not been demonstrated for wavelength ratiometric ap proaches. In particular, by judicious choice of excitation and emissio n wavelengths, the concentration range over which Zn(II) may be determ ined accurately can be increased by approximately 2 orders of magnitud e. As ABD-M also exhibits significant changes in excitation and emissi on spectra as well as lifetime upon binding to the active-site Zn(II) in holocarbonic anhydrase, it should also be useful for wavelength rat iometric and lifetime-based determinations.