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Biotechnology and Applied Biochemistry (2002) 36, (155–161) (Printed in Great Britain)

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

Elen A. Perpetuo*, Luis Juliano†, Sally M. Prado‡, Fernando Fratelli‡, Irene Fernandes§ and Ivo Lebrun*¹

*Laboratory of Biochemistry and Biophysics, Center of Applied Toxinology, CEPID, Av. Vital Brasil, 1500-Butantan, 05503-900 São Paulo, SP, Brazil, †Department of Biophysics, Center of Applied Toxinology, CEPID, UNIFESP, Av. Vital Brasil, 1500-Butantan, 05503-900 São Paulo, SP, Brazil, ‡Section of Anaerobic Vaccine, Butantan Institute, Av. Vital Brasil, 1500-Butantan, 05503-900 São Paulo, SP, Brazil, and §Laboratory of Immunopathology, Butantan Institute, Av. Vital Brasil, 1500-Butantan, 05503-900 São Paulo, SP, Brazil

Key words: Clostridium tetani, enzymic assay, metalloprotease.

Abbreviations used: Abz, o-aminobenzoyl; EDDnp, ethylenediamine dinitrophenyl; FL, flocculation limit; HC, heavy chain; LC, light chain; MMD, minimal mortal dose; syn*73–82, Abz-synaptobrevin_73–82-EDDnp fluorescent substrate; TTx, tetanus neurotoxin.

¹To whom correspondence should be addressed (e-mail Lebrun@butantan.gov.br).

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).

Received 11 February 2002/14 May 2002; accepted 24 July 2002

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