Electrophoretic characterisation of the venom samples obtained from various Anatolian snakes

13 Pages·2008·0.75 MB·English

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,
Nurşen ALPAGUT-KESKİN1, Çetin ILGAZ2 and Mehmet-Zülfü YILDIZ1,3

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:[phone]/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.

North-West J Zool, 4, 2008

Oradea, Romania
Electrophoretic characterisation of venoms obtained from some Anatolian snakes 17
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
(Ordu Province), Vipera kaznakovi – Hopa
(Colubridae) group snakes are mainly
(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
1, each specimen has a peculiar electro-
separation.
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
Gel photographs showing the
7.5% polyacrylamide, together with a pH 8.3
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
in the colubrid species Malpolon
containing separated proteins were stained
monspessulanus, protein fractions could
with 0.5% Amido Black (Naphtol Blue Black
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
viperid venom protein samples from
curves of the separations were obtained by
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-

North-West J Zool, 4, 2008
Electrophoretic characterisation of venoms obtained from some Anatolian snakes 19
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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Electrophoretic characterisation of venoms obtained from some Anatolian snakes 21

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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22 Arikan, H. et al.

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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Electrophoretic characterisation of venoms obtained from some Anatolian snakes 23

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.

North-West J Zool, 4, 2008
24 Arikan, H. et al.

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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Electrophoretic characterisation of venoms obtained from some Anatolian snakes 25

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.

North-West J Zool, 4, 2008