Intraspecific variation in the venom electrophoretic profile of saw-scaled viper, Echis carinatus of Central Punjab, Pakistan
A Feroze, S Malik, J Qureshi
echis carinatus, snake venoms, sodium dodecyl sulfate - poly acrylamide gel electrophoresis
A Feroze, S Malik, J Qureshi. Intraspecific variation in the venom electrophoretic profile of saw-scaled viper, Echis carinatus of Central Punjab, Pakistan. The Internet Journal of Veterinary Medicine. 2008 Volume 6 Number 2.
The use of Polyacrylamide gel electrophoresis (PAGE) for basic proteins may be a useful auxiliary tool for the characterization of whole snake venoms and for the taxonomic classification of snakes. The electrophoretic characterization of the basic venom proteins has emerged as an efficient method for taxonomic studies of the families Elapidae and Viperidae. The present study comprises the intra-specific protein profiling of venom of saw-scaled Viper (
Snake venom is defined as a highly potent, biologically active mixture of chemical constituents produced or secreted by the venom gland and injected into the prey to paralyze and digest it or for defense. It has been a powerful tool to elucidate biological processes of vital importance (1). Snake venoms are a mixture of complex proteins, which have many physical and pharmacological properties. Venoms are being explored for invaluable proteins, most of which are enzymes and toxins. Many enzymes and toxins have been located, isolated and purified from different snake venoms and the mechanism by which they induce pharmacological effects has been investigated (2). Each snake species has a unique venom with different components and different amounts of toxic and nontoxic compounds. The more closely related species of snakes exhibit more similarities in their venom composition. A number of investigators all over the world have studied the various components of venoms of many groups of snakes, using the Tiselius electrophoresis, paper electrophoresis as well as PAGE. Attempts have also been made to elucidate the distinctive biological activities of the venom toxic factors (3).
The electrophoretic method is very effective not only for identification of the venom components but also for comparative biochemical studies. The reproducibility of this technique is very high and more than ten protein bands can be detected (4). Many researchers have used the electrophoretic methods to study the protein components of venoms (5) & (6). Polyacrylamide gel has been widely utilized in the study of protein components of venoms from elapid snakes (7). Populations of some vipers have also been examined for morphology, hemipenes and electrophoretic patterns of venom proteins (8). It has been suggested that the use of electrophoresis is an auxiliary tool for the morpho- taxonomic studies along with venom identification of some species of the Elapidae and Viperidae families. (9).
Even though the relationships between species can be reflected in major toxic components of their venom, the evolution of venom is not fully understood. while studying the intra -specific variation in the components of venom it has been found that some components are conserved while others are variable. According to Glenn & Straight the geographically and ecologically isolated populations have a high potential for genetic variations. Further, the presence or absence of particular proteins in individual samples of whole venom can be attributed to differences in genes encoding these proteins, which in turn is determined by various processes that control gene frequencies in a population (1).
In Pakistan, studies on intra-specific differences in the venoms from these two snake species have been broadly carried out, keeping in view a few variables including age, sex and localities. Comparative biochemical and biological studies on the venoms of cobra (
Materials And Method
A total number of twelve adult saw-scaled vipers were captured from different regions of Central Punjab, Pakistan and the venom was extracted. After the completion of experiments, eight specimens were released back in the wild with the help of the snake charmer. Four specimens were brought to the Pakistan Museum of Natural History (PMNH) and after preserving in 70% ethanol, were added to the reptile repository located in the Zoological Sciences Division (ZSD) of the Museum.
Extraction & storage of Venom
The head of the each snake was seized, the posterior part was held firmly between the forefinger and the thumb of the left hand; the other fingers hold the neck against the palm of the hand. The body of the snake was then placed under the left axilla in order to prevent the animal from pulling free. The mouth of the snake was opened and its poisonous fangs were placed on the inner edge of a sterilized plastic vial. The venom was extracted by pressing the fangs very gently on the edge of the vial without using anesthetics. All the venom samples were stored at 4 Co to avoid any disruption of their natural toxic properties.
Assay& quantification of venom proteins
Venom samples were analyzed quantitatively as well as qualitatively for proteins. Protein estimation was done by Lowry (16) using Bovine Serum Albumin as standard. Protein concentration was measured spectro-photometrically at a wavelength of 595 nm. Qualitative analysis was done by SDS-PAGE. Electrophoresis was performed as described by Laemmli
Gel preparation & Electrophoresis
Venom samples were resolved on 10, 12.5 % SDS-polyacrylamide gels. About 10-30 µl of high and wide range protein Ladder (Sigma Cat. Nos. M-3788 & M-4038) was loaded as standard in a well and 10 µl
Staining & Destaining
Gel was placed in a tray long with bromophenol blue stain and with constant agitation on shaking bath. Duration of staining was 4-6 hours. After staining, gel was shifted to destaining solution. It was destained with constant agitation until the background became transparent and protein bands become visible in the form of blue colored bands.
Photography & gel drying
After destaining, the gel was photographed with the help of camera for permanent recording of results. Gel was the dried in vacuum gel drier (Heto Dry GDI, Heto Lab. Equipments. Denmark) at a temperature of 60 C for 2-3 hours.
Determination of Molecular Weights by SDS-Page
The standard curve was plotted by calculating the
Physical appearance of venom
The fresh venom of all snakes, right after extraction was fairly transparent however after some time it became translucent. The colour of all venom samples was yellow. Imperative characteristics and physical properties of venoms from some selected snake specimens are given in the table III. For convenience the readings have been given from the selected and healthier snake specimens.
General descriptions of venom proteins
The electrophoretic patterns of viper venom proteins are shown in figures 1 to 2. The molecular weights of the venom proteins given in the tables I & II. A maximum of 17 protein bands appeared in the one of the viper samples (fig 1). Great variations in molecular weights were observed in the individual venom samples of the species.
With 10% gel there were only few low molecular bands in most samples. As expected, however, a larger range of low molecular weight bands appeared on 12.5 % gel (fig 2). In the entire samples, the protein bands were found almost uniformly distributed in the higher, middle and the lower portions of the gel.
Proteins having the molecular weights 100,000 ±3000, 75000±3000, 67,000 ±2000, 63,000± 2000, 52000± 2000,14,000± 2000, 11,000± 500, 5500± 500 and 4,300± 300 were found in all the available venom samples and these proteins can be the species-specific for the given Viper species (
An important aspect of the individual variations was the absence of an apparently species-specific protein in any one or two samples. Proteins having the molecular weights 123,000 ±3000, 110,000± 3000, 58000±2000, 46,000± 2000, 38,500±2000, 36,000± 1000, 31,000 ± 2000, 23,000± 500, 18,000± 1000, 9,000± 500, 8,500± 500, 6,500± 500 and 3,200± 1000 were found in all except one venom sample (Table I &II). Proteins having the molecular weights 85,000/± 3000, 72,000± 2000, 56,000± 2000, 13000± 1000 and 21,000± 1000 were also found in most (but not all) of the venom samples (Fig 1 & 2).
One protein having an approximate molecular weight 92,000 ± 3000 (Table 2) was found in only three samples. Similarly another protein 104700± 4000 was found in only two venom samples (Table 1& 2). Such proteins appear in the venom owing to the individual differences that are common among the venoms of the snakes of all species. Regarding the classification and purification of any individual specific venom proteins for
In this study no variables, for instance, age, season of the year, sex, diet, captivity or seasonality of snakes were not controlled. All the snakes were captured from various localities of Central Punjab province during the early monsoon of 2007, without any discrimination of gender, however all snakes encountered and captured were male. With respect to age, all studied animals were adult, though it was not possible to determine their precise age. The venom samples were collected on different days, which did not allow for providing similar diet to all specimens as a few specimens were captured just prior to the experiment.
The absence of any, otherwise most frequent, protein in any one or two samples should not deprive that protein of its probability of being species-specific along with those proteins that are found in all samples. Some examples are such proteins are 123,000 ±3000, 110,000± 3000, 58000±2000, 46,000± 2000, 38,500±2000, 36,000± 1000, 6,500± 500 and 3,200± 1000. These were found in all except one venom sample (Table I &II). Any such nonappearance of a protein may rather signify the diverse individual variations that usually appear as a result of the various factors affecting the composition of venoms of almost all snake species. Venom composition can vary among individuals of the same species, and even in the same litter, but variation is greater among geographically different populations. Variation in snake venom composition even in natural conditions has been associated with factors such as geographical origin, season, sex, age and diet (181519).
Extrinsic individual variations in venom composition have also been observed even under experimental conditions in which these factors are well under control (9). Such variations could be the result of intrinsic or genetic factors. According to Daltry et al, variation in venom composition results from natural selection and not from changes in diet. Venom composition may also vary over time in the same individual. The proposition that a particular protein is specific for the species is due to the fact that any such protein suggested to be specific for one species is either generally absent or is less frequent in all the samples of the other species. Regarding the classification and purification of any individual specific venom proteins for
The results of this study show great individual variations in the protein profile of the venom of
While species-specific protein bands can be useful in the profiling of that species, locating the intra-specific protein bands of a venomous snake can help to establish its species status on a more reliable and definitive level. Such work may sequentially result in the identification, classification and segregation of any two venomous snake species by examining and evaluating their respective protein-banding patterns. Such protein profiling can, therefore, be an effective supplementary tool to the taxonomic studies of venomous organisms bringing authenticity to their identification and classification.