ac302997n_si_001.pdf (170.61 kB)
Development of a Fluorescence Internal Quenching Correction Factor to Correct Botulinum Neurotoxin Type A Endopeptidase Kinetics Using SNAPtide
journal contribution
posted on 2012-12-18, 00:00 authored by Thomas
M. Feltrup, Bal Ram SinghBotulinum neurotoxins (BoNTs), which are highly toxic
proteins responsible for botulism, are produced by different strains
of Clostridium botulinum. These various strains of
bacteria produce seven distinct serotypes, labeled A–G. Once
inside cells, the zinc-dependent proteolytic light chain (LC) degrades
specific proteins involved in acetylcholine release at neuromuscular
junctions causing flaccid paralysis, specifically synaptosomal-associated
protein 25 (SNAP-25) for botulinum neurotoxin type A (BoNT/A). BoNT
endopeptidase assays using short substrate homologues have been widely
used and developed because of their ease of synthesis, detection limits,
and cost. SNAPtide, a 13-amino acid fluorescence resonance energy
transfer (FRET) peptide, was used in this study as a SNAP-25 homologue
for the endopeptidase kinetics study of BoNT/A LC. SNAPtide uses a
fluorescein isothiocyanate/4-((4-(dimethylamino)phenyl)azo) benzoic
acid (FITC/DABCYL) FRET pair to produce a signal upon substrate cleavage.
Signal quenching can become an issue after cleavage since quencher
molecules can quench cleaved fluorophore molecules in close proximity,
reducing the apparent signal. This reduction in apparent signal provides
an inherent error as SNAPtide concentrations are increased. In this
study, fluorescence internal quenching (FIQ) correction factors were
derived using an unquenched SNAPtide peptide to quantify the signal
quenching over a range of SNAPtide concentrations and temperatures.
The BoNT/A LC endopeptidase kinetics at the optimally active temperature
(37 °C) using SNAPtide were studied and used to demonstrate the
FIQ correction factors in this study. The FIQ correction factors developed
provide a convenient method to allow for improved accuracy in determining
and comparing BoNT/A LC activity and kinetics using SNAPtide over
a broad range of concentrations and temperatures.