Fluorescence-based biosensing of zinc using carbonic anhydrase

被引:68
作者
Fierke, CA
Thompson, RB [1 ]
机构
[1] Univ Maryland, Sch Med, Dept Biochem & Mol Biol, Baltimore, MD 21201 USA
[2] Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA
[3] Univ Michigan, Dept Biochem, Ann Arbor, MI 48109 USA
关键词
biosensor; carbonic anhydrase; fluorescence; hippocampus; wavelength ratiometric; zinc;
D O I
10.1023/A:1012980628412
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Measurement of free zinc levels and imaging of zinc fluxes remains technically difficult due to low levels and the presence of interfering cations such as Mg and Ca. We have developed a series of fluorescent zinc indicators based on the superb sensitivity and selectivity of a protein, human apo-carbonic anhydrase II, for Zn(II). These indicators transduce the level of free zinc as changes in intensity, wavelength ratio, lifetime, and/or anisotropy; the latter three approaches permit quantitative imaging of zinc levels in the microscope. A unique attribute of sensors incorporating biological macromolecules as transducers is their capability for modification by site-directed mutagenesis. Thus we have produced variants of carbonic anhydrase with improved affinity for zinc, altered selectivity, and enhanced binding kinetics, all of which are difficult to modify in small molecule indicators.
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页码:205 / 222
页数:18
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