Isolation And Biological Properties Of Neurotoxin From Sea Anemone (Stichodactyla mertensii, S. haddoni)
A Veeruraj, M Arumugam, T Ajithkumar, T Balasubramanian
cytolysins, hemolysin, marine toxin, neurotoxin, sea anemone
A Veeruraj, M Arumugam, T Ajithkumar, T Balasubramanian. Isolation And Biological Properties Of Neurotoxin From Sea Anemone (Stichodactyla mertensii, S. haddoni). The Internet Journal of Toxicology. 2007 Volume 5 Number 2.
Neurotoxic proteins obtained from sea anemones Stichodactyla mertensii and S. haddoni were assess for neurotoxic activity of the isolated venom and several bioassays were carried out, and the effect of toxin was determined in vivo using sea shore crabs (Ocypode macrocera) the dose causing 50% lethality (LD50). The LD 50 of these two crude extract was obtained as 0.47 mg/kg and 0.71 mg/kg of crude toxic protein was used. The Brine shrimp lethality (LC50) bioassay was performed with Artemia salina nauplii. The LC50 of the S. mertensii was showed higher cytotoxicity (LC 50 = 0.625 mg/ml) than that of S. haddoni (LC 50 = 0.852 mg/ml). The protein patterns of isolated toxin were also analyzed by SDS-PAGE with an apparent molecular weight of 216 kDa (SmNt1), 193 kDa (SmNt2), 95 kDa (SmNt3), 60 kDa (SmNt4) & 46 kDa (SmNt5) and 103 kDa (ShNt1), 82 kDa (ShNt2) & 63 kDa (ShNt3). Neurotoxic activity was estimated by inject a small amount of the crude toxic protein into the third walking leg of a crab and observed the typical convulsions, paralysis, color changes and death provoked by neurotoxins. Hemolytic activity was also estimated as 13 mg/ml, 7 mg/ml in S. mertensii, S. haddoni respectively. The hemolytic values was estimated as 2.46, 4.92, 27.38& 9.85 HT/mg and 2.29, 4.57, 18.29 and 9.14 HT/mg respectively in chicken, cow, goat and human erythrocytes.
Sea anemones possess toxins are poisonous substances which are produced by nematocyst. Cnidarians produce polypeptide toxins which functions as Neurotoxin or Cytotoxin. Sea anemones are rich sources of pore forming toxins, which are used for prey acquisition or may act as chemical signals by repelling predators. Cytolytic toxins are secreted by intact contracting sea anemones on mechanical stimulation and four major groups of cytolysins from a number of sea anemones have been characterized extensively.
The most widely studied cytotoxic and cytolytic protein from sea anemone is equinatoxin II (EqT II), a basic protein from
Materials And Methods
The specimens were collected from Coral reefs at Mandapam near Kurusadai island (78 o 11' to 79 o 15' E longitude and from 8 o 49' to 9 o 15' N latitude) in the Gulf of Mannar, Tamilnadu, India. The animals were brought to the laboratory as live and immediately washed, kept in the sterile clean saline water and stored at -20 o C until further use.
Preparation of the crude extract
The crude extract was collected according to the standard methods . Briefly 100 g of the freeze–dried animal pieces was homogenized in an electrically driven tissue grinder/ blender for 5 min, the homogenate was dissolved in 100 ml normal saline (0.9% NaCl) and this was centrifuged at 10,000g for 10 min. Finally, the supernatant was collected into sterile test tubes. The desired test concentrations of the extract were always prepared from this stock by serial dilution.
Estimation of the protein content of the crude extract
Amount of protein in the samples were estimated according to the method of  with bovine serum albumin was used as a standard.
Molecular weights of the partially purified active fractions of crude extracts were analyzed by SDS-PAGE . Running gels contained 10.0% acrylamide, and electrophoresis was conducted at 50 V for 3 h in Tris–glycine buffer, maintained the pH 8.3. After run, gel was stained with 0.3% Coomassie blue(R-250) and their molecular weights were determined in gel documentation system (Lark, India) by comparison with protein Standards (GeNei TM ) Myosin, Rabbit Muscle 205 kDa; Phosphorylase b 97 kDa; Bovine Serum Albumin 66 kDa; Ovalbumin 43 kDa; Carbonic anhydrase 29 kDa.
The acute toxicity study was carried out for the isolated toxin extract using adult sea shore crabs
The cytotoxicity assay was evaluated using the standard method of  with
The hemolytic activity of the crude toxin was assessed by the micro hemolytic method . Chicken, Cow, Goat and Human blood (A1+ve) was collected from slaughter house and Government hospital, Parangipettai, Tamilnadu, with EDTA solution (2.7g/ 100 ml) as anticoagulant and brought to the laboratory. The blood was centrifuged at 5,000 rpm for 5 minutes; the supernatant are discarded, the pellet was suspended in normal saline (pH 7.4). The mixture was further centrifuged at 5,000 rpm for 5 min, the supernatant was discarded and pellet resuspended in normal saline (pH 7.4.). This procedure is repeated thrice. From these, 1% erythrocyte suspension was prepared by adding 99 ml normal saline to 1ml of packed RBC.
The micro hemolytic test was performed in 96 well ‘U' bottom microtitre plates. Different rows were selected for these different blood samples. Serial dilution of the crude toxin were made in 100 µl then also discarded. 100µl of 1% RBC was added into all the wells with appropriate controls were included in the test. 1% RBC suspension, 100 µl of distilled water was added, which served as a positive control and 100 µl of normal saline, which served as, negative control. The plate was gently shaken and allowed to stand for three hour at room temperature and the results were recorded. Uniform red color suspension in the wells considered as positive hemolysis and a butten formation in the bottom of the wells was considered as lack of hemolysis. Reciprocal of the highest dilution of the crude extract showing the hemolytic pattern was taken as one Hemolytic Unit (HU).
Estimation of the protein content of the crude extract
A total of 30 g of crude extracts were obtained from the samples. The protein concentration of crude was 130 µg/mg and 70
The protein patterns and molecular weight of the crude extracts of
The results showed that crude extract of both species (
The bioassay results
The hemolytic assay of sea anemones was represented in fig.4 and 5. The
In the last two decade, the importance of the biologically active compounds present in the venom of different marine organisms has become evident. The study of these compounds has permitted an improved understanding in the fields of pharmaceutical, neural, and biological sciences, and has allowed for the development of novel drugs.
It is known that the toxic compounds in sea anemones are proteins and it is possible to determine their structural properties. Previously, a toxic protein from the sea anemone
In earlier demonstrated that the three different kinds of nematocysts such as Microbasic amastigophores, Macrobasic amastigophores and Spirocysts . Nematocysts can be discharged the toxin using chemical or mechanical stimuli. The nematocyst immediately contact with a victim in order to cause pain, urtications, paralysis or death .
The hemolytic activities of these sea anemones are comparatively significant with earlier findings ; Hemolytic activities of Heteractis magnifica [16, 17] are indicating the cytolytic activity of venom through pore-formation onto biological membranes and this has amply been demonstrated. In the present study the extensive level of membrane breakdown in the various tissues. The
The acute toxicity study shows a dose- mortality relationship which was apparently sigmoidal condition. A Probit values (% mortality) of extract gave a straight line from which the LD50 was extrapolated. In the present investigation of neurotoxicity test for crude extract from
The present results indicate that the S
For the forgoing account, the tropical sea anemone