Common Sea Snake Research Articles

Preparation and detection of sea snake antisera raised in rabbits

Antivenoms are currently the most effective medication used in the treatment of snakebites. However, there were relatively few studies on preparation of antivenoms targeting sea snakes, especially common sea snakes in China. In this study, we sought to prepare and detect mono- and bispecific antisera raised in rabbits against venoms of two sea snakes, Hydrophis cyanocinctus and H. curtus. The results of enzyme-linked immunosorbent assay showed that the rabbit antisera generally showed clearly detectable immunological cross-reactions after the third immunization and indicated that the strength of cross-reactions increased with an increase in the immunizing dose. Proteins within the H. cyanocinctus and H. curtus venoms showed similar profiles and were mainly concentrated in the low-molecular-weight region (8–25 kDa). Western blotting results revealed that the bands of these low-molecular weight proteins were dense and showed strong immunogenicity. Although we detected comparatively few bands of the high-molecular-weight proteins, these also showed strong immunogenicity. Our results indicate that both mono- and bispecific antisera both can neutralize H. cyanocinctus and H. curtus venoms, and in this regard, the monospecific H. curtus and bispecific antiserum were found to be superior to the H. cyanocinctus antiserum. Given the increasing frequency of snakebites worldwide, we believe that the findings of this study will have high practical applicability.

Antivenom cross-neutralization of the venoms of Hydrophis schistosus and Hydrophis curtus, two common sea snakes in Malaysian waters.

Sea snake envenomation is a serious occupational hazard in tropical waters. In Malaysia, the beaked sea snake (Hydrophis schistosus, formerly known as Enhydrina schistosa) and the spine-bellied sea snake (Hydrophis curtus, formerly known as Lapemis curtus or Lapemis hardwickii) are two commonly encountered species. Australian CSL sea snake antivenom is the definitive treatment for sea snake envenomation; it is unfortunately extremely costly locally and is not widely available or adequately stocked in local hospitals. This study investigated the cross-neutralizing potential of three regionally produced anti-cobra antivenoms against the venoms of Malaysian H. schistosus and H. curtus. All three antivenoms conferred paraspecific protection from sea snake venom lethality in mice, with potency increasing in the following order: Taiwan bivalent antivenom < Thai monocled cobra monovalent antivenom < Thai neuro polyvalent antivenom (NPAV). NPAV demonstrated cross-neutralizing potencies of 0.4 mg/vial for H. schistosus venom and 0.8 mg/vial for H. curtus, which translates to a dose of less than 20 vials of NPAV to neutralize an average amount of sea snake venom per bite (inferred from venom milking). The cross-neutralization activity was supported by ELISA cross-reactivity between NPAV and the venoms of H. schistosus (58.4%) and H. curtus (70.4%). These findings revealed the potential of NPAV as a second-line treatment for sea snake envenomation in the region. Further profiling of the cross-neutralization activity should address the antivenomic basis using purified toxin-based assays.

Effect of a sea snake (Enhydrina schistosa) venom on the ganglionic nicotinic actions of acetylcholine.

Abstract The effects of venom from a common sea snake, Enhydrina schistosa, on the actions of acetylcholine in the superior cervical ganglion, the adrenal medulla and in the atropinized cat were investigated. The crude venom reduced nictitating membrane responses to preganglionic nerve stimulation and intra-arterial injection of acetylcholine, without lowering the responses to postganglionic nerve stimulation or intra-arterial adrenaline. In the eviscerated cat, the venom also antagonized rises in heart rate and blood pressure induced by in-injections into the coeliac artery of acetylcholine but not those of histamine. Pressor effects due to splanchnic nerve stimulation and carotid artery occlusion were blocked. In the atropinized cat, the venom depressed heart rate and pressor responses to intravenous acetylcholine, but not the responses to adrenaline. It was concluded that the venom antagonized the actions of acetylcholine at autonomic ganglia and the adrenal medulla.

The use of enzyme-linked immunosorbent assay for the quantitation of Enhydrina schistosa (common sea snake) venom: N.-H. Tan, K.-K. Lim and M. I. Nik Jaafar (Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia)

The use of enzyme-linked immunosorbent assay for the quantitation of Enhydrina schistosa (common sea snake) venom: N.-H. Tan, K.-K. Lim and M. I. Nik Jaafar (Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia)

Acidic phospholipases A 2 from the venom of common sea snake enhydrina schistosa

Two acidic phospholipases A have been purified from the venom of common sea snake ( Enhydrina schistosa) by chromatography on Sephadex G-75 gel media, Bio-Rex 70 ion-exchanger followed by repeated Chromatography on DEAE-Sephadex A-25 ion-exchanger. The two preparations were shown to be homogeneous by polyacrylamide gel electrophoresis and ion-exchange chromatography. The enzymes were shown to be specific for the ‘two’ position of egg yolk lecithin. The molecular weight of both enzymes determined by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis was approx. 14000. Both enzymes were non-lethal. Amino acid composition data indicated high contents of aspartic acid, glycine and alamine in both enzymes.

Amino acid sequence of a lethal myotoxic phospholipase A 2 from the venom of the common sea snake ( Enhydrina schistosa)

The complete amino acid sequence of E. schistosa VI:5b, a strongly myotoxic phospholipase A 2 from the venom of the common sea snake, Enhydrina schistosa, has been determined. The sequence determination was done exclusively by automated Edman degradation on constituent peptides. The main fragmentation was done by cyanogen bromide cleavage yielding two fragments of approximately equal size. Peptides derived by cleavage with trypsin and Staphylococcus aureus V8 protease were used to complete and align the sequence. The results indicate that the myotoxin consists of a single chain of 119 amino acids cross-linked by 7 disulfide bridges and is closely homologous to other phospholipases A 2 of elapid origin.

Isolation and characterization of a lethal myotoxic phospholipase a from the venom of the common sea snake Enhydrina schistosa causing myoglobinuria in mice

A strongly myotoxic phospholipase A was isolated from the venom of Enhydrina schistosa by gel filtration on Sephadex G-75 and ion exchange chromatography on Bio-Rex 70. The myotoxin is a basic protein containing 120 amino acid residues and seven disulfide bridges. The formula weight is 13,500. The release of myoglobin diagnostic of muscle necrosis was monitored by placing injected mice on sheets of white paper. Affected mice stained the paper red. The myotoxin has an acute ld 50 of 110 μg/kg and causes myoglobinuria at doses down to 30 μg/kg. The molecule lacks trytophan, indicating that this amino acid is not required either for toxicity or catalytic activity. A single histidine residue was modified by reaction with p-bromophenacyl bromide, resulting in > 99% loss of enzymatic and toxic activities. The N-terminal sequence shows homology with nontoxic and neurotoxic phospholipases A 2 of vertebrate origin. The myotoxicity may be of greater clinical importance than the curarimimetic toxins that have received so much attention heretofore.

Amino acid sequence of pelamitoxin a, the main neurotoxin of the sea snake, Pelamis platurus

The amino acid sequence of pelamitoxin a, the main neurotoxin of the sea snake, Pelamis platurus, has been completed. Analysis was done principally by means of Edman degradation of constituent tryptic peptides; chymotryptic and thermolytic peptides were also studied to assist in the sequence alignment. The results indicate that pelamitoxin a is identical to schistosa toxin 5 in amino acid sequence. These results indicate the feasibility of producing a common antivenin against toxins from some of the most common sea snakes.

Laser Raman scattering of neurotoxins isolated from the venoms of sea snakes Lapemis hardwickii and Enhydrina schistosa.

The venoms of sea snakes (family: Hydrophiidae) contain potent neurotoxins which bind to the acetylcholine receptor in the neuromuscular junction. A major toxin was isolated from the venoms of the sea snakes Lapemis hardwickii and Enhydrina schistosa according to previously published methods. These pure toxins were studied by laser Raman spectroscopy to elucidate further the structure-function relationship to the neurotoxin. Spectra were obtained from the powder, aqueous solution, and deuterated derivatives of each toxin. The peptide backbone conformation of these neurotoxins was found to be of "anti-parallel beta configuration," as distinct amide I and III bands appeared at 1672 and 1240 cm-1, respectively. No indication of alpha helical structure in the neurotoxins was observed from amide I and III bands. This was further confirmed by the spectra of the neurotoxins after deuterium exchange. The peaks due to a single tyrosine residue appeared at 644, 834, and 846 cm-1. The intensity ratios of the toxin from L. hardwickii venom were 0.92, 1.0, and 0.84 at 644, 834, and 846 cm-1. It is concluded that the tyrosine residue is involved in some unusual intramolecular interactions and not readily accessible to water molecules. Similar results were obtained for the toxin of Enhydrina schistosa (common sea snake). The fact that only 50% of the tyrosine molecule is modified by nitration is in complete agreement with laser Raman result. The lack of a sharp Raman line at 1361 cm-1 suggested that the single tryptophan residue may be "exposed." The previous demonstration that the tryptophan residue can be modified readily with different reagents confirms these results. A relatively symmetrical disulfide peak at 512 cm-1 indicates that the geometry of the C-C-S-S-C-C linkage is nearly identical for all four disulfide bonds in the molecule. The absence of phenylalanine was established by the lack of a phenylalanine peak in the laser Raman spectra and by amino acid analysis.

Isolation and partial characterization of pelamitoxin a from Pelamis platurus venom

The principal toxin, named pelamitoxin a, was isolated from venom of the sea snake Pelamis platurus caught in Northern Formosan waters. Pelamitoxin a is a polypeptide containing 60 amino acid moieties, including 9 cysteines, with N-terminal methionine and C-terminal asparagine. Its amino acid composition appears to be identical to that of hydrophitoxin b ( Liu and Blackwell, Toxicon 12, 543, 1974) from venom of the sea snake Hydrophis cyanocinctus as well as schistosa 5 toxin from venom Enhydrina schistosa ( Fryklund et al., Biochemistry 11, 4633, 1972). These results indicate excellent prospects for a rational approach to production of a common antivenin effective against major toxins from several of the common sea snakes.

Sea Snake Investigation in the Gulf of Thailand

Species of sea snakes collected in the Gulf of Thailand on 3 field trips during the summers of 1967, 1969, and 1972 are described. In all, 14,282 sea snakes were collected. Only one species of non-poisonous, aquatic snake, Acrochordus granulatus (false sea snake), was collected. The most common sea snake in the Gulf of Thailand was Lapemis hardwickii, which accounts for 81 per cent of all the catch in 1969 and 1972. Other sea snakes captured were Hydrophis cyanocinctus, H. torquatus diadema, H. ornatus, Enhydrina schistosa (Disteira schistosa), Kerilia jerdonii siamensis, Praescutata viperina (Thalassophina viperina, Lapemis viperinus), Microcephalophis gracilis (Hydrophis gracilis), Thalassophis anomalus, Pelamis pla- turus, Acalyptophis peronii, and Aipysurus eydouxii. Acalyptophis peronii previously thought to be found mainly in Australian waters was found in the Gulf of Thailand where four specimens were obtained. Venoms were extracted from the venomous glands of some of the species, venom yield and toxicity are reported. * * *

Chromatographic separation of Enhydrina schistosa (common sea snake) venom and the characterization of two principal neurotoxins

Chromatographic separation of Enhydrina schistosa (common sea snake) venom and the characterization of two principal neurotoxins

Isolation and Characterization of the Toxic Component of Enhydrina schistosa (Common Sea Snake) Venom

Abstract The toxic component from the venom of the common sea snake (Enhydrina schistosa) was isolated by passage of the venom through a carboxymethyl cellulose column. The preparation, which was shown to be homogeneous by zonal electrophoresis, ultracentrifugation, isoelectric focusing, and gel filtration, had a median lethal dose 50% (LD50) toward mice of 0.044 µg per g of body weight. The molecular weight of the toxic protein, as determined by sedimentation velocity and diffusion experiments, was found to be 7200. The minimum molecular weight calculated from amino acid analysis was 7000. The isoelectric point, determined by isoelectric focusing, was 9.2. Although hyaluronidase, alkaline phosphatase, phosphodiesterase, phospholipase A, acetylcholinesterase, DNase, leucine amino peptidase, and clotting activity could all be detected in unfractionated venom; no enzymatic activity could be found in the purified toxin. The compound N-bromosuccinimide completely destroyed the toxicity of the preparation, which, under the conditions employed, strongly suggests that tryptophan is necessary for toxicity.