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Applied and Environmental Microbiology, July 2009, p. 4382-4390, Vol. 75, No. 13
0099-2240/09/$08.00+0     doi:10.1128/AEM.00091-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Detection and Quantification of Botulinum Neurotoxin Type A by a Novel Rapid In Vitro Fluorimetric Assay ^,

Hervé Poras,^1, Tanja Ouimet,^1, Sou-Vinh Orng,¹ Marie-Claude Fournié-Zaluski,¹ Michel R. Popoff,² and Bernard P. Roques^1*

Pharmaleads, Paris BioPark, 11 Rue Watt, 75013 Paris, France,¹ CNR Anaérobies et Botulisme, Unité Bactéries Anaérobies et Toxines, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France²

Received 14 January 2009/ Accepted 3 May 2009

Botulinum neurotoxin type A (BoNT/A), the most poisonous substance known to humans, is a potential bioterrorism agent. The light-chain protein induces a flaccid paralysis through cleavage of the 25-kDa synaptosome-associated protein (SNAP-25), involved in acetylcholine release at the neuromuscular junction. BoNT/A is widely used as a therapeutic agent and to reduce wrinkles. The toxin is used at very low doses, which have to be accurately quantified. With this aim, internally quenched fluorescent substrates containing the fluorophore/repressor pair pyrenylalanine (Pya)/4-nitrophenylalanine (Nop) were developed. Nop and Pya were, respectively, introduced at positions 197 and 200 of the cleavable fragment (amino acids 187 to 203) of SNAP-25 (with norleucine at position 202 [Nle²⁰²]), which is acetylated at its N terminus and amidated at its C terminus. Cleavage of this peptide occurred between positions 197 and 198, as in SNAP-25, and was easily quantified by the strong fluorescence emission of the metabolite. To increase the assay sensitivity, the peptide sequence of the previous substrate was lengthened to account for exosite binding to BoNT/A. We synthesized the peptide PL50 (SNAP-25-NH₂ acetylated at positions 156 to 203 [Nop¹⁹⁷, Pya²⁰⁰, Nle²⁰²]) and its analogue PL51, in which all methionines were replaced by nonoxidizable Nle. Consistent with a large increase in affinity for BoNT/A, PL50 and PL51 exhibit catalytic efficiencies of 2.6 x 10⁶ M^–1 s^–1 and 8.85 x 10⁶ M^–1 s^–1, respectively, and behave as the best fluorigenic substrates of BoNT/A reported to date. Under optimized assay conditions, they allow simple quantification of as little as 100 and 60 pg of BoNT/A, respectively, within 2 h with a classical fluorimeter. Calibration of the method against the mouse 50% lethal dose assay unequivocally validates the enzymatic assay.

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* Corresponding author. Mailing address: Pharmaleads, Paris BioPark, 11 Rue Watt, 75013 Paris, France. Phone: (33) 1 43 25 50 45. Fax: (33) 1 43 26 69 18. E-mail: Bernard.roques{at}pharmaleads.com

Published ahead of print on 8 May 2009.

Supplemental material for this article may be found at http://aem.asm.org/.

These authors contributed equally to this work.

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Applied and Environmental Microbiology, July 2009, p. 4382-4390, Vol. 75, No. 13
0099-2240/09/$08.00+0     doi:10.1128/AEM.00091-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.