Development of a Fluorescence Internal Quenching Correction Factor to Correct Botulinum Neurotoxin Type A Endopeptidase Kinetics Using SNAPtide

Abstract

Botulinum 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 SNAP-25 for 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 FRET peptide, was used in this study as a SNAP-25 homologue for the endopeptidase kinetics study of BoNT/A LC. SNAPtide uses a 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.

A graphene oxide based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of botulinum neurotoxin A (BoNT/A) enzymatic activity

“…The limit of detection (LOD) for BoNT-LcA protease activity detection is around 1 fg/mL based on the background signal plus 3 times of standard derivation. These results are superior to the current detection methods including mouse bioassay (Shapiro et al, 1998), immunoassays (Cai et al, 2007;Lindstrom and Korkeala, 2006;Sapsford et al, 2008), organic fluorophore based FRET biosensors and the commercial available FRET kits (Dong et al, 2004;Gilmore et al, 2011;Mangru et al, 2005;Sun et al, 2009;Feltrup and Singh, 2012), whose BoNT LODs are in the range from 1 pg/mL to 1 ng/mL.…”

High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics

“…The calculated kinetic parameters of rLC-TD/A (Fig. 7(A)) was compared with that of rLC/A (earlier published by our group, [22]) and BoNT/A, with experiments performed under similar conditions. The results showed 2.8 - fold increase in Km of rLC-TD/A compared to the rLC/A (Table 2).…”

“…The enzyme kinetics was performed at 37 °C for the first ten minutes using 50 nM rLC-TD/A and various concentrations (0–8 µM) of SNAPtide® 521 and the kinetic parameters were calculated according to the published protocol [22]. The hydrolysis of the substrate was monitored with 490 nm excitation and 523 nm emission.…”

“…The increase in fluorescence signal, with respect to blank, indicates the extent of cleavage of the substrate or increase in activity of the protein. The RFU values were converted into µM using the method described in the published work [22]. Kinetic parameters were determined using the Lineweaver-Burk plot.…”

An Ultrasensitive Gold Nanoparticle-based Lateral Flow Test for the Detection of Active Botulinum Neurotoxin Type A

“…1a). The sequence of specific cleavage site was based on SNAPtide®, a very short SNAP-25 homologue with a unique cleavage site for BoNT/A [12, 13]. After incubation with BoNT/A-spiked serum sample for a certain time duration, the biotin at the amino terminus of the peptide was cleaved off, resulting in that gold nanoparticle-labeled streptavidin-substrate peptide complexes could not be captured on the test zone of LFTS, thus, LFTS presented a different coloration from BoNT/A-negative result (Fig.…”