COMPARISON OF PROTEINS , LETHALITY AND IMMUNOGENIC COMPOUNDS OF Androctonus crassicauda

Scorpions are venomous arthropods of the class Arachnida and are considered relatives of spiders, ticks and mites. There are approximately 1,500 species of scorpions worldwide, which are characterized by an elongated body and a segmented tail that ends in a venomous stinger. No specific treatment is available for scorpion envenomation, except for the use of antivenom. The current study aimed at comparing protein content and lethality of Androctonus crassicauda venom extracted by two different methods (electric stimulation and maceration of telsons). The LD50 calculated by probit analysis was 1.1mg/kg for venom obtained by electric stimulation and 39.19mg/kg for venom obtained by maceration of telsons. In the electrophoretic analysis, protein bands of the venom sample obtained by electric stimulation were between 12 and 53kDa (total: five bands), and those of venom extracted by maceration appeared as multiple protein bands, relative to the other venom sample. Low-molecular-weight proteins, revealed by western blotting, played an important immunogenic role in the production of antivenom. Lethality and protein levels varied according to the extraction method; venom obtained by the maceration technique showed lower toxicity than that obtained by electric stimulation.


INTRODUCTION
The three most important orders of Arachnida are Araneae (spiders), Scorpiones (scorpions) and Acari (ticks and mites) (10,19,25).Scorpions are terrestrial arachnids that are easily recognized by their characteristic elongated body and segmented tail ending in a bulbous sac and in a stinger (telson); there are approximately 1,500 scorpion species worldwide (4,19,24,26).Scorpion venom, which has lethal and paralytic effects, is a secretion composed of water, salts and simple, low-molecular-weight proteins (5,10,23).It is a unique defense and feeding weapon.Scorpions also employ this valuable tool, in several sophisticated ways, for subduing prey, deterring predators, probably during mating, and most of the times for protection against humans (2,4,9,12).Thus, scorpion envenomation still remains a major health problem in many tropical and subtropical countries (2,4,18,22,30).
Antivenom is still widely used in the treatment of envenomation as there are no vaccines or other effective agents available against animal venoms (31).Hence, representatives of 21 laboratories around the world, both public and private, have presented the methodologies used for producing antivenom (30).It is clear that availability of effective antivenom constitutes the cornerstone of envenomation treatment and requires the use of scorpion venom, which can be obtained by different methods such as manual extraction, electric stimulation and maceration (4,17,23,24,30).In Turkey, scorpion venom has been obtained by maceration of telsons since 1942 at Refik Saydam Hygiene Center (RSHC), Ankara, Turkey (20).
In the current work, scorpion venom samples obtained by two different methods were compared as to protein content and lethality and evaluated in order to verify (i) which method was the best for immunization, (ii) which compounds present in these venom samples have a relevant role in the production of antivenom, and (iii) which of these compounds can be considered immunogens.

Animals
Scorpions: Androctonus crassicauda (Figure 1) specimens were collected in Sanliurfa and Mardin provinces (Southeastern Anatolia), Turkey.They received water daily and were fed with crickets or cockroaches after every milking.Mice: Male Swiss albino mice (20±2g) were used in all experiments.They were bred and maintained at the Department for Laboratory Animals of RSHC.Mice were kept under room temperature and 60±10% humidity and fed with commercial rodent food pellets ad libitum throughout the experiment.

Venom samples
Electric stimulation method: Venom from mature scorpions was obtained by electric stimulation of their telsons, as previously described (24).It was mixed with sterile double distilled water and centrifuged at 10000g for 15 min at 4°C.Supernatant was stored at -20°C in the dark until use.
Maceration method: Three hundred and seventy-six telsons (Figure 2) of A. crassicauda were used.Venom solution was prepared by macerating telsons as previously described (23,24).All telsons were weighed by using an electronic balance with sensitivity of 0.001mg.Briefly, telsons were removed from dead scorpions at the point of their articulation with the last caudal segment.Then, they were ground to a fine powder which was dissolved in physiologic saline solution (PSS; 0.9% w/v NaCl) and kept at 4°C for 72h.The venom solution was centrifuged at 10000g for 10 min at 4°C.Supernatant was removed and immediately lyophilized and stored at -20°C until use.

Antivenom
The used antivenom was produced at RSHC by maceration of A. crassicauda telsons followed by administration to horses.

Protein assay
Protein content of the venom samples was determined by reading absorbance at 280nm and expressed as mg protein/ml (16,29).

Lethality assay
The median lethal dose (LD 50 ) for A. crassicauda venom extracted by both electric stimulation and maceration was calculated in mice.The used doses covered the full range of mortality from 0 to 100%.Venom was subcutaneously injected, at increasing doses, into groups of at least five mice each.An equivalent volume of PSS was injected into a group of five mice as negative control for each test.Following treatment with venom solution, the animals were monitored for 24h and LD 50 was then calculated using the probit method (7).

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE (7.5% gel) analyses of both venom samples and their protein bands were carried out according to Laemmli (14).Proteins were stained with 0.1% Coomassie Blue R-250 Silver (6).Molecular mass standard (Sigma, S8445) were run in parallel in order to calculate the molecular weights of proteins.Then, gel was photographed and the molecular weights were calculated using Molecular Imaging Software (MIS, Kodak).
Strips of the membranes were washed 3 times with TBST and exposed to diluted antivenom (1:1000) for 1h.Then, they were again washed three times with TBST and incubated with horseradish peroxidase-conjugated anti-horse antibody (1:5000) for 60min.Membranes were washed with TBST for 10min and antigens were visualized using 3,3'-diaminobenzidine (DAB; Sigma, SD0426) substrate; after development of the bands, reaction was stopped with PBS.

SDS-PAGE and Western Blotting
Protein content of venom obtained by maceration of telsons was 7.34mg/ml and that of venom obtained by electric stimulation was 0.561mg/ml.In the electrophoretic analysis, the venom sample obtained by electric stimulation showed protein bands between 12 and 53kDa (Figure 3; Lane A), and the venom sample obtained by maceration of telsons showed multiple protein bands (compared with the profile of the other venom sample) mostly between 12 and 162kDa (Figure 3, Lane B).For venom obtained by electric stimulation, five protein bands were observed, whereas for venom obtained by maceration, nine bands were noticed, and when compared, they showed only three similar protein bands -12kDa, 29kDa, and 35kDa (Figure 3).
Androctonus crassicauda antivenom strongly reacted with the venom obtained by the maceration method.Western blotting indicated 36, 53, 58 and 74kDa proteins as immunogens, contrasting to the results of the electrophoretic analysis.Specific antibody reacted with venom obtained by electric stimulation (Figure 4) for 36, 42, 53, 58, 74, 86, 135 and 150kDa proteins, which were considered immunogens.
Comparison of western blotting profiles of both venom samples showed proteins with similar molecular weights (36, 53 and 58kDa).

DISCUSSION
Scorpion venom contains short neurotoxin polypeptides consisting of simple, lowmolecular-weight proteins which have lethal and paralytic effects (10).Approximately 100,000 distinct peptides are estimated to be present in scorpion venom but only a limited number of these peptides have been described so far (28).The lethal fraction

Figure 2 .
Figure 2. Telson removed from a dead Androctonus crassicauda at the point of its articulation with the last caudal segment.

Table 1 .
Median lethal dose (LD 50 ) of Androctonus crassicauda venom obtained by two different methods (maceration and electric stimulation) and subcutaneously injected into mice.Similar symptoms were observed in mice during LD 50 determination.