Age-dependent variations in the venom proteins of Vipera kaznakovi Nikolsky, 1909 and Vipera ammodytes (Linnaeus, 1758) (Ophidia: Viperidae)

by i and Atatur (1979) as previously described in detail (Arikan et al., 2006). Briefly, venom samples were loaded onto the polyacrylamide gels and separated using a Canalco model 1200 electrophoresis apparatus. Gels were stained with 0.5% Amido black to visualize the protein bands and densitometric curves were obtained using a Gelman ACD-15 model 39430 densitometer at 500 nm.The authors received ethical permission for venom sampling (Ege University Animal Experiments Ethics Committee, 2010-43) and special permission for field studies (from Republic of Turkey Ministry of Forestry and Water Affairs). 3. ResultsThe venom extracts of Vipera kaznakovi and Vipera ammodytes specimens had a viscosity higher than that of water. Although venom secretions of 2 specimens of V. kaznakovi (16.5 and 30 cm total lengths) were colorless, they were light yellow in the 55-cm-long specimen. In contrast, the venom extracts of 3 specimens of V. ammodytes were light yellow.The gel photographs of the venom protein samples of V. kaznakovi specimens are presented in Figure 1. According to our results, the venom proteins of V. kaznakovi were separated into 12 fractions or fraction groups in the 2 shorter individuals (16.5 and 30 cm), while there were 13 fractions or fraction groups in the gel of the 55-cm-long individual (the longest one). Our comparative results of electrophoretic density curves (protein fractions) revealed significant quantitative differences especially between the 2 shorter individuals, while similar electrophoretic mobility patterns and fraction numbers were observed (Figures 1–4). Additionally, the densities of the venom proteins of the shortest individual differed strongly from the others (Figure 1). A B C Figure 1. Polyacrylamide gel electrophoresis of venoms of V. kaznakovi of different lengths. A. 16.5 cm, B. 30 cm, C. 55 cm (S: Start, junction between the stacking and separation gels). 0.225 0.200 0.175 0.150 0.125 0.100 0.075 0.050 0.025 O.D. + s Figure 2. Gel photograph showing the electrophoretic separation of the venom protein sample obtained from the 16.5-cm-long Vipera kaznakovi specimen, together with its densitometric tracing curve (O.D.: Optical density, S: Start, junction between the stacking and separation gels). 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 O.D. + s Figure 3. Gel photograph showing the electrophoretic separation of the venom protein sample obtained from the 30-cm-long Vipera kaznakovi specimen, together with its densitometric tracing curve. For further explanation, see caption of Figure 2.The gel photographs of the venom protein samples of V. ammodytes are presented in Figure 5. Similar results were also found in V. ammodytes. According to our results of the electrophoretic and densitometric analyses, the venom proteins of the 28.5-, 36.7-, and 51.5-cm-long V. ammodytes individuals were separated into 13, 14, and 13 fractions or fraction groups, respectively. Our comparative results of electrophoretic protein fractions and densities revealed significant quantitative differences especially between the shortest (28.5-cm-long) and the longest (51.5-cm-long) individuals, while similar electrophoretic mobility patterns and fraction numbers were observed (Figures 5–8). 4. DiscussionMany reports in the literature have documented variation in venom between different individuals of the same population of one species, during different ages of the animal (Chippaux, 2006; Mackessy, 2010). Various studies on agedependent variations in snake venom proteins showed that younger individuals had more potent venom in terms of toxicity, and procoagulant and defibrinating activity (Bonilla and Horner, 1969; Fiero et al., 1972; Minton, 1975; Theakston and Reid, 1978; Meier and Freyvogel, 1980; Glenn and Straight, 1985; Furtado et al., 1991; Tun-Pe et al., 1995; Guercio et al., 2006; Mackessy et al., 2006). Several parameters of venom are affected during ontogenetic development. As a result of the increased head size and so venom gland size, venom yields and total dry weight of adult snakes are generally greater than those of neonate or juvenile individuals and this can be regarded as a general trend for all venomous reptiles (Klauber, 1956; Meier and Freyvogel, 1980; Mackessy, 1985, 1988; Furtado et al., 1991; Mackessy et al., 2003, 2006, 2010). The total + S Figure 4. Gel photograph showing the electrophoretic separation of the venom protein sample obtained from the 55-cm-long Vipera kaznakovi specimen, together with its densitometric tracing curve. For further explanation, see caption of Figure 2.Figure 5. Polyacrylamide gel electrophoresis of venoms of V. ammodytes of different lengths. A. 28.5 cm, B. 36.7 cm, C. 51.5 cm (S: Start, junction between the stacking and separation gels). 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 O.D. + s Figure 6. Gel photograph showing the electrophoretic separation of the venom protein sample obtained from the 28.5-cm-long Vipera ammodytes specimen, together with its densitometric tracing curve. For further explanation, see caption to Figure 2. 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 O.D. + s Figure 7. Gel photograph showing the electrophoretic separation of the venom protein sample obtained from the 36.7-cm-long Vipera ammodytes specimen, together with its densitometric tracing curve. For further explanation, see caption to Figure 2.