Development of an operational synaptobrevin-based fluorescent substrate for tetanus neurotoxin quantification

Development of an operational synaptobrevin-based fluorescent substrate for tetanus neurotoxin quantification

Author Perpetuo, Elen Aquino Google Scholar
Juliano, Luis Autor UNIFESP Google Scholar
Prado, Sally M. Google Scholar
Fratelli, Fernando Google Scholar
Fernandes, Irene Google Scholar
Lebrun, Ivo Google Scholar
Institution CEPID
Butanan Inst
Universidade Federal de São Paulo (UNIFESP)
Abstract Tetanus neurotoxin (TTx), produced by Clostridium tetani, is a two-chain polypeptide with a heavy molecular chain (HC; 100 kDa) and a light molecular chain (LC; 50 kDa) linked by a disulphide bridge. the low-molecular-mass chain is classified as a zinc metalloprotease (EC 3.4.24.68) with specific hydrolysis on synaptobrevin. With the known enzymic activity for the LC of TTx, we developed a quantification method using a quenched fluorescence peptide substrate based on the synaptobrevin sequence (fragments 73-82), suitable for direct determination of the whole TTx (HC+LC) even in crude production batches, without the necessity of purification and reduction steps to isolate the LC of TTx. the rate of substrate hydrolysis was 200 nmol/min and it was totally inhibited by EDTA, anti-recombinant fragment C antibody, and the cleavage was in a single bond (Gln-Phe) with purified and crude TTx. Besides, ELISA applied to the anti-TTx serum produced at our Institute showed cross-reaction with every fraction of the crude TTx extract. Another aspect is that TTx activity depends on the storage time, reaching a maximum on day 10. the results obtained suggest that the use of the new fluorescent substrate, Abz-synaptobrevin(73-82)-EDDnp, enables easy and quick determination of TTx. It is a good alternative to some of the existing methods such as flocculation assay, and it can replace, under some conditions, the biological assays (minimal mortal dose).
Keywords Clostridium tetani
enzymic assay
metalloprotease
Language English
Date 2002-12-01
Published in Biotechnology and Applied Biochemistry. London: Portland Press, v. 36, p. 155-161, 2002.
ISSN 0885-4513 (Sherpa/Romeo, impact factor)
Publisher Portland Press
Extent 155-161
Origin http://dx.doi.org/10.1042/BA20020011
Access rights Closed access
Type Article
Web of Science ID WOS:000179819000001
URI http://repositorio.unifesp.br/handle/11600/27067

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