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Fluorescence analysis of sulfonamide binding to carbonic anhydrase
Author(s) -
Wang Sheila C.,
Zamble Deborah B.
Publication year - 2006
Publication title -
biochemistry and molecular biology education
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.34
H-Index - 39
eISSN - 1539-3429
pISSN - 1470-8175
DOI - 10.1002/bmb.2006.494034052656
Subject(s) - fluorophore , chemistry , fluorescence , carbonic anhydrase ii , ligand (biochemistry) , carbonic anhydrase , förster resonance energy transfer , binding constant , fluorescence spectroscopy , tryptophan , affinities , binding site , biophysics , enzyme , stereochemistry , biochemistry , biology , receptor , amino acid , physics , quantum mechanics
A practical laboratory experiment is described that illustrates the application of fluorescence resonance energy transfer to the study of protein‐ligand binding. The affinities of wild‐type and mutant human carbonic anhydrase II for dansylamide were determined by monitoring the increase in ligand fluorescence that occurs due to energy transfer from tryptophan residues near the enzyme active site. In a subsequent experiment, the binding constant of azetazolamide, a weaker fluorophore but a stronger ligand, is measured by competition with dansylamide. This simple experiment introduces students to the widely used technique of fluorescence spectroscopy and to the determination of protein‐ligand binding constants.