The Biochemistry of Snake Venom.

Overview

Snake venom is the poison fluid normally secreted by venomous snakes when biting. It is produced in the glands, and injected by the fangs. Snake venom is used to immobilize and/or kill prey, and used secondarily in defence. It is a clear, viscous fluid of amber or straw colour.

There are two main types of venom produced by snakes, containing primarily either:

*Neurotoxins - these attack the nervous system.

*Hemotoxins - these attack the circulatory system.

While most snakes' venom contains primarily either one or the other, there are some snakes which have a combination of both in their venom.

The snake's poison is a combination of biologically active agents: ferments or enzymes as proteases and hyaluronidase (including 20 digestive enzymes), metal ions, biogenic amines, lipids, free amino acids, and more than 80 large and small proteins and polypeptides that have only been partially identified. While it is a complex recipe, snake venom is made up of mainly proteins and enzymes. The primary constituents of snake venom are as follow:

*Enzymes - Spur physiologically disruptive or destructive processes.

*Proteolysins - Dissolve cells and tissue at the bite site, causing local pain and swelling.

*Cardiotoxins - Variable effects, some depolarise cardiac muscles and alter heart contraction, causing heart failure.

*Harmorrhagins - Destroy capillary walls, causing haemorrhages near and distant from the bite.

*Coagulation - Retarding compounds prevent blood clotting.

*Thromboses - Coagulate blood and foster clot formation throughout the circulatory system.

*Haemolysis - Destroy red blood cells.

*Cytolysins - Destroy white blood cells.

*Neurotoxins - Block the transmission of nerve impulses to muscles, especially those associated with the diaphragm and breathing.

Every snake has a different amount of the aforementioned agents its venom, hence the differing levels of toxicity.

Throughout this report, the examination of the venom of only the most deadly snake in the world: the 'Inland Taipan', will be carried out. The report contains an analysis of the venom of the Inland Taipan, along with its medical uses.

The Inland Taipan

The Oxyuranus microlepidotus (inland taipan) is a member of the family Elapidae (elapid snakes), and belongs to the Genus Oxyuranus. The back, sides and tail are of a buff brown colour, and it's eyes are of average size, with a blackish brown iris. It is found only in the central, and central western desert regions of Australia.

Although the inland taipan has the most lethal venom of any snake in the world, it is placid and shy. However, if cornered and/or provoked, it holds it's body in low, flat, S-shaped curves with it's head pointed straight at the disturber. It usually makes a single bite, or a few fast ones.

The venom of the inland taipan is primarily neurotoxic. However, while the myotoxic and procoagulative proteins are present to a lesser degree, they too contribute to the bite pathology.

Neurotoxins

The neurotoxins contained in the inland taipan's venom are as follow:

*Taipoxin - presynaptic neurotoxin, phospholipase A2 based,

moderately acidic sialo-glycoprotein, MW 45,600, as a

ternary complex 1:1:1 with a , b , g subunits. a and b

subunits are 120 amino acids long, with 7 disulphide

bridges. g subunit has 135 amino acids and 8disulphide

bridges. Only the very basic (pI >10) g-subunit has lethal

neurotoxicity. LD50 of complete molecule is 2 mg/kg (IV

mouse). 17% of venom.

*Paradoxin - presynaptic neurotoxin, phospholipase A2 based,

essentially identical to taipoxin. It accounts for 12% of

crude venom, is a sialo-glycoprotein with three subunits and

has an LD50 of 2 mg/kg (IV mouse). Amino acid analysis of

paradoxin and taipoxin, both in whole form and as subunits,

shows close homology.

*O. scutellatus fraction III - minimal data. Presumed

postsynaptic neurotoxin. LD50 100 mg/kg (IV mouse). 47% of

venom.

*O. scutellatus fraction IV - minimal data. Presumed

postsynaptic neurotoxin. LD50 100 mg/kg (IV mouse). MW

approximately 8,000. 10% of venom.

[http://www.inchem.org/documents/pims/animal/taipan.htm]

Composition of this mixture may not be uniform throughout all populations of taipans.

The presynaptic constituents are much more potent than those which are postsynaptic. They work by affecting the terminal axon. On reaching the neuromuscular junction the presynaptic neurotoxin must bind to the terminal axon membrane, damage the membrane, and then exert its toxin effects. Initially this may cause release of acetylcholine (Ach), with some muscle twitching, rarely noticed clinically, before destroying vesicles and blocking further Ach release. This process takes from 60 to 80 minutes. Following the process, the neuromuscular block becomes detectable, and quickly becomes complete paralysis. This is associated with a reduction in cholinergic synaptic vesicle number, fusion of vesicles, and damage of intracellular organelles such as mitochondria. There is an increase in the level of free calcium in the nerve terminal, so the neurotransmitter Ach appears to be progressively removed or made unavailable for release, which causes paralysis.

The postsynaptic neurotoxins cause blockade of the acetylcholine receptor on the muscle end-plate at the neuromuscular junction by binding to, or adjacent to the acetylcholine receptor protein on the muscle end plate, effectively blocking the signal arrival at the muscle. They can begin acting immediately after the reach the neuromuscular junction, so they cause paralysis before the presynaptic neurotoxins do. As this action is extracellular, these toxins are more readily reached by antivenom.

Neurotoxins also neutralize the enzyme Acetylcholinesterase, which brings the nervous system to a halt, causing paralysis. Diisopropylphosphorofluoridate is one reagent which has this deactivating property, and is present in the venom of the inland taipan.

Denrotoxins are another class of neurotoxin, which acts on the neuromuscular junction. They are presynaptic, but are different from those discussed earlier. They block some potassium channels on the terminal axon membrane, which causes an over-release of Ach, resulting in initial stimulation, then blockade, causing flaccid paralysis.

Procoagulants

Procoagulants have been isolated from O. scutellatus venom and O. microlepidotus venom. They are proteins, with a MW of about 200,000 D, and achieve their action in a manner analogous to factor Xa, causing conversion of prothrombin, through intermediates, to thrombin. However, they are direct prothrombin converters, working largely independent of cofactors in the absence of factor V, calcium and phospholipid. The thrombin product then converts fibrinogen to fibrin clots in vitro. [Walker et al, 1980, Speijer et al, 1986]

In human envenomation there is widespread consumption of fibrinogen resulting in defibrination and hypocoagulable blood. Any damage to blood vessels then causes increased bleeding, although spontaneous bleeding is not often seen. Usually platelets are not consumed, but factors V, VIII, Protein C and plasminogen all show acute reductions in human envenomation. While major clots are not seen in man, some fibrin cross linkage and stabilisation does occur in vivo, as XDP levels rise sharply in human envenomation. [White 1983c; White 1987c; White unpublished data]

The procoagulation toxins which activate the prothombin processes remain unknown. It is thought that these unknown components, which promote the formation of thrombin from prothrombin, without even the need of the cofactors calcium, factor V or phospholipids. As these cofactors are replaced by the unknown component, the production of thrombin is accelerated. As the clotting of blood requires the formation of fibrin, which is made from thrombin, to occur, the acceleration of thrombin production in turn accelerates fibrin clotting; the remainder fibrinogen molecule, from the splitting action of thrombin, polymerises to form insoluble fibrin; the structure of the clot. Added strength is given to these fibrin strands through covalent bonds between adjacent fibrin monomers.

The effects that are induced by due to the action of procoagulative venom on humans include vomiting or the expectoration of blood.

In the inland taipan, if antivenom is not given to the victim, the coagulopathy will usually be prolonged. Major haemorrhage associated with snakebite coagulopathy is not very common, nor is it rare, with intracranial bleeding a concern.

Myotoxins

Myotoxins interact with calcium-activated Ca 2+ -ATPase (CaATPase), a membrane protein found in the muscle sarcoplasmic reticulum, causing vacolation and eventual destruction of skeletal muscle. CaATPase is responsible for maintaining calcium balance within the muscle cell.

The Calcium Channels are opened by an electrical nerve signal. The Ca ions enter the Cytoplasm, releasing neurotransmitters. However, when myotoxins are present, they interfere with the opening of the Ca channel, reducing the amount of neurotransmitters released, slowing down the nervous system.

In the muscle cell, Calcium is constantly being pumped out of the Ca-pump. However, myotoxins can interfere with this, stopping the regeneration of Ca inside the cell, thus stopping the release of Ca. With the absence of Ca in the cell, chemical messages are unable to cross the synapse (because ordinarily, the Ca carries the message across). This leads to weakness and paralysis of the prey.

Another way that the myotoxins from the venom of the inland taipan cause paralysis are through the break up of the phospholipid compounds of the surface membranes of muscle cells. It is the enzyme Phospholipase A2 which destroys the muscle tissue. This enzyme exhibits two separate actions: a non-lethal esterase activity and a toxic neurological activity. The phospholipase in the venom of the inland taipan reacts in the form of a hydrolysis reaction.

In this way, the myotoxic effects of paralysis and weakness are caused.

Medical Uses

Venom is produced by a pair of large venom glands, situated on either side of the head. The snake delivers its venom by injecting it with fans; teeth with a canal through the centre, through which venom flows.

Some snakes spit their venom, although this will not be discussed as the inland taipan is not capable of this.

Antivenom

Antivenom is a serum that is commercially produced to neutralize the effects of envenomation by venomous snakes. The fresh snake venom used to produce antivenom is obtained either by manually milking a sinkae or by electrical stimulation. Venom is extracted from captive snakes every twenty or thirty days. In manual milking, the snake is held behind its head and induced to bite a thin rubber diaphragm covering a collecting vessel while the handler applies pressure to the snake's venom glands. The pressure is maintained until no more venom is discharged. In electrical stimulation, electrodes are touched to the opposite sides of the snake's head, causing the muscles around the venom gland to contract, expelling venom into a collection containe . The venom is freeze-dried (the preferred method), or dried with the help of a drying agent or a vacuum. [R.Zug & Carl H. Ernst & Harrison's Principles Of Internal Medicine]

Venom as a Medicine

Snake venom has great potential use as a medicine, because of all the compounds it contains, and their specific actions. In Asia, South America, and Europe, components of snake venom are used to treat blood disorders. Snake venom as a whole is not used, but the individual compounds are used.

Two analgesics derive from cobra venom: Cobroxin is used like morphine to block nerve transmission, and Nyloxin reduces severe arthritis pain. Arvin, an extract of the Malayan pitviper (Calloselasma), is an effective anticoagulant (it inhibits the formation of blood cloths).

Venom compounds are also used in research in such fields as Physiology, biochemistry, and immunology. By retarding or accelerating a biochemical or cellular process, venom components allow researchers to examine the process and to develop drugs to counter malfunctions.

Diseases for which snake venoms have been used in research include nerve diseases, such as epilepsy, multiple sclerosis, myasthenia gravis (Lou Gehrig's disease), Parkinson's disease, and poliomyelitis; musculoskeletal disease, including arthritis and rheumatism; cardiovascular disease , such as hypotension, hypertension, angina, and cardiac arrhythmias, and visual disorders, including neuritis, conjunctivitis, and cataracts. [R. Zug & Carl H. Ernst & Harrison]

The procoagulants in the venom of the inland taipan are used to activate prothrombin to alpha thrombin. The anticoagulants are used to prevent interference of immunoglobins which interfere with phospholipid dependent in vitro coagulation tests.

Considering that the components of snake venom are still largely unknown, there is great possibility for more medical uses of these compounds.

Conclusion

Snake venom consists of many compounds, although the main constituents are proteins and enzymes. These poisons cause muscle paralysis, internal bleeding, and degeneration of muscle tissues.

Because we do not yet have a full understanding of the biochemistry of snake venom, the medical uses of its compounds go largely untapped. However, this will soon change, as the research into snake venom is expanding, especially in Australia.

Bibliography

CD-Roms:

*Encyclopaedia Britannica

*Encarta

*Webster's

*World Book

Books:

*Chamber's Biology Dictionary [1990, Peter. M.B. Walker]

*Biochemistry [1978, A. L. Lehninger]

*McGraw-Hill Encyclopaedia of Science and Technology [1997, The Lakeside Press]

*Venoms & Victims [1988, J. Pearn & J. Covacevich, Queensland Museum and Ampion Press]

*Encyclopaedia of Life Sciences [1996, Marshall Cavendish Corp]

Websites:

*users.esc.net.au/~whitters/

*www.wch.sa.gov.au/paedm/clintox/ cslavh_antivenom_taipan.html

*www.aqua.org/animals/species/venom/venfaq.html

*www.reptileallsorts.com/bites-venom.htm

*www.pharmacology.unimelb.edu.au/ pharmwww/avruweb/snakebi.htm

*www.kingsnake.com/toxinology/snakes/ Oxyuranus/Oxyuranus.html

*www.inchem.org/documents/pims/animal/taipan.htm

*www.worthington-biochem.com/manual/P/PLA.html

*www.kordia.nl/pentapharm/snavenenz/snavenenz.html

*coloherp.org/cb-news/cbn-0103/Venom.html

*www.gov.sg/moh/mohiss/poison/covnomco.html

*www.ayurtoxicology.com/sv.html

*www.neuroguide.com/ache.html

*srv2.lycoming.edu/~newman/courses/ bio43799/acetylcholinesterase

*www.weizmann.ac.il/~jsgrp/AChE_ribbon.html

*itech.pjc.cc.fl.us/jkaplan/zootech/Course%20Materials/herplec24.htm

*mysite.mweb.co.za/residents/net12980/toxins.html