Elsevier

Analytical Biochemistry

Volume 396, Issue 2, 15 January 2010, Pages 188-193
Analytical Biochemistry

Rapid product analysis and increased sensitivity for quantitative determinations of botulinum neurotoxin proteolytic activity

https://doi.org/10.1016/j.ab.2009.09.034Get rights and content

Abstract

The ultimate molecular action of botulinum neurotoxin (BoNT) is a Zn-dependent endoproteolytic activity on one of the three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. There are seven serotypes (A–G) of BoNT having distinct cleavage sites on the SNARE substrates. The proteolytic activity is located on the N-terminal light chain (Lc) domain and is used extensively as the primary target toward therapeutic development against botulism. Here we describe an improved method using ultra-performance liquid chromatography (UPLC) whereby quantitative data were obtained in 1/10th the time using 1/20th the sample and solvent volumes compared with a widely used high-performance liquid chromatography (HPLC) method. We also synthesized a VAMP (vesicle-associated membrane protein)-based peptide containing an intact V1 motif that was efficiently used as a substrate by BoNT/D Lc. Although serotype C1 cleaves the serotype A substrate at a bond separated by only one residue, we were able to distinguish the two reactions by UPLC. The new method can accurately quantify as low as 7 pmol of the peptide substrates for BoNT serotypes A, B, C1, and D. We also report here that the catalytic efficiency of serotype A can be stimulated 35-fold by the addition of Triton X-100 to the reaction mixture. Combining the use of Triton X-100 with the newly introduced UPLC method, we were able to accurately detect very low levels of proteolytic activity in a very short time. Sensitivity of the assay and accuracy and rapidity of product analysis should greatly augment efforts in therapeutic development.

Section snippets

Materials

Recombinant BoNT Lc protease of serotype A (LcA) and serotype B (LcB) were purified as described previously [11], [12], [13], and a purification similar to that of serotype D (LcD) will be published elsewhere. LcC1 was purified as described previously [14]. A SNAP-25 (synaptosomal-associated protein of 25 kDa) sequence-derived substrate peptide for LcA (SNKTRIDEANQRATKML) [6], [15], a VAMP (vesicle-associated membrane protein) sequence-derived substrate peptide [16] for LcB

UPLC analyses of substrate and products

The endopeptidase activity of BoNT Lc can be followed by measuring the disappearance of substrate or the appearance of either product. However, any method that allows simultaneous detection and measurement of both substrate depletion and product accumulation allows an assessment of the possibility of substrate used in any other contaminating reaction. Because peptides strongly absorb in the ultraviolet (UV) region centered at 210 nm, continuous monitoring of chromatographic eluate of

Acknowledgments

The research described here was supported by the Joint Science and Technology Office for Chemical and Biological Defense (JSTO–CBD, 3.10011_06_RD_B and 3.10012_06_RD_B). We thank Stephen I. Toth, Matthew L. Ludivico, and Trista Haupt for assistance with the UPLC setup and enzyme assays; Ernst Brueggemann for the mass spectrometry data; and John Cardellina for detergent structures. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily

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