J Arthropod-Borne Dis, December 2013, 7(2): 139–146 M Khoobdel et al.: Purification of the… http://jad.tums.ac.ir Published Online: August 31, 2013 Original Article Purification of the Immunogenic Fractions and Determination of Toxicity in Mesobuthus eupeus (Scorpionida: Buthidae) Venom Mehdi Khoobdel 1, Taghi Zahraei-Salehi 2, Bahar Nayeri-Fasaei 2, *Mohammad Khosravi 1, Zahra Omidian 3, Mohammad Hassan Motedayen 4, Abolfazal Akbari 4 1Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran 2Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran 3Department of Parasitology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran 4Razi Vaccine and Serum Research Institute-Karaj Branch, Karaj, Iran (Received 25 Jun 2012; accepted 22 Jan 2013) Abstract Background: Scorpions stings are a health problem in many parts of the world. Mesobuthus eupeus (Buthidae) is the most prevalent species in the Middle East and Central Asia. Definition of toxicogenic and immunogenic characteris- tics of the venom is necessary to produce antidote. In this study, the noted properties of M. eupeus venom were eval- uated. Methods: Venom was obtained by milking M. eupeus scorpions for lyophilization. Toxicity was determined after injecting the venom to albino mice and calculating LD50. Polyclonal antibodies against M. eupeus venom were ob- tained from immunized rabbits. The CH-Sepharose 4B column was used for isolating the specific antibodies. 10 mg of the affinity-purified antibodies were conjugated with a CH-Sepharose 4B column and M. eupeus venom was ap- plied to the column. The bound fragments were eluted using hydrogen chloride (pH: 2.5). Crude venom and affinity- purified fractions of the venom were analyzed by SDS-PAGE technique. Results: Lethal dose (LD) was 8.75, 11.5 and 4.5 mg/kg for IP, SC and IV respectively. The LD50 of M. eupeus venom was 6.95 mg/kg. The crude venom had 12 detectable bands with molecular weights of 140, 70, 50, 33, 30, 27, 22, 18, 14, 10 kDa and two bands less than 5 kDa. The affinity-purified venom presented eight bands. The 27 kDa band was clearly sharper than other bands but 70, 18, 10 and one of the less than 5 kDa bands were not observed. Conclusions: Contrary to popular belief, which know scorpion venom as non-immunogenic composition, the current study was shown that the most fractions of the M. eupeus are immunogenic. Keywords: Mesobuthus eupeus, Scorpion, Venom, Immunogenic, Toxicogenic Introduction Scorpions have existed on earth about 400 million years ago (Ozkan et al. 2007). The scorpion stings are a major threat to human and animal health especially in tropi- cal regions (Bawaskar et al. 2012, Warrell 2012). Annual rate of scorpion stings is 1.2 million, and the mortality rate is about 3250 per year. Children are more vulnerable to scorpion envenomation and the highest death rate is observed in this age group (Chippaux and Goyffon 2008). Scorpions belong to the phylum Arthropoda, class Arachnida, order Scorpiones. 1500 discribed species of scorpions are inclued 70 genera and 6 families. 50 species are dan- gerous for human (Keskin and Koc 2006) where Buthidae family is the most ven- omous of them (Shirmardi et al. 2010). Iranian scorpion (sting agents) species are classified in Buthidae and Scorpionidae fam- ilies with 16 genera and 25 species (Dehgani et al. 2009). The limited number of danger- ous species are found in Iran (Sagheb et al. 2012). Mesobuthus eupeus is a species be- *Corresponding author: Dr Mohammad Khosravi, E-mail: khosravi.m@ut.ac.ir 139 J Arthropod-Borne Dis, December 2013, 7(2): 139–146 M Khoobdel et al.: Purification of the… http://jad.tums.ac.ir Published Online: August 31, 2013 longing to the Buthidae family and com- monly known as the lesser asian scorpion or the mottled scorpion. It was found in the Middle East and Central Asia and is respon- sible for many cases of envenomation in these regions (Karatas 2003, Sadeghian 2003, Dehghani and Khamehchian 2008). Mesobuthus eupeus is the most common species in Iran. Its venom contains several toxin fractions, which may cause a number of scorpion sting symptoms (Tuuri and Reynolds 2011, Sagheb et al. 2012). Scorpion venom consists of many bio- logical compounds which affects vertebrate and invertebrate organisms (Upadhyay and Ahmad 2008). Scorpion venom composes of short-chain peptides with low molecular weight (Adiguzel 2010), which elicit a strong immunogenic reaction in the host (Corzo et al. 2001). As yet, about 400 toxic peptides have been detected in scorpion venoms but it has been estimated that 100.000 distinct peptides exist in scor- pion venom (Karatas 2003). Serotherapy is the only effective treat- ment against scorpion stings and has been an issue of discussion in the last decade (Boyer et al. 2009, Duarte et al. 2010). Based on previous reports, approximately 42500 scorpion stings occur in Iran annually (Dehghani and Fathi 2012). In Iran, the scor- pion antivenom is made through the process of injecting horses with a mixture of six different scorpion venoms including: Hemiscorpius lepturus, Buthotus saulcyi, B. schach, Odon- tobuthus doriae, M. eupeus and Androctonus crassicauda (Razi Vaccine and Serum Re- search Institute, Karaj, Iran). Many investigations were performed to improve the quality of antidote against scor- pion venom. Study of the immunological prop- erties of venom is critical for antivenom de- velopment as much as better (Inceoglua et al. 2006). Moreover the detection of anti- genic proteins is very important in the field of toxicology and parasitology (Kalapothakisa et al. 2001). So development of specific anti- bodies against immunogenic fragments of the venom can effectively improve therapeu- tic alliance. Gel electrophoresis, electro-fo- cusing or liquid chromatography are used to detect protein patterns of venoms (Escoubas et al. 2002, Pimento et al. 2003). The current study was conducted to in- vestigate the immunogenic and toxicogenic properties of the M. eupeus venom. Materials and Methods Venom preparation Mesobuthus eupeus scorpions were col- lected with UV light at night from different parts of the Khuzestan Province (31°19′– 32°73′N , 48°41′–49°4′ E, with an area of 63,238 km²) in South West of Iran and were milked by electric stimulation at the end of the tail. The freeze-dried venom was dis- solved in distilled water and then dialyzed against distilled water at 4 °C for 48 hours. After dialysis, the venom solution was cen- trifuged at 1500rpm for 15 minutes, and the supernatant was collected. Protein assay The protein content of venoms was de- termined by the absorbance at 280nm with Bovine Serum Albumin (BSA) as standard. Toxicity determination All experiments were performed accord- ing to the guidelines of the ethical committee of the Faculty of Veterinary Medicine of Tehran University, Iran (National Ethics Ad- visory Committee 2006). For toxicity determination, increasing con- centrations of the venom were injected sub- cutaneously (SC), intraperitoneally (IP) and intravenously (IV) to albino mice. Following treatment with venom solution, animals were monitored for 24 hours, and the number of dead animals was recorded at the end of the 140 J Arthropod-Borne Dis, December 2013, 7(2): 139–146 M Khoobdel et al.: Purification of the… http://jad.tums.ac.ir Published Online: August 31, 2013 experiment, then, LD was calculated. LD50 was determined using the Spearman-Kaerber method. Briefly, 35 mice were divided into 7 groups of 5 mice each. Appropriate venom concentrations were prepared to cover the full range between zero and 100% of in- duced animal mortalities. Different doses (175, 160, 145, 130, 119, and 109µ g) of the venom stock solution were prepared and in- jected intraperitoneally (IP). An equivalent volume of buffer was injected into 5 mice as a negative control group. Deaths were scored up to 24h and LD50 was then calculated. Production of polyclonal antibody Outbreed New Zealand white male rab- bits were acclimatized to room temperature at 18 °C for two weeks former to immuniza- tion. Preimmune sera was attained through- out this period. The immunization plan and programmes of immunization were the alike as those detailed previously. In initial im- munization, three rabbits were each injected intradermally with 250 µg of venom in 0.5 ml of PBS emulsified with 0.5 ml of complete Freund’s adjuvant by a multiple injection method (10 sites/ rabbit) (Inceoglua et al. 2006). These first injections were pursued by three sets of booster injection. Booster in- jections were made at 2nd, 4th and 6th weeks with 130 µ gr of immunogen, 0.5ml of PBS and 0.5ml of incomplete Freund’s adjuvant at two sites in both thighs intramuscularly. The existence of antibodies in serum was de- termined through immunodiffusion and As- coli's test. Finally, after 10 days, the immun- ization blood was directly collected into ster- ilized glass tubes without any anti-coagu- lants and allowed to clot in cold. Serum was pipette out and centrifugated at 1500 rpm for 10 minutes and then isolated in a sterilized vial and stored at 4 °C for bioassay tests. Purification of polyclonal antibody against venom Polyclonal antibody against venom was first purified by ammonium sulfate precipitation (50% saturation for the final solution) and dialyzed in PBS and then subjected to an affinity column conjugated with venom. The column was prepared by conjugating 20mg of venom with 7ml of activated CH-Sepharose 4B. Cyanogen bromide activation was per- formed by the method of Cuatrecasas (March et al. 1974). Antibody was eluted from the column with 0.1M glycine pH 2.5 and fractions were collected and neutralized immediately by adding an appropriate amount of 1 M tris-pH 9 to each fraction. Purification of immunogenic peptides of venom The fractions, including the exact anti- bodies were merged, dialyzed against Borate buffer pH 8.4, overnight and used for an- other affinity column. Ten mg of this affinity purified antibody conjugated with a CH- Sepharose 4B column and 5mg of M. eupeus venom were applied to it. The bound proteins were eluted as before. SDS-PAGE analysis of the venom The protein profiles of crude venom as well as the affinity fractions (purified venom) were analyzed by SDS-PAGE (Laemmli 1970), the concentration of acrylamide was 15%. Pro- teins were stained with 1% coomassie blue R 250. Molecular mass standard (Vivantis, prod- uct No: PR0602) was run in parallel in order to calculate molecular weights of the proteins. Then, the gels were photographed and molec- ular weights of the proteins were calculated. Results Venom lethal dose (LD) was assessed by either subcutaneous, intraperitoneal or IV in- jection using 18±2g albino mice. LD was 8.75, 11.5 and 4.5mg/ kg of the body weight of albino mice for IP, SC and IV, respectively. 141 J Arthropod-Borne Dis, December 2013, 7(2): 139–146 M Khoobdel et al.: Purification of the… http://jad.tums.ac.ir Published Online: August 31, 2013 The median lethal dose (LD50) of M. eupeus venom was 6.95mg/ kg with IP injection. Proteins of the venom were determined to be between 5 and 140 kDa on electrophoresis on 15% polyacrylamide gel. The crude venom had 12 detectable bands with molecular weights of 140, 70, 50, 33, 30, 27, 22, 18, 14, 10 kDa and two bands less than 5 kDa. The affinity-purified venom presented eight bands. The 27 kDa band was clearly sharper than other bands but 70, 18, 10 and one of the less than 5 kDa bands were not observed (Fig. 1). Fig. 1. The SDS-PAGE analysis of Mesobuthus eupeus scorpion venom. From right Lane 1: Marker proteins (175, 130, 95, 70, 62, 51, 42, 29, 22 and14 respectively). Lanes 2 and 3: Electrophoretic pattern of the immunogenic frac- tions present in the venom (140, 50, 33, 30, 27, 22, 14, ≤5 kDa) and crude venom (140, 70, 50, 33, 30, 27, 22, 18, 14, 10, ≤5, ≤5 kDa) respectively. Table 1. The variations of protein in Mesobuthus eupeus venom Protein bands (kDa) 140 70 50 33 30 27 22 18 14 10 Fever than 5 Total number of protein bands A + + + + + + + + + + ++ 12 B + - + + + + + - + - + - 8 A) Venom samples B) Immunogenic fractions of venom Discussion In the present investigation, we deter- mined the in vivo toxic effects of the venom of M. eupeus. The venom of M. eupeus ap- pears to be more toxic when injected intra- venously. This phenomena could be associ- ated to different toxicokinetics of the three injection methods. Additionally, we studied the electropho- retic protein pattern of the crude venom, and immunogenic fractions of the venom. The results clearly displayed that most of M. eupeus venom fragments were immuno- genic. Our results also showed that scorpion toxins were proteins with various molecular 142 J Arthropod-Borne Dis, December 2013, 7(2): 139–146 M Khoobdel et al.: Purification of the… http://jad.tums.ac.ir Published Online: August 31, 2013 weights, which induce both toxicological and immunological reactions invivo. We also developed an approach toward application and refining of the immunogenic fractions of M. eupeus venom. Previous studies in Iran determined 4.5 mg/kg (Zayerzadeh et al. 2012) and 1.45 mg/ kg (Hassan 1984) as the median lethal dose (LD50) of M. eupeus venom. Another study was calculated the median lethal dose of M. eupeus venom 0.18mg/ kg via intracere- broventricular (ICV) injection (Ozkan and Carhan 2008). In our study, LD50 of the venom was 6.95mg/ kg via IP injection. Diverse studies reported various numbers of protein bands with different molecular weights for scorpion venoms. Molecular weights of Iurusdufoureius asiaticus specie venom were determined 14–205 kDa with individual variations (Turkey) (Keskin and Koc 2006). A similar study on Tityus pachyurus specie suggested 14–97 kDa venom proteins using electrophoresis (SDS-PAGE) method (Latin America) (Barona et al. 2004). They developed three anivenom which prominent- ly reacted with low molecular weight frag- ments. The most of venom proteins molecu- lar weights of M. eupeus were 12–112 kDa (Ozkan and Carhan 2008). We determined protein fragments from 5 to 140 kDa. One study showed that the venom of M. gibbosus consisted of 19 protein bands with molecular weight from 6.5 to 210 kDa (Ucar and Tas 2003). Protein bands with molecular weight of 28, 30, 33, 68 and 98 kDa were detected in the venom of the captive male M. gibbosus from the same biotope during the summer (Tur- key, Mugla Province) (Ozkan and Ciftci 2010). The causes of disagreement between studies may be due to the effects of the sex, geography, and hormonal condition of scorpions, which all alter feeding manners and result in venom creation with diverse molecular weights. In the current study, 12 protein bands were de- tected in M. eupeus scorpion venom. Variations in the biochemical and immu- nological contents of the various scorpion ven- oms must be considered to realize clinical signs, produce efficient antivenoms and de- termine optimal dosage (El-Hafny et al. 2002, Calvete 2010). Recognition and comparing of the MesoLys-C amino acid sequence of three major species scorpion is used for detecting phylogenetic relationships of various scor- pion species. For example, MesoLys-C iso- lated from M. eupeus of Khuzestan exhibited the highest and the lowest sequence similar- ities with M. gibbosus and M. cyprius, re- spectively (Eskandari and Khoonmirzaei 2011). The ability of heminecrolysin to suppress- ing the major physiopathological effects of H. lepturus envenomation may be due to elicit high titer of specific IgGs (Borchani et al. 2011). The antigenicity studies of iberiotoxin of Eastern Indian scorpion demonstrated whole protein was not necessary to stimulate the im- mune system, because a small fragment of the venom protein called the antigenic determinant was adequate for eliciting the immune response (Gomase et al. 2009). A study performed by Garcia et al. (2003) approved this statement. Gazarian et al. (2005) realized that no im- munity was developed against scorpion ven- om during evolution. Because of no evolu- tionary relationship between humans im- munity and scorpion venom, scorpion ven- oms can be suitable candidates for immuno- genic probes (March et al. 1974). Because of completely distinct phylogenetics properties of two noted entities, any structural changes of scorpion venoms can followed and prob- ably manipulated for inactivation of their antigenic activity (Gazarian et al. 2005). Contrary to popular belief, which know scorpion venom as non-immunogenic com- position, the current study was shown that the most fractions of the M. eupeus were immunogenic. Further investigations are nec- essary to explain more details of these immu- 143 J Arthropod-Borne Dis, December 2013, 7(2): 139–146 M Khoobdel et al.: Purification of the… http://jad.tums.ac.ir Published Online: August 31, 2013 nogenic fractions and to detecting lesser tox- icant fragments, which, improves the quality of the antidotes and helps vaccines designing. Acknowledgements This study was financially supported by the Health Research Center, Baqiyatallah Uni- versity of Medical Sciences with grant num- ber BMSU/HRC/2011/2-442. We should thank to personnel of Dr Rasteghar laboratory in Faculty of Veterinary Medicine of Tehran Uni- versity, for their kind cooperation. The authors declare that there is no conflict of interest. References Adiguzel S (2010) In vivo and in vitro ef- fects of scorpion venoms in Turkey: a mini-review. J Venom Anim Toxins incl Trop Dis. 16(2): 198–211. Barona J, Otero R, Nunez V (2004) Toxico- logical and immunological aspects of scorpion venom (Tytius pachyurus), neu- tralizing capacity of antivenoms pro- duced in Latin America. Biomedica. 24(1): 42–49. Barral-Netto M, Vinhas V, Schriefer A, Barral A, Santos SB, Almeida AR, Novaes G (1991) Immunological studies with the venom of the scorpion Tityus serrulatus. Brazilian J Med Bio Res. 24(2): 171– 180. Bawaskar HS, Bawaskar PH (2012) Scor- pion sting: Update J Assoc Physicians India. 60(1): 46–55. Borchani L, Sassi A, Yekhlef RB, Safra I, Ayeb ME (2011) Heminecrolysin, a poten- tial immunogen for monospecific anti- venom production against Hemiscorpius lepturus scorpion. Toxicon. 58(8): 681– 688. Boyer LV, Theodorou AA, Berg RA, Mallie J (2009) Antivenom for critically ill children with neurotoxicity from scor- pion sting. N Engl J Med. 360: 2090– 2098. Calvete JJ (2010) Antivenomics and venom phenotyping: A marriage of conven- ience to address the performance and range of clinical use of antivenoms. Toxicon. 56(7): 1284–1291. Chippaux JP, Goyffon M (2008) Epidemio- logy of scorpionism: A global app- raisal. Acta Trop. 107(2): 71–79. Corzo G, Escoubas P, Villegas E, Barnham KJ, He W, Norton RS, Nakajima T (2001) Characterization of unique am- phipathic antimicrobial peptides from venom of the scorpion Pandinus impe- rator. J Biochem. 359: 35–45. Dehghani R, Fathi B (2012) Scorpion sting in Iran: A review, Toxicon, Available at: http://dx.doi.org/10.1016/j. toxicon. 2012.06.002. Dehghani R, Djadid ND, Shahbazzadeh D, Bigdelli S (2009) Introducing Comp- sobuthus matthiesseni (Birula, 1905) scorpion as one of the major stinging scorpions in Khuzestan, Iran. Toxicon. 54(3): 272–275. Dehghani R, Khamehchian T (2008) Scro- tum Injury by Scorpion Sting. J Ar- thropod-Borne Dis. 2(1): 49–52. Duarte CG, Alvarenga LM, Lopes CD, Avila RA, Nguyen C, Molina F (2010) In vivo protection against Tityus serrulatus scorpion venom by antibodies raised against a discontinuous synthetic epitope. Vaccine. 28: 1168–1176. El-Hafny B, Chgoury F, Adil N, Chen N, Nassar M (2002) Intraspecific variability and pharmacokinetics, characteristics of Androctonus mauretanicus scorpion venom. Toxicon. 40(11): 1609–1616. Escoubas P, Corzo G, Whiteley BJ, Celerier ML, Nakajima T (2002) Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and high-performance liquid chromatography study of quan- titative and qualitative variation in ta- 144 J Arthropod-Borne Dis, December 2013, 7(2): 139–146 M Khoobdel et al.: Purification of the… http://jad.tums.ac.ir Published Online: August 31, 2013 rantula spider venoms. Rapid Commun Mass Spectrom. 16(5): 403–413. Eskandari G, Khoonmirzaei AN (2011) Phy- logenetic Analysis of Lysozyme C from the Scorpion Mesobuthus eupeus Venom Gland. J Bio Sci. 6(1): 9–11. Garcia C, Calderón-Aranda ES, Anguiano GA, Becerril B, Possani LD (2003) Analysis of the immune response induced by a scorpion venom sub-fraction, a pure pep- tide and a recombinant peptide, against toxin Cn2 of Centruroides noxius Hoff- mann. Toxicon. 41(4): 417–427. Gazarian KG, Gazarian T, Hernández R, Possani LD (2005) Immunology of scorpion toxins and perspectives for generation of anti-venom vaccines. Vac- cine. 23(26): 3357–3368. Gomase VS, Phadnis AC, Somnath W (2009) Proteomics based prediction of anti- genicity of iberiotoxin from eastern Indian scorpion. Inter J Drug Discov. 1(1): 10–13. Hassan F (1984) Production of scorpion antivenom. In: Tu A (ed) Handbook of Toxins, Insect Poisons, Allergens and other Invertebrates Venoms. Marcel Dekker, New York, pp. 577–605. Inceoglua B, Langob J, Rabinovicha A, Whetstonea P, Hammock BD (2006) The neutralizing effect of a polyclonal antibody raised against the N-terminal eighteen-amino acid residues of bir- toxin towards the whole venom of Parabuthus transvaalicus. Toxicon. 47: 144–149. Kalapothakisa E, Jardima SA, Magalha˜esa C, Mendesa T, Marcoa L, De Afonsob, Chavez-Olo´rteguic LCC (2001) Screen- ing of expression libraries using ELISA: identification of immunogenic proteins from Tityus bahiensis and Tityus serrulatus venom. Toxicon. 39: 679–685. Karatas A (2003) Mesobuthus eupeus (Koch, 1839) (Scorpiones: Buthidae) in Ana- tolia. Euscorpius. 7: 1–7. Keskin NA, Koc HA (2006) Study on venom proteins of Iurusdufoureius asiaticus Birula, 1903 (Scropiones: Iuridae). Acta Parasitol Turcica. 30(1): 60–62. Laemmli K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680–685. March SC, Parikh I, Cuatrecasas P (1974) A simplified method for cyanogen bro- mide activation of agarose for affinity chromatography. Anal Biochem. 60(1): 149–152. National Ethics Advisory Committee (2006) Ethical Guidelines for Observational Studies: Observational research, audits and related activities. Ministry of Health, Wellington, New Zealand. Available at: http://www.newhealth.govt.nz/neac/. Ozkan O, Ciftci G (2010) Individual varia- tion in the protein profile of the venom of Mesobuthus gibbosus (Brullé, 1832) (Scorpiones: Buthidae) from Turkey. J Venom Anim Toxins Incl Trop Dis. 16(3): 505–508. Ozkan O, Carhan A (2008) The neutralizing capacity of Androctonus crassicauda antivenom against Mesobuthus eupeus scorpion venom. Toxicon. 52: 375–79. Ozkan O, Adiguzel S, Kar S, Kurt M, Yakistiran S, Cesaretli Y, Orman M, Karaer KZ (2007) Effects Of And- roctonus crassicauda (Olivier, 1807) (Scorpiones: Buthidae) venom on rats: correlation among acetyl cholinester- ase activities and electrolytes levels. J Venom Aanim Toxins Trop Dis. 13(1): 69–81. Pimenta AM, Almeida D, Delima MF, Eauclaire ME, Bougis PE (2003) Indi- vidual variability in Tityus serrulatus (Scorpiones, Buthidae) venom elicited by matrix-assisted laser desorption/ ionization time-of-flight mass spectrom- etry. Rapid Commun Mass Spectrom 2003. 17(5): 413–418. 145 J Arthropod-Borne Dis, December 2013, 7(2): 139–146 M Khoobdel et al.: Purification of the… http://jad.tums.ac.ir Published Online: August 31, 2013 Sadeghian H (2003) Transient ophtalmoplegia following envenomation by the scor- pion Mesobuthus eupeus. Neurology. 60(2): 346–347. Sagheb MM, Sharifian M, Moini M, Sharifian AH (2012) Scorpion bite prevalence and complications: report from a referral cen- tre in southern Iran. Trop Doct. 42(2): 90–91. Shirmardi SP, Shamsaei M, Gandomkar M, Saniei E, Ghannadi M, Zare A (2010) Comparison of two purified toxic fractions from Mesobuthus eupeus scorpion venom. J Venomous Animals Toxins include Trop Dis. 16(4): 639– 646. Tuuri RE, Reynolds S (2011) Scorpion en- venomation and antivenom therapy. Pediatr Emerg Care. 27(7): 667–672. Ucar G, Tas C (2003) Cholin esterase inhib- itory activities of the scorpion Mes- obuthus gibbosus (Buthidae) venom pep- tides. FABAD J Pharm Sci. 28: 61–70. Upadhyay RK, Ahmad S (2008) Isolation, pu- rification and characterization of venom toxins from Indian Red Scorpion, Mesobuthus Tamulus. J Cell Tissue Res. 8(1): 1297–1302. Warrell DA (2012) Venomous stings, stings, and poisoning. Infect Dis Clin North Am. 26(2): 207–223. Zayerzadeh E, Koohi MK, ZareMirakabadi A, Fardipoor A, Kassaian SE, Rabbani S, Anvari MS (2012) Amelioration of cardio-respiratory perturbations follow- ing Mesobuthus eupeus envenomation in anesthetized rabbits with commercial polyvalent F(ab′)2 antivenom. Toxicon. 59(2): 249–256. 146