A dual-step fluorescence resonance energy transfer-based quenching assay for screening of caspase-3 inhibitors
| Autor: | Heidi Appelblom, Tero Soukka, Antti Valanne, Pauliina Niemelä, P. Malmi |
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| Rok vydání: | 2007 |
| Předmět: |
Streptavidin
Time Factors Biophysics chemistry.chemical_element Nanoparticle Biochemistry Fluorescence Substrate Specificity chemistry.chemical_compound Fluorescence Resonance Energy Transfer Biotinylation Enzyme Inhibitors Molecular Biology Alexa Fluor Quenching (fluorescence) Dose-Response Relationship Drug Cell Biology Acceptor Caspase Inhibitors Förster resonance energy transfer chemistry Nanoparticles Europium |
| Zdroj: | Analytical biochemistry. 375(1) |
| ISSN: | 0003-2697 |
| Popis: | The control of cell death is an intricate process involving a multitude of intracellular modulators. Among these molecules, the caspases have a central role and have become an interesting group of enzymes in the current pharmaceutical industry. We have developed a novel dual-step fluorescence energy transfer-based separation-free assay method for the primary screening of caspase-3 inhibitors in vitro. This method relies on fluorescent europium(III)-chelate-doped nanoparticle donors coated with streptavidin in conjunction with a dual-labeled (N-terminal Alexa Fluor 680 fluorescent acceptor and C-terminal BlackBerry Quencher 650) caspase-3-specific peptide substrate modified with a biotinyl moiety. In the assay, the nanoparticle donor excites the fluorescent acceptor, whose emission is monitored with time-resolved measurements. The intensity of the acceptor reflects the activity of the enzyme because the intensity is controlled by the proximity of the quencher. Owing to the dual-step fluorescence resonance energy transfer, this method enables a sensitized fluorescence signal directly proportional to the extent of enzymatic activity with relatively background fluorescence-free measurements in the event of complete enzyme inhibition. The generic nanoparticle donors further promote versatility and cost-efficiency of the method. The performance evaluated as the inhibitor (Z-DEVD-FMK) dose-response curve (IC(50) value of approximately 12 nM) was in good agreement with that of the recent methods found in literature. This assay serves as a model application proving the feasibility of the europium-chelate-doped nanoparticle labels in a homogeneous assay for proteolytic activity. |
| Databáze: | OpenAIRE |
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