Site-specific mutants of carbonic anhydrase for fluorescence energy-transfer-based metal-ion biosensing

Richard B. Thompson; Zhengfang Ge; Marcia W. Patchan; Laura L. Kiefer; Carol A. Fierke

doi:10.1117/12.221726

29 September 1995 Site-specific mutants of carbonic anhydrase for fluorescence energy-transfer-based metal-ion biosensing

Richard B. Thompson, Zhengfang Ge, Marcia W. Patchan, Laura L. Kiefer, Carol A. Fierke

Author Affiliations +

Proceedings Volume 2508, Chemical, Biochemical, and Environmental Fiber Sensors VII; (1995) https://doi.org/10.1117/12.221726
Event: European Symposium on Optics for Environmental and Public Safety, 1995, Munich, Germany

Abstract

In order to gain wavelength and analyte flexibility, we have recently altered the transduction approach of our fluorescence-based biosensor. Briefly, binding of metal ions such as zinc to the active site of carbonic anhydrase is transduced by metal-dependent binding of a colored inhibitor to a fluorescent derivative of the enzyme; in the absense of metal the inhibitor does not bind and the label fluorescence is unquenched, but at higher metal concentrations the inhibitor binds, energy transfer occurs with moderate efficiency and the fluorescent label exhibits reduced intensity and lifetime. Inasmush as Forster energy transfer is distance dependent the position of the fluorescent label on the surface of the enzyme has some impact on the performance of the sensor. We designed, produced, and expressed site-selective mutants of carbonic anhydrase which could be unambiguously derivatized with suitable fluorescent labels, and which gave much improved responses to zinc ion compared with randomly derivatized wild type enzyme.

Citation Download Citation

Richard B. Thompson, Zhengfang Ge, Marcia W. Patchan, Laura L. Kiefer, and Carol A. Fierke "Site-specific mutants of carbonic anhydrase for fluorescence energy-transfer-based metal-ion biosensing", Proc. SPIE 2508, Chemical, Biochemical, and Environmental Fiber Sensors VII, (29 September 1995); https://doi.org/10.1117/12.221726

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