Class-selective drug detection: fluorescently-labeled calmodulin as the biorecognition element for phenothiazines and tricyclic antidepressants
| Autor: | Lyndon L.E. Salins, Phillip M. Douglass, Emre Dikici, Sylvia Daunert |
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| Rok vydání: | 2002 |
| Předmět: |
Time Factors
Calmodulin Protein Conformation Biomedical Engineering Pharmaceutical Science Gene Expression Bioengineering Biosensing Techniques Antidepressive Agents Tricyclic Sensitivity and Specificity Substrate Specificity Drug detection chemistry.chemical_compound Microtiter plate Mutant protein Phenothiazines Phenothiazine Fluorescent Dyes Pharmacology chemistry.chemical_classification biology Organic Chemistry Spectrometry Fluorescence chemistry Biochemistry Mutation biology.protein Biosensor Biotechnology Tricyclic Cysteine Protein Binding |
| Zdroj: | Bioconjugate chemistry. 13(6) |
| ISSN: | 1043-1802 |
| Popis: | A small-scale, homogeneous, rapid sensing system for phenothiazines and tricyclic antidepressants (TCAs) has been developed by employing fluorescently labeled mutant calmodulin (CaM) as the recognition element. A calmodulin mutant containing a unique cysteine residue at position 109 on the 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. The response of labeled CaM in the presence of calcium to increasing concentrations of chlorpromazine hydrochloride (CPZ), as well as other phenothiazines and structurally related antipsychotics and antidepressants, was investigated. Fluorescence measurements were performed on benchtop and microtiter plate fluorometers. The responses were characterized as a change in the signal intensity of the 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, with limits of detection in the micromolar range. Selectivity studies indicated negligible response of the biosensing system to structurally unrelated compounds. This work represents a proof-of-concept assay for rapid, homogeneous detection of drugs employing binding proteins as the biorecognition element. |
| Databáze: | OpenAIRE |
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