| Abstract
| - A small-scale, homogeneous, rapid sensing system for phenothiazines and tricyclic antidepressants(TCAs) has been developed by employing fluorescently labeled mutant calmodulin (CaM) as therecognition element. A calmodulin mutant containing a unique cysteine residue at position 109 onthe protein was expressed in Escherichia coli. Following purification, the environment-sensitive, thiol-specific fluorophores N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC),6-acryloyl-2-dimethylaminonaphthalene (acrylodan), and 4-[N-(2-(iodoacetoxy)ethyl)-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD ester) were coupled to the C109 site of the mutant protein. Theresponse of labeled CaM in the presence of calcium to increasing concentrations of chlorpromazinehydrochloride (CPZ), as well as other phenothiazines and structurally related antipsychotics andantidepressants, was investigated. Fluorescence measurements were performed on benchtop andmicrotiter plate fluorometers. The responses were characterized as a change in the signal intensity ofthe labeled protein upon ligand binding, and the stability of the system was monitored over a nine-month period. The assay showed specificity for the phenothiazine and TCA classes of drugs, withlimits of detection in the micromolar range. Selectivity studies indicated negligible response of thebiosensing system to structurally unrelated compounds. This work represents a proof-of-concept assayfor rapid, homogeneous detection of drugs employing binding proteins as the biorecognition element.
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