India is a habitat for nearly one thousand four hundred forty-seven species of spiders under three hundred and sixty-five genera and sixty families. Our initial survey on toxic bite by spider revealed severe edema, itching, acute pain, and hemorrhage following tissue necrosis, which are the general symptoms of envenomation, but there are no reports of mortality. Significantly, Hippasa partita spider, commonly called “funnel web spider,” which is endemic in hilly regions of the Western Ghats is responsible for envenomation. In this study, a nonenzymatic neurotoxin has been purified from H. partita venom gland extract. Gel filtration and ion exchange chromatography were used to purify the toxin into homogeneity as shown by SDS-PAGE, RP-HPLC, and MALDI-TOF. Neurotoxin is devoid of enzymatic activities but causes intense neurotoxic symptoms. Neurotoxin is found to inhibit the twitch response of sciatic nerve gastrocnemius muscle preparation and is found to be postsynaptic in action. Neurotoxin is devoid of coagulant activity, edema, and hemorrhage and is nonlethal to mice (up to 5？mg/kg body weight). In conclusion, a neurotoxin, which is a principle agent in whole venom responsible for induced neurotoxic symptoms, has been purified and characterized. 1. Introduction Spider venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Spider venoms exhibit a wide spectrum of biological activities that results in both local [1, 2] and systemic toxicities [3]. The main purpose of the venom is to immobilize and to digest the prey. H. partita spider is endemic in hilly regions of the Western Ghats [4] and is commonly called “funnel web spider” which is mainly responsible for toxic bites. Severe edema and itching, acute pain, and hemorrhage following tissue necrosis are the general symptoms of envenomation, but there are no reports of mortality. Spider bites are often treated with folk medicine. Thus, less attention is paid towards documentation, and no documentation is available in hospitals on clinical pathology. Spider venoms have been extensively studied for understanding the neurotoxic property [5–7]. Neurotoxins with varied potency have been isolated and studied [6, 7]. Such neurotoxins are acylpolyamines, proteins, and peptides in nature. The venoms from Agelenopsis aperta of the American (USA) subcontinent [8–11] and Phoneutria nigriventer of the Brazilian subcontinent [12, 13] are the most explored species for neurotoxicity among the spider venoms. Initial
References
[1]
S. Nagaraju, K. S. Girish, J. W. Fox, and K. Kemparaju, “‘Partitagin’ a hemorrhagic metalloprotease from Hippasa partita spider venom: role in tissue necrosis,” Biochimie, vol. 89, no. 11, pp. 1322–1331, 2007.
[2]
S. Nagaraju, S. Devaraja, and K. Kemparaju, “Purification and properties of hyaluronidase from Hippasa partita (funnel web spider) venom gland extract,” Toxicon, vol. 50, no. 3, pp. 383–393, 2007.
[3]
S. Nagaraju, Biochemical and pharamacological characterization of spider venom [Ph.D. thesis], University of Mysore, Mysore, India, 2007.
[4]
M. Siliwal and S. Molur, “Checklist of spiders (Arachnida: Araneae) of South Asia including the 2006 update of Indian spider checklist,” Zoos' Print Journal, vol. 22, no. 2, pp. 2551–2597, 2007.
[5]
E. Grishin, “Polypeptide neurotoxins from spider venoms,” European Journal of Biochemistry, vol. 264, no. 2, pp. 276–280, 1999.
[6]
P. Escoubas, S. Diochot, and G. Corzo, “Structure and pharmacology of spider venoms neurotoxins,” Biochimie, vol. 82, no. 9-10, pp. 893–907, 2000.
[7]
R. O. Beleboni, R. O. G. Carolino, A. B. Pizzo et al., “Pharmacological and biochemical aspects of GABAergic neurotransmission: pathological and neuropsychobiological relationships,” Cellular and Molecular Neurobiology, vol. 24, no. 6, pp. 707–728, 2004.
[8]
W. S. Skinner, M. E. Adams, G. B. Quistad et al., “Purification and characterization of two classes of neurotoxins from the funnel web spider, Agelenopsis aperta,” Journal of Biological Chemistry, vol. 264, no. 4, pp. 2150–2155, 1989.
[9]
M. E. Adams, V. P. Bindokas, L. Hasegawa, and V. J. Venema, “ω-Agatoxins: novel calcium channel antagonists of two subtypes from funnel web spider (Agelenopsis aperta) venom,” Journal of Biological Chemistry, vol. 265, no. 2, pp. 861–867, 1990.
[10]
I. M. Mintz, M. E. Adams, and B. P. Bean, “Use of spider toxins to discriminate between neuronal calcium channels,” Society for Neuroscience Abs, vol. 18, p. 9, 1992.
[11]
R. Llinás, H. Moreno, M. Sugimori, M. Mohammadi, and J. Schlessinger, “Differential pre- and postsynaptic modulation of chemical transmission in the squid giant synapse by tyrosine phosphorylation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 5, pp. 1990–1994, 1997.
[12]
L. Rezende Jr., M. N. Cordeiro, E. B. Oliveira, and C. R. Diniz, “Isolation of neurotoxic peptides from the venom of the “armed” spider Phoneutria nigriventer,” Toxicon, vol. 29, no. 10, pp. 1225–1233, 1991.
[13]
M. D. N. Cordeiro, S. G. De Figueiredo, A. D. C. Valentim et al., “Purification and amino acid sequences of six TX3 type neurotoxins from the venom of the Brazilian “armed” spider Phoneutria nigriventer (Keys.),” Toxicon, vol. 31, no. 1, pp. 35–42, 1993.
[14]
S. Nagaraju, Y. H. Mahadeswaraswamy, K. S. Girish, and K. Kemparaju, “Venom from spiders of the genus Hippasa: biochemical and pharmacological studies,” Comparative Biochemistry and Physiology C, vol. 144, no. 1, pp. 1–9, 2006.
[15]
B. K. Tikader and M. S. Malhotra, “Lycosidae (Wolf spiders),” Fauna India (Araneae), vol. 1, pp. 248–447, 1980.
[16]
R. B. da Silveira, J. F. dos Santos Filho, O. C. Mangili et al., “Identification of proteases in the extract of venom glands from brown spiders,” Toxicon, vol. 40, no. 6, pp. 815–822, 2002.
[17]
U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature, vol. 227, no. 5259, pp. 680–685, 1970.
[18]
G. Bohm, R. Muhr, and R. Jaenicke, “Quantitative analysis of protein far UV circular dichroism spectra by neural networks,” Protein Engineering, vol. 5, no. 3, pp. 191–195, 1992.
[19]
A. L. Harvey, R. J. Marshall, and E. Karlsson, “Effects of purified cardiotoxins from the Thailand cobra (Naja naja siamensis) on isolated skeletal and cardiac muscle preparations,” Toxicon, vol. 20, no. 2, pp. 379–396, 1982.
[20]
J. Meier and R. D. G. Theakston, “Approximate LD50 determinations of snake venoms using eight to ten experimental animals,” Toxicon, vol. 24, no. 4, pp. 395–401, 1986.
[21]
A. J. Quick, “Prothrombin time (one stage procedure),” in Haemorrahgic Diseases and Thrombosis, L. Fediger, Ed., pp. 391–395, Philadelphia, Pa, USA, 2nd edition, 1996.
[22]
B. S. Vishwanath, R. M. Kini, and T. V. Gowda, “Characterization of three edema-inducing phospholipase A2 enzymes from habu (Trimeresurus flavoviridis) venom and their interaction with the alkaloid aristolochic acid,” Toxicon, vol. 25, no. 5, pp. 501–515, 1987.
[23]
H. . Kondo, S. Kondo, H. Ikezawa, and R. Murata, “Studies on the quantitative method for determination of haemorrhagic activity of Habu snake venom,” Japanese Journal of Medical Science and Biology, vol. 13, pp. 43–52, 1960.
[24]
H. G. Boman and U. Kaletta, “Chromatography of rattlesnake venom A separation of three phosphodiesterases,” Biochimica et Biophysica Acta, vol. 24, pp. 619–631, 1957.
[25]
O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, “Protein measurement with the Folin phenol reagent,” The Journal of Biological Chemistry, vol. 193, no. 1, pp. 265–275, 1951.
[26]
D. Zhang and S. Liang, “Assignment of the three disulfide bridges of huwentoxin-I, a neurotoxin from the spider Selenocosmia huwena,” Journal of Protein Chemistry, vol. 12, no. 6, pp. 735–740, 1993.
[27]
W. D. Branton, L. Kolton, Y. N. Jan, and L. Y. Jan, “Neurotoxins from Plectreurys spider venom are potent presynaptic blockers in Drosophila,” Journal of Neuroscience, vol. 7, no. 12, pp. 4195–4200, 1987.
[28]
Q. Shu and S. P. Liang, “Purification and characterization of huwentoxin-II, a neurotoxic peptide from the venom of the Chinese bird spider Selenocosmia huwena,” Journal of Peptide Research, vol. 53, no. 5, pp. 486–491, 1999.
[29]
V. P. Bindokas and M. E. Adams, “ω-Aga-I: a presynaptic calcium channel antagonist from venom of the funnel web spider, Agelenopsis aperta,” Journal of Neurobiology, vol. 20, no. 4, pp. 171–188, 1989.
[30]
S. P. Liang, D. Zhang, X. Pan, Q. Chen, and P. Zhou, “Properties and amino acid sequence of huwentoxin-I, a neurotoxin purified from the venom of the Chinese bird spider Selenocosmia huwena,” Toxicon, vol. 31, no. 8, pp. 969–978, 1993.
