North-Western Journal of Zoology Vol. 4, No. 1, 2008, pp.16-28 [Online: Vol.4, 2008: 06]

Electrophoretic characterisation of the venom samples obtained from various Anatolian snakes (Serpentes: Colubridae, Viperidae, Elapidae)

Hüseyin ARIKAN1, Bayram GÖÇMEN1*, Yusuf KUMLUTAŞ2, 1 2 1,3 Nurşen ALPAGUT-KESKİN , Çetin ILGAZ and Mehmet-Zülfü YILDIZ

1- Ege University, Faculty of Science, Department of Biology, Zoology Section, 35100 Bornova, Izmir, Turkey 2 - Dokuz Eylül University, Faculty of Education, Department of Biology, Buca, Izmir, Turkey 3 - Harran University, Faculty of Art and Science, Department of Biology, Zoology Section, Osmanbey Campus, Sanliurfa, Turkey * Corresponding author: Ege University, Faculty of Science, Department of Biology, Zoology Section, 35100 Bornova, Izmir, Turkey, Tel:+90 (232) 388 40 00/1795, E-mail: [email protected]

Abstract. The venoms extracted from a colubrid snake [Malpolon monspessulanus (Hermann)], seven viperids [Montivipera xanthina (Gray), Montivipera wagneri Nilson &Andrén, Vipera ammodytes (Linnaeus), Vipera kaznakovi (Nikolsky), Vipera eriwanensis (Bonaparte), Vipera barani Böhme & Joger, Macrovipera lebetina (Linnaeus)] and an elapid snake [Walterinnesia aegyptia (Lataste)] collected from various regions of Anatolia were compared using polyacrylamide gel disc electrophoresis and densitometry analysis methods. The electrophoretic patterns (protein bands) of the examined venom snakes were demonstrated. The resulting electropherograms showed important qualitative differences amongst the colubrid snake Malpolon monspessulanus, the elapid species Walterinnesia aegyptia and the viperid snakes. In M. monspessulanus and W. aegyptia samples the total protein fraction numbers were 11 and 12, respectively, while in viperid samples the number was between 10 and 14, indicating a higher venom complexity in viperids compared to that of opistoglyph- colubrid and proteroglyph-elapid snakes. Electrophoretic data support the phylogenetic argument previously outlined for the family Viperidae. Moreover, it is concluded that the Macrovipera wagneri and Walterinnesia aegyptia are closely related taxa with front-fanged delivery systems in the light of protein band analogies.

Key words: Colubrid, viperid and elapid snake venoms, polyacrylamide gel disc electrophoresis, densitometry.

Introduction Basoglu & Baran 1980, Nilson et al. 1988, Nilson et al. 1999, Nilson & Andrén 2001, The venomous snakes have been Ugurtas et al. 2001, Budak & Göçmen widespread in tropical and subtropical 2005, Joger 2005) 15 venomous snake regions of the world (Edstrom 1992, species [14 in the family Viperidae Arikan et al. 2005). Studies related to (Solenoglypha) and one in the family snakes in Anatolia often directly deal Elapidae (Proteroglypha)] and four with the taxonomy and distribution of semi-venomous snake species of the species. According to studies con- family Colubridae (Opisthoglypha) live ducted in Anatolia (Baran 1976, in Anatolia.

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The polyphyletic family Colubridae foraging strategy (sit-wait-strike). produces venom in a specialised According to Lenk et al. (2001), Eurasian cephalic and oral gland, a Duvernoy’s vipers could be unambiguously divided gland, located in the temporal region into four monophyletic groups. (Mackessy 2002). Although the Only a few electrophoretical studies Duvernoy’s gland is a homologue of have been conducted on venom proteins the venom glands of viperid and elapid of different viperid and colubrid snakes snakes, it is anatomically and from Anatolia as well as their clinical, functionally distinct (Kardong 2002). physiological and serological effects on Venoms obtained from snakes of the human (Arikan et al. 2005, Arikan et al. Viperidae, Colubridae and Elapidae 2006, Göçmen et al. 2006). families in various parts of the world, The present study deals with the have been used for research in the venom proteins of some viperid pharmacological, biochemical, immu- snakes, a colubrid species (Malpolon nological and toxicological fields monspessulanus) and a single elapid (Hageman 1961, Mebs 1968, Tu & species (Walterinnesia aegyptia) living in Ganthavorn 1968, Basu et al. 1969, Anatolia. The aim of the present study Bonilla & Horner 1969, Robertson & was to establish the venom electro- Delpierre 1969, Young & Miller 1974, phoretic patterns of various venomous McKinstry 1983, Kochva 1987, Minton snakes from different parts of Turkey & Weinstein 1987, Tun-Pe et al. 1995, and, to make proper comments on the Kardong 2002, Mackessy 2002, Arikan phylogenies and taxonomies of the et al. 2005). Snake venoms are mixtures examined taxa in the light of obtained of biologically active substances most findings. of which are enzymes or non- enzymatic polypeptides. They affect victims in different ways depending on Materials and Methods their enzyme ingredients. They can be The viperid, colubrid and elapid neurotoxic or haemolytic. In the light of specimens used in the study were collected the conducted studies mentioned from different parts of Anatolia at different above, it has been reported that dates [Montivipera xanthina – Gumuldur (Izmir venoms of solenogyph (Viperidae and Province), Montivipera wagneri – Karakurt Crotalidae) and opistoglyph (Kars Province), Vipera ammodytes – Persembe (Colubridae) group snakes are mainly (Ordu Province), Vipera kaznakovi – Hopa (Artvin Province), Vipera eriwanensis – haemolytic-proteolytic while the Ardahan (Ardahan Province), Vipera barani – venoms of proteroglyph (Elapidae and Sakarya (Sakarya Province), Macrovipera Hydrophiidae) group snakes are lebetina – Ceylanpinar (Sanliurfa Province), fundamentally neurotoxic. Lenk et al. Walterinnesia aegyptia – Tektek Mountain (2001) have stated that the evolutionary (Sanliurfa Province), Malpolon monspessulanus - Cigli (Izmir Province)]. All specimens radiation at least in Viperidae might captured were mature. The specimens were have been driven by possession of an taken to the laboratory alive and their venoms effective venom apparatus and a were extracted without applying any pressure

North-West J Zool, 4, 2008 18 Arikan, H. et al. on their venom glands, as described by Tare et (Malpolon monspessulanus) and one al. (1986). To remove dead cells, venom elapid species (Walterinnesia aegyptia) extracts were centrifuged for 5 minutes at 600 were given in Fig.1. As seen in Figure g and stored at -20°C until electrophoretic separation. 1, each specimen has a peculiar electro- During electrophoretic analysis, a 5 µl phoretic pattern. Significant differences venom sample was used for each separation. in fraction numbers (Table 1), electro- The venom proteins were separated according phoretic mobilities and densities of the to Davis (1964), slightly modified by Özeti and venom proteins were observed among Atatür (1979). In detail, a pH 6.7 stacking gel the taxa. was layered above the pH 9 separation gel of 7.5% polyacrylamide, together with a pH 8.3 Gel photographs showing the tris-glycine buffer system. The “loading gel” electrophoretic separation of each of Davis (1964) was omitted. Electrophoretic venom sample together with their separations were run at room temperature densitometric tracing curves, are given (approx. 20-25°C) using a Canalco Model 1200 in Figs. 2 to 10. As can be seen in Fig. 2, disc electrophoresis apparatus. Gels containing separated proteins were stained in the colubrid species Malpolon with 0.5% Amido Black (Naphtol Blue Black monspessulanus, protein fractions could 10-B) and excess stain was removed passively only be divided into 11 fractions, one of in 7% acetic acid baths. Then, the stained gels which was in the albumin zone and 10 were photographed. Qualitative evaluation of were in the globulin zone. the gels was done directly from the The electrophoretic patterns of the electropherograms and the densitometric curves of the separations were obtained by viperid venom protein samples from means of a Gelman ACD-15 Model 39430 Anatolian specimens (Fig.3-10) showed densitometer at 500 nm. qualitative differences among them; which suggest that all of the taxa have clearly distinct venom proteins. These Results venom proteins could be separated into The venom secretions of both 10-14 fractions. Among the seven Malpolon monspessulanus and Wal- vipers examined, it was found that the terinnesia aegyptia which have an total protein fraction number was opisthoglyph and a proteroglyph lowest in Montvipera wagneri and venom apparatus, respectively, were highest in Montivipera xanthina. The colorless. In contrast, the venom venom protein fractions were found to extracts of the viperid snakes, which be 10 in Montvipera wagneri, 12 in Vipera have a solenoglyph venom apparatus kaznakovi, Macrovipera lebetina and were light yellow in color and had a Walterinnesia aegyptia, 13 in Vipera higher viscosity than those of the ammodytes, Vipera eriwanensis and colubrid and elapid venom secretion. Vipera barani, and 14 in Montvipera Gel photographs of the venom xanthina. protein samples of the seven viperid The venom protein distribution species belonging to three different patterns of two Montivipera species genera (Vipera, Montivipera and (wagneri and xanthina) are distinctly Macrovipera), one colubrid species different. The main qualitative diffe-

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rences were: (a) in wagneri the number and eriwanensis, Euro-Asian ammodytes of discernible globulin fractions was 8 and Anatolian barani) consist of a total compared to 12 in xanthina, (b) there of 12-13 electrophoretic fraction (Figs. 5- was a discernible pre-albumin fraction 8), with 11-12 fractions found in the in xanthina compared to a discernible globulin and 1-2 fraction(s) in the post-albumin fraction in wagneri. albumin zone. A discernible pre-albu- The venom proteins of four species min fraction before the main albumin of the genus Vipera (Caucasian kaznakovi fraction was only seen in V. eriwanensis.

Table 1. Distribution of the numbers of fractions obtained from the venom protein electrophoresis of different Anatolian snakes. G: Globulin fraction number, PoA: Post-albumin fraction number, A: Albumin fraction number, PreA: Pre-albumin fraction number.

Species G PoA A PreA Total Malpolon monspessulanus 10 - 1 - 11 Montivipera xanthina 12 - 1 1 14 Montivipera wagneri 8 1 1 - 10 Vipera kaznakovi 11 - 1 - 12 Vipera ammodytes 12 - 1 - 13 Vipera eriwanensis 11 - 1 1 13 Vipera barani 12 - 1 - 13 Macrovipera lebetina 9 - 2 1 12 Walterinnesia aegyptia 10 1 1 - 12

The other viperid species which wagneri (Fig. 4), a post-albumin fraction belongs to the different genera, was also present, but was more Macrovipera lebetina is clearly distinct prominent in Walterinnesia aegyptia. from the all other investigated viperid species by the presence of 9 fractions in the globulin zone and three albumin Discussion fractions which one of them is pre- albumin (Fig. 9). In many previous studies (Tu & The densitometric curves of venom Ganthavorn 1968, Basu et al. 1969, proteins of Afro-Asiatic elapid species, Bonilla & Horner 1969, Young & Miller Walterinnesia aegyptia were observed for 1974), it has been reported that the the first time and had a total of 12 secretions of Duvernoy’s gland in fractions (Fig.10). The numbers of various colubrid snakes have im- globulin/albumin fractions were portant characteristic electrophoretic detected as 10/2. As seen in Montivipera patterns and more similar complexities

North-West J Zool, 4, 2008 20 Arikan, H. et al. compared to elapid and viperid and the venom gland secretion of the venoms. Minton and Weinstein (1987) elapid species Walterinnesia aegyptia pointed out that the colubrid venoms had a total of 11 and 12 fractions they analyzed using SDS-PAGE (protein bands) respectively, while in electrophoresis, contained 7-10 protein the viperid samples they numbered bands and that the Duvernoy’s gland between 10-14. Therefore, it can be secretion was as complex as most concluded that the venom of viperid proteroglyph (Elapidae & Hydrophi- snakes is more complex compared to dae) venoms. However, the total the Duvernoy’s gland secretion in number of protein bands was lower Malpolon monspessulanus (a colubrid than those of viperid snakes. Our snake) and the venom gland secretion results, based on the polyacrylamide in Walterinnesia agyptia (an elapid gel electrophoresis, indicated that the snake). This finding is in accordance Duvernoy’s gland secretion of the with those of Minton and Weinstein colubrid snake Malpolon monspessulanus (1987) and Mackessy (2002).

Figure 1. Venom protein electropherograms of a colubrid (Malpolon monspessulanus), eight viperid and an elapid (Walterinnesia aegyptia) snakes (S: Start, junction between the stacking and separation gels).

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Figure 2. Gel photograph showing the electrophoretic separation of the venom protein sample obtained from an Afro-Euro-Asiatic Malpolon monspessulanus, together with its densitometric tracing curve. OD: Optical density, S: Start (junction between the stacking and separation gels).A: Albumin fraction zone, G: Globulin fraction zone.

Figure 3. Gel photograph showing the electrophoretic separation of the venom protein sample obtained from one Anatolian Ottoman Viper, Montivipera xanthina, together with its densitometric tracing curve. PreA: Pre-albumin fraction. For further explanation, see legend to Fig. 2.

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Figure 4. Gel photograph showing the electrophoretic separation of the venom protein sample obtained from one Irano-Anatolian Wagner’s Adder, Montivipera wagneri, together with its densitometric tracing curve. PoA: Post-albumin fraction. For further explanation, see legend to Fig. 2.

Nilson & Andrén (1986) have investigators showed that the venoms proposed a phylogenetic relationship from the youngest (smallest) snakes tree based on the morphological have fewer protein bands, the number characters amongst the mountain of bands increased as the snakes aged, vipers (Montivipera species, formerly the venom of young snakes had a high known as Vipera xanthina complex) of lethal potency and as snakes aged, this the Middle East. According to this potency decreased. Similar results were theory Montivipera wagneri is more obtained for Ottoman Viper, Monti- primitive than M. xanthina. Our results vipera xanthina by Arikan et al. (2006). obtained from the gel elecrophoresis of All specimens used in the present venom proteins support this theory study had already reached sexual since the total fraction number of maturity. So, the study material did not venom proteins was very low in M. allow us to make a comparative study wagneri. on the intra-specific variations based Tun-Pe et al. (1995) studied Russell's on age or size. However, we detected viper (Daboia russelli siamensis) venoms remarkable differences among the taxa using SDS-PAGE electrophoresis in examined, both in the total number of different sized specimens. These protein bands and the density of each

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Figure 5. Gel photograph showing the electrophoretic separation of the venom protein sample obtained from one Anatolian-Caucasian Viper, Vipera kaznakovi, together with its densitometric tracing curve. For further explanation, see legend to Fig. 2.

Figure 6. Gel photograph showing the electrophoretic separation of the venom protein sample obtained from one Euro-Anatolian-Caucasian Nose-horned Viper, Vipera ammodytes, together with its densitometric tracing curve. For further explanation, see legend to Fig. 2.

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Figure 7. Gel photograph showing the electrophoretic separation of the venom protein sample obtained from one Anatolian-Caucasian Small Viper, Vipera eriwanensis, together with its densitometric tracing curve. PreA: Pre-albumin fraction. For further explanation, see legend to Fig. 2.

Figure 8. Gel photograph showing the electrophoretic separation of the venom protein sample obtained from one Anatolian Baran’s Viper, Vipera barani, together with its densitometric tracing curve. For further explanation, see legend to Fig. 2.

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Figure 9. Photograph showing the electrophoretic separation of the venom protein sample obtained from one Afro-Asiatic Levantine Viper, Macrovipera lebetina, together with its densitometric tracing curve. PreA: Pre-albumin fraction. For further explanation, see legend to Fig. 2.

Figure 10. Gel photograph showing the electrophoretic separation of the venom protein sample obtained from one Afro-Asiatic Desert Cobra, Walterinnesia aegyptia, together with its densitometric tracing curve. PoA: Post-albumin fraction. For further explanation, see legend to Fig. 2.

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protein band. Montivipera wagneri was Montivipera xanthina (14 bands). These found to have the lowest total number electrophoretic venom protein patterns of proteins with 10 fractions, while are consistent with the findings of Lenk Montivipera xanthina had the highest et al. (2001). with 14 fractions. Accordingly, the Although there was a general venom of the Ottoman viper, Monti- consensus in the literature that the vipera xanthina appears said to have a Duvernoy’s “venom” gland in more complex composition from the colubrids is homologous to the venom viewpoint of venom proteins compared glands of viperid snakes, Kardong to all the other viperid, colubrid and (2002) and Mackessy (2002) have stated elapid taxa examined in this study. that it is functionally and anatomically Similarities such as the low number distinct. In addition it has many other globulin fractions and the presence of a physical (viscosity, color) and chemical post-albumin fraction between Monti- (composition, toxicity, enzymatic vipera wagneri and Walterinnesia ae- activity) differences in its venom. There gyptia, which is recognized as the most is no information available on the primitive elapid species (Keogh 1998), biochemistry and pharmacology of indicate a close relationship between venoms and also on the anatomy, the two snake families; Viperidae and histology and cytology of the venom Elapidae, both with front-fanged glands from the species distributed in venom delivery systems. Anatolia. Future studies should focus Lenk et al. (2001) used the nucleo- on the above mentioned fields in order tide sequences of mitochondrial cyto- to gain more information and a deeper chrome b and 16S rRNA genes to understanding of both the functional reconstruct a molecular phylogeny of and evolutionary relationships bet- the family Viperidae. According to ween the venom components and the their analysis, Eurasian vipers have snakes that produce them. been divided into four monophyletic groups. The radiation might have been driven by the possession of an effective References venom apparatus. Our results indicate the Eurasian vipers living in Anatolia Arikan, H., Göçmen, B., Mermer, A., Bahar, H. could be divided into four groups in (2005): An electrophoretic comparison of the terms of venom protein bands: (a) venoms of a colubrid and various viperid snakes from Turkey and Cyprus, with some Irano-Caucasian Montivipera wagneri taxonomic and phylogenetic implications. (10 bands); (b) Middle East-Caucasian Zootaxa 1038: 1-10. Macrovipera lebetina plus V. kaznakovi Arikan, H., Alpagut Keskin, N., Çevik, I. E., Ilgaz, (12 bands); (c) Euro-Anatolian- Ç. (2006): Age-dependent variations in the venom proteins of Vipera xanthina (Gray, 1849) Caucasian V. ammodytes plus Vipera (Ophidia: Viperidae). Turkiye Parazitoloji s.str [Vipera barani and V. eriwanensis] Dergisi (Acta Parasitologica Turcica) 30 (2): (13 bands) and (d) Anatolian 163-165.

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