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List Labs POSTER - Presented at the 5th International Conference on Basic and Therapeutic Aspects of Botulinum and Tetanus Toxins, June 2005 in Denver
Christian, T.; Shine, N.; Eaton, L.; Crawford, K.
The botulinum neurotoxins are composed of two subunits, a 100 kD heavy chain and a 50 kD light chain, which are linked by a single disulfide bond. The heavy chain is responsible for binding and translocation while the light chain domain contains the enzymatic activity. The light chain is a zinc dependent metalloprotease. The eukaryotic substrate for botulinum neurotoxin type A is the synaptosomal protein SNAP-25. As previously reported, we have developed fluorescence resonance energy transfer (FRET) substrates based on SNAP-25 that are readily recognized and cleaved by the type A holotoxin and light chain. One of the substrates contains an oAbz/DNP FRET pair and the other a FITC/DABCYL FRET pair (US patent #6504006). In this study, the specific activity of the holotoxin and recombinant light chain were determined using both FRET substrates. Specific activity is expressed in terms of moles of SNAPtide cleaved per minute per milligram of enzyme. For comparison, the specific activity of holotoxin and light chain were ascertained under optimal condition for the holotoxin. The specific activity was also calculated for the light chain under its optimal conditions. A series of ZnCl2 concentrations were tested in several buffer systems to determine whether addition of ZnCl2 would improve the enzymatic activity of the light chain. In order to determine the ultimate sensitivity of this method for detecting the presence of the protease, the limit of detection (LOD) of the SNAPtide assay was established using both substrates with the holotoxin and the light chain.