Fluorescence-based biosensing of zinc using carbonic anhydrase

Measurement of free zinc levels and imaging of zinc fluxes remains technica lly difficult due to low levels and the presence of interfering cations suc h as Mg and Ca. We have developed a series of fluorescent zinc indicators b ased on the superb sensitivity and selectivity of a protein, human apo-carb onic anhydrase II, for Zn(II). These indicators transduce the level of free zinc as changes in intensity, wavelength ratio, lifetime, and/or anisotrop y; the latter three approaches permit quantitative imaging of zinc levels i n the microscope. A unique attribute of sensors incorporating biological ma cromolecules as transducers is their capability for modification by site-di rected mutagenesis. Thus we have produced variants of carbonic anhydrase wi th improved affinity for zinc, altered selectivity, and enhanced binding ki netics, all of which are difficult to modify in small molecule indicators.