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 as a coercive extraction usually results in the addition of some superfluous material from the fangs and mouth of the snake into the venom. Twenty two snakes were milked for their venom without anesthetics. All the venom samples were stored at 4C o to avoid any disruption of their natural toxic properties.

Venom samples of the two snake species were analyzed quantitatively as well as qualitatively for proteins. Quantitatively, the protein estimation was done by (19) using Bovine Serum Albumin as standard. Protein concentration was measured spectro-photometrically at a wavelength of 595 nm. Qualitative analysis was done by Sodium-Dodecyl-Sulphate- Poly Acrylamide Gel Electrophoresis (SDS-PAGE). Electrophoresis was performed (18) using a discontinuous buffer system.

Venoms samples were resolved on 10, 12 and 15% SDS-polyacrylamide gels. About 10-30 ul of high (Cat. No. M-3788 from Sigma) and wide range protein Ladder (from Sigma Cat. No. M-4038) was loaded as standard in a well and 10 ul supernatant of each of the samples was loaded with the help of Hamilton syringe in different wells. Gel was run at a current of 30-40 volts for overnight.

Gel was placed in a tray long with bromophenol (staining solution) 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.

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.

The standard curve was plotted by calculating the Rf values of each standard protein against the log₁₀ of its molecular weight. Finding its Rf value on the standard curve and reading the log10 molecular weight from the ordinate determined the molecular weight of the unknown polypeptide or protein. The antilog of this number was the molecular weight of the protein. Low (6,500 to 66,000) and High molecular weight protein makers (14,600 to 215,940 Daltons) from Sigma Chemicals and from Life Technologies were used in this study.

Saw-scaled viper (Echis carinatus) is a rough scaled snake with large eyes, wider head than neck and stocky body. The scales are heavily keeled. In Pakistan it is found mostly in Thar and Cholistan deserts in Sind and Punjab and also Astola Island off Makran coast in Baluchistan. This snake is considered to be the world’s most dangerous snake because of its highly toxic venom, its abundance near cultivated areas, and its aggressive, easily excitable temperament. Its venom is predominantly hemotoxic and quite potent (16).

The Common Cobra (Naja naja) is a smooth-scaled snake with black eyes, wide neck and head and medium body. Coloring varies form black or dark brown to yellowish white. Naja naja is found in eastern areas of Pakistan as far west as Karachi. It may be found in flat grasslands, among scattered trees, near rice fields and other cultivated areas, near settlements. Usually not found in deserts or rainforests. It occurs at sea levels and higher elevations. This species is found in Punjab, Baluchistan and Sindh where it is quite common. When aroused or threatened, the cobra will lift its head off the ground and spread its hood, making it more menacing. Its venom is highly neurotoxic, causing respiratory paralysis with some tissue damage (16).

In this study variables related to geographical origin, age, season of the year sex diet and seasonality of snakes were not controlled the animals of each species were captured from different localities and the number of animals each locality was small. This did not allow a precise analysis of this variable. In relation to age, all studied animals were adult, being impossible to determine their precise age. In addition, the venom samples were collected on different days, which did not permit to control the effect of diet and season in venom composition.

The fresh venom of all snakes, right after milking was fairly transparent however after some time it became translucent. The color of all viper venom samples was dark yellow whereas the color of all cobra venom samples was light yellow. Imperative characteristics and physical properties of venoms from some selected snake specimens are given in the table 5. For convenience the readings have been given from the selected and healthier snake specimens.

The electrophoretic patterns of snake venom proteins are shown in tables 1 to 4 and from figures 1 to 4. The analysis of molecular weights revealed that a maximum of 17 protein bands appeared in the viper samples (table 1) and 12 protein bands in the cobra samples (table 1). A great variation in relation to the molecular weights was observed in the individual venom samples in both species.

10% SDS-PAGE was run for both species. With the 10% gel there were molecular bands in both the species. For that reason 12.5 % and 15% gel was very few low run to get better resolution of the snake venom proteins. As expected, a larger range of low molecular weight bands appeared on 12.5 % (table 2 and 3) and still larger number on the 15 % gel (table 1 and 4). In the entire samples of cobra and viper venoms, the protein bands were found almost uniformly distributed in the higher, middle and the lower portions of the gel. A larger number of bands appeared on 12.5 % gel. Most of these bands had not appeared in the 10 % gel.

Proteins having the molecular weights 100,000 ± 3000, 75000 ± 3000, 67,000 ± 2000, 63,000 ± 63,000 ± 2000, 52000 ± 2000, 14,000 ± 2000, 11,000 ± 500, 5500 ± 500 and 4,300 ± 300 were found in all the available viper samples and these proteins can be undeniably considered as species-specific for the given Viper species (Echis carinatus) (Table 1- 4). An exciting aspect of the individual variations was the absence of an apparently species-specific protein in any one or two samples of that species. 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 but one viper sample. 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 of the viper samples (Table 1-4).

One protein having an approximate molecular weight 92,000 ± 3000 (Table 4) was found in only three viper samples. Two other proteins were found in only two viper samples. Some proteins appeared in only a single sample. These like proteins showed the individual-to-individual differences that are common among the venoms of the snakes of all species. As the presence of any individual specific protein is most uncertain and unpredictable in any venom sample, scanty literature is available regarding the classification and purification of any individual specific proteins especially for those species.

Proteins having the molecular weights 23,000± 1000, 11,500± 1000, 10,500± 500, 9,000± 500 and 6,000± 500 were found in all the available cobra samples These proteins can be considered as species-specific for the given cobra species (Naja naja). Proteins having the molecular weights 37,000± 1000, 30,000± 1000, 21500± 500, 19,000± 1000, 5500± 500, 4000± 500 and 3,100± 500 were found in all but one cobra sample. Similarly Proteins having the molecular weight 72,000± 3000 and 62,000± 2000 were found in most of the cobra samples (Table 1 & 4).

One protein having an approximate molecular weight 46000± 500 was found in only one cobra sample. Some other proteins were found in only a few cobra samples. (Table 4). Still many others appeared in only a single sample. Such proteins showed the individual-to-individual differences that are common among the venoms of the snakes of all species.

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. Any such nonappearance of a protein may rather signify the diverse individual-to-individual variations that usually appear as a result of the presence of various variables affecting the composition of the 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 (4, 11 and 13). However, it was demonstrated that, even under experimental conditions, in which these factors were controlled, extrinsic individual variations in venom composition were also observed. (24) The author suggested that variations resulted from intrinsic or genetic factors. It was also suggested (6) that 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 results of this study showed a great protein variation in venom samples of the two species. These data are in agreement with other works that showed individual variation in snake venom composition (9, 11, 12, 22, and 18). The proposition that a particular protein is specific for the species is also maintained by 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. For example all the proteins that were found in all samples of viper were absent in all the cobra samples. Almost all the proteins that could be the species-specific for the viper samples did not appear in the cobra samples. Similarly almost all the proteins that were found in all samples of cobra were absent in the entire viper samples. Although any of these aforementioned proteins may be found in one or two samples of the other species but this frequency is much less than that what is required for a protein to be identified as specific for that species.

Compared to morphological data, molecular markers, including the protein and nucleotide data have the benefit of giving an estimate of phylogeny reasonably free of the confounding effects of differing natural selection pressures on the external phenotype. Moreover molecular sequence data also have the advantage that they can give at least an approximate estimate of times of divergence between lineages, although the interpretation of molecular clocks is subject to various analytical problems (18).

This study has contributed to our understanding of the biochemical characteristics and diversity of venom composition of two medically important viper species of Pakistan. Nevertheless, some of our conclusions are tentative and await confirmation by future studies due to the absence of comparative studies of the venoms of Pakistani vipers and the scarcity of detailed information on the biology of the two species of interest. When this knowledge becomes available, these viper species of Pakistan will continue to prove a fruitful subject for investigating patterns of evolutionary and ecological divergence in the vertebrate predators. The detailed and comparative investigations of the venom proteins of these viper species in future will also lead to knowing better evolutionary and ecological significance of the vipers of Pakistan.

It may be concluded that locating the intra-specific proteins for a venomous snake can help to establish its species status on a more reliable and definitive level. Species-specific proteins for the two species can be useful in the protein fingerprinting and profiling of these species. 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. This protein fingerprinting can, therefore, be an effective supplementary tool to the modern taxonomic studies of the venomous organisms bringing authenticity to their identification and classification.

Figure 1

Table 1: =15% Approximate molecular weights of venom proteins from the and (Resolution of figure 1)

Figure 2

Table 2: =12.5% Approximate molecular weights of venom proteins from the (Resolution of figure 2)

Figure 3

Table 3: =12.5% Approximate molecular weights of venom proteins from the (Resolution of figure 3)

Figure 4

Table 4: = 15% Approximate molecular weights of venom proteins from the and (Resolution of figure 4)

Figure 5

Table 5: Some Physical characteristics of the venoms of two experimental snake species

Figure 6

Figure 1: SDS-PAGE analysis of snake venom proteins from and

Figure 7

Figure 2: SDS-PAGE analysis of snake venom proteins from

Figure 8

Figure 3: SDS-PAGE analysis of snake venom proteins from

Figure 9

Figure 4: SDS-PAGE analysis of snake venom proteins from and