RESEARCH
Sea snake venom structure probed Biochemists isolate, characterize toxic components, find tryptophan residue to be key to toxicity Dr. Tu holds sea snake used in study of venom structure and properties C&EN:
Sea snakes are among the most feared creatures that fishermen in many parts of the world encounter. More deadly than the rattle snake, and more numerous, it's not uncommon in tropical and subtropical regions bordering the Indian and Pacific Oceans for several hundred of the reptiles to spill on deck with a fish catch each time a net is hauled on board. Yet a single bite of a sea snake's fangs can bring death within hours through action of its toxins on the neuromuscular junctions. Recent analyses of sea snake venom toxins reveal that they are proteins whose chemical structures bear a remarkably close resemblance among different species from different areas. Significant, too, is the finding of Dr. Anthony T. Tu, biochemistry professor at Colorado State University in Fort Collins, that simple chemical modification of the single tryptophan residue in a purified sea snake toxin detoxifies the toxin without appearing to affect its antigenicity. An important potential outcome of this work—which was carried out under contract to the Office of Naval Research—is that modified toxin may make possible more rapid production of antivenom serum of high potency, Dr. Tu told the annual meeting of the American Association for the Advancement of Science in Chicago last week. Hardest part. All told, the Colorado biochemist has collected more than 9000 sea snakes of various species from the Philippines, Thailand, and Malaysia. The hardest part of sea snake research, he says, is gathering enough sample to work with: one sea snake yields at most only a few milligrams of venom. However, he now has enough venom on hand "to provide work for several Ph.D/s." For his study of the lethal toxin of Laticauda semifasciata from the Phil-
ippines, Dr. Tu extracts venom from the glands of some 600 snakes. Using water to dissolve the venom from the pulverized venom glands, he and his coworkers, Dr. Bor-shyue Hong and Paul M. Toom, remove the insoluble tissue debris and lyophilize. Column chromatography of the sample dissolved in buffer solution yields five fractions, only one of which is toxic when injected intravenously into mice. Twice as lethal as the impure material, it has an LD 5 0 of 0.13 microgram per gram animal weight, versus 0.28 for the crude venom. This fraction comprises two active molecules, which Dr. Tu refers to as toxin a and toxin b. They differ only slightly from each other in their amino acid content. Both have a molecular weight of about 6800. Zonal electrophoresis, isoelectric focusing, and crystallization of the two toxin functions, as well as evidence from sedimentation velocity and sedimentation equilibrium studies, point to the individual purity of toxin a and b. Amino acid analyses show the two toxins to be very similar. Toxin a consists of 62 amino acid residues, and toxin b of 61. In both, arginine is the N-terminal amino acid, with either aspartic acid or asparagine the Cterminal one. Both have internal disulfide bridges at four locations. Stable. Presence of the four S-S bonds points to the molecular configuration's being tightly held in place, Dr. Tu surmises. Bolstering this contention is the unusually high degree of physical stability of the toxin molecules. For example, solutions of the toxins can withstand heating at 100° C. for 30 minutes, and exposure to pH extremes of 1 to 11, without undergoing any loss of toxicity. The single tryptophan residue found in L. semifasciata toxins a and b—
Dermot 0'Sullivan
which seemingly is a common feature of sea snake toxins—is vital for the physiological activity of the molecules. Interaction with N-bromosuccinimide (NBS), for instance, completely detoxifies the protein. Furthermore, Dr. Tu and his coworkers observe that a progressive decrease in toxicity accompanies addition of NBS to solutions of the toxins until all available tryptophans are modified. The solution is then essentially nonlethal. Dr. Tu's work corroborates previous findings by Dr. Nobuo Tamiya and his group at Tohoku University, Sendai, Japan, on venom from the sea snake Erahu unagi. Dr. Tamiya found tryptophan to be a key amino acid residue in causing toxicity. He has now made observations on L. semifasciata similar to Dr. Tu's. A similar detoxifying effect accompanies treatment with 2-nitrophenylsulfenyl chloride and 2-hydroxy-5nitrobenzyl bromide, both of which— like NBS-interact with tryptophan's indole group. On the other hand, modification of the arginine residues with 1,2-cyclohexanedione, or of lysine residues with O-methylisourea, brings about no change in toxicity. Dr. Tu plans to study the effect, if any, of modifying some of the other amino acid residues. He also is working on the amino acid sequence. Antigenicity. Although tryptophan residue is essential for toxicity, it may not be essential for antigenicity of the molecule, Dr. Tu says. For example, immunodiffusion of the tryptophan-modified and -unmodified L. semifasciata toxins yields a single fused precipitin band. In vivo experiments will yield further evidence on this point. This suggests to Dr. Tu that tryptophan-modified toxin may prove superior to untreated venom for inducing JAN. 4, 1971 C&EN 25
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the production of antivenin in horses— the usual commercial method of mak ing serum to counteract snakebites. Because of the sea snake venom's ex treme toxicity, antivenin production now is a slow process. Small quan tities of the venom are injected into the horses over a period of about a year to avoid risk of killing the horses. The process could be speeded up, he suggests, by using nontoxic tryptophan-modified venom. Dr. Tu and his associates already have examined the toxins of two other sea snakes, Enhydrina schistosa col lected in the Straits of Malacca off the Malayan coast, and Lapemis hardwickii from the Gulf of Thailand. Amino acid profiles of toxins from these reptiles closely resemble those of toxins a and b of L. semifasciata. NBS modification of the single trypto phan residues also completely neu tralizes toxicity of the venom proteins. Sea snakes abound in the coastal waters of Baja, Calif., Mexico, Central and South America, Southeast Asia, and East Africa. Cold waters at the southern tips of Africa and South America have prevented the snakes from migrating into the Atlantic Ocean, Dr. Tu explains. But he fore sees a grave danger ahead should a sea-level canal be built in Panama, per mitting poisonous snakes to enter the Caribbean and the Atlantic.
Laminar air flow tents keep lab animals healthy When Dr. Gaiy L. Enold pitched his nylon tent in the animal-care facility at Atlas Chemical a couple of years ago, he came in for a fair amount of good-natured ribbing. But "Enold the Tentmaker" jokes notwithstanding, the former University of Pennsylvania veterinarian began housing laboratory animals under his "laminar flow" tents and he now has the healthiest animals around. The key to Dr. Enold's success is a high-volume, low-velocity curtain of filtered air, introduced through the tente perforated ceiling. The effect is much like that of a shower. Air jets constantly "wash" the animals and force pathogen-bearing particles out the bottom of the tent, which is sus pended a few inches from the floor. The result is a nearly germ-free atmos phere. Animal morbidity and mortal ity rates have dropped sharply, signif icantly reducing costs and enhancing the scientific value of experiments. Air to the tent is prefiltered to re move large particulate matter such as animal hair, then passed through a secondary high efficiency particulate air (HEPA) filtration system. The
