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Some chemical properties of the venom of the scorpionfish Scorpaena guttata
Toxicon, 1971, Vol. 9, pD. 69-78. Pvaamon Prea. Printed in Great Britain.
SOME CHEMICAL PROPERTIES OF THE VENOM OF THE SCORPIONFISH SCORPAENA GUITATA* R1cHARn C. ScHAEFFER, JR.,t RICHARD W. CARrsoNt and FiNDLAY E. RUSSELL Laboratory of Neurological Research, University of Southern California School of Medicine, Los Angeles, California, U.S .A. (Acceptedforpub&atlon 1 July 1970) Abstract-A method for extracting and stabilizing a fish venom is proposed . Extracts from fresh venomous spines of Scorpaena guttata were prepared in distilled water at 5°, lyophilized, reconstituted in 0-05 M sodium phosphate buffer, pH 7-4, with 0-9 per cent NaCl and 10 -3 M Cleland's reagent at 5°, and centrifuged. Using this technique, extracts were found to be stable up to 21 days . Fractionation indicated that the lethal property was associated with protein(s) having a molecular weight of more than 50,000 and less than 800,000. The semi-purified lethal fraction had an intravenous LD . in mice of 0-9 mg protein per kg body weight, while the LD+e of the crude venom was 2-6 mg protein per kg . This is the first report on the separation of a fraction of a fish venom which is more lethal than the crude venom.
members of the family Scorpaenidae, are a varied group of about 350 species. Included in this family are the stonefishes, lionfishes or zebrafishes, bullrout and waspfish . They are widely distributed throughout all tropical and most temperate seas. The greatest number of species is found in the tropical Indo-Pacific . For the most part they are shallow water fishes living in or around rocks, coral reefs or kelp beds, with which they often blend favorably. Some species bury themselves in the sand and may lie motionless for long periods of time . Thus, they are often handled or trod upon unknowingly, that is, until envenomation occurs. The stings of these fish lead to pain, localized swelling, discoloration and paresthesis around the wound, and in some cases to lymphadenitis, lymphadenopathy, nausea, vomiting, weakness, pallor and syncopy. In the more severe cases there may be intense pain, respiratory distress, shock, coma and death (RUSSELL, 1965). The venom of the scorpionfishes is contained within the tissues which envelop certain of the dorsal, pelvic and anal fin spines . The number of venomous spines varies with the different scorpaenid species, as does the structure of the venom gland or venom-containing tissues . Envenomation occurs through mechanical pressure on the spine, which tears the integumentary sheath encasing the spine, permitting the venom to escape into the wound. The toxins of these various piscine species appear to have certain chemical and physiopharmacological properties in common. These have been reviewed in detail elsewhere (SAUNDERS, 1959; RUSSELL, 1965; RUSSELL, 1969). The California scorpionfish, Scorpaena guttata Girard, commonly called the sculpin in California, is the only member of the genus Scorpaena found in Southern California THE scomoNFIsims,
*Supported by ONR contracts NR 305-786 and NR 104-793 to F. E. Russell. fFellows, USPHS Training Program GM01700. 69
70
R. C. SCHAEFFER, 7R., R. W. CARLSON and F. E. RUSSELL
waters . This fish may reach a length of 40 cm and ranks among the most popular fish taken by rod and reel fishermen, accounting for the frequency of stings by this animal (HALsTEAD, 1951 ; RussELL, 1965). The venom apparatus of this species consists of 12 dorsal spines, 3 anal spines, 2 pelvic spines and their enveloping integumentary sheath (HAISTEAD, 1955). The venom of the California scorpionfish is contained within certain specialized secretory cells in the slender, fusiform strand of greyish tissue found lying within the distal one-half or two-thirds of the glandular grooves on either side of the sting. Extracts of these tissues have been found to be opalescent, of neutral pH, very unstable and nondialyzable. Treatment with a chelating agent (EDTA), or reduced glutathione (GSH), or with a proteinase inhibitor (diisopropylfluorophosphate) did not preserve the extract's toxicity. Partial purification of the extract was achieved by salting-out with ammonium sulfate, and by DEAF-cellulose column chromatography. With the latter method, purification of the toxin to 11 - 0,ug protein per mouse LDP was sometimes obtained . A toxic fraction was also obtained adjacent to the origin of the small pore polyacrylamide gel in which the venom extract was resolved by disc electrophoresis . The lethal fraction was thought to be a high molecular weight protein and moderately negatively charged at pH 7-4 (Taylor, unpublished thesis, 1963 ; Saunders, unpublished data). The present study was initiated to determine certain of the chemical and physiopharmacological properties of the venom of Scorpaena guttata . This report deals with attempts to stabilize the toxin and to define certain of its chemical properties . A subsequent paper will treat of the physiopharmacology of this toxin. MATERIALS AND METHODS
Collection and venom extraction
The fish were caught on hook and line off San Pedro, California and transported to the laboratory in aerated containers . They were then sacrificed and the first 12 dorsal, three anal and two pelvic spines quickly removed. Two methods of venom extraction were studied (a) Aspiration method. The integumentary sheath covering each spine was stripped to the base of the spine and the venom was aspirated from the glandular grooves with a micropipette. The pipette was connected to a vacuum source and a collection flask maintained at 5° . The extract was then centrifuged at 5000 rev per min for 10 min and the supernatant lyophilized . (b) Batch method. Stripped spines were immersed in a beaker of distilled water at 5° . The beaker was gently agitated for 5 min and the solution decanted . This procedure was carried out three times and the washings were combined and lyophilized .
Stability studies
The lyophilized crude venom, obtained by both methods, was reconstituted with the various stabilizing solutions, then centrifuged at 5000 rev per min for 10 min and used for the subsequent chemical and physiopharmacological studies. A pH stability test was done using 5 mg dry weight of crude venom (aspiration method) per ml of 0-05 M sodium phosphate buffer solution at pH 5-7, 6-5, 7-0 and 8.0, with a control in distilled water. All solutions were maintained at 5° and tested for lethality at designated intervals.
Venom of the Scorpionflsh
71
To determine the temperature stability of the crude venom, 100 mg dry weight (batch method) was added to 10 ml of the phosphate buffer containing 0-2 M NaCl. The extract was centrifuged and the supernatant divided into four samples. The samples were maintained at 80°, 50°, 25°, and a control at 5°. These were assayed for lethality at 30 min intervals as described elsewhere. The 27° and 5° samples were tested up to 72 hr. The effect of 10-4 M reduced glutathione (GSH), cysteine and parachloromercuribenzoate (PCMB) on the lethality of crude venom extracts was determined by adding these to separate samples of 5 mg dry weight of crude venom (aspiration method) in 1 -0 ml of 0-05 M phosphate buffer, pH 8-0, at 5°. A control sample was maintained in phosphate buffer. Samples were assayed for lethality on preparation, and at one, four and seven days. Cleland's reagent, EDTA, and a mixture of the two, all at 10-3 M, were added to samples containing 5 mg dry weight of crude venom (aspiration method) per ml of 0-05 M phosphate buffer, pH 7-4, at 5°. A control sample was maintained in phosphate buffer. Samples were periodically tested for lethality up to 21 days. Gelfiltration
Sephadex G-50 and G-200 were swollen in 0-05 M sodium phosphate buffer, pH 7-4, containing 0-9 per cent NaCl and 10-3 M Cleland's reagent. The gels were equilibrated at 5°. Dried crude venom (batch method), 200 mg, was reconstituted with 4 ml of 0-05 M phosphate buffer, pH 7-4, containing 0-9 per cent NaCl and 10-3 M Cleland's reagent. The extract was centrifuged and the supernatant applied to a 28x2-5 cm Sephadex G-50 column . The first peak from the column (tubes 8-16) was lyophilized, reconstituted with 3 ml of deionized water and rechromatographed on a 2-5 x 20 cm Sephadex G-200 column . Dried crude venom (batch extraction), 350 mg, containing 100 mg total protein, was reconstituted with 4 ml of the stabilizing solution, centrifuged, and the supernatant applied to Sephadex G-200 column of 2-5x28 cm. Fractions were collected in a Beckman 132 fraction collector or an automatic Warner Chilcott time-drop fraction collector and analyzed on a Beckman DB-G grating spectrophotometer at 260 and 280 mp. A Beckman transistorized hydrogen lamp power supply and a GME automatic transferator were used . All operations were carried out at 5°. Ion exchange chromatography
Diethylaminoethyl cellulose (DEAE-C, Bio-Rad Lab) was washed with 0.5 M HCI and 0-5 M NaOH, and then with deionized water until the pH reached 7-0 . It was further treated with 0-05 M sodium phosphate buffer, pH 7-4, until the effluent reached pH 7-4. The DEAE-C slurry was equilibrated at 5° and poured into a 2-5 cm column to a height of 30 cm. Dried crude venom (aspiration method), 50 mg, was applied to the column and eluted with 0-05 M sodium phosphate. A flow rate of 10 ml per hr was established. After the first peak was eluted the salt concentration was raised stepwise from 0-1 M to 0-3 M NaCl. Chromatography was conducted at 5°, and fractions were collected and assayed for lethality. Electrophoresis
The Millipore phoroslide system was used for the electrophoresis of both the crude venom and its fractions. Electrophoresis of 10 mg per ml of venom samples was carried out for 15 min in triaminoacetic acid buffer, pH 4-0, and sodium barbital buffer, pH 8-6. The cellulose acetate strips were stained with buffalo black solution and destained with 10 per cent acetic acid .
72
R. C. SCHAEFFER, JR., R. W. CARLSON and F. E. RUSSELL
Protein
The LowRY et al. (1951) method was employed to determine the protein concentrations of the crude venom and its fractions. A calibration curve was prepared with crystalline bovine albumin, and concentrations were read at 500 and 750 m1.. Bioassay
Venom extracts were assayed for lethality by i.v. injection into 18-24 g male Swiss albino mice . The FjmD--MuENcH (1938) method was used for specific LD50 determinations of the crude venom. The DIXON (1965) method was used in determining lethality before and during all physiopharmacological and chemical experiments. A 10 min survival or death end point was chosen for the lethality determinations . RESULTS
Venom extraction and assay Table 1 shows the LD50 determinations of the crude venom. The aspiration method gave a product which was relatively more pure than that obtained by the batch method but it appeared to be less stable . This may be due, in part, to the effects of the trauma of aspiration which no doubt damages most of the cells within the glandular grooves. Although the batch method gave a greater number of impurities it also gave more venom protein (8-3 mg dry weight as compared to 0-6 mg per spine) . The weights the inorganic salts contributed to the doses cited for the LD 50 were not calculated, but they would appear to be similar for the two extraction methods, and relatively insignificant . TABLE 1 . Dose (mg/kg) 0-5 0-75 1.00 1-25 6-0 7-0 8-0 9.0
LDyo DETERMINATIONS OF LYOPHILIZED
Scorpaena guttata vENOM
Aspiration method Batch method Deaths/mice tested 0/10 2/10 4/10 7/10
0/10 3/10 5110 10/10
Aspiration method LD so : 1-1 mg/kg (1-0 mg protein/kg) body weight . Batch method LDw : 7-7 mg/kg (2-6 mg protein/kg) body weight.
Stability studies
Venom extracts prepared by either the aspiration or batch method were extremely unstable . In addition to the findings related to their instability at different pH and temperatures, and to trauma of the venom-containing cells, it was found that repeated freezing and thawing, changes in dilution and heavy metal contamination also affected stability. Freshly prepared extracts were more stable than extracts prepared from frozen spines, even when the spines had been maintained at -60°. Some toxicity was lost on lyophilization and lyophilized samples lost their lethal property faster than freshly prepared extracts .
Venom of the Scorpionfish
73
Figure 1 shows the relative stability of a venom extract at different pH values. It can be seen that only at pH 8-0 was the crude venom relatively stable at 5°. At a lower pH the extract was no more stable than a control extract in distilled water. Figure 2 shows the relative stability of a venom sample extracted in Cleland's reagent at different temperatures over a 72-hr period . The venom was extremely heat labile, even in Cleland's reagent, losing all of its lethality in 30 min at 50°. Venom maintained at 5° pH STABILITY TEST a-* e- " -~ -~
CONTROL pH - 8.0 pH-6 .5 p H-7.0 g2-g3 p H-5.7
8
24
48
72
TIME- hn .
88
FIG. 1 . STABILITY IN MICE OF THE LETHAL PROPERTY OF Scorpaena guttata VENOM AT DIFFERENT pH VALUES AS DETERMINED IN MICE.
HEAT STABILITY TEST
200 100. Go .
0-0
&--"
27'
M-B Bdand 8d
20i 10 Oâ
.
d
10
15 .
TIME -bru .
20
72
FIG. 2. STABILITY OF THE LETHAL PROPERTY OF THE VENOM AT DIFFERENT TEMPERATURES AS DETERMINED IN MICE.
Original sample (batch extraction) was extracted in Cleland's reagent.
74
R. C. SCHAEFFER, JR., R. W. CARLSON and F. E. RUSSELL CHEMICAL STABILITY TEST S
so. j.40~
~=CONTROL -~ CYSTEINE- I0 IA -~SSH-10M ,-, P C M S-1 d.4M
ô 30 "
0
to10~
a-
aa
as
HOURS
164
FIG. 3. STABILITY OF THE LETHAL PROPERTY OF THE VENOM, AS DETERMINED IN MICE, IN THE PRESENCE OF DIFFERENT CHEMICALS. CHEMICAL STABILITY TEST 3
M-BCONTROL &-A EDTA-10 M O--OCLELAND'StEDTA-Id=M O-OCLELAND'S- 16aM .
DAYS LETHAL PROPERTY OF THE VENOM, AS DETERMINED IN MICE, IN THE FIG. 4. STABILITY OF THE PRESENCE OF STABn1zING SOLUTIONS.
was active throughout the test period of 14 days. It seems likely that the use of Cleland's reagent during the extraction procedure reduces the loss in the lethal property of the venom. Preliminary studies indicated that without this reagent the loss in lethality was much quicker, even at low temperatures . Figure 3 shows the effects of GSH, cysteine and PCMB on the lethal property of a venom extract at pH 8-0 and at 5°. None of these reagents provided any stabilizing effect on the venom extract over long periods of time . However, GSH and cysteine could possibly provide some stabilizing effect during the first 4 days. After that lethality was quickly lost. Figure 4 shows the effects of EDTA, Cleland's reagent, and EDTA and Cleland's reagent on the lethality of the venom . It can be seen that Cleland's reagent lends considerable stability to the extract up to 21 days, the end of the test period . The combination
Venom of the Scorpionfish
75
of Cleland's and EDTA also helped stabilize the lethal activity, as did EDTA alone, although to a lesser extent . During these and other stability tests it was observed that extracts in 0-05 M sodium phosphate buffer tended to precipitate unless 0-9 per cent NaCl was added. Thus, in these and all other tests where extracts were held for long periods of time, saline was routinely added to each sample . Gelfiltration Figure 5 shows the elution profile from a Sephadex G-50 run. The first peak (tubes 8-16) was lethal to mice at a dose of 1-5 mg protein per kg body weight . The second and third peaks did not contain material lethal to mice. They had a greater absorbance at 260 mp than at 280 mp. Figure 6 shows the elution profile when tubes 8-16 of the G-50 column were rechromatographed on a G-200 column . The first peak represents large molecular weight material which did not enter the gel. This material did not produce death or illness in mice . The second and third peaks, representing fractions below 800,000 molecular weight, as determined by Sephadex G-200 characteristics, did not cause death when injected into mice, but they did produce an acute illness characterized by tachypnea, hyperpnea and transient prostration. Figure 7 shows the elution profile of crude venom (batch extraction) on Sephadex G-200. The venom was resolved into four fractions ; the second fraction (tubes 28--37) contained material which was lethal to mice. The LD6p of tube 33 was 0-9 mg protein per kg body weight . From an analysis of the optical density of the elution profile it was estimated that the second peak contained approximately 20 per cent of the total protein. The other peaks did not contain material lethal to mice. Ion exchange chromatography Figure 8 shows the elution profile of crude venom (aspiration method) chromatographed on DEAE-C. After a small initial peak was eluted, the NaCl concentration was 8EPHAOtX 6-
eo
-
t " Oayt --- : 1 600p t---I Lethal Freatlen a z W a
l,0
=Ô TUBE NUMBER
Fia. 5. ELu71oN PRoFILz of ZOO mg of LYoPHII1z® CRUD£ v£Nou (BATCH 1JrmACTIoN) CHROMATOORAPIID ON S£PHAD£x G-50 .
76
R. C. SCHAEFFER, JR ., R. W. CARLSON and F. E. RUSSELL 6EPHADEX 8-200
2o
FIG. 6.
a0
TUBE
NUMBER
ELUTION PRoFuz OF THE tzrHAr FRACTION (TUBES 8-16) FROM SEPARATION sHOwN IN FIG . 5, REcHRomAToGRAPHED oN SEPHADEx G-200.
SEPHADE%
6-200
- 260 MN . . . .. 260 MN ---i
Lethality
u
F d O
10
20
30
TUBE
NUMBER
40
FIG. i. ELUTION PROFILE of 350 mg OF LYOPHIiizED cRUDE vENOM, coNTAINING 100 PROTEIN, cHROmATOGRAPHm ON SEPHADEx G-200.
mg
raised stepwise . Three additional fractions were resolved but no lethal activity was detected in any fraction. However, only 50 mg of crude venom was used and Cleland's reagent was not employed in the experiment . Electrophoresis
Although cellulose acetate strip electrophoresis did not give as distinct bands as would be desired, it did provide a method for rapid bipolar analysis of the crude venom and its fractions. Extracts prepared by either the batch or aspiration method displayed three
Venom of the Scorpionfish
Fm . 8.
ELUTION PROFILE of
77
50 mg LYopHIL=D CRUDE VENOM (ASPIRATION ExmACnoN) cHRomAror,RAPHED oN DEAE-C . .
to four anionic and two to three cationic bands at pH 8-6. A fast moving anionic band appeared to be related to the non-lethal low molecular weight material obtained with the Sephadex separation . At pH 4-0 the crude venom showed four cationic bands. The lethal fraction from the Sephadex G-200 separation showed two indistinct cationic bands. At pH 8-6 the lethal fraction from the Sephadex G-200 showed one slow moving anionic band and two slow moving cationic bands. In all of the electrophoretic studies a considerable amount of material remained at the origin . DISCUSSION
It is obvious that the lethal property of S. guttata venom is extremely unstable . From the results with PCMB (Fig. 3) it would appear that sulfhydryl groups are required for the lethal action of the venom. Cysteine and GSH appear to have no significant effect on this activity until several days have passed ; then it is possible that they may catalyze the oxidation of sulfhydryl groups, causing a rapid decline in lethality. Cleland's reagent is a much more effective sulfhydryl reagent. The various stabilization studies indicate that the present preferred method for obtaining a stable extract of the crude venom is to (1) Extract from fresh spines by the batch method, using distilled water at 5° (2) Immediately lyophilize
(3) Reconstitute in 0-05 M sodium phosphate buffer, pH 7-4, with 0-9 per cent NaCl and 10-8 M Cleland's reagent at 5°, and (4) Centrifuge.
If the extract must be held for any length of time it should be kept at as low a temperature as possible without freezing . Under these conditions the lethal activity of the extract should remain stable up to 21 days . Fractionation of the venom indicates that the lethal property is associated with protein(s) having a molecular weight of more than 50,000 and less than 800,000. Following separation on Sephadex G-200 the semi-purified lethal fraction has an i.v. LD50 in mice of 0-9 mg protein per kg body weight, while the LD50 of the crude venom (batch method)
78
R. C. SCHAEFFER, JR., R. W. CARLSON and F. E. RUSSELL
is 2-6 mg protein per kg . The Sephadex peak containing the lethal activity contains approximately 20 per cent of the total protein, when the batch method of extraction is used . When studied on cellulose acetate strips, this peak was found to consist of more than one protein. Acknowledgements-The authors thank Dr . J. W. DUBNOFF and Dr . P. R. SAUNDEits for technical advice, and R. ScHNrroER, R. LrNsKY and T. COOKE for their assistance in obtaining many of the fish . REFERENCES CAmERoN, A M. and ENDEAN, R. (1966) The venom apparatus of the scorpionfish Notesthes robusta. Toxicon 4, 111. CLELAND, W. W. (1964) Dithiothreitol, a new protective reagent for SH groups. Biochemistry 3, 480. DixoN, W. J. (1%5) The up and down method for small samples. l. Am. statist. Ass. 60, 967. HALSt7AD, B. W. (1951) Injurious effects from the sting of the scorpionfish, Scorpaena guttata Girard . Calif. Med. 74, 395. HAtsrEAD, B. W. (1955) The venom apparatus of the California seorpionfish, Scorpaena guttata. Trans. Am . microscop. Soc. 74,145 . LowRY, H., RosBBROUGH, N. J., FARR, A. L. and RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. l. Biochem. 193, 265. REED, L. H. and MUENcH, H. (1938) A simple method of estimating 50% end points . Am . .1. Hyg. 27, 493. RussELL, F. E. (1965) Marine toxins and venomous and poisonous marine animals. In : Advances in Marine Biology, Vol. 2, p. 256 (RussELL, F. S., Ed .) . London : Academic Press. RussE.L, F. E. (1967) Comparative pharmacology of some animal toxins . Fedn Proc. Fedn Am. Soc. exp. Biol . 26, 1206. SAUNDEm, P. R. (1959) Venoms of scorpionfishes . Proc. west. Pharmacol. Soc. 2, 47 . SAUNDERs, P. R. (1960) Pharmacological and chemical studies of the venom of the stonefish (genus SynanceJa) and other scorpionfishes . Ann. N.Y. Acad . Scl. 90, 798.
SOME CHEMICAL PROPERTIES OF THE VENOM OF THE SCORPIONFISH SCORPAENA GUITATA* R1cHARn C. ScHAEFFER, JR.,t RICHARD W. CARrsoNt and FiNDLAY E. RUSSELL Laboratory of Neurological Research, University of Southern California School of Medicine, Los Angeles, California, U.S .A. (Acceptedforpub&atlon 1 July 1970) Abstract-A method for extracting and stabilizing a fish venom is proposed . Extracts from fresh venomous spines of Scorpaena guttata were prepared in distilled water at 5°, lyophilized, reconstituted in 0-05 M sodium phosphate buffer, pH 7-4, with 0-9 per cent NaCl and 10 -3 M Cleland's reagent at 5°, and centrifuged. Using this technique, extracts were found to be stable up to 21 days . Fractionation indicated that the lethal property was associated with protein(s) having a molecular weight of more than 50,000 and less than 800,000. The semi-purified lethal fraction had an intravenous LD . in mice of 0-9 mg protein per kg body weight, while the LD+e of the crude venom was 2-6 mg protein per kg . This is the first report on the separation of a fraction of a fish venom which is more lethal than the crude venom.
members of the family Scorpaenidae, are a varied group of about 350 species. Included in this family are the stonefishes, lionfishes or zebrafishes, bullrout and waspfish . They are widely distributed throughout all tropical and most temperate seas. The greatest number of species is found in the tropical Indo-Pacific . For the most part they are shallow water fishes living in or around rocks, coral reefs or kelp beds, with which they often blend favorably. Some species bury themselves in the sand and may lie motionless for long periods of time . Thus, they are often handled or trod upon unknowingly, that is, until envenomation occurs. The stings of these fish lead to pain, localized swelling, discoloration and paresthesis around the wound, and in some cases to lymphadenitis, lymphadenopathy, nausea, vomiting, weakness, pallor and syncopy. In the more severe cases there may be intense pain, respiratory distress, shock, coma and death (RUSSELL, 1965). The venom of the scorpionfishes is contained within the tissues which envelop certain of the dorsal, pelvic and anal fin spines . The number of venomous spines varies with the different scorpaenid species, as does the structure of the venom gland or venom-containing tissues . Envenomation occurs through mechanical pressure on the spine, which tears the integumentary sheath encasing the spine, permitting the venom to escape into the wound. The toxins of these various piscine species appear to have certain chemical and physiopharmacological properties in common. These have been reviewed in detail elsewhere (SAUNDERS, 1959; RUSSELL, 1965; RUSSELL, 1969). The California scorpionfish, Scorpaena guttata Girard, commonly called the sculpin in California, is the only member of the genus Scorpaena found in Southern California THE scomoNFIsims,
*Supported by ONR contracts NR 305-786 and NR 104-793 to F. E. Russell. fFellows, USPHS Training Program GM01700. 69
70
R. C. SCHAEFFER, 7R., R. W. CARLSON and F. E. RUSSELL
waters . This fish may reach a length of 40 cm and ranks among the most popular fish taken by rod and reel fishermen, accounting for the frequency of stings by this animal (HALsTEAD, 1951 ; RussELL, 1965). The venom apparatus of this species consists of 12 dorsal spines, 3 anal spines, 2 pelvic spines and their enveloping integumentary sheath (HAISTEAD, 1955). The venom of the California scorpionfish is contained within certain specialized secretory cells in the slender, fusiform strand of greyish tissue found lying within the distal one-half or two-thirds of the glandular grooves on either side of the sting. Extracts of these tissues have been found to be opalescent, of neutral pH, very unstable and nondialyzable. Treatment with a chelating agent (EDTA), or reduced glutathione (GSH), or with a proteinase inhibitor (diisopropylfluorophosphate) did not preserve the extract's toxicity. Partial purification of the extract was achieved by salting-out with ammonium sulfate, and by DEAF-cellulose column chromatography. With the latter method, purification of the toxin to 11 - 0,ug protein per mouse LDP was sometimes obtained . A toxic fraction was also obtained adjacent to the origin of the small pore polyacrylamide gel in which the venom extract was resolved by disc electrophoresis . The lethal fraction was thought to be a high molecular weight protein and moderately negatively charged at pH 7-4 (Taylor, unpublished thesis, 1963 ; Saunders, unpublished data). The present study was initiated to determine certain of the chemical and physiopharmacological properties of the venom of Scorpaena guttata . This report deals with attempts to stabilize the toxin and to define certain of its chemical properties . A subsequent paper will treat of the physiopharmacology of this toxin. MATERIALS AND METHODS
Collection and venom extraction
The fish were caught on hook and line off San Pedro, California and transported to the laboratory in aerated containers . They were then sacrificed and the first 12 dorsal, three anal and two pelvic spines quickly removed. Two methods of venom extraction were studied (a) Aspiration method. The integumentary sheath covering each spine was stripped to the base of the spine and the venom was aspirated from the glandular grooves with a micropipette. The pipette was connected to a vacuum source and a collection flask maintained at 5° . The extract was then centrifuged at 5000 rev per min for 10 min and the supernatant lyophilized . (b) Batch method. Stripped spines were immersed in a beaker of distilled water at 5° . The beaker was gently agitated for 5 min and the solution decanted . This procedure was carried out three times and the washings were combined and lyophilized .
Stability studies
The lyophilized crude venom, obtained by both methods, was reconstituted with the various stabilizing solutions, then centrifuged at 5000 rev per min for 10 min and used for the subsequent chemical and physiopharmacological studies. A pH stability test was done using 5 mg dry weight of crude venom (aspiration method) per ml of 0-05 M sodium phosphate buffer solution at pH 5-7, 6-5, 7-0 and 8.0, with a control in distilled water. All solutions were maintained at 5° and tested for lethality at designated intervals.
Venom of the Scorpionflsh
71
To determine the temperature stability of the crude venom, 100 mg dry weight (batch method) was added to 10 ml of the phosphate buffer containing 0-2 M NaCl. The extract was centrifuged and the supernatant divided into four samples. The samples were maintained at 80°, 50°, 25°, and a control at 5°. These were assayed for lethality at 30 min intervals as described elsewhere. The 27° and 5° samples were tested up to 72 hr. The effect of 10-4 M reduced glutathione (GSH), cysteine and parachloromercuribenzoate (PCMB) on the lethality of crude venom extracts was determined by adding these to separate samples of 5 mg dry weight of crude venom (aspiration method) in 1 -0 ml of 0-05 M phosphate buffer, pH 8-0, at 5°. A control sample was maintained in phosphate buffer. Samples were assayed for lethality on preparation, and at one, four and seven days. Cleland's reagent, EDTA, and a mixture of the two, all at 10-3 M, were added to samples containing 5 mg dry weight of crude venom (aspiration method) per ml of 0-05 M phosphate buffer, pH 7-4, at 5°. A control sample was maintained in phosphate buffer. Samples were periodically tested for lethality up to 21 days. Gelfiltration
Sephadex G-50 and G-200 were swollen in 0-05 M sodium phosphate buffer, pH 7-4, containing 0-9 per cent NaCl and 10-3 M Cleland's reagent. The gels were equilibrated at 5°. Dried crude venom (batch method), 200 mg, was reconstituted with 4 ml of 0-05 M phosphate buffer, pH 7-4, containing 0-9 per cent NaCl and 10-3 M Cleland's reagent. The extract was centrifuged and the supernatant applied to a 28x2-5 cm Sephadex G-50 column . The first peak from the column (tubes 8-16) was lyophilized, reconstituted with 3 ml of deionized water and rechromatographed on a 2-5 x 20 cm Sephadex G-200 column . Dried crude venom (batch extraction), 350 mg, containing 100 mg total protein, was reconstituted with 4 ml of the stabilizing solution, centrifuged, and the supernatant applied to Sephadex G-200 column of 2-5x28 cm. Fractions were collected in a Beckman 132 fraction collector or an automatic Warner Chilcott time-drop fraction collector and analyzed on a Beckman DB-G grating spectrophotometer at 260 and 280 mp. A Beckman transistorized hydrogen lamp power supply and a GME automatic transferator were used . All operations were carried out at 5°. Ion exchange chromatography
Diethylaminoethyl cellulose (DEAE-C, Bio-Rad Lab) was washed with 0.5 M HCI and 0-5 M NaOH, and then with deionized water until the pH reached 7-0 . It was further treated with 0-05 M sodium phosphate buffer, pH 7-4, until the effluent reached pH 7-4. The DEAE-C slurry was equilibrated at 5° and poured into a 2-5 cm column to a height of 30 cm. Dried crude venom (aspiration method), 50 mg, was applied to the column and eluted with 0-05 M sodium phosphate. A flow rate of 10 ml per hr was established. After the first peak was eluted the salt concentration was raised stepwise from 0-1 M to 0-3 M NaCl. Chromatography was conducted at 5°, and fractions were collected and assayed for lethality. Electrophoresis
The Millipore phoroslide system was used for the electrophoresis of both the crude venom and its fractions. Electrophoresis of 10 mg per ml of venom samples was carried out for 15 min in triaminoacetic acid buffer, pH 4-0, and sodium barbital buffer, pH 8-6. The cellulose acetate strips were stained with buffalo black solution and destained with 10 per cent acetic acid .
72
R. C. SCHAEFFER, JR., R. W. CARLSON and F. E. RUSSELL
Protein
The LowRY et al. (1951) method was employed to determine the protein concentrations of the crude venom and its fractions. A calibration curve was prepared with crystalline bovine albumin, and concentrations were read at 500 and 750 m1.. Bioassay
Venom extracts were assayed for lethality by i.v. injection into 18-24 g male Swiss albino mice . The FjmD--MuENcH (1938) method was used for specific LD50 determinations of the crude venom. The DIXON (1965) method was used in determining lethality before and during all physiopharmacological and chemical experiments. A 10 min survival or death end point was chosen for the lethality determinations . RESULTS
Venom extraction and assay Table 1 shows the LD50 determinations of the crude venom. The aspiration method gave a product which was relatively more pure than that obtained by the batch method but it appeared to be less stable . This may be due, in part, to the effects of the trauma of aspiration which no doubt damages most of the cells within the glandular grooves. Although the batch method gave a greater number of impurities it also gave more venom protein (8-3 mg dry weight as compared to 0-6 mg per spine) . The weights the inorganic salts contributed to the doses cited for the LD 50 were not calculated, but they would appear to be similar for the two extraction methods, and relatively insignificant . TABLE 1 . Dose (mg/kg) 0-5 0-75 1.00 1-25 6-0 7-0 8-0 9.0
LDyo DETERMINATIONS OF LYOPHILIZED
Scorpaena guttata vENOM
Aspiration method Batch method Deaths/mice tested 0/10 2/10 4/10 7/10
0/10 3/10 5110 10/10
Aspiration method LD so : 1-1 mg/kg (1-0 mg protein/kg) body weight . Batch method LDw : 7-7 mg/kg (2-6 mg protein/kg) body weight.
Stability studies
Venom extracts prepared by either the aspiration or batch method were extremely unstable . In addition to the findings related to their instability at different pH and temperatures, and to trauma of the venom-containing cells, it was found that repeated freezing and thawing, changes in dilution and heavy metal contamination also affected stability. Freshly prepared extracts were more stable than extracts prepared from frozen spines, even when the spines had been maintained at -60°. Some toxicity was lost on lyophilization and lyophilized samples lost their lethal property faster than freshly prepared extracts .
Venom of the Scorpionfish
73
Figure 1 shows the relative stability of a venom extract at different pH values. It can be seen that only at pH 8-0 was the crude venom relatively stable at 5°. At a lower pH the extract was no more stable than a control extract in distilled water. Figure 2 shows the relative stability of a venom sample extracted in Cleland's reagent at different temperatures over a 72-hr period . The venom was extremely heat labile, even in Cleland's reagent, losing all of its lethality in 30 min at 50°. Venom maintained at 5° pH STABILITY TEST a-* e- " -~ -~
CONTROL pH - 8.0 pH-6 .5 p H-7.0 g2-g3 p H-5.7
8
24
48
72
TIME- hn .
88
FIG. 1 . STABILITY IN MICE OF THE LETHAL PROPERTY OF Scorpaena guttata VENOM AT DIFFERENT pH VALUES AS DETERMINED IN MICE.
HEAT STABILITY TEST
200 100. Go .
0-0
&--"
27'
M-B Bdand 8d
20i 10 Oâ
.
d
10
15 .
TIME -bru .
20
72
FIG. 2. STABILITY OF THE LETHAL PROPERTY OF THE VENOM AT DIFFERENT TEMPERATURES AS DETERMINED IN MICE.
Original sample (batch extraction) was extracted in Cleland's reagent.
74
R. C. SCHAEFFER, JR., R. W. CARLSON and F. E. RUSSELL CHEMICAL STABILITY TEST S
so. j.40~
~=CONTROL -~ CYSTEINE- I0 IA -~SSH-10M ,-, P C M S-1 d.4M
ô 30 "
0
to10~
a-
aa
as
HOURS
164
FIG. 3. STABILITY OF THE LETHAL PROPERTY OF THE VENOM, AS DETERMINED IN MICE, IN THE PRESENCE OF DIFFERENT CHEMICALS. CHEMICAL STABILITY TEST 3
M-BCONTROL &-A EDTA-10 M O--OCLELAND'StEDTA-Id=M O-OCLELAND'S- 16aM .
DAYS LETHAL PROPERTY OF THE VENOM, AS DETERMINED IN MICE, IN THE FIG. 4. STABILITY OF THE PRESENCE OF STABn1zING SOLUTIONS.
was active throughout the test period of 14 days. It seems likely that the use of Cleland's reagent during the extraction procedure reduces the loss in the lethal property of the venom. Preliminary studies indicated that without this reagent the loss in lethality was much quicker, even at low temperatures . Figure 3 shows the effects of GSH, cysteine and PCMB on the lethal property of a venom extract at pH 8-0 and at 5°. None of these reagents provided any stabilizing effect on the venom extract over long periods of time . However, GSH and cysteine could possibly provide some stabilizing effect during the first 4 days. After that lethality was quickly lost. Figure 4 shows the effects of EDTA, Cleland's reagent, and EDTA and Cleland's reagent on the lethality of the venom . It can be seen that Cleland's reagent lends considerable stability to the extract up to 21 days, the end of the test period . The combination
Venom of the Scorpionfish
75
of Cleland's and EDTA also helped stabilize the lethal activity, as did EDTA alone, although to a lesser extent . During these and other stability tests it was observed that extracts in 0-05 M sodium phosphate buffer tended to precipitate unless 0-9 per cent NaCl was added. Thus, in these and all other tests where extracts were held for long periods of time, saline was routinely added to each sample . Gelfiltration Figure 5 shows the elution profile from a Sephadex G-50 run. The first peak (tubes 8-16) was lethal to mice at a dose of 1-5 mg protein per kg body weight . The second and third peaks did not contain material lethal to mice. They had a greater absorbance at 260 mp than at 280 mp. Figure 6 shows the elution profile when tubes 8-16 of the G-50 column were rechromatographed on a G-200 column . The first peak represents large molecular weight material which did not enter the gel. This material did not produce death or illness in mice . The second and third peaks, representing fractions below 800,000 molecular weight, as determined by Sephadex G-200 characteristics, did not cause death when injected into mice, but they did produce an acute illness characterized by tachypnea, hyperpnea and transient prostration. Figure 7 shows the elution profile of crude venom (batch extraction) on Sephadex G-200. The venom was resolved into four fractions ; the second fraction (tubes 28--37) contained material which was lethal to mice. The LD6p of tube 33 was 0-9 mg protein per kg body weight . From an analysis of the optical density of the elution profile it was estimated that the second peak contained approximately 20 per cent of the total protein. The other peaks did not contain material lethal to mice. Ion exchange chromatography Figure 8 shows the elution profile of crude venom (aspiration method) chromatographed on DEAE-C. After a small initial peak was eluted, the NaCl concentration was 8EPHAOtX 6-
eo
-
t " Oayt --- : 1 600p t---I Lethal Freatlen a z W a
l,0
=Ô TUBE NUMBER
Fia. 5. ELu71oN PRoFILz of ZOO mg of LYoPHII1z® CRUD£ v£Nou (BATCH 1JrmACTIoN) CHROMATOORAPIID ON S£PHAD£x G-50 .
76
R. C. SCHAEFFER, JR ., R. W. CARLSON and F. E. RUSSELL 6EPHADEX 8-200
2o
FIG. 6.
a0
TUBE
NUMBER
ELUTION PRoFuz OF THE tzrHAr FRACTION (TUBES 8-16) FROM SEPARATION sHOwN IN FIG . 5, REcHRomAToGRAPHED oN SEPHADEx G-200.
SEPHADE%
6-200
- 260 MN . . . .. 260 MN ---i
Lethality
u
F d O
10
20
30
TUBE
NUMBER
40
FIG. i. ELUTION PROFILE of 350 mg OF LYOPHIiizED cRUDE vENOM, coNTAINING 100 PROTEIN, cHROmATOGRAPHm ON SEPHADEx G-200.
mg
raised stepwise . Three additional fractions were resolved but no lethal activity was detected in any fraction. However, only 50 mg of crude venom was used and Cleland's reagent was not employed in the experiment . Electrophoresis
Although cellulose acetate strip electrophoresis did not give as distinct bands as would be desired, it did provide a method for rapid bipolar analysis of the crude venom and its fractions. Extracts prepared by either the batch or aspiration method displayed three
Venom of the Scorpionfish
Fm . 8.
ELUTION PROFILE of
77
50 mg LYopHIL=D CRUDE VENOM (ASPIRATION ExmACnoN) cHRomAror,RAPHED oN DEAE-C . .
to four anionic and two to three cationic bands at pH 8-6. A fast moving anionic band appeared to be related to the non-lethal low molecular weight material obtained with the Sephadex separation . At pH 4-0 the crude venom showed four cationic bands. The lethal fraction from the Sephadex G-200 separation showed two indistinct cationic bands. At pH 8-6 the lethal fraction from the Sephadex G-200 showed one slow moving anionic band and two slow moving cationic bands. In all of the electrophoretic studies a considerable amount of material remained at the origin . DISCUSSION
It is obvious that the lethal property of S. guttata venom is extremely unstable . From the results with PCMB (Fig. 3) it would appear that sulfhydryl groups are required for the lethal action of the venom. Cysteine and GSH appear to have no significant effect on this activity until several days have passed ; then it is possible that they may catalyze the oxidation of sulfhydryl groups, causing a rapid decline in lethality. Cleland's reagent is a much more effective sulfhydryl reagent. The various stabilization studies indicate that the present preferred method for obtaining a stable extract of the crude venom is to (1) Extract from fresh spines by the batch method, using distilled water at 5° (2) Immediately lyophilize
(3) Reconstitute in 0-05 M sodium phosphate buffer, pH 7-4, with 0-9 per cent NaCl and 10-8 M Cleland's reagent at 5°, and (4) Centrifuge.
If the extract must be held for any length of time it should be kept at as low a temperature as possible without freezing . Under these conditions the lethal activity of the extract should remain stable up to 21 days . Fractionation of the venom indicates that the lethal property is associated with protein(s) having a molecular weight of more than 50,000 and less than 800,000. Following separation on Sephadex G-200 the semi-purified lethal fraction has an i.v. LD50 in mice of 0-9 mg protein per kg body weight, while the LD50 of the crude venom (batch method)
78
R. C. SCHAEFFER, JR., R. W. CARLSON and F. E. RUSSELL
is 2-6 mg protein per kg . The Sephadex peak containing the lethal activity contains approximately 20 per cent of the total protein, when the batch method of extraction is used . When studied on cellulose acetate strips, this peak was found to consist of more than one protein. Acknowledgements-The authors thank Dr . J. W. DUBNOFF and Dr . P. R. SAUNDEits for technical advice, and R. ScHNrroER, R. LrNsKY and T. COOKE for their assistance in obtaining many of the fish . REFERENCES CAmERoN, A M. and ENDEAN, R. (1966) The venom apparatus of the scorpionfish Notesthes robusta. Toxicon 4, 111. CLELAND, W. W. (1964) Dithiothreitol, a new protective reagent for SH groups. Biochemistry 3, 480. DixoN, W. J. (1%5) The up and down method for small samples. l. Am. statist. Ass. 60, 967. HALSt7AD, B. W. (1951) Injurious effects from the sting of the scorpionfish, Scorpaena guttata Girard . Calif. Med. 74, 395. HAtsrEAD, B. W. (1955) The venom apparatus of the California seorpionfish, Scorpaena guttata. Trans. Am . microscop. Soc. 74,145 . LowRY, H., RosBBROUGH, N. J., FARR, A. L. and RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. l. Biochem. 193, 265. REED, L. H. and MUENcH, H. (1938) A simple method of estimating 50% end points . Am . .1. Hyg. 27, 493. RussELL, F. E. (1965) Marine toxins and venomous and poisonous marine animals. In : Advances in Marine Biology, Vol. 2, p. 256 (RussELL, F. S., Ed .) . London : Academic Press. RussE.L, F. E. (1967) Comparative pharmacology of some animal toxins . Fedn Proc. Fedn Am. Soc. exp. Biol . 26, 1206. SAUNDEm, P. R. (1959) Venoms of scorpionfishes . Proc. west. Pharmacol. Soc. 2, 47 . SAUNDERs, P. R. (1960) Pharmacological and chemical studies of the venom of the stonefish (genus SynanceJa) and other scorpionfishes . Ann. N.Y. Acad . Scl. 90, 798.