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|Title:||Development of an operational synaptobrevin-based fluorescent substrate for tetanus neurotoxin quantification|
|Authors:||Perpetuo, Elen Aquino|
Juliano, Luis [UNIFESP]
Prado, Sally M.
Universidade Federal de São Paulo (UNIFESP)
|Citation:||Biotechnology and Applied Biochemistry. London: Portland Press, v. 36, p. 155-161, 2002.|
|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 184.108.40.206) 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).|
|Appears in Collections:||Artigo|
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