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.
Department of Cell Biology and Biochemistry, Integrated Toxicology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA.
Benjamin
B
Rowe
James J
JJ
Schmidt
Leonard A
LA
Smith
S Ashraf
SA
Ahmed
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