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Background: Peptide substrates which are specific for Botulinum neurotoxin type B (BoNT/B) have been designed by taking advantage of the fact that each serotype of the neurotoxin selectively cleaves one of the SNARE membrane fusion proteins at a unique site. Substrates for BoNT/B, VAMPtide, are based on the native target SNARE protein, synaptobrevin-2 or VAMP2. Three VAMPtide substrates with similar peptide sequences but different fluorophores quenched by an appropriate chromophore are compared. Data demonstrating the efficiency of cleavage of these peptides by BoNT/B holotoxin and light chain as well as studies of the minimum concentration of enzyme detectable by each peptide substrate, is presented. Methods: VAMPtide (o-Abz/Dnp), VAMPtide (FITC/DABCYL), and VAMPtide (PL 150, Pya/Nop) are product numbers 540, 541, and 542, respectively, from List Biological Laboratories, Inc. VAMPtide(PL 150, Pya/Nop) is the original product of and is also marketed by Pharmaleads, Paris, France. For each BoNT/B substrate, the time-dependent increase in fluorescence intensity was monitored as a function of BoNT/B holotoxin or light chain concentration using the optimum buffer and temperature conditions established for each in prior experiments. The excitation and emission wavelengths were set to 321 nm and 418 nm, respectively, for the o-Abz-substrate, 490 nm and 523 nm using a cutoff filter of 495 nm for the FITCsubstrate, and 340 nm and 400 nm for the PL 150, Pya/Nop substrate. Results: Evaluation of all three peptides indicated that VAMPtide(PL 150, Pya/Nop) was most efficiently cleaved by BoNT/B. This substrate was cleaved at least 20 times faster than the other two VAMPtide substrates using approximately 10 times less enzyme. Conclusions: Of the three BoNT/B substrates compared in this evaluation, VAMPtide (PL 150, Pya/Nop), Prod #542, is the preferred peptide substrate for use in both detection and high throughput studies of BoNT/B.