Rapid product analysis and increased sensitivity for quantitative determinations of botulinum neurotoxin proteolytic activity.
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.
Rowe
Benjamin
B
Department of Cell Biology and Biochemistry, Integrated Toxicology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA.
Schmidt
James J
JJ
Smith
Leonard A
LA
Ahmed
S Ashraf
SA
D000595:Amino Acid Sequence; D001905:Botulinum Toxins / Q000145:classification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057230:Limit of Detection; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000145:classification / Q000378:metabolism; D012703:Serotyping; D013379:Substrate Specificity
D010455:Peptides; D011994:Recombinant Proteins; D017830:Octoxynol; D001905:Botulinum Toxins