An alternative bioassay for botulinum neurotoxin type A based on its endopeptidase activity

Recent studies have shown that botulinum toxin type A (BoNT/A) is a zinc-endopeptidase specific for SNAP-25. We aimed to compare kinetic parameters of this reaction by using two substrates and reverse phase-HPLC (RP-HPLC). Recombinant SNAP-25(1-506), and a synthetic peptide spanning the toxin cleavage site (SNAP-25(137-206)), were used to characterize the proteolytic activity of BoNT/A. Toxin activity was measured by monitoring the peak area of the product of reaction (SMAP-25(198-206)) by RP-HPLC. Reaction specificity was confirmed by mass-spectrometry and N-terminal sequence analysis. SNAP-25(1-206) and SNAP-25(137-206) were cleaved at position Gln(197)-Arg(198), with respective Km values of 79 and 353 mu M. Zinc ions, EDTA, O-phenanthroline and DTT were inhibitory, with respective IC50 values of 0.04 mu M, 0.4, 1.8 and 25 mM. Classical inhibitors of metalloproteases, such as phosphoramidon and captopril, had no effect on toxin activity. The RP-HPLC assay is suitable for the estimation of BoNT/A activity in vitro, although in its current form it lacks the sensitivity required for estimation of BoNT/A in clinical formulations. SNAP-25(1-206) was found to be a better substrate for BoNT/A.