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The Malayan krait (Bungarus candidus) is a neurotoxic Elapid snake which is commonly found in Southeast Asia. The venom of krait contains two classes of toxin, that are the post-synaptically active α-bungarotoxins and the pre-synaptically active β-bungarotoxins. The similarity of neurological symptoms has been reported in victims envenoming by B. candidus from different geographical areas.
The authors are trying to identify the major postsynaptic neurotoxin in the venom of Javan B. candidus. They isolate and purify the snake venom from Javan B. candidus and Bungarus multicinctus from Taiwan by high performance liquid chromatography (HPLC), SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Electrospray-mass spectrometry (ES-MS). By comparing the mass (7983.75 Da), lethal toxicity (LD50) (0.23 mg/g in mice), affinity to nicotinic acetylcholine receptors, and by 40 N-terminal amino acid residues as determined by Edman degradation of B. multicinctus, they conclude that the toxin in B.candidus is indistinguishable from a toxin originally isolated from B. multicinctus , that is α-bungarotoxin (A31). Besides, they also confirm their points by cloning and sequencing a genomic DNA from B. candidus which encodes the 74 amino acid sequence of A31 and part of its signal peptide, revealing complete identity to the A31 gene in exon and 98.9% identity in intron sequences. Furthermore, they also sequence the entire mitochondrial cytochrome b gene of B. candidus and B. multicinctus for comparison. Thus I think the authors are succeeded to determine that the two snakes are closely related. 1
However, there is some incompleteness in this experiment. Although they manage to identify the neurotoxin in B. candidus as α-bungarotoxins, they do not prove that the toxin is the major postsynaptic neurotoxin. In order to strengthen their points, I will extract other minor postsynaptic toxin to compare with the power of the toxin. There is some similar toxin in the venoms of all elapid snakes and their close relatives, the sea snakes. For example, k-Bungarotoxins which is a minor component of the venom is found exclusively in the venom of kraits. They are structurally similar to the A31 and bind to neuronal acetylcholine receptor.
Furthermore, the authors do not explain how α-bungarotoxins can cause paralysis in victim post-synaptically. They should explain more about it to strengthen their points. A31 which is a long neurotoxin causes a failure of neuromuscular transmission for 2-3 hours by binding to post-synaptic nicotinic acetylcholine receptor at the neuromuscular junction (NMJ) in a relatively irreversible manner. This will deplete the release of synaptic vesicles from nerve terminal boutons. Muscle contraction is inhibited and caused paralysis of the mice.2
I think by determining the LD50 of the toxin is not enough to confirm that A31 is the major postsynaptic neurotoxin. LD50 is unreliable to measure toxicity of the venom and results may be influenced by genetic characteristics of the snake population, animal species tested, environmental factors and mode of administration. Other reason is it can only measure acute toxicity and does not consider toxic effect that cannot cause death but are nonetheless serious like brain damage. LD50 is also different between species which means that it is safe for mice but it can be extremely toxic for humans and vice versa. 3 Hence I think their methods are not suitable. Different amounts of venom should be taken to determine the LD50 when measuring toxicity as even though it is the most poisonous venom, if the amount of venom is little, it can also do not cause death or have any effects to the victims. 4
The authors also fail to determine the dissociation constant (KDs) accurately by competition with the initial association rate of 125I-α-bungarotoxin. These may be due to several reasons. The calculated KD or the differences in affinity is depended on the method used. Some toxins and receptors may be degraded when the toxins are left incubated overnight at the room temperature to make sure the toxins had reached equilibrium. Furthermore, this equilibrium competition binding assays may lead to slightly lower affinities than determined by other methods in the case of very high affinity ligands such as α-bungarotoxin due to ligand depletion. Sample purity can also affect the binding affinity. In this experiment, the degree of purity of the toxin from B. candidus venom is determined by ES-MS analysis, but the commercial sample of α-bungarotoxin is unknown. The authors think about these factors and just assume the affinities of the α-toxin from B. candidus venom and α-bungarotoxin to be indistinguishable in this experiment. In my opinion, KDs should be determined accurately to prove that α-bungarotoxin has the highest affinity to the acetylcholine receptors (AchR) compare to other compound. The toxin should not be left overnight for incubation to prevent degradation of both toxin and receptor. As we know, the degrees of sample purity may influence the binding affinity. Thus, the degree of purity of the commercial sample of α-bungarotoxin should also be determined by ES-MS. 1
In order to improve this experiment, I will collect the venom of B. candidus from different geographic areas to confirm that α-bungarotoxins is the major postsynaptic neurotoxin as after envenoming with snake, some patients are managed to survive while some patients are died. For determining the LD50, 0.2 ml of serially 1.4 fold-diluted venom solutions is intravenous injected into the tail vein of mice. Different areas of snakes and their venom are tested. The control group is performed using normal saline solution. The endpoint of lethality of the mice is determined after 24 hours observation and LD50 is also calculated like the authors do. 5
A major class of nicotinic receptors in the nervous system is the one that binds α-bungarotoxin and contains the α7 gene product. Besides, I will use PC12cells to study nicotinic receptors and determine the affinity of the toxin because they can bind to the toxin. The number of the toxin binding sites on the surface of PC12 cells is determined. PC12 cells are plated on 24 well plates for 24-48 hours before the assay is conducted. Culture medium is removed and replaced with identical medium that included 10 nM 125I-α-Bungarotoxin with and without either 1 mM α-Bungarotoxin or 250 mM nicotine. The cells are incubated for 1 hour at 37°C and then are rinsed four times with 2 ml aliquots of HEPES solution. Cells were solubilized and scraped in 0.5 ml of 1N NaOH. Determinations are done in triplicate within each experiment. 6