High-throughput fluorogenic assay for determination of botulinum type B neurotoxin protease activity

Botulinum neurotoxins are responsible for botulism, a flaccid muscular para lysis caused by inhibition of acetylcholine release at the neuromuscular ju nction, This occurs by cleavage of conserved proteins involved in exocytosi s such as synaptobrevin by the zinc metallopeptidase activity of the light chain of some botulinum neurotoxins. Botulism, for which there is presently no therapy available, is a relatively widespread disease that may result i n death. Consequently, the development of drugs able to inhibit the hydroly tic activity of these neurotoxins is of great interest, Design and screenin g of such inhibitors could be largely facilitated by using high-throughput assays, With this aim, a novel in vitro test for quantifying the proteolyti c activity of botulinum type B neurotoxin was developed. The substrate is t he 60-94 fragment of human synaptobrevin-1 which was modified by introducti on of the fluorescent amino acid L-pyrenylalanine in position 74 and a p-ni trophenylalanyl residue as quenching group in position 77, The cleavage of Syb 60-94 [Pya(74), Nop(77)] by th, toxin active chain occurs selectively b etween residues 76 and 77 as in the case of the unmodified synaptobrevin an d is directly quantified by measuring the strong fluorescence of the formed metabolite Syb 60-76 [Pya(74)], This is the easiest, quickest, and cheapes t assay described to date for measuring the proteolytic activity of botulin um type B neurotoxin, It can be easily automated for high-throughput screen ing, Moreover, amounts of about 3.5 pg/ml of botulinum type B neurotoxin co uld be detected by this method, (C) 2001 Academic Press.