title: Depolarization after resonance energy transfer (DARET): a sensitive fluorescence-based assay for botulinum neurotoxin protease activity.
abstract: 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.
authors.affiliation: Neurotoxin Research Program, Department of Biological Sciences, Allergan, Irvine, CA 92612, USA.
authors.forename: Marcella A
authors.initials: MA
authors.lastname: Gilmore
authors.forename: Dudley
authors.initials: D
authors.lastname: Williams
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Okawa
authors.forename: Bret
authors.initials: B
authors.lastname: Holguin
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: James
authors.forename: Justin A
authors.initials: JA
authors.lastname: Ross
authors.forename: K
authors.initials: K
authors.lastname: Roger Aoki
authors.forename: David M
authors.initials: DM
authors.lastname: Jameson
authors.forename: Lance E
authors.initials: LE
authors.lastname: Steward
mesh_terms: D001905:Botulinum Toxins / Q000378:metabolism; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D050825:Synaptosomal-Associated Protein 25; C409504:blue fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E