Age-dependent variation in the venom of Australian common tiger snake (Notechis scutatus)

Report and Abstracts

539

The edema inducing activity of Ophiophagus hannah (king cobra) venom t,-amino acid oxidase. N.-H. Tax and S.-K. G~ov (Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia). ~-A~rto acid oxidase (EC 1 .4 .3 .2) occurs widely in snake venoms . Snake venom t.-amino acid oxidase is moderately lethal to mice . The toxic action of L-amino acid oxidase has been the subject of much discussion but the mode of action is still unclear. In this report, we examined the edema-inducing activity of two L-amino acid oxidases (OH-LAAO-1 and OH-LAAO-2) from Ophiophagus bannah (king cobra) venom. Using mouse paw assay, both enzymes were found to be strong edema inducers . The minimum edema doses of OH-LAAO-1 and OH-LAAO-2 were determined to be 6.8 pg (5 .4-9.1 pg) and 6.l pg (4.8-7 .9 pg), respectively. At a 5 pg dose, OH-LAAO-1 produced edema that peaked 1 h after the injation and the level sustained for up to 6 hr, while at 12 .5 N g dose, the edema sustained for more than 24 hr. Unlike many other venom edema inducers (e .g . processes and phospholipase AZ enzymes) the edema inducing activity of O. hannak t.-amino acid oxidase was not inhibited by diphenhydramine nor dexamethasone. Administration of glutathione to the mouse, however, did inhibit the edema inducing activity of the enzyme, indicating that the edema inducing activity of ~-amino acid oxidase involves hydrogen peroxide which is formed by the oxidative deamination of the enzyme . The amino acid sequences of two Malayan cobra venom neurotoxins. M. C. M. CtiiUNa,' A. ARrtuaAStZ and N. H. Terr' (' Bioprooessing Technology Unit, National University of Singapore, Singapore; and z Department of Biochemistry, University of Malaya, Faculty of Medicine, Kuala Lumpur, Malaysia). T~ ~rwo major neurotoxins, sputa-toxin l (SN1) and sputa-toxin 2 (SN2) of Malayan cobra (Naja raja sputatrix) venom were isolated by Sephadex G-75 gel filtration chromatography followed by SP-Sephadex C-25 cation exchange chromatography and reverse-phase high performance liquid chromatography . Hoth SN1 and SN2 are short chain postsynaptic neurotoxins. The purified neurotoxins were subjected to microsequencing using peptides derived from glu-C endoproteinase digestion . The sequence for SN2 is: LrE-C-H-D-Q-Q-S-S-Q-T-P-T-T-T-G-CS-G-G-)rT-N-C-Y-K-K-R-W-R-D-H-R-G-Y-R-T-E-R-G-C-G-C-P-S-V, and is highly homologous to many other cobra venom short-chain neurotoxins, in particular the neurotoxin of Philippines cobra (Naja naja philippinensis) venom. The sequence for SNl is also highly homologous to SN2, as well as cobrotoxin, the neurotoxin isolated from Taiwan cobra (Naja raja afro) venom. Sequence analysis of the major hemorrhagin from the venom ojMalayan pit viper (Calloselasma rhodostoma) . M. C. M. C~tuxo,' G. Poxxtrntnw~ and N. H. T~niz (' Bioprocesaing Technology Unit, National University of Singapore, Singapore; and ~Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia). Rxooosroxtrr is the major hemorrhagin isolated from Malayan pit viper (Calloselasma rhodostoma) venom. It has 203 amino acid residues with 8 half-cystine and is a glycoprotein . The amino acid sequence of rhodostoxin was determined by automated Edman microsequencing of peptides derived from digest with endoproteinase Lys C. The N-terminal sequence was determined to be : N-H-E-I-K-R-H-V-D-I-V-V-V-V-D-S-R-F-C-T-K, and the sequence is homologous to that of several well-characterized aotalid venom hemonhagins such as hemorrhagic toxin e from Crotalus atrox venom. Recently, some authors predicted the existence of a hemorrhagic protein in the venom of Malayan pit viper, and that the putative hemorrhagic protein shares a common precursor with rhodostomin, a platelet aggregation inhibitor from the same venom. A comparison of our amino acid sequence with the translated sequence of the putative hemorrhagic protein shows nearly l00% homology. Rhodostoxin has at least one asparagine-linked oligosaccharide chain at position 181, which contains mannose and N-acetyl-o-glucosamine type of sugars . Age-dependent variation in the venom of Australian common tiger snake (Notschis scutatus). N.-H. Tax,' G. PoxNUUUrw' and P. J. M~alscx~x2 (' Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia ; and zVenom Supplies, P.O. Box 547, Tanunda, South Australia). Mwxv authors have reported variations in venom biological properties due to differences in the age of snake. The age~ependent variation in snake venom composition is presumably due to developmental processes with increasing age of the snakes. In this study, we compared the biological properties of venoma of the adult and juvenile Australian elapid Notschis scutatus (common tiger snake) from Lake Alexandrina, South Australia. The PAGE and SDS-PAGE patterns indicated some minor differences in the protein wmposition of thejuvenile and adult venoms . The adult and juvenile venoms, however, exhibited similar protease, phosphodiesterase, alkaline phosphomoncesterase, hyaluronidase, phospholipase A, acetylcholinesterase, 5'-nucleotidase and t-amino acid

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Report and Abstracts

oxidase activities and similar content of prothrombin activator. The i.v . co,ss of the juvenile and adult venoms were determined to be 0.060 pg/g (0 .042-0 .084 ug/g) and 0.030 pgjg (0 .021-0 .042legJg), respectively . In conclusion, our results show that for the common tiger snake, venom of the juvenile snake exhibited biological properties generally similar to that of the adult snake, although electrophoretic studies did indicate some minor differences in the venom protein composition . The similarity in biochemical composition of the adult andjuvenile common tiger snake venoms is probably related to the prey types of the snake. This is in contrast to the results of many comparative studies on the biological properties of the juvenile and adult crotalid snake venoms that demonstrated substantial age-dependent variation in the venoms. Studies on theprocoagulant from the venom of Australian brawn snakes (Pseudonaja spp.) . V. WILLIAMS,' J. Warren and P. MIR'rSCHIN 3 ('Haematology Department, and ZState Toxinology Services, Adelaide Children's Hospital, North Adelaide SA 5006, Australia; and ~Venom Supplies, Tanunda SA, Australia) . BxowN snakes of the genus Pseudonaja are the leading cause of snakebite and snakebite deaths in Australia. A significant feature of systemic envenoming by these snakes, is a defibrination type coagulopathy . Associated cerebral haemorrhage, though uncommon, is a leading cause of snakebite death in Australia. We have an ongoing research program into the procoagulants and related effects of these venoms. In the portion of these studies reported here, we compared the isolated procoaglutants from four clinically significant species, P. a,~nis, P. injramacula, P . nuchalis, P. textilis . The procoagulants were isolated from crude venom by anion exchange and gel filtration chromatography . The elution profile of the procoagulants were virtually identical and a standard mol. wt marker indicated the procoagulants had a mol. wt around 80,000 . The specific activity of the procoagulants was determined ; P. textilis, P. inframacula showed twice the activity of P. affinis, P. nuchalis. Ouchterlony immunodiffusion showed precipitin lines for all four procoagulants against CSL Brown Snake Antivenom. PAGE showed a single diffuse band for each of the venom procoagulants, with only sight inter-species variability in mol. wt . The procoagulant activity was completely inhibited by benzamidine and dansyl-GGACK, with partial inhibition by DFP, leupeptin, and to a lesser extent, aprotinin. These results suggest that the procoagulant is a serine protease, although, surprisingly, PMSF was without inhibitory effect . The procoagulants are glycoproteins, partially inhibited by neuraminidase and NANA, indicating an important role for the carbohydrate in procoagulant activity . The precise function of the carbohydrate portion has not yet been elucidated. Partial cloning of stonustoxin gene using RT-PCR . F. J. GHADFSSY, K. JEYASEELAN, H. E. Kxoo and R. Yu>:x (Department of Biochemistry, National University of Singapore, Kent Ridge Crescent, Singapore 0511). Tt~ L~~r, factor from the venom of the stonefish (Svnanceia horrifia) consists of two protein subunits (a and ß) with mol. wts of 71,000 and 79,000, respectively . Reverse transcription of the venom gland total RNA followed by PCR (RT-PCR) yielded two DNA products of molecular sizes 1 .7 and l .8 kb. When subcloned and partially sequenced, these enabled us to identify a region within the nucleotide sequence which corresponded to the amino acid sequence of the toxin determined separately . Current work is aimed at characterising these PCR products completely . Isolation of plotoxin andplotolysin from the skin secretion of the cat fish Plotosus camas. J. Ortut~x,' K. NGALIe,' B. K. Lows and M. J. NtK J~xxZ (' School of Applied Science, MARA Institute of Technology, Shah Alam, Selangor, Malaysia ; and zDepartment of Biochemistry, Faculty of Medicine, University of Malaya, 59100 Kuala Lumpur, Malaysia). T~ car fish, Plotosus camas or 'ikan sembilang', is found in waters surrounding Malaysia . It is known to be venomous with its sting causing skin necrosis, edema and severe pulsating pain. Several enzymes including phosphomonoesterase, acetylcholinesterase, and protease have been isolated and purified from the skin secretion of the fish . The soluble skin secretion extract of Plotosus camas had an ~w of 7 .01lg/g in mice and exhibited haemolytic activity of 71 HU/mg protein. The crude extract was fractionated by Sephadex G-50 chromatography followed by DEAE-Sephacel chromatography to yield partially purified tonic fraction that exhibited haemolytic activity and ~w of 0.02I+gJg. In IEF, the partially purified preparation exhibited two major protein bands with pIs of 5.7 and 5.9, respectively . These two protein bands had identical mol. wt of 33,000 on SDS-PAGE, suggesting that the partially purified preparation contained two proteins with two different physiological activities, namely, neurotozic and haemolytic activities . The two proteins were designated plotozin (the neurotoxic component) and plotolysin (the haemolytic component) respectively . Attempts to separate these two proteins were not yet successful and thus subsequent studies to characterise these activities were carried out using the partially purified preparation . The plotoxin component caused a wmplete irreversible blockade of muscle