Optical Investigation of Snake Venom of Vipera Lebetina Obtusa

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1 Optical Investigation of Snake Venom of Vipera Lebetina Obtusa 1 S.A.TOPCHIEVA, 2 H.A.ABIYEV, 2 M.A.MEHRABOVA 1 Institute of Zoology, Azerbaijan National Academy of Sciences 2 Institute of Radiation Problems, Azerbaijan National Academy of Sciences 1 AZ 1073, Baku, Pass.1128, block AZ1143, Baku, 9, B.Vakhabzadeh str. Azerbaijan Republic shafiga.topchiyeva@mail.ru, mehrabova@mail.ru Abstract: - Chemical analyses showed, that in structure of venom of Vipera Lebetina obtusa founded out significant concentration of heavy metal, such as Cd, Ni, Zn, Cr, Pb. As a result by cooling of samples up to nitric temperature 77К it is appears the new narrow maxima at 440 and 470 nm of wavelength which is connected by metals taking place in structure of venom of Vipera lebetina obtusa. As a result of the experimental researches it is established, that under influence of small doses of γ-radiation at the doses of 2.7, 4.05, 5.4 kgy within 1.0, 1.5 and 2.0 hours, toxicity decrease in gradual and it is possible pharmacological activity of snake venom enzymes is decrease. Key-Words: - snake, venom, solution, absorption, spectra, irradiated, photoluminescence, emission 1 Introduction In territory of Azerbaijan live 23 kinds of snakes, 4 kinds of them are venomous. Research of toxins of various kinds of snakes, and also huge attention to zootoxins is defined not only inquiries of medical practice, but their studying and use represents the big interest for various branches of biology, physiology, bioorganic chemistry, biophysics, toxicology and other areas of sciences. Studying of venomous products of biosynthesis of the snakes, what are unique on a chemical compound and physiological action group of biologically active substances, revealing electrophysical properties of snake venom Vipera lebetina obtusа is an actual problem of a medical and biologic science. These problems still are not perfect, demand deep theoretical and practical analyses and are still actual. In references the review on studying of physical and chemical properties of solutions of venom of the Central Asian snakes is resulted. Factor of a superficial tension, viscosity and density factor is spent in the range of temperatures К with use densitometries and viscosimetrics methods of the physical and chemical analysis. At the analysis at the temperature range К deviations of these parameters on viscosity and density isotherms are noted. Studying of isotherms has given the chance to ascertain, that the observable effect is connected with own intermolecular interactions in them [1]. Relative density of venom of the cobra which value corresponded is experimentally defined. It is noticed, that reaction of snake venoms -is sour. Water solutions of venom of snakes are unstable and lose toxicity in some days after mixing. Snake venoms (Echis carinatus, Vipera lebetina Obtusa, Naja naja) in the physiological solution containing 50 % of glycerin, at storage in a refrigerator within 6 months do not reduce toxicity. More proof to influence of factors of an environment there are venoms only after drying over steams chloride calcium or after liofils drying. Venom of a cobra, at storage on a cold in the soldered ampoule has kept toxicity more than 20 years. Snake venoms are thermostabilite and in the sour environment maintain heating to 120 С without loss of activity [2]. Destroy operate on venoms some chemical agents, such as permanganate cilium, chloroform, ethanol, methyl dark blue. Snake venoms activation under the influence of physical factors, for example, at influence of an ultra-violet irradiation and X-rays [3]. So, in 7 days after a venom irradiation of Filipina cobras by γ radiation ( 60 Со) in doses 0.25, 0.5, 1 mrad, toxicity of snake venom made 83, 66 and 43 %, accordingly from not irradiated venom [4]. The considerable variation in the maintenance of sulfur proteases and metal proteases is marked in snake venoms. For example, in venom of an ordinary viper (Vipera berus) 75 % of all proteolysis ISBN:

2 activity is metal proteases. 25 % - sulfur proteases. In venom of Vipera lebetina on a share of metal proteases have to 15 % of total proteolysis activity, thus sulfur proteases provide 85 % of activity of venom [5]- [8]. The fraction which entirely reproduces all displays of an intoxication is identified and allocated In venom of Vipera lebetina. Venom of Vipera lebetina is a part of medical products. It is applied as a source of reception of commercial preparations FGN, phosphorus distresses and oxygen dazes L-amino acids, and also as a diagnostic preparation at illnesses of curtailing system of blood [9]. On literary data snake venom in the dried up kind keeps pharmacological and toxic properties till 22 years and even more. At cultivation of venom by water by physiological solution or glycerin toxic properties of snake venoms are not lost. Short-term influence of heats also does not render essential influence on toxicity of venom. Considerable influence on toxicity and quantity of allocated venom is rendered various biological, ecological factors, and also chemical agents and some physical factors. The basic component of venom responsible for its toxic properties are biologically active components of the aluminous nature and including various enzymes [10]- [13]. Small doses of venom do not cause any clinical displays of a venoming and long ago used at treatment of many heavy illnesses. Venom of Vipera lebetina renders anti-inflammatory and analgeting action. It is considered possible to use of venoms of Vipera libetina, Echis carinatus and cobras in otolaryngology [14]-[16]. Snake venoms are a great value for medicine and biology. Venoms of snakes are used at preparation of mono-and polyvalent whey. Venom of Vipera libetina is applied as blood the stopping means in otolaryngology at operations during removing of glands, at nasal bleedings etc. [17]-[21]. Into structure of venom of snakes enter protoletics enzymes destroying fibers, enzymes proteases and esterase, curtailing blood, and variety of others. So, in venom of rattle snakes Grotalus durissus are found out ions of some metals: Na, Ca, K, Mg, Zn, Fe, Cu, Co, Mn which activate a number of enzymes of venom or temporarily break their action [22], [23]. Influence of ecological factors on a chemical compound of venom Caucasian Vipera lebetina obtusа is revealed. Elementary structure of venom of Vipera lebetina obtusа caught of the various areas of Azerbaijan polluted by technogenic emissions of the industrial enterprises is studied by the atom -absorption spectrophotometer method. In venom of Vipera lebetina obtusа are defined the maintenance of heavy metals: Cr, Pb, Cd, Ni and Zn [24]-[26]. Researches on revealing of influence of γ -radiation on toxicity and on pharmacological properties of venom of Vipera lebetina obtusа are carried out in [27]. Thus, the considerable number of works has been devoted all-round studying of venom of venomous snakes. The picture of toxic and pharmacological action of snake venoms on an organism is in detail tracked. Inconsistent and many-sided characteristics of snake venoms have been resulted. Influence of snake venom on various functional systems of an organism has been revealed. Toxicity, snake venom have been studied. The comparative pharmacological and biochemical characteristic of venom of snakes has been given. There are literary dates on research of the snake venoms, however many questions still remain not mentioned and demand the deep analysis and studying [1], [16], [23], [25]. Among huge number of biologically active substances of an animal origin the important place taken snake venom is the unique by the chemical nature and physiological action to group of connections. Snake venom is thermostabile and in the sour environment, maintains heating to C without activity loss. Destroy chemical agents operate on venoms: permanganate calium, chloroform, ethanol, methyl dark blue. Snake venom inactivated also under the influence of some physical factors: an ultra-violet irradiation, x-rays and others. Despite available achievements in diagnostics and treatment of poisonings by snake venom studying of a chemical compound, their physical and chemical properties, toxicity pharmacokinetics and a metabolism of snake venom the general laws of influence of electromagnetic radiation on physical and chemical and biophysical characteristics of Transcaucasian Vipera Lebetina obtusa venom are not established. In the literature data on research of the snake venom are cited, however many questions still remain not mentioned and demand the deep analysis and studying. Influence of temperature on electrophysical parameters of venom and photoconductivity of zootoxins is not studied. Proceeding from the above-stated, research of electrophysical parameters of snake venom with a research objective of a photo-and thermal stability of crystals of snake venom is represented rather actual. ISBN:

3 Research of toxins of snakes, and also huge attention to zootoxins is defined not only inquiries of medical practice, but their studying and use represents the big interest for various branches of biology, physiology, bioorganic chemistry, biophysics, toxicology and other areas of sciences. Proceeding from the above-stated, the purpose of the present work was complex studying of influence of electromagnetic radiation on physical and chemical, biophysical properties of Transcaucasian Vipera Lebetina obtusa venom. 2 Experiment material of research venom vipera, dried up in exicator above pairs chloride calcium and the venom irradiated with small dozes served γ- radiations. For experiment tests of dry venom, and also their water solutions have been taken. Water solutions of venom have been subjected γ radiation up to dozes 1.35, 2.7, 4.05, 5.4 kgy during 0.5, 1.0, 1.5, 2.0 hours. Simultaneously dry venom vipera was exposed γ-radiations in continuation 2.0 hours a dose 5.4 kgy. In comparative aspect action scale of radiation on spectral characteristics of venom Caucasian viper Vipera lebetina obtusа was studied. Irradiation of venom vipera small dozes scale of radiation (γ - radiation D=0.75kG/sec) carried out on K-25 to isotope installation with application 60 Со. Ultra-violet spectra passing of venom have been removed on device Specord UV-VIS. On Fig. 1 it is resulted UV-VIS a spectrum of absorption of a water solution of venom vipera. Wave number in sm -1 Fig. 1 UV-VIS a spectrum of absorption of venom vipera. Apparently from figure to 1 venom vipera characteristic maxima of absorption are peculiar in the field of 285 nm and 800 nm. Absorption of group in these molecules represents the big resounding structures with the big number of the connected communications Electrons, carrying out chemical communication are not located, but are the general for all structure. Therefore it is possible to speak about characteristic for zootoxins spectra of absorption. With the help of measurement of a spectrum of absorption of venom vipera, after an irradiation, it is possible to define places of attack and to establish chemical changes zootoxin, both in the irradiated solutions, and in a firm phase. On Fig. 2 spectra of absorption of samples of venom vipera, irradiated are resulted γ radiation (water solutions and dry venom). Wave number in sm - 1 Fig.2 Spectra of absorption of samples of venom vipera, irradiated by γ - radiation (1, 2, 3, 4 - water solutions, 5-dry venom): 1-control; 2, 3, 4 - irradiated (D =1.35, 2.7, 4.05 kgy), 5 (D=5.4kGy) Apparently from figure 2 at increase in a dose of an irradiation γ-radiation of solutions of venom vipera observes characteristic changes of optical density of researched samples of snake venom. The revealed reduction of intensity of absorption at 260 and 300 nm testifies to course of biochemical reactions in a firm phase of separate enzymes zootoxins As a result of the lead experimental researches it is established, that under influence of small dozes γ- radiations at dozes 2.7, 4.05, 5.4 kgy within 1.0, 1.5 and 2.0 hours results in gradual decrease of toxicity and it is possible pharmacological activity of enzymes of snake venom. It is necessary to note, that at comparing intensity of absorption of control samples venom with ISBN:

4 samples zootoxin irradiated up to dozes 1.35 kgy it has not been revealed essential changes. It is necessary to note, that at comparison intensity of absorption of control samples (not irradiated) poison with samples zootoxin irradiated up to dozes 1.35 kgy it has not been revealed essential changes. From above-stated follows, that influence on venom vipers small dozes γ-radiation up to dozes 1.35 kgy within 0.5 hours does not result in reduction of intensity of absorption and according to toxicity, and including pharmacological activity. The subsequent increase in a dose scale of radiation up to 2.7, 4.05, 5.4 kgy within 1.0, 1.5, 2.0 hours promotes proportional reduction of intensity of absorption of snake venom and according to reduction, both toxicity, and pharmacological activity. At low temperatures researches and at revealing prostrations an effect in organic and biological systems of an organism from effective methods is radiotermoluminence. The method radiotermoluminence will allow receiving data on structural properties of system, on the centers of stabilization of charges of initial products radiolis venom, about ways of migration of energy absorbed at an irradiation and so on. The sample were excited a Nitrogen Laser diode emitting at nm with a pulse width of 0.6 ns. The spectra were analyzed with a Jobin-Yvon HR 360 monochromator coupled with cooled CCD detector. The visible emitted light was collected from the same side. Luminescent properties at liquid N2 temperature measured using a cold finger cryostat under vacuum. Results of chemical analyses shown, that in structure of venom (Vipera lebetina obtusa) is found out significant concentration of heavy metals (Cd, Ni, Zn, Cr, Pb). Intensity (rel.) λ (nm) λ exc = nm T = 300K Fig.3 Photoluminescence spectra of venom at 300 K In Fig. 3 and Fig.4 emission spectra of Vipera lebetina obtusa under λ exc = 337.1nm at room and 77 K temperature are present. Fig.4 Pphotoluminescence spectra of venom at 77 K It is visible, that PL specter of venom covers the area nm and consists of the wide maximum at 520 nm and poorly appreciable maxima covering wavelengths of nm at 300K. As a result of cooling samples up to nitric temperature (77К) appears new narrow maxima at 440 and 470 nm which is connected by metals taking place in structure of venom of Vipera lebetina obtusa Samples of venom were irradiated in special a ditch with scales - beams at 77К up to doses of % 5 кgr. Before an irradiation samples were cleared of traces of oxygen. The irradiation was spent on air and in vacuum. Curves lighting registered with a speed ~ 5 0 /l.min. It is shown, that in an interval of temperatures К the curve lighting radiotermoluminence venom of vipera irradiated at 77К up to dozes 3 кgr is characterized not by a symmetric maximum at temperature 172К. Warming up to temperatures 320К results in monotonous decrease of intensity of a luminescence. It is established, that intensity of peak at 172К increases depending on doze D=0-15 кgr gamma irradiation under the linear law. The irradiation in air results in additional weak peak at 185К and to downturn of intensity at 172К. It is shown, that the peak of a luminescence at 172К can be dosimetric for an estimation of the absorbed doze. ISBN:

5 The opportunity of change of medicinal properties of venom of vipera is discussed at influence of small doses of scale - radiation. 4 Results and Conclusion Thus, as a result of the lead researches it is revealed that under influence of small dozes γ- radiations up to dozes 2.7, 4.05, 5.4 kgy during 1.0, 1.5 and 2.0 hours results in reduction of intensity of absorption at 260 and 300 nm that testifies to course of biochemical reactions in a firm phase of separate enzymes zootoxin, and in turn results in decrease of toxicity and it is possible pharmacological activity of enzymes of snake venom. However influence of γ-radiation on a snake venom solution up to dozes 1.35 kgy within 30 minutes promotes stabilization as to toxicity, and pharmacological activity with simultaneous increase accordingly a period of storage of water solutions of snake venom that can be used at sterilization of water solutions of snake poison, and in turn is important for a farmaceutical industry by manufacture of injections on a basis zootoxins. References: [1] А.Т.Berdiyeva, Ashgabat, Ylym, 163 pp [2] B.Lomonte, G.Lean, L.A.Hanson, J. Toxicon, v. 34, 10, pp , 1996 [3] Sh.A.Topchiyeva, H.A.Abiyev, Ecoenergenika, Baku, 1, pp.21-23, 2004 [4] Sh.A.Topchiyeva, A.M.Magerramov, N.N.Musayeva,L.Z.Allahverdiyeva, R.Z.Allahverdiyeva, H.A.Abiyev, 40 th IUPOC International Symposium on Macromol, World Polymer Congress Macro, Paris-France, 5.3, pp.120, Juli 4-9, 2004 [5] E.Winkler, M.Chovers, S.Almog, J. Lab. Clin. Med., v.1, 6, pp , 1993 [6] A.Gasmi, H.Louzir, H.Karoui, M. Ayeb, Nat Toxins, v.2, 1, pp.44-48, 1994 [7] M.Menashe, H.Rochat, E.Zlotkin, Insect. Biochem., v.10, 6, pp , 1980 [8] K.H.Trummal Vija, J.Subbi, J.Siigur, Biochim. Biophys. Acta, v.1476, 2, pp.331, 2000 [9] L.М.Mitelman, Questions Hematology radiobiol of magnetic fields, Tomsk, pp , 1965 [10] Pavlovski Е.N. Venomous Animals of the USSR, Moscow, pp. 35, 1931 [11] M.A.McLane, E. E.Sanchez, A.Wong, Paquette-Straub, C., Perez, J. C.,Disintegrins. Current Drug Targets-Cardiovascular and Haematological Disorders v.4, pp , 2004 [12] M.S.Ramirez, E.E.Sanchez, C.Garcia-Prieto, J.C.Perez, G.Rodriguez- Chapa, M.R.McKeller, R.Ramirez, Y.De Anda, Comp. Biochem Physiol, Part C.v.124, pp.91-98, 1999 [13] J.Siigur, K.Tnismgi, A.T.Tu, E.Siigur J. Toxicon, v.34, 5, pp , 1996 [14] Sh.A.Topchiyeva, H.A.Abiyev, Ecoenergy, Baku, 1, pp.21, 2004 [15] А.Т.Berdiyeva, Izvestia TSSR Ser. Biol., 5, pp.88-91, 1962, [16] J.A.Galán, E.E.Sánchez, S.Bashir, J.C.Pérez, Canadian J. Chem. v.83, pp , 2005 [17] А.Ahunov, Some physico-chemical and biological properties of venom proteases of the Central Asian vipera. Uzb. Bio. Journal. 2, pp.75-76, 1974 [18] Z.S.Barkagan, I..А.Valseva, L.Sh.Mitelman, Anticoagulating and toxic properties of Central Asian cobra venom. Izvestia Akad. Biol., 1, pp , 1967 [19] Z.S. Barkagan, Е.Y.Suhoveeva, V.I.Shevchenko, Abstracts III All-Union Herpetological Conference, pp.30-31, 1973 [20] I.А.Valseva, Е.N.Pavlovski, F.F.Talizin, Akad, v. 145, 2, pp , 1962 [21] Y.B. Ishaki, Bulletin of Otorhinolaryngology,1959, 5, p [22] E.S.Salafranca, Int. J. Appl. Radiatand Isotop., v.4, 1, pp.60, 1973 [23] E.E.Sanchez, J.A.Galan, W.K.Russell, D.H.Russell, J.G.Soto, J.C.Perez, Toxinol. Applied Pharmacol., In Press, 2005 [24] Sh. A. Topchieva, М.А.Меhrabova, H. A. Abiyev, Investigation of electrophysical parameters of snake venom, WSEAS EUROPMENT - EUROSIAM Intern. Conferences, Vouliagmeni, Athens, Greece, December 29-31, 2010, p.85-89, RTH-ATLANTIC-UNIVERSITY-UNION, [25] Sh.A.Topchiyeva, H.A.Abiyev, J. Izvestia, NASA, Series of Biological Sciences, Baku, 5-6, pp , 2006 [26] Topchiyeva Sh.A., Mehrabova M.A., Abiyev H.A. Influence of ecological factors on electrophysical parameters of snake venom. Inter. Conf. Physics of Insulators, Saint- Petersburg, 2011, ISBN:

6 d/program2011.pdf [27] Topchiyeva Sh.A., M.A.Mehrabova Abiyev H.A., The effect of environmental pollution factors on Macrovipera Lebetina obtuse venom. WSEAS International Conference on CLIMATE CHANGES, GLOBAL WARMING, BIOLOGICAL PROBLEMS, Catania, Sicily, Italy November 3-5, pp.75, TANIA.pdf, ISBN: