title: A capillary electrophoresis technique for evaluating botulinum neurotoxin B light chain activity.
abstract: Botulinum neurotoxin B (BoNT/B) produces muscle paralysis by cleaving synaptobrevin/vesicle-associated membrane protein (VAMP), an 18-kDa membrane-associated protein located on the surface of small synaptic vesicles. A capillary electrophoresis (CE) assay was developed to evaluate inhibitors of the proteolytic activity of BoNT/B with the objective of identifying suitable candidates for treatment of botulism. The assay was based on monitoring the cleavage of a peptide that corresponds to residues 44-94 of human VAMP-2 (V51) following reaction with the catalytic light chain (LC) of BoNT/B. Cleavage of V51 generated peptide fragments of 18 and 33 amino acids by scission of the bond between Q76 and F77. The fragments and parent peptide were clearly resolved by CE, allowing accurate quantification of the BoNT/B LC-mediated reaction rates. The results indicate that CE is suitable for assessing the enzymatic activity of BoNT/B LC.
authors.affiliation: Neurotoxicology Branch, Pharmacology Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010, USA. Michael.Adler@amedd.army.mil
authors.forename: Michael
authors.initials: M
authors.lastname: Adler
authors.forename: Harlan F
authors.initials: HF
authors.lastname: Shafer
authors.forename: Heather A
authors.initials: HA
authors.lastname: Manley
authors.forename: Brennie E
authors.initials: BE
authors.lastname: Hackley
authors.forename: James D
authors.initials: JD
authors.lastname: Nicholson
authors.forename: James E
authors.initials: JE
authors.lastname: Keller
authors.forename: Michael C
authors.initials: MC
authors.lastname: Goodnough
mesh_terms: D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism*; D019274:Botulinum Toxins, Type A; D019075:Electrophoresis, Capillary / Q000379:methods*; D005615:Freezing; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D009994:Osmolar Concentration; D050683:R-SNARE Proteins; D012492:Salts / Q000494:pharmacology; D013696:Temperature; D015032:Zinc / Q000494:pharmacology
chemical_list: D008565:Membrane Proteins; D050683:R-SNARE Proteins; D012492:Salts; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D015032:Zinc