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SNAP-Etide, a FRET Substrate for Botulinum Toxin Type E

Shine, N.; Christian, T.; Primak, Y.

Botulinum neurotoxins have seven distinct serotypes, four of which (types A, B, E and F) cause botulism is humans. The neurotoxins are composed of a 100 kDheavy chain, and an enzymaticallyactive zinc-dependent light chain (50 kD). The metalloproteasepresents an ideal target for the development of potential therapeutic inhibitors if botulism. One of the most efficient methods for rapid high throughput screening of potential inhibitory compounds is based on the use of intramolecularlyquinched fluorescent substrates. As previously reported, the fluorescence resonance energy transfer (FRET) peptides, SNAPtideand VAMPtideTM, were devised at List as substrates for botulinumtoxin type A and B, respectively. Recently, a FRET substrate for botulinumneurotoxin type E, SNAP EtideTM, has been designed and evaluated. This peptide, based on the eukaryotic target SNAP-25, contains an o-aminobenzoicacid fluorophore(o-Abz) and a 2,4-dinitrophenol (Dnp) acceptor chromophore. It is readily cleaved by botulinumneurotoxin type E, light chain (LcE). Tests of the activity ofLcE with SNAP EtideTMas a function of pH, ZnCl2and Tween-20 concentrations indicate that the optimum buffer for the cleavage of the substrate is 50 mMHEPES, pH 7.8, 0.1% Tween-20. As observed for both type A and type B light chains, the addition of ZnCl2is inhibitory to the FRET substrate cleavage reaction. The hydrolysis of SNAP EtideTMby LcE shows a linear response to the enzyme concentration. A total enzyme digest of increasing concentrations of SNAP EtideTMusing trypsin, indicates a linear increase in fluorescence up to 40 M substrate. At higher concentrations a significant inner filter effect is observed. A value for Kmwas estimated using o-Abz-Lysto correct for the inner filter effect. Substrate inhibition is observed for concentrations greater then 100 M SNAP EtideTM.