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Low molecular weight peptides in the venom of Odontobuthus odontrous (Scorpiones: Buthidae)
Kinza Zafar, Hafiz Muhammad Tahir, Rabia Mishal, Muhammad Arshad, Muhammad Khalid Mukhtar, Muhammad Mohsin Ahsan, Azhar Abbas Khan & Shafaat Yar Khan 1. Department of Biological Sciences, University of Sargodha, Pakistan 2. Department of Entomology, University College of Agriculture, University of Sargodha. hafiztahirpk1@yahoo.com ABSTRACT Odontobuthus odontrous is one of the medically important and dangerous scorpions belonging to family Buthidae. Present study was aimed to evaluate the presence of low molecular weight peptides in the venom of the O. odontrous. Low molecular weight peptides are therapeutically important and they have significant role in ion channel blocking and tumor growth impairing. Venom fractions were separated by using High Performance Liquid Chromatography. The results of present study confirmed the presence of at least five low molecular weight peptides in the range of 2-8kDa.The recognition of these low molecular weight peptides can not only be an important step in producing effective anti-venom against such dangerous scorpion species, but also these peptides are probes for identifying specific types of ion channels. These peptides can also be the important tools for understanding ion channel physiology. Moreover, this study can be helpful in more advancement in drug development, cancer treatment, or other pharmaceuticals. Key words: Odontobuthus odontrous, low molecular weight peptides, scorpion venom. INTRODUCTION Scorpions are most primitive arthropods belonging to order scorpiones and class Arachnida (Devila et al., 1996; Ruming et al., 2010). They are classified into 18 families and about 1500 different species and subspecies (Prendini & Wheeler, 2005; Bawaskar & Bawaskar, 2012). The most fatal and medically important scorpions belong to Buthidae family (Michael, 2003).

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The venom of scorpion is water soluble and composed of mucosa, oligopeptides, nucleotides, amino acids, ions, neurotransmitters and low molecular weight peptides (Possini et al., 1999). It also includes enzymes such as phospholipase, hyaluronidase, lipase (Zouari et al., 2005; Park et al., 2007) alkaline phosphotases and proteolytic enzymes (Incesu et al., 2005). In excitable membranes the low molecular weight peptides which are cysteine rich, directly affect the Na+, K+, Cl, and Ca2+ ion channels (DeBin al., 1993). The blocking activity of certain toxins on the voltage gated channels for impairing the growth of tumors has been studied (Fiske et al., 2006; Gomez-Varela et al., 2007; Pillozzi et al., 2009). Especially the K+ and Cl- channels stop proliferation of neoplastic cells (Pardo, 2004; Kunzelmann, 2005). Being an active blocker of gated channels, it can be first made safer and then employed to target certain cancerous cells (Fiske et al, 2006). Certain glioma cells in the brain can be prevented by utilizing toxins from scorpion venom (Lyons et al., 2002). The venom of scorpions contains toxins which after modulation can be utilized for synthesis of eco-friendly insecticides (Leng et al., 2011), in cancer treatment (Incesu et al., 2005), protein-engineering scaffolds (Angeletti, 1998), and production of biopesticides (Blake, 2007; Gurevitz, 2010). Because of the high affinity and selective interaction with ion channels scorpion toxins can be used as a probe with the structures of different ion channels and also evaluate their physiological effect to the different cells (Wanke & Restano-Cassulini, 2007; Massensini et al., 2002). Peptides of the some scorpion venom are potential blocker of active channels and can destroy the cancerous cells actively (Fiske et al., 2006). The cytotoxic and anti-proliferative effects of scorpion venoms have also been confirmed (Salarian et al., 2012). Scorpion venom has been proved to be beneficial in the treatment of different neurological disorders, cancers, cardiovascular diseases, leukemia, autoimmune diseases, HIV, carcinomas, haemorragic, pancreatitus, epilepsy and transplant rejections

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(Wang et al., 2005; Zargon et al., 2010; Gupta et al., 2007; Chen et al., 2012; Sarzaeem et al., 2012; Song et al., 2012). Scorpion venom is also being used for the production of different painkillers, vaccines and protein engineering scaffold (Fabiano et al., 2008). Aim of present project was to recognize the low molecular weight peptides (if any) in the venom of O. odontrous. The outcome of the study will be helpful to pave the ways of future potential applications of scorpion venom in different industries of health, agriculture and pharmaceuticals. MATERIALS AND METHODS Scorpion collection The study was conducted at Department of Biological Sciences, University of Sargodha, Pakistan from March 2012 to September 2013. For the study scorpions were collected from sandy areas of Sargodha District (32 03 00 24 N) (73 36 01 54 E), using portable UV lamps. Collection was done during nigh. Extraction of Venom Venom of the scorpions was milked by electrical stimulation of the telsons. The venom was collected in 1.5ml ependroffs and frozen at -20 C until use. Sixty adult scorpions of same size and weight were milked to obtain the venom. Sample venom preparation The lyophilized crude venom was dissolved in (100%) HPLC graded acetonitrile and centrifuged at 14,000 rpm for 15 minutes. The mucous material and un-dissolved residues were separated apart. The supernatant was collected in 1.5 ml eppendrofs for loading over organic column of HPLC. Characterization of Venom

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To characterize the venom supernatant was filtered with the help of 0.45 nylon syringe filter papers of and was loaded over organic column equilibrated with acetonitrile. The flow rate was adjusted to 1ml/mins. The temperature was kept 37 C and the maximum pressure of pump was 400bar. Molecular peaks were recorded on refractive index detector (1200 series Agilant). Acetonitrile was used as a mobile phase and the run for each sample was approximately 1 hour. RESULTS AND DISCUSSION The results of the HPLC showed five, peaks each corresponding to a different peptide fraction. Molecular weights of the lowest and highest peaks were 2.76 kDa and 8.64 kDa respectively. The detail of the peptide fractions and their molecular weights is given in the Figure 1. Gomes et al. (2010) reported in their study that the venom of many scorpion species contain low molecular weight peptides (less than 10 kDa), as also apparent from our findings. These low molecular weight peptides distinctly alter the Na+, Ca2+, K+, Cl- ion channels (Possani et al., 2000; Rodrguez & Possani, 2005). The short-chain neurotoxins with 3 to 4.4 kDa act on potassium or chloride channels and long-chain neurotoxins, which have 6.5 to 8.5 kDa, mostly act on sodium channels (Possani et al., 2000; Rodrguez & Possani, 2004; Rodrguez & Possani, 2005). It has also been observed that low molecular weight peptides are excellent membrane blockers and control the signal transduction pathways in malignant tumor series (Hayden et al., 2006). This distinction can be employed in tumor growth impairing i.e. by blocking voltage gated channels (Fiske et al., 2006; Gomez-Varela et al., 2007). As the low molecular weight peptides are actively involved in K+ channel blocking so they can be employed in tumor treatment. According to Smith et al. (2012) buthid venom contains peptides mostly between the range of 7 and 8 kDa while non-buthids venom contains peptides less than 5 kDa. However, the results of present study are contrary to smith et al. (2012) as we recorded at least four peptides with molar masses less than 5 kDa from the O. odontrous (Buthid scorpion) and only one peptide was with molar mass greater than 5kDa.

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Ozkan et al. (2011) reported four protein bands with molecular masses of 4, 6, 31, and 46 kDa in the venom of Leiurus abdullahbayrami. Among them protein bands with molecular masses of 4 and 6 kDa were more strongly detected than other protein bands. Amir et al. (1994) have also found four peptide fractions in the venom of Isometrus vittatus (buthids scorpion) having molecular weights 80kDa, 75.5 kDa, 19 kDa and 17 kDa. Dyason et al. (2002) have also reported two major groups of peptide fractions from the venom of Parabuthus scorpions, the short chain K+ peptides in the range of 3-4.5 kDa and long chain Na+ toxins are in the range of 6.5-8 kDa. CONCLUSION Here we report the presence of low molecular weight peptides in the venom of Odontobuthus odontrous. The most important characteristic of these low molecular weight peptides is their application for cancer treatment and tumor growth impair i.e., in medicine. Although scorpion venom is composed of extremely diverse active peptides which induce both toxicological and immunological responses but also contain low molecular peptides that offer an excellent resource for use in drug development. REFERENCES Amir, R., Alam, J. M. and Khan, M.A.J. (1994). Isolation and biological properties of scorpion venom:IV Comparative study on four proteinases isolated from the venom of Isometrus Vittatus. Pak. J. Pharma. Sci., 7(1): 21-32. Bawaskar, H. S. and Bawaskar, H. P. (2012). Scorpion Sting: Update. J. Assoc. Physicians India., 60: 46-53. Blake, K. (2007). Scorpion Toxin Makes Fungus Deadly to Insect Pests. Ellen Ternes, 301: 405-4076. Chen, Y., Cao, L., Zhong, M., Zhang, Y. and Han, C. (2012). Anti-HIV-1 Activity of a new Scorpion Venom Peptide Derivative Kn2-7. PLoS ONE, 7: 34947.

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Song, X., Zhang, G., Sun, A., Guo, J., Tian, Z., Wang, H. and Liu, Y. (2012). Scorpion venom component III inhibits cell proliferation by modulating NF-B activation in human leukemia cells. Exp. Therap Med., 4: 146-150. Wang, W. X. and Ji, Y. H. (2005). Scorpion venom induces glioma cell apoptosis in vivo and inhibits glioma tumor growth in vitro. J. Neurooncol., 73: 17. Wanke, E. and Restano-Cassulini, R. (2007). Toxins interacting with ether-a` -go-gorelated gene voltage-dependent potassium channels. Toxicon, 49: 239248. Zargon, J., Umar, S, Sajad S., Naime, M., Ali S, Khan H A et al. (2010).Scorpion venom (Odontobuthus doriae) apoptosis by depolarization of mitochondria and reduces Sphase population in human breast cancer cells (MCF-7). Toxicon In Vitro. 25: 9. Zouari, N., Miled, N., Cherif, S., Mejdoub, H. and Gargouri, Y. (2005). Purification and characterization of a novel lipase from the digestive glands of a primitive animal: The scorpion. Biochimica et Biophysica Acta, 1726: 67 74.

Figure 1. Fractions and their molar masses extracted from the venom of Odontobuthus odontrous.

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10 9 8 Molar mass (kDa) 7 6 5 4 3 2 1 0 I II III Fractions IV V 2.76 2.94 3.34 4.9 8.64

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