Enzymatic Profiling of Tetanus and Botulinum Neurotoxins Based on Vesicle-Associated-Membrane Protein Derived Fluorogenic Substrates

Enzymatic Profiling of Tetanus and Botulinum Neurotoxins Based on Vesicle-Associated-Membrane Protein Derived Fluorogenic Substrates

Author Perpetuo, Elen Aquino Google Scholar
Juliano, Luis Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Fratelli, Fernando Google Scholar
Prado, Sally M. A. Google Scholar
Pimenta, Daniel C. Google Scholar
Lebrun, Ivo Google Scholar
Institution Butantan Inst
Universidade Federal de São Paulo (UNIFESP)
Abstract Botulinum (BoNT) and tetanus (TeNT) neurotoxins are bacterial zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. There are seven serotypes of BoNT, while TeNT is found in one serotype. in order to characterize their enzymatic activities and to propose serotype-differentiation an enzymatic assay based on their metalloprotease activity was developed. the assays were conducted with FRET peptides derived from SNAP-25, synaptobrevin and syntaxin. the substrates were cleaved by 2 ng/mL of toxin at different rates (K-cat /K-M from 0.028 to 75.9 mu M.s(-)) at a single bond, as confirmed by Q-TOF mass spectrometry. Inhibition of the hydrolysis was obtained with EDTA or with specific antibodies directed to each neurotoxin. Different substrate selectivities, especially by BoNT-A and E, suggest that these substrates can be used as a putative method for clostridial toxin quantification and serotype differentiation and could be easily adapted to a high-throughput protocols.
Keywords Botulinum neurotoxin
tetanus neurotoxin
fluorogenic substrates
proteolytic activity
enzymatic assay
Language English
Sponsor Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Fundacao Butantan
Date 2008-10-01
Published in Protein and Peptide Letters. Sharjah: Bentham Science Publ Ltd, v. 15, n. 10, p. 1100-1106, 2008.
ISSN 0929-8665 (Sherpa/Romeo, impact factor)
Publisher Bentham Science Publ Ltd
Extent 1100-1106
Origin http://dx.doi.org/10.2174/092986608786071166
Access rights Closed access
Type Article
Web of Science ID WOS:000260058100011
URI http://repositorio.unifesp.br/handle/11600/30964

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