## 4 Results and Discussion  
### The Biomolecular Toxicity and Activity of Botulinum Neurooxins (BoNTs)  
(a) The physical-chemical featuresThe BoNT [Chemical Abstracts Service Registry Numbers (CASRNs): 107231-15-2 (botulin b > f), 93384-43-1 (botulin b > a; botulin b > um toxin a), 93384-45-3 (toxins botulin b > c)] exotoxins are produced and secreted under anaerobic conditions by the _C. botulinum_ (categorized based on what BoNTs produce and not on their phylogenetic affinity) [1,18]. BoNTs are the most potent biotoxins found in nature (in mice LD50 = 1 ng/kg for type A) and therefore are 100 billion times more toxic than cyanide. BoNTs belong to the zinc endopeptidases that are proteins with a molecular weight of about 150 kDa. The toxin is released during bacterial cell lysis and each strain of _C. botulinum_ usually produces only one type. Its structure consists of two fractions, M and L (larger), linked by a disulfide bond. An additional hemoglutin is also attached in the L chain [19]. Thus, BoNT-A and BoNT-B contain two polypeptide chains, the A and B chains, that play different roles and are linked together by a disulfide bond and a nontoxic hemagglutinin. The light chain A has a molecular weight of about 50 kDa and the heavy chain B has a molecular weight of about 100 kDa [20]. It is the heavy polypeptide chain that binds to the acetylcholine synapses of peripheral nerve cells. Light chain is a zinc-containing protease that acts by binding to proteins that play a role in the mechanism of acetylcholine release (SNAP-25, syntaxine synaptobevin-VAMP). In cultures, BoNTs bind to other proteins to form protein complexes weighing 300-900 kDa [21,22]. Some BoNTs are not inactivated by enzymes in the intestine but are absorbed in the upper part of the neuromuscular junction and inhibit the release of acetylcholine. There are seven types of _C. botulinum_ toxins in terms of their antigenicity, denoted by the Latin letters A, B, C, D, E, F, and G and a number of subtypes (e.g., for type A there are five subtypes A\({}_{1}\), A\({}_{2}\), A\({}_{3}\), A\({}_{4}\), A\({}_{5}\)) [1,22]. The neurotoxins of each type differ up to 70% in amino acid level, while the subtypes differ between 2.6 and 32%. The most common types that affect humans are A, B, E, and rarely F, and the types C, D, and E can cause diseases in other mammals, birds, and fish [10,23]. Thus, there are four _C. botulinum_ groups I, II, III, and IV. Group I proteolytic C. _botulinum_ produces toxins of types A, B, or F and the nonproteolytic group II C. _botulinum_ produces toxins of types B, E, or F. These two strains differ significantly genetically and are responsible for most cases of botulism [24]. These bacteria are present in soil and in aquatic sediments worldwide, growing and producing botulinum toxins under anaerobic conditions. The genetic information that encodes their production can be chromosomal or plasmid [25]. Modality of the toxic action  
BoNTs are the most potent toxins found in nature (e.g., in mice the LD50 = 1 ng/kg for type A) and are 100 billion times more toxic than hydrogen cyanide and Sarin (an organophosphorus nerve gas compound) [26]. Since even a small amount of BoNTs, entered through ingestion, inhalation or absorption through the eye or a continuous solution of the skin can cause serious illness, all materials suspected of containing the contaminating toxin require particular attention in handling. BoNTs are active by either ingestion or inhalation (they do not occur naturally) and the clinical effect on the patient is similar in both ways of entering the body. Samples should be placed in unbreakable sterile sealed refrigerated containers and examined as soon as possible. Samples taken from wounds are an exception and should not be refrigerated [1,9].