Depolarization after resonance energy transfer (DARET): a sensitive fluorescence-based assay for botulinum neurotoxin protease activity
| Autor: | Dudley H. Williams, Justin A. Ross, Yumiko Okawa, K. Roger Aoki, Marcella A. Gilmore, B. Holguin, Nicholas G. James, Lance E. Steward, David M. Jameson |
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| Rok vydání: | 2010 |
| Předmět: |
Botulinum Toxins
Synaptosomal-Associated Protein 25 Recombinant Fusion Proteins Green Fluorescent Proteins Biophysics Fluorescence Polarization Biochemistry Green fluorescent protein Substrate Specificity Fluorescence Resonance Energy Transfer Enzyme kinetics Botulinum Toxins Type A Molecular Biology Chemistry Hydrolysis Substrate (chemistry) Depolarization Cell Biology Fluorescence Endopeptidase Kinetics Luminescent Proteins Förster resonance energy transfer Fluorescence anisotropy |
| Zdroj: | Analytical biochemistry. 413(1) |
| ISSN: | 1096-0309 |
| Popis: | The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)-fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134-206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm. Energy transfer from the BFP to the GFP in the intact substrate results in a substantial depolarization of the GFP emission. The energy transfer is eliminated when the fluorescent domains separate on cleavage by the endopeptidase, and emission from the directly excited GFP product fragment is then highly polarized, resulting in an overall increase in polarization. This increase in polarization can be monitored to assay the proteolytic activity of BoNT/A and BoNT/E in real time. It allows determination of the turnover rate of the substrate and the kinetic constants (V(max) and k(cat)) based on the concentration of cleaved substrate determined directly from the measurements using the additivity properties of polarization. The assay is amenable to high-throughput applications. |
| Databáze: | OpenAIRE |
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