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.

Capillary electrophoresis laser-induced fluorescence for screening combinatorial peptide libraries in assays of botulinum neurotoxin A

“…With UV detection the absorbance of product, P2, was routinely masked by the background absorbance of the HNTE running buffer although peaks for substrate, S, and product, P1, were visible (data not shown). Similar masking effects were reported for UV detection in CE separations of reaction products of BoNT/B LC in HEPES buffer [21]. Hence in order to facilitate detection and quantitation of products, detection methods based on laser-induced fluorescence were developed.…”

“…The same labeling reagent has been used in CE-LIF for characterization of VAMP-thioredoxin fusion protein, developed as a potential substrate for BoNT/B [20]. A detailed CE-based study of BoNT/B light chain (LC) activity using UV detection has been reported, although no results were given for quantitation in the presence of potential modulator compounds [21].…”

Capillary electrophoresis of proteins 2003–2005

“…CZE was used to study b-amyloid and its aggregation [275], botulinum neurotoxin B light chain activity [276], nonenzymatic posttranslation modifications on BSA [277], aminoacyl-tRNA synthetase localized in cell nucleus [278], protein-protein interactions [279], aggregation and denaturation of antibodies [280], glycosylation of murine erythropoietin [281], PEGylation of proteins [282], glutamine deamidation in proteins [283], complexation of apomyoglobin with Coumarin 153 [284], reaction of FQ with ovalbumin [285], interaction of GFP with calcium [286], noncovalent complexation in myoglobin [287], DNA-protein interactions [288][289][290], binding fucoidan to antithrombin [291], retinoic acid to b-lactoglobulin B [292], and binding phospholipid and glycosaminoglycan to human b 2 -glycoprotein [293].…”

Micellar electrokinetic chromatography and laser induced fluorescence detection of botulinum neurotoxin type A activity using a dual-labelled substrate

“…Capillary electrophoresis (CE) has been emerging as another promising way for analysing BoNT's activity because of the extremely small sample volume needed per analysis and high throughput potential [19,22]. During the assay, CE is used to separate the product from the peptide substrate after reaction with BoNTs.…”

“…During the assay, CE is used to separate the product from the peptide substrate after reaction with BoNTs. Since the toxin activity is assayed in solution phase, the CE format allows kinetic analyses of toxin activity [22]. However, due to the small light-path length in CE, it is necessary to use sensitive detectors to detect the low concentrations of BoNT.…”