Differential Characterization of Botulinum Toxins (types A and E ) Using Quenched Fluorescent Substrates

Perpetuo E A¹ , Juliano L,² Fratelli F³, Prado SMA³ , Lebrun, I.¹

1 Laboratory of Biochemistry and Biophysics, 2 Department of Biophysics-Center of Applied Toxinology-CEPID, UNIFESP, 3 Section of Anaerobic Vaccines, Butantan Institute, São Paulo-SP, Brasil.

Botulinum neurotoxins (BoNT) are zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. This occurs by cleavage of toxin light chain on conserved proteins involved in exocytosis of neuron vesicles, such as synaptobrevin. In order to develop a sensitive assay to quantify the proteolytic activity of BoNT A and E, we designed fluorescent substrates based on target proteins sequences hydrolysed by them. The substrates are the fragments of rat synaptobrevin, sintaxin IA and SNAP-25 which were modified introducing the fluorescent o-aminobenzoyl (Abz) in N-terminal and ethylenediamine dinitro-phenyl (EDDnp) in C-terminal position. The cleavage of a single peptidic bond by toxin active chain is directly quantified by measuring the strong fluorescence of the formed N-terminus peptide fragment metabolite. This is a rapid and quick assay to measure the proteolytic activity of BoNT A and E. Moreover, amounts of ng of neurotoxin could be detected by this method. Solutions of holotoxin of BoNT A and E (10 ng) obtained from Section of Anaerobic Vaccines from Butantan Institute, were analysed by SDS-PAGE to verify their purity and after incubated with the fluorescent substrates (5 mM) in reaction buffer (50mM Tris-HCl pH 7,5) at 37°C for 10 minutes. A hydrolysis curve was plotted and the fragments were collected and submitted to aminoacid analysis and mass spectrometry to verify the cleavage point. The cleavage of SNAP-25 by the toxin active chain was only between residues Gln ₁₉₇-Arg ₁₉₈ by BoNT A and Arg ₁₈₀-Ile ₁₈₁ by BoNT E.The substrate hydrolysed by BoNT A was not by BoNT E. This fact could be usefull to distinguish the active toxin. Immunoprecipitation assays of BoNT proteolytic activity by immunocomplex formation with protein A -Sepharose were performed. Protein- A Sepharose (10 mg/ml) was incubated for 30, 60 and 90 min at 37°C with mice antibody raised against BoNT A and E. At the end of this time, 10 ng of BoNT A in Tris buffered saline was added and incubated for 5 min at to obtain the immunocomplex sandwich. The solution was filtered through a 0.45µ Millipore membrane and transferred to the cuvette of the spectrofluorimeter. Finally, 5 µM of  the fluorescent substrate was added to this solution and readings were taken. The BoNT proteolytic activity was significantly inhibited ( 98%).The same method was used to measure the tetanus toxin activity (BiotechnolAppl Biochem. 36:155-61, 2002).  The fluorescent assays were selective and gave low background readings. Besides, this method did not employ reverse-phase extraction steps, so could be easily adapted to a production process or other quantitative diagnostic assays.

Supported by FAPESP, CNPq and Butantan Foundation.

Correspondence to: perpetuoe@hotmail.com or lebrun@butantan.gov.br