Comp. Biochem.Physiol.Vol. 99B, No. 4, pp. 793-798, 1991
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PURIFICATION AND PARTIAL CHARACTERIZATION OF STONUSTOXIN (LETHAL FACTOR) FROM S Y N A N C E J A HORRIDA VENOM C. H. POH,* R. YUEN,*t H. E. KHOO,* M. CHUNG,* M. GWEE~ and P. GOPALAKRISHNAKONE§ *Departments of Biochemistry, :~Pharmacology and §Anatomy, Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore 0511
(Received 30 January 1991) Abstract--1. The lethal factor of the stonefish (Synanceja horrida) venom, designated as the stonustoxin, was purified to homogeneity by a two-step procedure on Sephacryl S-200 High Resolution (HR) gel permeation and DEAE Bio-Gel A anion exchange chromatography. 2. Stonustoxin has a native mol. wt of 148,000 and an isoelectric point of 6.9. 3. SDS-polyacrylamide gel electrophoresis revealed two subunits (designated ~, and fl) with mol. wts of 71,000 and 79,000, respectively. 4. The amino acid composition of both subunits and the N-terminal amino acid sequence of the fl subunit were also determined. 5. Purified stonustoxin had an LDs0 of 0.017 #g/g which is 22-fold more potent than that of the crude venom. 6. The toxin exhibited potent haemolytic activity in vitro and endema-inducingactivity with a minimum edema dose (MED) of 0.15 Pg in mouse paw. The edema effect was not antagonized by diphenhydramine.
INTRODUCTION
(electrophoresis grade) was purchased from Schwarz/Mann Biotech (Cleveland, OH, USA), diphenhydramine (Merck) was a gift from Dr N. H. Tan, Department of Biochemistry, University of Malaya and the chromogenic substrates used for proteinase assays were supplied by Boehringer Mannheim (Far East). All other chemicals were from Sigma Chemical Company (St Louis, MO, USA). Male Swiss mice were supplied by the Animal Centre of the National University of Singapore. All other chemicals were of analytical grade, and were the best grades available.
Stonefish of the genus Synanceja have been responsible for a n u m b e r of deaths and severe poisoning to humans through stings from the spines (Saunders et al., 1962). The crude venom exuded from the venom glands attached to the spines was reported to be myotoxic which resulted in the paralysis of cardiac, involuntary and skeletal muscles (Saunders et aL, 1962; Austin et al., 1961; Russell, 1965). It was also shown to possess hyaluronidase and haemolytic activities (Austin et al., 1965; Duhig and Jones, 1928a; 1928b). Hitherto there has been only one report on the attempted purification of one of the bioactive components, namely the lethal fraction from the crude venom of Synanceja horrida (Deakins and Saunders, 1967). However, no detailed chemical and biological properties of this lethal fraction have been published. In this paper we report on the purification and partial characterization of the lethal factor, designated as stonustoxin (stonefish National University of Singapore) from the venom of Synanceja horrida with the view to fully elucidate its mode of action.
Protein determination Protein was determined by the method of Bradford (I 976) using the protein assay kit from Bio-Rad Laboratories and bovine serum albumin as standard.
Lethality assay The LDs0 values were determined by intravenous injection of the venom fractions into the caudal vein of male Swiss mice (20 + 2 g) and subsequently calculated by the method of Spearman-Kaber (WHO, 1981). Four mice were tested at each of the seven dose levels.
Extraction of venom The dorsal spines of the stonefish were excised from the surrounding tissue. The crude venom was aspirated out from the venom sacs by using a I ml syringe fitted with a 21G needle. The venom was lyophilized immediately after collection and stored at -70°C until use.
MATERIALS AND METHODS Live stoneflsh (Synanceja horrida) were obtained by local fishermen. Sephacryl S-200 HR (high resolution) and SDS-PAGE mol. wt markers were obtained from Pharmacia AB (Uppsala, Sweden); DEAE-Bio-Gel A (100-200 mesh) and protein assay kits were purchased from Bio-Rad Laboratories (Richmond, CA, USA). Acrylamide and tris(hydroxymethyl)-aminoethane (electrophoresis grade) were obtained from Polysciences (Warrington, PA, USA), giycine i'Author to whom correspondence should be addressed.
Purification All operations were carried out at 4°C. One hundred and seventy milligrams of crude venom were reconstituted in 2.5ml of 0.05 M sodium phosphate buffer, pH7.4 and subsequently centrifuged at 3000g for 5 min to remove insoluble material. Two miUilitres of this solution (containing about 35 mg/ml of protein) was then applied to a Sephacryl S-200 HR gel permeation column (1.5 × 84 cm) previously equilibrated with the same buffer. Elution was
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C.H. POH et al.
carried out at a constant flow rate of 12ml/hr and the absorbance of the column eluant was monitored at 280 urn. The lethal fractions (as determined by the lethality assay) from the gel permeation column were pooled and loaded onto a DEAE-Bio-Gel A (100-200 mesh) anion-exchange column (2.0 x 9 era) pre-equilibrated with 0.05 M sodium phosphate buffer, pH 7.4. After thorough washing of the column with start buffer, the bound protein fractions were eluted with a linear buffer gradient (0-0.15 M sodium chloride in start buffer, 600 ml) at a flow rate of 8.0 ml/hr. Three miUilitre fractions were collected.
Polyacrylamide gel electrophoresis (PAGE) Native gel electrophoresis was carried out in a 5% gel according to the method of Davis (1964) while SDS-PAGE (6%) was performed as described by Laemmli (1970). Protein bands were detected either by silver or Coomassie Blue staining.
Molecular weight determination The native mol. wt of stonustoxin was determined by high performance liquid chromatography at pH 7.0 using 10 mM sodium phosphate buffer with 0.2 M sodium sulphate in a gel permeation column (Bio-Sil SEC-400, Bio-Rad Labs, 300 x 7.5 ram) at a flow rate of 1.0 ml/min. Protein was monitored at 280 nm using a Shimadzu SPD-7AV u.v. detector. The column was calibrated with thyroglobulin (669,000 mol. wt), IgG (160,000 tool. wt), ovalbumin (43,000 tool. wt), myoglobin (18,000 tool. wt) and cyanocobalamin (1400 tool. wt) from Bio-Rad Laboratories.
Isoelectric point determination Isoelectric focusing was carried out using a 110 ml column according to the method of Vesterberg and Svensson (1966). One hundred milligrams of crude venom were first loaded onto the column, following which focusing was carried out at 300 V and 4°C. After 72 hr, electrofocusing was stopped, and 2 mi fractions were collected to assay for lethal activity and for pH determination.
Protein blotting Six per cent gels (14 x 16 era, 1.5 mm thick) were used for SDS-PAGE. After electrophoresis, the gel was electroblotted for 1 hr at 90 V based on the method of Matsudaira (1987). Following staining and destaining, the membrane (Problott, Applied Biosystems) was rinsed with glass distilled water (3 x ), and the protein bands were then excised with a clean scalpel and stored in Eppendorf vials at - 20°C.
(various final concentrations) at 37°C for 60 rain. The degree of haemolysis was determined as a percentage of haemoglobin released (measured at 540 nm) by the toxin compared with a 100% control effected by the addition of 10 mg/ml heparin. Edema-inducing activity was measured by injecting 20 #1 stonustoxin (0.15-0.4#g) in 0.05M phosphate buffer, pH 7.4 into the subplantar muscle of the right hind paw of male Swiss mice (20-22 g). The left paw served as control. The edema-inducing activity was expressed as a percentage of swelling in the right hind paw as measured with calipers after different time intervals (Otterness and Moore, 1988). The effect of the antihistamine, diphenhydramine, on edema-inducing activity was tested by injecting the diphenhydramine (50 mg/kg i.p.) 30 min before injection of 0.5 #g stonustoxin. Hyaluronidase activity was measured using hyaluronic acid from human umbilical cord (Ferrante, 1956) and phospholipase A2 according to the method of Tan and Tan (1988). Proteinase activities were assayed as described by Yuen et al. (1987). RESULTS
Toxicity o f the crude venom The crude venom is a colourless fluid with a p H of 6.8. The protein content accounted for 20% o f the dry weight of the lyophilized venom. The crude venom is highly toxic to the mouse. The estimated L D ~ was 0.36/zg/g. A lethal dose (i.v.) of the venom caused convulsive movements, respiratory arrest, urination, hypersalivation and death within 3-30 min of injection.
Purification o f lethal factor Isolation of the lethal factor of S. horrida was achieved in two steps. The first step involved fractionation of the crude venom in a Sephacryl S-200 H R column (Fig. 1). Three major peaks were evident but only fraction 1 exhibited lethal activity and accounted for 24% of the total venom protein loaded. The LDs0 (i.v.) of fraction 1 was found to be 0.035 #g/g. This is 11-fold more toxic than the crude venom. Fraction 1 was then further resolved into four subfractions by D E A E Bio-Gel (Fig. 2). Based on the lethality assay, the lethal factor was located in peak 4. This accounted for 36% of the protein content of
N- Terminal sequencing Amino-terminal sequencing of the electroblotted subunits of stonustoxin was carried out using an Applied Biosystems 471-02 protein/peptide sequenator. Prior to sequencing, the membrane pieces were carefully loaded into the upper cartridge block of the sequenator, followed by a precycied polybrene-conditioned filter before final assembly.
Amino acid analysis The amino acid compositions were determined using on-membrane amino acid analysis protocol (Dupont et al., 1988). The membrane bands were first hydrolyzed by a manual vapour hydrolysis method using a mixture of HCI/TFA (2: 1, v/v) at 165°C for 45 rain. The membranes were then transferred directly to an Applied Biosystems Model 420A Dcrivatizer/Amino Acid Analyzer for on-line PTC-amino acid analyses.
Biological activities Direct haemolytic activity was assayed on washed erythrocytes (2%) and whole blood (diluted with 2 vols of 0.9% saline) of the rabbit, mouse, rat, guinea pig and human as described by Wu et al. (1982) and Ho and Ko (1988). The erythrocytes were incubated with stonustoxin
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Fig. 1. Elution profile for the separation of lethal factor from Synanceja horrida crude venom on a Sephacryl S-200 High Resolution (HR) column (1.5 x 84cm) in 0.05 M sodium phosphate buffer, pH 7.4. Fraction l contained the lethal factor.
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Fig. 2. Elution profile for chromatography of lethal factor on a DEAE-Bio-Gel A column (2.0 × 9 era) equilibrated with 0.05 M sodium phosphate buffer, pH 7.4. Bound proteins were eluted with a linear salt gradient (0-0.15 M sodium chloride in start buffer, 600 ml). The lethal factor (stonustoxin) is located in peak 4.
Fig. 4. HPLC gel permeation chromatography of stonustoxin at pH 7.0 (10 mM sodium phosphate buffer with 0.2 M sodium sulphate) using a Bio-Sil, SEC-400 column (300 × 7.5 mm Bio-Rad Labs). The retention times (RT) for the tool. wt markers that were used to calibrate the column are marked accordingly.
fraction 1, and thus 9% of the crude venom protein. The LDs0 (i.v.) value of this fraction was found to be 0.017#g/g, which represented a 22fold increase in toxicity over the crude venom. In addition, this fraction gave only a single band upon electrophoresis in a 5% non-denaturing polyacrylamide gel (Fig. 3). This fraction is designated as the stonustoxin.
Amino acid analysis
Molecular weight and isoelectric point High performance size exclusion liquid chromatography of the purified lethal factor showed a single protein peak with a mol. wt of 148,000 (Fig. 4) but SDS gel electrophoresis of the same fraction revealed two bands with mol. wts of 71,000 and 79,000 (designated as ~ and # subunits, respectively) (Fig. 5). The isoelectric point (pI) was found to be at pH 6.9.
The amino acid compositions of the ~ and # subunits of stonustoxin are shown in Table 1. No major differences in the amino acid compositions of the two subunits were evident.
N-Terminal amino acid sequence The N-terminal sequence of the fl subunit is shown in Fig. 6. Under the same conditions of the experiment, we were unable to obtain the N-terminal amino acid sequence of the ~ subunit. This suggests that the ~ subunit may have a blocked N-terminus.
Biological properties Stonustoxin exhibited high haemolytic activity on washed erythrocytes or diluted blood. The toxin lysed erythrocytes of rat, guinea pig and rabbit but at
Fig. 3. Electrophoresis of stonustoxin in 5% non-denaturing polyacrylamide gel Lanes 1, 2 and 3 are stonustoxin at different concentrations. CBPB 9~/4--F
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Fig. 6. N - t e r m i n a l sequence o f the fl s u b u n i t o f stonustoxin.
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Fig. 5. SDS-PAGE of stonustoxin in 6% gel. Lane 1: stonustoxin a and fl subunits (71 and 79 k, respectively); Lane 2: mol. wt markers. different concentrations. However, it failed to lyse mice and human erythrocytes even at a concentration of 200 #g/ml (Table 2). The action of stonustoxin in vivo as revealed by the post-mortem examination of mice injected with stonustoxin, also showed that blood collected after death was not haemolysed. Stonustoxin exhibited potent edema-inducing activity which was dose dependent (Fig. 7). The minimum edema dose (MED) was 0.15/~g and maximum swelling occurred within 60 min of injection. The presence of diphenhydramine (50 mg/kg) as an antiinflammatory agent failed to inhibit the edema-inducing effect of stonustoxin (Fig. 8). No phospholipase A2, hyaluronidase and proteinase activities could be detected in stonustoxin. DISCUSSION
The stonefish (Synanceja horrida) is considered to be a dangerous species of marine fish because there have been 267 reported cases of severe stonefish Table 1. Amino acid compositions of the ~t and // subunits of stonustoxin* Amino acid
~t Subunit (71,000)
fl Subunit (79,000)
Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met lie Leu Phe Lys Total number
46 62 47 80 13 37 31 56 29 21 60 4 36 70 40 32 663
64 82 52 73 14 32 34 54 26 24 59 6 41 77 45 37 720
*Trp and Cys were not determined.
envenomation over the period 1965-1981 (Sutherland, 1983). The LDs0 (i.v.) of the crude venom was 0.36/~g/g. This is highly potent when compared with the related venomous fishes of the genus Scorpaenidae and Pterois, which were reported to have a LDs0 (i.v.) of 2.6/~g/g (Schaeffer et al., 1971) and 1.1/~g/g, respectively (Saunders and Taylor, 1959). After purification, the lethal factor (stonustoxin) from S. horrida had an LD50 of 0.017/~g/g which is 22-fold more potent than the crude venom. We have reported here for the first time, the complete purification of the lethal factor (stonustoxin) from the stonefish, Synanceja horrida by a simple two-step chromatographic procedure. Most earlier investigators have hitherto managed to obtain only partially purified samples for their studies on the biological properties of this molecule (Saunders and Tokes, 1961; Austin et al., 1965; Deakins and Saunders, 1967). For example, Deakins and Saunders (1967) obtained a 10-fold purified lethal factor from S. horrida by using starch gel electrophoresis. However, they were unable to recover any lethal fractions from DEAE--ceUulose columns at pH 8 by gradient elution up to 0.75 M NaC1. In fact, most of their venom proteins did not seem to bind to the column, and were eluted with start buffer. This result is baffling as we were able to obtain the lethal factor in good yield at pH 7.4 using DEAE-Bio-Gel A (Fig. 2). However, our results seem to correlate with the observations of Austin et al. (1965). These workers also used a two-step procedure to fractionate the venom proteins of S. trachynis, but their protocol differed from ours in the choice of ion exchangers. They used CM cellulose at pH6.08 instead of DEAE-Bio-Gel at pH 7.4. The lethal factor is bound to both ion exchangers at the given pHs because it has an pI of 6.9 (present work). However, the choice of pH 7.4 for the anion exchange experiment is probably more favourable since the lethal factor had been shown to be more stable at neutral pH (Saunders and Tokes, 1961). Table 2. Haemolytic activity of stonustoxin on diluted blood and washed erythroeytes of different animal species; data show means ± SE from three experiments Species Rat
Stonustoxin (~g/ml)
Haemolysis(%) 2% RBC Dilu~dblood
0.01 0.03 0.05 0,10
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13±2 46.4±2.5 75.9±3.4 1~%
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0.03 0.03 0.10 10.0
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4.4±0.3 37±1 69.7±4.2 1~
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7.5±0.8 20.0±0.3 70.0±3 100
6.0±0.5 16.3±2.3 36.4±0.8 1~
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200 100
Nil Nil
Nil Nil
Stonustoxin
Stonustoxin is shown by HPLC to have a native mol. wt of 148,000 (Fig. 4). This result is in accord with the values reported by Austin et al. 0965) who showed that the lethal fractions from the venom of S. trachynis and S. verrucosa had sedimentation coefficients of 8.4--9.0 S, or mol. wts of about 150,000. Stonustoxin is probably a heterodimer since SDS-PAGE showed that it consisted of two different 71 and 79 kDa bands. The amino acid compositions of these bands have been determined (Table l) and they seemed to have fairly similar compositions. However, it is interesting to find that the 71 kDa band (~ subunit) has a blocked N-terminal residue. We attempted to unblock this polypeptide directly on the membrane by the method of Wellner et al. (1990) but subsequent sequencing gave a high background that prevented us from reading the sequence correctly. The 79 kDa ~ subunit), on the other hand, was amenable to N-terminal sequencing. The first 39 residues of this polypeptide chain are shown in Fig. 6. However, this sequence did not seem to show any homology with other toxins or lethal factors when a search was conducted against the various protein sequence databases (GENBANK: Genpept and Swissprot and EMBL: PIR and NERF). Therefore, stonustoxin may represent a new class of toxin whose structure-function relationship remains to be fully elucidated. Stonustoxin was found to be devoid of phospholipase A2, proteinase and hyaluronidase activities. The most prominent biological activities of stonustoxin were its haemolytic and edema-inducing effects. Rat, guinea pig and rabbit erythrocytes were most susceptible to the haemolytic action of stonustoxin. Our present finding agrees well with that of other investigators (Duhig and Jones, 1928b; Weiner, 1959a; Halstead et al., 1956). The lack of an in vivo haemolytic effect in mice injected with stonustoxin may suggest that the direct cause of death is unlikely to be haemolysis. Reported local effects caused by stonefish envenomation were extreme pain, ischaemia and intensive swelling at the site of the stings (Phelps, 1960).
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Fig. 8. Effect of diphenhydramine on edema formation induced by stonustoxin injection into mouse hind paw. Diphenhydra~ine (50 mg/kg) was administered 30 rain before stonustoxin (0.5 #g in 0.02 mi) injection. Each point represents the mean (+SE) of five mice.
The swelling persists for 2--4 days even after treatment with antivenin. In agreement with this, we have found stonustoxin to be a potent inflammatory toxin. The edema effect in mice lasted for more than 24hr after injection. These effects were not inhibited by diphenhydramine, thus suggesting that the increase in vascular permeability might not have been mediated by histamine release. It seems certain that one single protein, (the stonustoxin), in the stonefish venom is able to exhibit different biological activities. This concept has been suggested by Weiner (1959a, b). Further studies are being conducted in our laboratory to ascertain the mechanism of action of stonustoxin and the roles played by its two subunits. Acknowledgements--We would like to express our gratitude to Miss Rani Mahendran for her technical assistance during the initial phase of the work. This work has been supported by the National University of Singapore (Grant No. 870309 awarded to R. Yuen of the NUS Multidisciplinary Research Group on Venoms and Toxins). Part of this work was presented at the 2nd Asia-Pacific Congress on Animal, Plant and Microbial Toxins, 19-22 February 1990, Varanasi, India.
REFERENCES Austin L , Cairncross K. D. and McCallum I. A. N. (1961)
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Fig. 7. Stonustoxin-induced swelling in mouse hind paw.
Different doses (0.02 ml) of stonustoxin and normal saline were injected into the hind paw (subplantar). Each point represents the mean (+ SE) of five mice. Edema volume change was measured with calipers with the left paw as control.
Some pharmacological actions of the venom of the stonefish "Synanceja horrida". Arch Int. Pharmacodyn. CXXXI, 339-347. Austin L., (3illis R. (3. and Youatt (3. (1965) Stonefish venom: some biochemical and chemical observations. Aust. J. exp. Biol. Med. Sci. 43, 79-90. Bradford M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analyt. Biochem. 72, 248-254. Davis B. J. (1964) Disc electrophoresis---II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427. Deakins D. E. and Saunders P. R. (1967) Purification of the lethal fraction of the venom of the stonefish Synanceja horrida (Linnaeus). Toxicon 4, 257-262.
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Duhig J. V. and Jones G. (1928a) The venom apparatus of the stonefish Synanceja horrida. Mem. Queensland Mus. 9, 136-150. Duhig J. V. and Jones G. (1928b) Haemotoxin of the stoneflsh Synanceja horrida. Aust. J. exp. Biol. 5, 173-179. Dupont D., Keim P., Chui A., Bozzini M. and Wilson K. (1988) Gas-phase hydrolysis for PTC-amino acid analysis. Applied Biosystems User Bulletin issue No. 2 (May), 10 pp. ABI, Foster City, USA. Ferrante F. D. (1956) Turbidimetric measurement of acid mucopolysaccharides and hyaluronidase activity. J. biol. Chem. 220, 303-306. Halstead B. W., Chitwood M. J. and Modglin F. R. (1956) Stonefish stings and the venom apparatus of Synanceja horrida (Linneaus). Trans. Am. Microscop. Soc. 75, 381-397. Ho C. L. and Ko J. L. (1988) Purification and characterization of a lethal protein with phospholipase A activity from the hornet (Vesper basalis) venom. Biochim. biophys. Acta 963, 414--422. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Matsudaira P. (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. biol. Chem. 262, 10035-10038. Otterness I. G. and Moore P. F. (1988) Carrageenan foot edema test. In lmmunochemical Techniques Part 1: Chemotaxis and Inflammation. Methods in Enzymology (Edited by DiSabato G.), Vol. 162, pp. 321-327. Academic Press, New York. Phelps D. R. (1960) Stonefish poisoning. Med. J. Aust. 20, 293-294. Russell F. E. (1965) Marine toxin: venomous and poisonous marine animals. In Advances in Marine Biology (Edited by Russell F. E.), Vol. 3, pp. 255-384. Academic Press, London. Saunders P. R., Rothman S., Medrano V. A. and Chin H. P. (1962) Cardiovascular actions of venom of the stonefish Synanceja horrida. Am. J. Physiol. 203, 429-432.
Saunders P. R. and Taylor P. B. (1959) Venom of the lionfish Pterois volitans. Am. J. Physiol. 197, 437--440. Saunders P. R. and Tokes L. (1961) Purification and properties of the lethal fraction of the venom of the stonefish, Synanceja horrida (Linneaus). Biochim. Biophys. Acta 52, 527-532. Sehaeffer R. C., Carlson R. W. and Russell F. E. (1971) Some chemical properties of the venom of the scorpionfish, Scorpaena guttata. Toxicon 9, 69-78. Sutherland S. K. (1983) Australian Animal Toxin, pp. 401-410. Oxford University Press, Melbourne. Tan N. H. and Tan C. S. (1988) Acidimetric assay for phospholipase A using egg yolk suspension substrate. Analyt. Biochem. 170, 282-288. Vesterberg O. and Svensson H. (1966) Isoclectric fractionation, analysis, and characterization of ampholytes in natural pH gradients. IV. Further studies on the resolving power in connection with separation of myogiobins. Acta chem. Scand. 20, 820-834. Weiner S. (1959a) Observations on the venom of the stonefish Synanceja trachynis. Med. J. Aust. 9, 620-627. Weiner S. (1959b) The production and assay of stonefish antivenin. Med. J. Aust. 14, 715-719. Wellner D., Panncersclvam C. and Horecker B. L. (1990) Sequencing of peptides and proteins with blocked N-terminal amino acids: N-acetylserine or N-acetylthreonine. Proc. natn. Acad. Sci. USA 87, 1947-1949. WHO (1981) Progress in the characterization of venoms and standardization of antiivenoms. WHO offset Publication No. 58, 23 pp. Wu S. H., Wang K. T. and Ho C. L. (1982) Purification and pharmacological characterization of a cardiotoxin-like protein from Formosan Banded Krait Bungarus multicintus venom. Toxicon 20, 753-764. Yuen R., Cheng L. Y. and Gopalakrishnakone K. (1987) Enzymic studies on the venom of stonefish (Genus Synanceja). Proceedings of the 1st AsiaPacific Congress on Animal Plant and Microbial Toxins, p. 452. International Society on Toxinology, UK.
0305-0491/91 $3.00+ 0.00 © 1991 PergamonPress pie
Printed in Great Britain
PURIFICATION AND PARTIAL CHARACTERIZATION OF STONUSTOXIN (LETHAL FACTOR) FROM S Y N A N C E J A HORRIDA VENOM C. H. POH,* R. YUEN,*t H. E. KHOO,* M. CHUNG,* M. GWEE~ and P. GOPALAKRISHNAKONE§ *Departments of Biochemistry, :~Pharmacology and §Anatomy, Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore 0511
(Received 30 January 1991) Abstract--1. The lethal factor of the stonefish (Synanceja horrida) venom, designated as the stonustoxin, was purified to homogeneity by a two-step procedure on Sephacryl S-200 High Resolution (HR) gel permeation and DEAE Bio-Gel A anion exchange chromatography. 2. Stonustoxin has a native mol. wt of 148,000 and an isoelectric point of 6.9. 3. SDS-polyacrylamide gel electrophoresis revealed two subunits (designated ~, and fl) with mol. wts of 71,000 and 79,000, respectively. 4. The amino acid composition of both subunits and the N-terminal amino acid sequence of the fl subunit were also determined. 5. Purified stonustoxin had an LDs0 of 0.017 #g/g which is 22-fold more potent than that of the crude venom. 6. The toxin exhibited potent haemolytic activity in vitro and endema-inducingactivity with a minimum edema dose (MED) of 0.15 Pg in mouse paw. The edema effect was not antagonized by diphenhydramine.
INTRODUCTION
(electrophoresis grade) was purchased from Schwarz/Mann Biotech (Cleveland, OH, USA), diphenhydramine (Merck) was a gift from Dr N. H. Tan, Department of Biochemistry, University of Malaya and the chromogenic substrates used for proteinase assays were supplied by Boehringer Mannheim (Far East). All other chemicals were from Sigma Chemical Company (St Louis, MO, USA). Male Swiss mice were supplied by the Animal Centre of the National University of Singapore. All other chemicals were of analytical grade, and were the best grades available.
Stonefish of the genus Synanceja have been responsible for a n u m b e r of deaths and severe poisoning to humans through stings from the spines (Saunders et al., 1962). The crude venom exuded from the venom glands attached to the spines was reported to be myotoxic which resulted in the paralysis of cardiac, involuntary and skeletal muscles (Saunders et aL, 1962; Austin et al., 1961; Russell, 1965). It was also shown to possess hyaluronidase and haemolytic activities (Austin et al., 1965; Duhig and Jones, 1928a; 1928b). Hitherto there has been only one report on the attempted purification of one of the bioactive components, namely the lethal fraction from the crude venom of Synanceja horrida (Deakins and Saunders, 1967). However, no detailed chemical and biological properties of this lethal fraction have been published. In this paper we report on the purification and partial characterization of the lethal factor, designated as stonustoxin (stonefish National University of Singapore) from the venom of Synanceja horrida with the view to fully elucidate its mode of action.
Protein determination Protein was determined by the method of Bradford (I 976) using the protein assay kit from Bio-Rad Laboratories and bovine serum albumin as standard.
Lethality assay The LDs0 values were determined by intravenous injection of the venom fractions into the caudal vein of male Swiss mice (20 + 2 g) and subsequently calculated by the method of Spearman-Kaber (WHO, 1981). Four mice were tested at each of the seven dose levels.
Extraction of venom The dorsal spines of the stonefish were excised from the surrounding tissue. The crude venom was aspirated out from the venom sacs by using a I ml syringe fitted with a 21G needle. The venom was lyophilized immediately after collection and stored at -70°C until use.
MATERIALS AND METHODS Live stoneflsh (Synanceja horrida) were obtained by local fishermen. Sephacryl S-200 HR (high resolution) and SDS-PAGE mol. wt markers were obtained from Pharmacia AB (Uppsala, Sweden); DEAE-Bio-Gel A (100-200 mesh) and protein assay kits were purchased from Bio-Rad Laboratories (Richmond, CA, USA). Acrylamide and tris(hydroxymethyl)-aminoethane (electrophoresis grade) were obtained from Polysciences (Warrington, PA, USA), giycine i'Author to whom correspondence should be addressed.
Purification All operations were carried out at 4°C. One hundred and seventy milligrams of crude venom were reconstituted in 2.5ml of 0.05 M sodium phosphate buffer, pH7.4 and subsequently centrifuged at 3000g for 5 min to remove insoluble material. Two miUilitres of this solution (containing about 35 mg/ml of protein) was then applied to a Sephacryl S-200 HR gel permeation column (1.5 × 84 cm) previously equilibrated with the same buffer. Elution was
793
794
C.H. POH et al.
carried out at a constant flow rate of 12ml/hr and the absorbance of the column eluant was monitored at 280 urn. The lethal fractions (as determined by the lethality assay) from the gel permeation column were pooled and loaded onto a DEAE-Bio-Gel A (100-200 mesh) anion-exchange column (2.0 x 9 era) pre-equilibrated with 0.05 M sodium phosphate buffer, pH 7.4. After thorough washing of the column with start buffer, the bound protein fractions were eluted with a linear buffer gradient (0-0.15 M sodium chloride in start buffer, 600 ml) at a flow rate of 8.0 ml/hr. Three miUilitre fractions were collected.
Polyacrylamide gel electrophoresis (PAGE) Native gel electrophoresis was carried out in a 5% gel according to the method of Davis (1964) while SDS-PAGE (6%) was performed as described by Laemmli (1970). Protein bands were detected either by silver or Coomassie Blue staining.
Molecular weight determination The native mol. wt of stonustoxin was determined by high performance liquid chromatography at pH 7.0 using 10 mM sodium phosphate buffer with 0.2 M sodium sulphate in a gel permeation column (Bio-Sil SEC-400, Bio-Rad Labs, 300 x 7.5 ram) at a flow rate of 1.0 ml/min. Protein was monitored at 280 nm using a Shimadzu SPD-7AV u.v. detector. The column was calibrated with thyroglobulin (669,000 mol. wt), IgG (160,000 tool. wt), ovalbumin (43,000 tool. wt), myoglobin (18,000 tool. wt) and cyanocobalamin (1400 tool. wt) from Bio-Rad Laboratories.
Isoelectric point determination Isoelectric focusing was carried out using a 110 ml column according to the method of Vesterberg and Svensson (1966). One hundred milligrams of crude venom were first loaded onto the column, following which focusing was carried out at 300 V and 4°C. After 72 hr, electrofocusing was stopped, and 2 mi fractions were collected to assay for lethal activity and for pH determination.
Protein blotting Six per cent gels (14 x 16 era, 1.5 mm thick) were used for SDS-PAGE. After electrophoresis, the gel was electroblotted for 1 hr at 90 V based on the method of Matsudaira (1987). Following staining and destaining, the membrane (Problott, Applied Biosystems) was rinsed with glass distilled water (3 x ), and the protein bands were then excised with a clean scalpel and stored in Eppendorf vials at - 20°C.
(various final concentrations) at 37°C for 60 rain. The degree of haemolysis was determined as a percentage of haemoglobin released (measured at 540 nm) by the toxin compared with a 100% control effected by the addition of 10 mg/ml heparin. Edema-inducing activity was measured by injecting 20 #1 stonustoxin (0.15-0.4#g) in 0.05M phosphate buffer, pH 7.4 into the subplantar muscle of the right hind paw of male Swiss mice (20-22 g). The left paw served as control. The edema-inducing activity was expressed as a percentage of swelling in the right hind paw as measured with calipers after different time intervals (Otterness and Moore, 1988). The effect of the antihistamine, diphenhydramine, on edema-inducing activity was tested by injecting the diphenhydramine (50 mg/kg i.p.) 30 min before injection of 0.5 #g stonustoxin. Hyaluronidase activity was measured using hyaluronic acid from human umbilical cord (Ferrante, 1956) and phospholipase A2 according to the method of Tan and Tan (1988). Proteinase activities were assayed as described by Yuen et al. (1987). RESULTS
Toxicity o f the crude venom The crude venom is a colourless fluid with a p H of 6.8. The protein content accounted for 20% o f the dry weight of the lyophilized venom. The crude venom is highly toxic to the mouse. The estimated L D ~ was 0.36/zg/g. A lethal dose (i.v.) of the venom caused convulsive movements, respiratory arrest, urination, hypersalivation and death within 3-30 min of injection.
Purification o f lethal factor Isolation of the lethal factor of S. horrida was achieved in two steps. The first step involved fractionation of the crude venom in a Sephacryl S-200 H R column (Fig. 1). Three major peaks were evident but only fraction 1 exhibited lethal activity and accounted for 24% of the total venom protein loaded. The LDs0 (i.v.) of fraction 1 was found to be 0.035 #g/g. This is 11-fold more toxic than the crude venom. Fraction 1 was then further resolved into four subfractions by D E A E Bio-Gel (Fig. 2). Based on the lethality assay, the lethal factor was located in peak 4. This accounted for 36% of the protein content of
N- Terminal sequencing Amino-terminal sequencing of the electroblotted subunits of stonustoxin was carried out using an Applied Biosystems 471-02 protein/peptide sequenator. Prior to sequencing, the membrane pieces were carefully loaded into the upper cartridge block of the sequenator, followed by a precycied polybrene-conditioned filter before final assembly.
Amino acid analysis The amino acid compositions were determined using on-membrane amino acid analysis protocol (Dupont et al., 1988). The membrane bands were first hydrolyzed by a manual vapour hydrolysis method using a mixture of HCI/TFA (2: 1, v/v) at 165°C for 45 rain. The membranes were then transferred directly to an Applied Biosystems Model 420A Dcrivatizer/Amino Acid Analyzer for on-line PTC-amino acid analyses.
Biological activities Direct haemolytic activity was assayed on washed erythrocytes (2%) and whole blood (diluted with 2 vols of 0.9% saline) of the rabbit, mouse, rat, guinea pig and human as described by Wu et al. (1982) and Ho and Ko (1988). The erythrocytes were incubated with stonustoxin
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FRACTION NUMBER
Fig. 1. Elution profile for the separation of lethal factor from Synanceja horrida crude venom on a Sephacryl S-200 High Resolution (HR) column (1.5 x 84cm) in 0.05 M sodium phosphate buffer, pH 7.4. Fraction l contained the lethal factor.
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Fig. 2. Elution profile for chromatography of lethal factor on a DEAE-Bio-Gel A column (2.0 × 9 era) equilibrated with 0.05 M sodium phosphate buffer, pH 7.4. Bound proteins were eluted with a linear salt gradient (0-0.15 M sodium chloride in start buffer, 600 ml). The lethal factor (stonustoxin) is located in peak 4.
Fig. 4. HPLC gel permeation chromatography of stonustoxin at pH 7.0 (10 mM sodium phosphate buffer with 0.2 M sodium sulphate) using a Bio-Sil, SEC-400 column (300 × 7.5 mm Bio-Rad Labs). The retention times (RT) for the tool. wt markers that were used to calibrate the column are marked accordingly.
fraction 1, and thus 9% of the crude venom protein. The LDs0 (i.v.) value of this fraction was found to be 0.017#g/g, which represented a 22fold increase in toxicity over the crude venom. In addition, this fraction gave only a single band upon electrophoresis in a 5% non-denaturing polyacrylamide gel (Fig. 3). This fraction is designated as the stonustoxin.
Amino acid analysis
Molecular weight and isoelectric point High performance size exclusion liquid chromatography of the purified lethal factor showed a single protein peak with a mol. wt of 148,000 (Fig. 4) but SDS gel electrophoresis of the same fraction revealed two bands with mol. wts of 71,000 and 79,000 (designated as ~ and # subunits, respectively) (Fig. 5). The isoelectric point (pI) was found to be at pH 6.9.
The amino acid compositions of the ~ and # subunits of stonustoxin are shown in Table 1. No major differences in the amino acid compositions of the two subunits were evident.
N-Terminal amino acid sequence The N-terminal sequence of the fl subunit is shown in Fig. 6. Under the same conditions of the experiment, we were unable to obtain the N-terminal amino acid sequence of the ~ subunit. This suggests that the ~ subunit may have a blocked N-terminus.
Biological properties Stonustoxin exhibited high haemolytic activity on washed erythrocytes or diluted blood. The toxin lysed erythrocytes of rat, guinea pig and rabbit but at
Fig. 3. Electrophoresis of stonustoxin in 5% non-denaturing polyacrylamide gel Lanes 1, 2 and 3 are stonustoxin at different concentrations. CBPB 9~/4--F
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Fig. 5. SDS-PAGE of stonustoxin in 6% gel. Lane 1: stonustoxin a and fl subunits (71 and 79 k, respectively); Lane 2: mol. wt markers. different concentrations. However, it failed to lyse mice and human erythrocytes even at a concentration of 200 #g/ml (Table 2). The action of stonustoxin in vivo as revealed by the post-mortem examination of mice injected with stonustoxin, also showed that blood collected after death was not haemolysed. Stonustoxin exhibited potent edema-inducing activity which was dose dependent (Fig. 7). The minimum edema dose (MED) was 0.15/~g and maximum swelling occurred within 60 min of injection. The presence of diphenhydramine (50 mg/kg) as an antiinflammatory agent failed to inhibit the edema-inducing effect of stonustoxin (Fig. 8). No phospholipase A2, hyaluronidase and proteinase activities could be detected in stonustoxin. DISCUSSION
The stonefish (Synanceja horrida) is considered to be a dangerous species of marine fish because there have been 267 reported cases of severe stonefish Table 1. Amino acid compositions of the ~t and // subunits of stonustoxin* Amino acid
~t Subunit (71,000)
fl Subunit (79,000)
Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met lie Leu Phe Lys Total number
46 62 47 80 13 37 31 56 29 21 60 4 36 70 40 32 663
64 82 52 73 14 32 34 54 26 24 59 6 41 77 45 37 720
*Trp and Cys were not determined.
envenomation over the period 1965-1981 (Sutherland, 1983). The LDs0 (i.v.) of the crude venom was 0.36/~g/g. This is highly potent when compared with the related venomous fishes of the genus Scorpaenidae and Pterois, which were reported to have a LDs0 (i.v.) of 2.6/~g/g (Schaeffer et al., 1971) and 1.1/~g/g, respectively (Saunders and Taylor, 1959). After purification, the lethal factor (stonustoxin) from S. horrida had an LD50 of 0.017/~g/g which is 22-fold more potent than the crude venom. We have reported here for the first time, the complete purification of the lethal factor (stonustoxin) from the stonefish, Synanceja horrida by a simple two-step chromatographic procedure. Most earlier investigators have hitherto managed to obtain only partially purified samples for their studies on the biological properties of this molecule (Saunders and Tokes, 1961; Austin et al., 1965; Deakins and Saunders, 1967). For example, Deakins and Saunders (1967) obtained a 10-fold purified lethal factor from S. horrida by using starch gel electrophoresis. However, they were unable to recover any lethal fractions from DEAE--ceUulose columns at pH 8 by gradient elution up to 0.75 M NaC1. In fact, most of their venom proteins did not seem to bind to the column, and were eluted with start buffer. This result is baffling as we were able to obtain the lethal factor in good yield at pH 7.4 using DEAE-Bio-Gel A (Fig. 2). However, our results seem to correlate with the observations of Austin et al. (1965). These workers also used a two-step procedure to fractionate the venom proteins of S. trachynis, but their protocol differed from ours in the choice of ion exchangers. They used CM cellulose at pH6.08 instead of DEAE-Bio-Gel at pH 7.4. The lethal factor is bound to both ion exchangers at the given pHs because it has an pI of 6.9 (present work). However, the choice of pH 7.4 for the anion exchange experiment is probably more favourable since the lethal factor had been shown to be more stable at neutral pH (Saunders and Tokes, 1961). Table 2. Haemolytic activity of stonustoxin on diluted blood and washed erythroeytes of different animal species; data show means ± SE from three experiments Species Rat
Stonustoxin (~g/ml)
Haemolysis(%) 2% RBC Dilu~dblood
0.01 0.03 0.05 0,10
80.9±1.2 94.7±2.3 1~ 1~
13±2 46.4±2.5 75.9±3.4 1~%
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0.03 0.03 0.10 10.0
~.7±0.1 71.9±0.6 88.8±0.7 1~
4.4±0.3 37±1 69.7±4.2 1~
Rabbit
0.05 0.10 0.20 10.~
7.5±0.8 20.0±0.3 70.0±3 100
6.0±0.5 16.3±2.3 36.4±0.8 1~
Mice Human
200 100
Nil Nil
Nil Nil
Stonustoxin
Stonustoxin is shown by HPLC to have a native mol. wt of 148,000 (Fig. 4). This result is in accord with the values reported by Austin et al. 0965) who showed that the lethal fractions from the venom of S. trachynis and S. verrucosa had sedimentation coefficients of 8.4--9.0 S, or mol. wts of about 150,000. Stonustoxin is probably a heterodimer since SDS-PAGE showed that it consisted of two different 71 and 79 kDa bands. The amino acid compositions of these bands have been determined (Table l) and they seemed to have fairly similar compositions. However, it is interesting to find that the 71 kDa band (~ subunit) has a blocked N-terminal residue. We attempted to unblock this polypeptide directly on the membrane by the method of Wellner et al. (1990) but subsequent sequencing gave a high background that prevented us from reading the sequence correctly. The 79 kDa ~ subunit), on the other hand, was amenable to N-terminal sequencing. The first 39 residues of this polypeptide chain are shown in Fig. 6. However, this sequence did not seem to show any homology with other toxins or lethal factors when a search was conducted against the various protein sequence databases (GENBANK: Genpept and Swissprot and EMBL: PIR and NERF). Therefore, stonustoxin may represent a new class of toxin whose structure-function relationship remains to be fully elucidated. Stonustoxin was found to be devoid of phospholipase A2, proteinase and hyaluronidase activities. The most prominent biological activities of stonustoxin were its haemolytic and edema-inducing effects. Rat, guinea pig and rabbit erythrocytes were most susceptible to the haemolytic action of stonustoxin. Our present finding agrees well with that of other investigators (Duhig and Jones, 1928b; Weiner, 1959a; Halstead et al., 1956). The lack of an in vivo haemolytic effect in mice injected with stonustoxin may suggest that the direct cause of death is unlikely to be haemolysis. Reported local effects caused by stonefish envenomation were extreme pain, ischaemia and intensive swelling at the site of the stings (Phelps, 1960).
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Fig. 8. Effect of diphenhydramine on edema formation induced by stonustoxin injection into mouse hind paw. Diphenhydra~ine (50 mg/kg) was administered 30 rain before stonustoxin (0.5 #g in 0.02 mi) injection. Each point represents the mean (+SE) of five mice.
The swelling persists for 2--4 days even after treatment with antivenin. In agreement with this, we have found stonustoxin to be a potent inflammatory toxin. The edema effect in mice lasted for more than 24hr after injection. These effects were not inhibited by diphenhydramine, thus suggesting that the increase in vascular permeability might not have been mediated by histamine release. It seems certain that one single protein, (the stonustoxin), in the stonefish venom is able to exhibit different biological activities. This concept has been suggested by Weiner (1959a, b). Further studies are being conducted in our laboratory to ascertain the mechanism of action of stonustoxin and the roles played by its two subunits. Acknowledgements--We would like to express our gratitude to Miss Rani Mahendran for her technical assistance during the initial phase of the work. This work has been supported by the National University of Singapore (Grant No. 870309 awarded to R. Yuen of the NUS Multidisciplinary Research Group on Venoms and Toxins). Part of this work was presented at the 2nd Asia-Pacific Congress on Animal, Plant and Microbial Toxins, 19-22 February 1990, Varanasi, India.
REFERENCES Austin L , Cairncross K. D. and McCallum I. A. N. (1961)
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Fig. 7. Stonustoxin-induced swelling in mouse hind paw.
Different doses (0.02 ml) of stonustoxin and normal saline were injected into the hind paw (subplantar). Each point represents the mean (+ SE) of five mice. Edema volume change was measured with calipers with the left paw as control.
Some pharmacological actions of the venom of the stonefish "Synanceja horrida". Arch Int. Pharmacodyn. CXXXI, 339-347. Austin L., (3illis R. (3. and Youatt (3. (1965) Stonefish venom: some biochemical and chemical observations. Aust. J. exp. Biol. Med. Sci. 43, 79-90. Bradford M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analyt. Biochem. 72, 248-254. Davis B. J. (1964) Disc electrophoresis---II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427. Deakins D. E. and Saunders P. R. (1967) Purification of the lethal fraction of the venom of the stonefish Synanceja horrida (Linnaeus). Toxicon 4, 257-262.
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Duhig J. V. and Jones G. (1928a) The venom apparatus of the stonefish Synanceja horrida. Mem. Queensland Mus. 9, 136-150. Duhig J. V. and Jones G. (1928b) Haemotoxin of the stoneflsh Synanceja horrida. Aust. J. exp. Biol. 5, 173-179. Dupont D., Keim P., Chui A., Bozzini M. and Wilson K. (1988) Gas-phase hydrolysis for PTC-amino acid analysis. Applied Biosystems User Bulletin issue No. 2 (May), 10 pp. ABI, Foster City, USA. Ferrante F. D. (1956) Turbidimetric measurement of acid mucopolysaccharides and hyaluronidase activity. J. biol. Chem. 220, 303-306. Halstead B. W., Chitwood M. J. and Modglin F. R. (1956) Stonefish stings and the venom apparatus of Synanceja horrida (Linneaus). Trans. Am. Microscop. Soc. 75, 381-397. Ho C. L. and Ko J. L. (1988) Purification and characterization of a lethal protein with phospholipase A activity from the hornet (Vesper basalis) venom. Biochim. biophys. Acta 963, 414--422. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Matsudaira P. (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. biol. Chem. 262, 10035-10038. Otterness I. G. and Moore P. F. (1988) Carrageenan foot edema test. In lmmunochemical Techniques Part 1: Chemotaxis and Inflammation. Methods in Enzymology (Edited by DiSabato G.), Vol. 162, pp. 321-327. Academic Press, New York. Phelps D. R. (1960) Stonefish poisoning. Med. J. Aust. 20, 293-294. Russell F. E. (1965) Marine toxin: venomous and poisonous marine animals. In Advances in Marine Biology (Edited by Russell F. E.), Vol. 3, pp. 255-384. Academic Press, London. Saunders P. R., Rothman S., Medrano V. A. and Chin H. P. (1962) Cardiovascular actions of venom of the stonefish Synanceja horrida. Am. J. Physiol. 203, 429-432.
Saunders P. R. and Taylor P. B. (1959) Venom of the lionfish Pterois volitans. Am. J. Physiol. 197, 437--440. Saunders P. R. and Tokes L. (1961) Purification and properties of the lethal fraction of the venom of the stonefish, Synanceja horrida (Linneaus). Biochim. Biophys. Acta 52, 527-532. Sehaeffer R. C., Carlson R. W. and Russell F. E. (1971) Some chemical properties of the venom of the scorpionfish, Scorpaena guttata. Toxicon 9, 69-78. Sutherland S. K. (1983) Australian Animal Toxin, pp. 401-410. Oxford University Press, Melbourne. Tan N. H. and Tan C. S. (1988) Acidimetric assay for phospholipase A using egg yolk suspension substrate. Analyt. Biochem. 170, 282-288. Vesterberg O. and Svensson H. (1966) Isoclectric fractionation, analysis, and characterization of ampholytes in natural pH gradients. IV. Further studies on the resolving power in connection with separation of myogiobins. Acta chem. Scand. 20, 820-834. Weiner S. (1959a) Observations on the venom of the stonefish Synanceja trachynis. Med. J. Aust. 9, 620-627. Weiner S. (1959b) The production and assay of stonefish antivenin. Med. J. Aust. 14, 715-719. Wellner D., Panncersclvam C. and Horecker B. L. (1990) Sequencing of peptides and proteins with blocked N-terminal amino acids: N-acetylserine or N-acetylthreonine. Proc. natn. Acad. Sci. USA 87, 1947-1949. WHO (1981) Progress in the characterization of venoms and standardization of antiivenoms. WHO offset Publication No. 58, 23 pp. Wu S. H., Wang K. T. and Ho C. L. (1982) Purification and pharmacological characterization of a cardiotoxin-like protein from Formosan Banded Krait Bungarus multicintus venom. Toxicon 20, 753-764. Yuen R., Cheng L. Y. and Gopalakrishnakone K. (1987) Enzymic studies on the venom of stonefish (Genus Synanceja). Proceedings of the 1st AsiaPacific Congress on Animal Plant and Microbial Toxins, p. 452. International Society on Toxinology, UK.
