SNAP Etide® Peptide Substrate (o-Abz/Dnp) for C. botulinum Type E Neurotoxin

• Product #550 – SNAP Etide Peptide Substrate for Botulinum Toxin Type E

Experimental SNAP-Etide assay:

Testing of compounds identified through the virtual screen was performed using a commercially available SNAP-Etide substrate (List Biological Inc., CA) as employed in prior work, ...

• SNAP Etide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type E Neurotoxin

Materials - 

SNAP-Etide^TMsubstrate (Product #550) and botulinum neurotoxin type E light chain, recombinant (Product #635A), are both products of List Biological Laboratories, Inc.

Methods - 

Fluorimentric Assay:

Continuous assays were performed on a SPECTRA max GEMINI XS fluorescence microplate reader (Molecular Devices, Sunnyvale, CA) using Greiner FLUO-TRAC black flat-bottomed plates (E&K Scientific, Campbell, CA).  Stock solutions of the FRET substrate was made in dimethylsulfoxide (DMSO).  Final dilutions were made in the appropriate buffer.  Plates were equilibrated at 37C for 15 min prior to initiation of the reaction.  For all experiments the time-dependent increase in fluorescence intensity was monitored at 37C. The excitation wavelength was set to 321 nm and emission to 418 nm. 

Buffer Optimization:

FRET assays were performed to test the activity of LcE with SNAP-Etide^TMas a function of pH, Tween-20 and ZnCl₂.  Three separate experiments were performed (Figure 1).  The cleavage reaction was initiated with addition of 5 nM LcE to the wells containing 10 M SNAP-Etide^TM in the appropriate buffer.  Initial velocities of cleavage in RFU/sec were evaluated and compared for each assay in order to determine the optimum buffer conditions for the reaction.

LcE Titration:

LcE titration experiment was performed in 50 mMHEPES, pH 7.8, 0.1% Tween-20, using 10 M SNAP-Etide^TM.  LcE was prepared at 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 M concentrations.  Following equilibration, the cleavage reaction was initiated with addition of 10 M SNAP-Etide^TM.  Initial velocities of cleavage were plotted against LcE concentration (Figure 2).

Trypsin Digest: 

Dilutions of SNAP-Etide^TMwere prepared in 50 mM HEPES, pH 7.8, 0.1% Tween-20 to achieve 70, 60, 50, 40, 30, 25, 15, 7.5, 3.75, 1.88, and 0.94 M concentrations.  The reaction was initiated with addition of 10 nM trypsin into each well.  End point readings were taken after 50 min.  A second round of 10 nM trypsin was added to each well in order to achieve total enzyme digestion.  The maximum fluorescence reached was graphed as RFU/5000 against SNAP-Etide^TM concentration (Figure 3A).  An identical experiment was run using 2.5 nM LcE for digestion of SNAP-Etide^TM.  Initial velocities of cleavage were graphed in RFU/sec against substrate concentration (Figure 3B).

Inner Filter Effect Correction: 

Dilutions of SNAP-Etide^TM were prepared in 50 mM HEPES, pH 7.8, 0.1% Tween-20 to achieve concentrations ranging from 250 M to 2 M.  Fluorescence end point readings of SNAP-Etide^TM at each concentration were recorded.  In order to determine theinner filter effect at each substrate concentration another set of end point fluorescence (RFU) readings were recorded after addition of 5.0 M free o-Abz-Lys.  Fluorescence intensity obtained for SNAP-Etide^TM was then subtracted from the fluorescence intensity obtained for SNAP-Etide^TMand o-Abz-Lysin order to obtain fluorescence for the free o-Abz-Lyspeptide.  The decrease in fluorescence of the o-AbzLysin the presence of SNAP-Etide^TM reflects the inner filter effect (Table 1).  A correction factor is obtained for each SNAP-Etide^TM concentration: 

correction factor = RFU (o-Abz-Lys) at each [SNAP-Etide^TM] RFU (o-Abz-Lys)

Initial reaction rates were obtained for each substrate concentration after addition of 2.5 nM LcE.  The rates were corrected as given in Table 1.  The plots of initial velocity versus SNAP-Etide^TM concentration (Figure 4)indicates a decreasing rate of cleavage at concentrations of substrate greater then 100 M.  This is consistent with substrate inhibition.  The kinetic data was analyzed using the substrate inhibition equation from Kaleida Graph software: 

ax b+(x(1+x/c)) , where a = Vmax, b = Km, and c = Ki, competitive inhibition constant

• Product #550 - SNAP-Etide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type E Neurotoxin
• Product #635A - Botulinum Neurotoxin Type E Light Chain, Recombinant

... Pertussis toxin (List Biological Laboratories, Cambell, CA) treatment of macrophages was carried out as described (Holian, 1986). ...

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:

• Product #180 - Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 - Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A - Pertussis Toxin from B. pertussis (in Glycerol)