A lethal myotoxin isolated from the venom of the Australian king brown snake (Pseudechis australis)

Toxlron, Vol . 17, pp . 549-533 . © Peryamon Press Ltd. 1979. Printed in Great Britain.

0041-0101/7911101-03498020010

A LETHAL MYOTOXIN ISOLATED FROM THE VENOM OF THE AUSTRALIAN KING BROWN SNAKE (PSEUDECHIS A USTRALIS)

T. M . LEONARDt,* M. E. H. HOWDEN * arid I. SPENCEf "School of Chemistry, Macquarie University, North Ryde, New South Wales, Australia 2113 and tpepartment of Pharmacology, Roche Research Tnstitute of Marine Pharmacology, Dee Why, New South Wales, Australia 2099 (Accepted jor publication 13 February 1979)

T. M. LEONARDI, M. E. H. HOWDEN and I. SPExce . A lethal myotoxin isolated from the venom of the Australian king brown snake (Pseudechis australis) . Tozicon 17, 549-555, 1979.-A lethal myotoxin, named mulgotoxin a, was isolated from the venom of the Australian king brown snake (Pseudechis australis) by chromatography on Biorex 70 and SP-Sephadex ion exchange resins . Mulgotoxin a was obtained from one of four lethal fractions which were present in the venom. Mulgotoxin a is a basic toxin consisting of a single polypeptide chain of 122 amino acid residues cross-linked by seven disulphide bridges. Its N- and C-terminal residues are aspartic acid and leucine respectively. Mulgotoxin a causes myoglobinuria in mice and has an approximate LDso of 200 Rg/kg body weight (mice) . Its activity appears to be specific for skeletal muscle, causing massive cell damage both in viva and in vitro. INTRODUCTION

MEMBERS of the Australian black snake family are found in many parts of Australia and in

Papua-New Guinea. The common black snake (Pseudechis porphyriacus) is distributed in the Eastern coastal areas, while the king brown or mulga snake (P. australis Gray), the largest member of the family, occupies the drier, inland regions of the Australian mainland (STACKHOUSE, 1970). P. australis is one of the largest snakes in Australia and is capable of injecting considerable quantities of venom, the largest amount obtained in a single `milking' being 600 mg (WORRELL, 1963) . Although few fatal encounters with humans have been reported for P. australis, its similarity to other dangerous species, such as the common brown snake (Pseudonaja t. textilis), or the taipan (Oxyuranus s. scutellatus), may be a cause of misidentification (GARNET, 1968). ROWLANDS et al. (1969) have described in remarkable detail the clinical and pathological features of a human fatality following envenomation by P, australis. Studies on the venom and its constituents havebeen restricted to its ef%cts on laboratory animals (KELLAWAY and THOMPSON, 1930 ; KELLAWAY et al., 1932) and to the preliminary fractionation of its haemolytic components on ion exchange resin (DOERY and PEARSON, 1961). KELLAWAY et aI. (1932) concluded that the venom of P. australis, unlike the venoms of other Australian elapid snakes, did not have a curarimimetic action . A number oftoxins from Australian snake venoms have been found to possess both enzymic and neurotoxic activity (GARNET, 1970 ; KARLSSON et al., 1972 ; FOHLMAN et al., 1976). Since no clear mode of action has been established in earlier research on the venom of P. australis, we decided to investigate the nature of its toxins . Venom

MATERIALS AND METHODS

Desiccated P . australis venom was obtained from the Australian Reptile Park, Gosford North, NSW 2250, Australia. 549

550

T. M. LEONARDT, M. E. H. HOWDEN and I. SPENCE

lon exchange chromatography oJcrude venom

Hiorex 70 200-400 mesh resin (Biorad Laboratories) was converted to the ammonium form as described by Kaxtssorr and E~tceti (1972) . The pretreated resin was suspended in 0~2 M ammonium acetate solution and packed in a 1~6 x 37 cm column . Two void volumes of 0~2 M buffer were passed through the column. Samples (185 mg) were applied to the column in 10 ml of 0~2 M buffer, and the column was eluted with 600 ml of a linear gradient of 0~2-1~4 M ammonium acetate solution at pH 6~8 at a flow rate of 20 ml/hr. Five ml/fractions were collected. SP-Sephadez C25 chromatography

SP-Sephadex gel (Pharmacia) was equilibrated for several days in 0~5 M ammonium acetate solution at pH 7~5. The gel was then converted to the ammonium form as described above for Biorex 70 resin. It was then packed under a wnstant Bow of 20 ml/hr in a 1~6 x 18 cm column . Samples were applied in S ml of 0~3 M ammonium acetate buffer at pH 7~5. The column was eluted with a 05-1 M gradient of ammonium acetate solution at pH 7~5 using a Gradipore gradient former (Gradipore Australia) . The column efliuent was monitored continuously at 280 nm . Ionic strength was determined with a platinum glass electrode conductivity bridge . Po/yacrylamide gel isoelectric focusing

Analytical gel electrofocusing was carried out by a procedure similar to that of WRIGLeY (1971) . Gels were prepared from a mixture of demineralised water, acrylamide (electrophoresis grade, final wncentration 7~5 ~ w/v) and Ampholines (LKB, final concentration 1 ~ v/v). Polymerisation was initiated by 10 mg/ml of analytical grade potassium persulphate . Acetic acid (0~1 M) and 0~1 M sodium hydroxide were the electrode solutions for the anode and cathode respectively. Samples (50-200 pg) were applied in 10 sucrose solution and then placed under a 5 ~ sucrose layer containing 1 ~ Ampholines . Gels were run at an initial current of 2 mA per gel to a maximum of 400 V for 2-4 hr. On completion the gels were stained with 004% (w/v) Coomassie blue G250 in 3~5 ~ perchloric acid (RetsNEx et al ., 197 . The gels were placed in 10 ~ trichloroacetic acid after staining to remove background colour, and finally stored in 7~ acetic acid . Protein co~rtent of crude venom

Protein determination was performed according to the method of Lowßv et al. (1951). The intensity of the resulting blue colour was measured spectrophotometrically at 750 nm . A standard curve was prepared using BSA Fraction 5 (Calbiochem) . Lethality assay

Approximate i.n,o's were determined by i.p. injection of adult white mice weighing 20 ~ 1 g. Three to five mice were injected at each dose level. The number of deaths was recorded over a 24 hr period . Freezedried samples were dissolved in 0~9 % saline . Myoglobinuria assay

The assay was similar to that used by Foxt .Mnx and EAKER (1977) . Urine from mice injected with the toxin was obtained in two ways . In the first method the animals were placed on white filter paper which covered the floor of their cages and the red-stained areas were eluted with demineralised water. In the second procedure the animals were killed approximately 20 hr after injection, their bladders were exposed and the contents removed with a syringe. The extracted urine was applied to a Sephadex G-50 column and eluted with 0~2 M ammonium acetate buffer . Only one band was observed. The absorption spectrum of this eluate between 400 and 600 nm (Hnx~xtn et al., 1966 ; KApEN, 1973) showed that the urine extract contained myoglobin . Neurophysiology

The mode of action of the crude venom and of the toxin was examined in mice using electromyographic techniques . Male Fullensdorf mice (50-55 g) were anaesthetized with sodium pentobarbitone (85 mg/kg) Electromyographic readings were made from the interdigital muscle of the hind limb in response to stimulation of the sciatic nerve. The electrocardiogram was monitored by two subcutaneous electrodes. Molecular weight determination

The molecular weight of the toxin was obtained by chromatography on Sephadex G-100 according to the method of Axnxews (1965). Sephadex gel was equilibrated and packed in a 1~6 x 37 cm column with 0~2 M ammonium acetate buffer. Samples were applied in 1-2 ml of the buffer. Standards used were alkaline phosphatase (Sigma Chemical Co.), BSA Fraction 5 (Calbiochem), pepsin (Sigma), ribonuclease A (Sigma) and cytochrome c (Mann Research Laboratories) . The logs of the molecular weights of the markers were plotted against the reciprocal of their elution volumes, and a linear regression analysis was applied to the data points." Amino acid analysis

Duplicate lyophilized peptide samples were hydrolyzed for 22, 48 and 72 hr at I10`C in 6 M hydro-

Lethal Myotoxin in Snake Venom

55 1

chloric acid containing 0~5 ~ phenol to allow quantitative recovery of tyrosine . These were then incubated at 110°C and analyzed on a Beckman 120 B amino acid analyser . Proline was determined from the 440 nm trace of the analyser effluent . Tryptophan was measured colorimetrically after reaction of the peptide with p-dimethylaminobenzaldehyde by the procedure of SFffs and CxAMHERS (1949). Absorbance readings were taken at 590 nm . The values for serine, threonine and cysteine were obtained by extrapolation to zero hydrolysis time . Disulphide bridge analysis

Reduction of the disulphide bridges in the toxin was accomplished as described by SCHERAGA (1962) in 8 M deionized urea solution at pH 8~6 containing ethylenediamine tetra-acetic acid and the appropriate quantity of dithiothreitol solution (40 mg/ml) . The reaction mixture was left for 4 hr at room temperature then chromatographed on a Sephadex G-50 column in order to remove any unreacted reagents . The reduced toxin was lyophilized and allowed to react with 5,5'-dithiobis-(2-nitrobenzoic acid) according to the method of ELLMAN (1959) . The absorbance of the product at 412 nm was measured after 30 min. An extinction cce~ICient of 13,600 was used for the carboxythiophenylate anion (HA9EEH, 1972). An unreduced sample, as a control, was subjected to the same processes. N- and C-Terminal analysis

N-Terminal analysis of the toxin was carried out by the method of GRAY (1972) involving polyamide sheets as described by WooD and WANG (1967). Ten nmole of toxin reacted with 10 pl of dansyl chloride at pH 100 for 1 hr at 37°C . The solvent was then removed in a vacuum line and the residue was hydrolyzed in 6 M hydrochloric acid for 17 hr at 100°C. The resulting residue was dissolved in ] 0 pl of ethyl acetate and identified by thin-layer chromatography (TLC). The TLC sheets were developed in 1 ~5 ~ formic acid, then in a perpendicular direction with benzene acetic acid 9 :1, and finally again in the latter direction with ethyl acetate-methanol-acetic acid 20 :1 :1 . The spots were examined under ultraviolet light. The N-terminal residue was identified by comparison with standard dansyl amino acids (Sigma). C-Terminal analysis was performed by the hydrazinolysis method of AXweoRI (NARII'A, 1970). Hydrazinolysis was carried out in a sealed Pyrex tube at 100°C for 14 hr. The free amino acid was separated from the amino acid hydrazides by absorbing the latter on Amberlite IR-120 resin (BRAUN and SCHROEDER, 1962). The Rf value of the free amino acid was determined after TLC on Kieselgel 60 F plates developed in 1-butanol-acetic acid-water 4 :1 :1 . RESULTS

The protein content of the crude venom was 68 ~. The approximate hDS o of crude venom was 055 mg/kg. Isolation of mulgotoxln a Fractionation of crude P. australis venom on a column of Biorex 70 ion-exchange resin produced eight major fractions (Fig . 1) . Only fractions 1 and 5 were lethal . Fraction 1 had an approximate Lnso greater than 1 mg/kg and was shown by analytical gel electrofocusing to be a complex mixture. Fraction 5, on the other hand, had an approximate I-US o of 025 mg/kg and electrofocused as four distinct bands. Fraction 5 was chromatographed on a

EFFLUENT ml

P. sustralis VENOM ON BIOREX 7O ION EXCHANGE RESIN. The column was eluted with a linear gradient of 0~2-1 ~4 M ammonium acetate buf%r at pH 6~8. The conductivity trace (O -- O) monitored the ammonium acetate (n concentration. The solid line indicates absorption at 280 nm . Shaded areas correspond to lethal fractions. FIG. I. FRACITONATION OF CRUDE

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T. M. LEONARDI, M. E. H. HOWDEN and 1. SPENCE

EFFLUENT ml FIG. 2. ELUTION PROFILE OF FRACTION S (FIG . 1) CHROMATOGRAPHED ON SULPHOPROPYLSEPHADEX C-2S ION EXCHANGE RESIN.

The column was eluted with a gradient of 05-1 M ammonium acetate buffer at pH 7~5. The conductivity trace (O -- O) monitored the ammonium acetate (I) concentration . The solid line indicates absorption at 280 nm . Lethal areas are indicated.

column of SP-Sephadex and gave the elution profile shown in Fig . 2. Fractions SP-1 and SP-2 from the latter chromatogram were lethal, and both produced myoglobinuria in mice. Fraction SP-1 made up 70 ~ by weight of fraction 5 and was homogeneous in Sephadex G-50 chromatography . Polyacrylamide gel electrofocusing of fraction SP-l showed a single band at an isoelectric point of 9~8. Its approximate L~so was 020 mg/kg. Fraction SP-1 represented 135 ~ of the crude venom protein . Fraction SP-2 showed one major and a minor band on gel electrofocusing . It constituted 5~5 ~ of the whole venom protein and had an approximate LDSo of 035 mg/kg . We have called fraction SP-1 mulgotoxin a after one of the popular names, mulga snake, of the species being studied (RowLnNns et a1., 1969). Partial characterization of mulgotoxin a Amino acid analysis and molecular height . Table 1 shows the amino acid composition TABLE 1 . AMINO ACID COMPOSITION OF MULGOTOXIN

Amino acid Lys His Arg Asx Thr Ser* Glx Pro Gly Ala Cys* Val Met Ile Leu Tyrt Phe Trp$

Number of residues 16 1 3 13 6 6 6 11 10 14 4 1 3 S 8 4

Total number of residues 122 Calculated formula weight 13,484 *From zero time extrapolation. tTaken from 22 hr hydrolysis run. $Obtained by colorimetric analysis .

a

Lethal Myotoxin in Snake Venom

553

found for mulgotoxin a. The value for tyrosine was obtained from the 22 hr hydrolysis . A large ammonia peak was observed during the elution of the amino acid hydrolysate . This was expected since asparagine and glutamine residues would be converted to the corresponding acids. Since the isoelectric point of the toxin was 9~8 it was therefore assumed that the majority of the acidic residues were in the amide form. The analysis showed that the native toxin possesses 122 amino acid residues, corresponding to a formula weight of 13,484. The molecular weight of mulgotoxin a was calculated from the results of gel permeation chromatography on Sephadex G-100 to be 13,710, which is in reasonable agreement with the figure derived from amino acid analysis . N- ai :d C-Terminal analysis . The N-terminal amino acid residue of mulgotoxin a was identified as aspartic acid. However, due to the conditions used in the hydrolysis reaction, there is a possibility that this residue may be present in the amide form. The C-terminal amino acid of the toxin is leucine . No other N- or C-terminal amino acids were detected, which is an indication of the purity of mulgotoxin a and that it consists of a single polypeptide chain . Disulphide bridge analysis . The results of reaction of the unreduced toxin with 5,5'dithiobis (2-nitrobenzoic acid) confirmed the absence offree sulphydryl groups . The reduced toxin analysed for 14 free sulphydryl groups . This value agreed with that obtained from amino acid analysis . The number of disulphide links present in native mulgotoxin a was therefore seven. Neurophysiology and lethality. Injections (i.p.) of crude venom into mice at doses between 065 and 5 mg/kg, and ofmulgotoxin a at doses between 025 and 1 mg/kg, caused death after periods ranging from 1 to 20 hr depending on the dose. Symptoms produced included paralysis at higher doses, respiratory distress and minor spasms at death, as well as myoglobinuria . If death did not occur within 36 hr the animal usually recovered . When neuromuscular transmission was examined in isolated diaphragm preparations, the crude venom at 100 ~g/ml caused no change in either spontaneous or evoked release of acetylcholine preceding depolarisation and destruction ofthe muscle fibres . Mulgotoxin a at 1 mg/kg depressed neuromuscular transmission in anaesthetized mice. The compound muscle action potential in the interdigital muscles of the hind foot in response to stimulation of the sciatic nerve was depressed 97 ~ in 1 hr, at which time the animals died, apparently from respiratory failure . The electrocardiogram showed no abnormalities until after the cessation of respiration. Examination, under a dissecting microscope, of skeletal muscles from these animals indicated massive destruction of fibres both in the diaphragm and in muscles from the leg (extensor digitorum longus and saleus). No macroscopic damage either to cardiac muscle or to smooth muscles close to the (i .p.) site of injection was seen. Other toxins

At least three other lethal components were found in the venom of P. australis. Together they constituted less than IO ~ by weight of the crude venom. Two of these were eluted in fraction 1 from the BioRex 70 chromatogram, and one lethal component was eluted after mulgotoxin a in the SP-Sephadex chromatogram. When fraction 1 was chromatographed on SP-Sephadex, two lethal fractions were separated . The fraction that was eluted first was heterogeneous in gel electrofocusing, while the second fraction showed one major band with only minor contamination . Both these lethal fractions displayed high haemolytic activity when tested on human erythrocytes (HOWDEN and LEONARDI, unpublished) .

554

T. M. LEONARDI, M. E. H. HOWDEN and I. SPENCE DISCUSSION

A number of phospholipases have been isolated from snake venoms in recent years . The majority of these enzymes are much less lethal than the neurotoxins . However several potent neurotoxins, e.g. notexin (KARLSSON et a1., 1972), taipoxin (FOHLMAN et al., 1976), crotoxin (BREITHAUPT et al., 1975) and ß-bungarotoxin (WERNICKES et al., 1975), have also shown high phospholipase activity. Some of these toxins, particularly notexin and taipoxin (HARxtS et al., 1977) exhibited myotoxic activity in vivo. FOHLMAN and FAKER (1977) have isolated from the sea snake Enhydrina schistosa a myotoxic phospholipase, myotoxin VI :S, which appears to possess both myotoxic and neurotoxic properties, the myotoxicity overshadowing the neurotoxicity . Although we have not yet determined whether mulgotoxin a displays phospholipase activity, it has a number of properties similar to these enzymatic neurotoxins, viz., molecular weight, amino acid composition and myotoxic activity . The obvious differences lie in the important sites of action and in the relatively lower lethality of mulgotoxin a. Its preference for attack of skeletal muscle membrane is indicated by the resulting leakage of myoglobin from the muscle fibres . Selectivity for skeletal muscle may reside in the substrate specificity, since the lipid contents, particularly 3-sn-phospholipads, in muscle membrane differ from those in erythrocyte membrane (Zwnnl, et al., 1975) . The cause of death from envenomation by most elapines has been shown to be due to a curarimimetic block at the neuromuscular junction (MELDRUM, 1965) . Most elapid venoms contain neurotoxins, either pre-or post-synaptically acting, as their main lethal components. In the case of P. australis we have shown that the venom contains a major myotoxin . No post-synaptic blocking action was observed for the venom as a whole prior to destruction of the muscle fibres . Acknowledgements-The authors thank J. T. BAKER, L. B. JA~s, P. B. H. O'CoxwELr., R. J. QutHx, K. M. TAYLOR and E. WORRELL for special assistance, and are grateful to the School of Chemistry, Macquarie University, for support of one of us (T.M .L.) . REFERENCES

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