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  • nisoldipine The different serotypes A G have unique receptor


    The different serotypes (A-G) have unique receptors to which they bind to produce the toxic effect on the nerve cells. (Lance, 2004, Rosetto et al., 2014). Intracellularly, the endopeptidase activity of different serotypes of BoNT target selectively a unique group of proteins, the SNARE nisoldipine (Kumar et al., 2014, Rosetto et al., 2014). BoNT/A and BoNT/E cleave SNAP-25 (synaptosomal associated protein), (Blasi et al., 1993, Schiavo et al., 1993, Binz et al., 1994), BoNT/C cleaves both syntaxin and SNAP-25 (Blasi et al., 1993) and BoNT/B, BoNT/D, BoNT/F and BoNT/G cleave VAMP (Niemann et al., 1994). Interestingly, cleavage sites for each of these BoNT serotypes are unique and very specific. It becomes necessary to detect the presence of this deadly bacterial toxin since it is capable of contaminating air, liquid and solid food. If fallen into the wrong hands BoNT could lead to bio-terrorism and fatal casualties (Franz et al., 1997, Altas, 2002). Appropriate biological assays are needed to detect, diagnose, and develop antidotes. There are a few assay platforms which have been developed for detection of botulinum neurotoxin. Mouse bioassay is the gold standard and most sensitive technique for the detection of biologically active BoNT, but there are several drawbacks (Cherington, 2004, Kukreja and Singh, 2014). This technique is expensive to perform, requires sacrificing of mice and is not specific for a BoNT serotype unless used in combination with antibodies. Moreover, it takes up to 4 days to complete, which makes it unsuitable for rapid detection and screening. Because of its disadvantages, various attempts are being made in order to replace the mouse bioassay. Another technique used for the detection of botulinum neurotoxins is ELISA, but this technique also is time-consuming. One of the other disadvantages of ELISA is that it is able to detect the presence of BoNT but it fails to distinguish if the toxin is in its active or inactive state (Moorthy et al., 2004). Therefore, it is important to have an assay that is cost-effective, easy to perform, robust, easy to handle and has high sensitivity. The cleavage of the BoNT endopeptidase substrate or its derived peptide (e.g., SNAPtide) is commonly used for assaying and detection (Wang and Singh, 2010, 2012; Feltrup and Singh, 2012). Peptide substrates of varying length (15–66 residues) have been tested for efficient cleavage and kinetics, with varying results (Schmidt and Bostian, 1995, Silhar et al., 2010). List Biologicals developed a 13-mer peptide by replacing the Glutamine-194 of SNAP-25 with glutamic acid SNAPtide, which showed catalytic efficiency (Kcat/Km) of LCA as 28.5 × 104 M−1sec−1 (Todd et al., 2005), which is comparable to full-length substrate GST-SNAP25 is 27.0 × 104 M−1 s−1 (Baldwin et al., 2004). Additionally, Breidenbach and Brunger (2004), demonstrated the involvement of α and β exocites in the endopeptidase activity of BoNT/A LC. These exocites are involved in the binding and recognition of the substrate. Therefore, the requirement of a longer peptide as a substrate is unique for the BoNT endopeptidase. It is also known that the size of the enzyme and substrate make a difference in the enzyme activity (Wang et al., 2010, Mizanur et al., 2013). The requirement of longer substrate raises the following questions: a) Does the size of enzyme and substrate play a role in the endopeptidase activity and kinetics of proteolysis? and b) What is the role of different domains and associated proteins in the endopeptidase activity of this enzyme? Additionally, the uniqueness of active site (20 Å deep negatively charged active site), the presence of exosites (α and β), and unique substrate recognition mechanism make this problem more interesting (Breidenbach and Brunger, 2004). In other words, detection of toxin is not just an analytical problem, it needs a careful consideration of the biochemical function of this molecule.