Development of an operational synaptobrevin-based fluorescent substrate for tetanus neurotoxin quantification.
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).
Perpetuo
Elen A
EA
Laboratory of Biochemistry and Biophysics, Center of Applied Toxinology, CEPID, Av. Vital Brasil, 1500-Butantan, 05503-900 São Paulo, SP, Brazil.
Juliano
Luis
L
Prado
Sally M
SM
Fratelli
Fernando
F
Fernandes
Irene
I
Lebrun
Ivo
I
D000595:Amino Acid Sequence; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000737:chemistry; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry*; D008666:Metalloendopeptidases / Q000032:analysis* / Q000737:chemistry* / Q000145:classification; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D050683:R-SNARE Proteins; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity; D013744:Tetanus Toxin / Q000032:analysis* / Q000737:chemistry* / Q000145:classification
D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010446:Peptide Fragments; D050683:R-SNARE Proteins; D013744:Tetanus Toxin; C035979:tetanospasmin; D008666:Metalloendopeptidases