Development of an operational synaptobrevin-based fluorescent substrate for tetanus neurotoxin quantification
| Autor: | Ivo Lebrun, L. Juliano, Elen Aquino Perpetuo, Sally M. A. Prado, Irene Fernandes, Fernando Fratelli |
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| Rok vydání: | 2002 |
| Předmět: |
Clostridium tetani
Synaptobrevin Molecular Sequence Data Biomedical Engineering Bioengineering medicine.disease_cause Sensitivity and Specificity Applied Microbiology and Biotechnology Substrate Specificity R-SNARE Proteins Hydrolysis Tetanus Toxin Drug Discovery medicine Amino Acid Sequence Peptide sequence Fluorescent Dyes chemistry.chemical_classification Chromatography Chemistry Process Chemistry and Technology Membrane Proteins Metalloendopeptidases Substrate (chemistry) General Medicine Peptide Fragments Enzyme Activation Spectrometry Fluorescence Enzyme Biochemistry Molecular Medicine Quantitative analysis (chemistry) Biotechnology |
| Zdroj: | Biotechnology and Applied Biochemistry. 36:155 |
| ISSN: | 0885-4513 |
| DOI: | 10.1042/ba20020011 |
| Popis: | 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). |
| Databáze: | OpenAIRE |
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