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Effect of proteolytic enzymes on the venom of the sea wasp Chironex fleckeri
T°xtc°n, 1971, Vol. 9, pp . 43133. P~amon Prey. Printed in drat Britain
EFFECT OF PROTEOLYTIC ENZYMES ON THE VENOM OF THE SEA WA5P CHIRONEX FLECKERI A. G. M. Maiut and E. H. BAXTER
Commonwealth Serum Laboratories, Parkville, Victoria 3052, Australia (Acceptedfor publkation 26 Apri11971)
Tim Box jellyfish (Chironex ,fileckeri, SovTxcrn-r, 1956) known as the sea wasp has been responsible for fatal stingings in Australian tropical waters (BARNES, 1966). Our studies on the venom properties (BAXTER and MARK, 1969) and immunogenicity (BAXTER et al., 1968 ; BAxTER et al., 1970) suggested that the active molecules are moderately large and probably proteinaceous. Further support for this view was given by the report of ENDEAN and HENDERSON (1969) that saline extracts of intracapsular material from the nematocysts of C. ,~feckeri lost their activity against muscle if incubated with a bacterial protease . It therefore was of interest to obtain evidence as to the nature of the components of the venom concerned with the lethal, haemolytic and dermonecrotic properties, by studying the effects of incubation of the venom with proteolytic enzymes. Venom extracts were kindly supplied by Dr . J. H. Barnes of Cairns, Queensland, and were kept frozen at -15°from time of receipt until immediately before use. Collection of the venom had been made by electrical `milking' techniques (BARNES, 1967). The lethal, haemolytic and dermonecrotic effects of the venom were assayed as previously described (BAXTER et al., 1968 ; BAXTER and Maxi, 1969). Crude trypsin was obtained from Difco (1 :300) and crystalline trypsin was lyophilized 2-times crystallized trypsin (Worthington Biochem. Corpn., Freehold, N.J., U.S .A.). The diluent for trypsin was Dulbecco phosphate buffered saline containing calcium and magnesium (Dui.BE000 and VocT, 1954). Pronase P (45,000 PUK, Sogo Boeki Kaisha Ltd., Japan) was made up in physiological saline . Crudepapain was Papain Solution (activated) a diagnostic productprepared by the Commonwealth Serum Laboratories for use in blood grouping serology. It contained cysteine as activator and was used diluted in M/15 phosphate buffer, pH 5~4 . Plasmin was a laboratory preparation obtained by activation of Cohn fraction III-6 in glycerol . Because of the heatlabile nature of the venom (BAXTER and MARR, 1969) all mixtures were incubated at 25°. Two control mixtures were incubated in all experiments and contained respectively venom without enzyme and enzyme without venom. The effect of trypsin on the venom haemolysin was tested using mixtures containing 390-12,500 hg crude trypsin/ml final mixture, which contained 10 per cent v/v venom. After 60 min incubation the residual haemolytic activity was determined. Results are shown in Fig. 1. It can be seen that incubation with increasing concentrations of crude trypsin caused increasing loss of haemolytic activity of the venom. As this suggests proteolytic digestion of the haemolytic component, theeffect of a range of enzyme preparations was tested (Table 1). The haemolytic, lethal and dermonecrotic activities of the venom were all reduced by incubation with pepsin, pronase or trypsin. To confirm that the reduction in all biologic TOSICON 1971 Yot. 9.
431
432
A . G. M. MARR and E . H. BAXTER ioo 90 80 70 60 50
2
3
log i o enzyme conceniratlon,
4
/cq/ml
FYO . 1 . EFFECT OF CRUDE TRYPSII~I ON Tf~ HAF.MOLYSIN OF SEA WASP VENOM .
TABLH 1 . EFFECT OF PROTEOLYTIC ENZYMES ON VENOM OF Tfn; SEA WASP
Enzyme
Enzyme concentration (L~g/ml)
Venom ( ~ v/v)
Incubation time (min)
Crude trypsin Cryst.trypsin Crude papain Crude papain Plasmin Pmnase .
11,250 500 2500 5000 " 500
10 5 10 10 5 5
240 60 60 1200 960 60
Residual activity ( ~ control) Haemolytic Lethal Dermonecrotic <1 <0~4 25 <0~4 <0~4 0~4
<0~4 0~9
<3
3
12
0~3
3
"At a strength of I S units/ml .
activities was due specifically to proteolysis, trypsin was mixed with an equal concentration of soya bean trypsin inhibitor before addition to the incubation flask. This resulted in a 100 per cent recovery of all these activities . These results suggest that the haemolytic, lethal and dermonecrotic components of the venom are all proteinaceous in nature . This agrees with their other properties of immunogenicity, molecular size, heat lability and precipitation by 40 per cent w/v ammonium sulphate (BAXTER et al., 1968 ; BAXTER and MAxR, 1969 ; BAXTER et al., 1970). The pharmacological activity of venom extracts on rat ileal preparations was reported by ENDEAN and HErmExsox (1969) to resist incubation with trypsin and thus this activity must be diûerent from the haemolytic, lethal and dermonecrotic factors described here. T10XICON1971 Yol.9.
Sea Wasp V~om
433
REFERENCES B~, J. H. (19C~ Studies on throe venomous cubomedusae . Symp . Zool. Soc. Land. i6, 307. B~utvAS, J. H. (1967) Extraction of caidarian venom from living tentacle. In : Animal Toxins, pp . 115-129, (Russl rt, F. E, and Swrmsas, P. R, Eds.). Oxford : Pergamon Press. Bexrrde, E. H., M~ex, A. C~ . M. and L~t~, W. R (1968) Immunity to the venom of the sea wasp Chirorux ,flecked. Toxicon 6, 45 . B~xrx~e, E. H. and Mnxn, A. d. M. (1969) Sea wasp (Chironcx flecked) venom: lethal, haemolytic and dermonecrotic properties. Taxiton 7, 195 . B~, E. H., M~ex, A. (I . M. and Lnx$, W. R. (1970) Sea wasp (Chironex Jleckcd) toxin-Experimental immunity. 2nd Int. Syrrlp . Animal and Plant Toxins, Israel, 1970. (Abstract in Toxicon 8, 121). Du~ecco, R. and VoaT, M. (1954) Plaque formation and isolation of pun lines with poliomyelitis viruses. J. exp . Med. 99, 167. Erro~v, R and Hsnro~esox, L. (1969) Furtherstudies of toxic material from nematocysts of thecubomedusan Chironex decked Southcott. Toxicon 7, 303. Sou~xarrr, R V. (1956) Studies on Australian cubomedusae including a new genus and species apparently harmful to man. Aunt. J, mar. Freshwat . Res. 7, 254.
TOXICON 1971 Yo1. 9.
EFFECT OF PROTEOLYTIC ENZYMES ON THE VENOM OF THE SEA WA5P CHIRONEX FLECKERI A. G. M. Maiut and E. H. BAXTER
Commonwealth Serum Laboratories, Parkville, Victoria 3052, Australia (Acceptedfor publkation 26 Apri11971)
Tim Box jellyfish (Chironex ,fileckeri, SovTxcrn-r, 1956) known as the sea wasp has been responsible for fatal stingings in Australian tropical waters (BARNES, 1966). Our studies on the venom properties (BAXTER and MARK, 1969) and immunogenicity (BAXTER et al., 1968 ; BAxTER et al., 1970) suggested that the active molecules are moderately large and probably proteinaceous. Further support for this view was given by the report of ENDEAN and HENDERSON (1969) that saline extracts of intracapsular material from the nematocysts of C. ,~feckeri lost their activity against muscle if incubated with a bacterial protease . It therefore was of interest to obtain evidence as to the nature of the components of the venom concerned with the lethal, haemolytic and dermonecrotic properties, by studying the effects of incubation of the venom with proteolytic enzymes. Venom extracts were kindly supplied by Dr . J. H. Barnes of Cairns, Queensland, and were kept frozen at -15°from time of receipt until immediately before use. Collection of the venom had been made by electrical `milking' techniques (BARNES, 1967). The lethal, haemolytic and dermonecrotic effects of the venom were assayed as previously described (BAXTER et al., 1968 ; BAXTER and Maxi, 1969). Crude trypsin was obtained from Difco (1 :300) and crystalline trypsin was lyophilized 2-times crystallized trypsin (Worthington Biochem. Corpn., Freehold, N.J., U.S .A.). The diluent for trypsin was Dulbecco phosphate buffered saline containing calcium and magnesium (Dui.BE000 and VocT, 1954). Pronase P (45,000 PUK, Sogo Boeki Kaisha Ltd., Japan) was made up in physiological saline . Crudepapain was Papain Solution (activated) a diagnostic productprepared by the Commonwealth Serum Laboratories for use in blood grouping serology. It contained cysteine as activator and was used diluted in M/15 phosphate buffer, pH 5~4 . Plasmin was a laboratory preparation obtained by activation of Cohn fraction III-6 in glycerol . Because of the heatlabile nature of the venom (BAXTER and MARR, 1969) all mixtures were incubated at 25°. Two control mixtures were incubated in all experiments and contained respectively venom without enzyme and enzyme without venom. The effect of trypsin on the venom haemolysin was tested using mixtures containing 390-12,500 hg crude trypsin/ml final mixture, which contained 10 per cent v/v venom. After 60 min incubation the residual haemolytic activity was determined. Results are shown in Fig. 1. It can be seen that incubation with increasing concentrations of crude trypsin caused increasing loss of haemolytic activity of the venom. As this suggests proteolytic digestion of the haemolytic component, theeffect of a range of enzyme preparations was tested (Table 1). The haemolytic, lethal and dermonecrotic activities of the venom were all reduced by incubation with pepsin, pronase or trypsin. To confirm that the reduction in all biologic TOSICON 1971 Yot. 9.
431
432
A . G. M. MARR and E . H. BAXTER ioo 90 80 70 60 50
2
3
log i o enzyme conceniratlon,
4
/cq/ml
FYO . 1 . EFFECT OF CRUDE TRYPSII~I ON Tf~ HAF.MOLYSIN OF SEA WASP VENOM .
TABLH 1 . EFFECT OF PROTEOLYTIC ENZYMES ON VENOM OF Tfn; SEA WASP
Enzyme
Enzyme concentration (L~g/ml)
Venom ( ~ v/v)
Incubation time (min)
Crude trypsin Cryst.trypsin Crude papain Crude papain Plasmin Pmnase .
11,250 500 2500 5000 " 500
10 5 10 10 5 5
240 60 60 1200 960 60
Residual activity ( ~ control) Haemolytic Lethal Dermonecrotic <1 <0~4 25 <0~4 <0~4 0~4
<0~4 0~9
<3
3
12
0~3
3
"At a strength of I S units/ml .
activities was due specifically to proteolysis, trypsin was mixed with an equal concentration of soya bean trypsin inhibitor before addition to the incubation flask. This resulted in a 100 per cent recovery of all these activities . These results suggest that the haemolytic, lethal and dermonecrotic components of the venom are all proteinaceous in nature . This agrees with their other properties of immunogenicity, molecular size, heat lability and precipitation by 40 per cent w/v ammonium sulphate (BAXTER et al., 1968 ; BAXTER and MAxR, 1969 ; BAXTER et al., 1970). The pharmacological activity of venom extracts on rat ileal preparations was reported by ENDEAN and HErmExsox (1969) to resist incubation with trypsin and thus this activity must be diûerent from the haemolytic, lethal and dermonecrotic factors described here. T10XICON1971 Yol.9.
Sea Wasp V~om
433
REFERENCES B~, J. H. (19C~ Studies on throe venomous cubomedusae . Symp . Zool. Soc. Land. i6, 307. B~utvAS, J. H. (1967) Extraction of caidarian venom from living tentacle. In : Animal Toxins, pp . 115-129, (Russl rt, F. E, and Swrmsas, P. R, Eds.). Oxford : Pergamon Press. Bexrrde, E. H., M~ex, A. C~ . M. and L~t~, W. R (1968) Immunity to the venom of the sea wasp Chirorux ,flecked. Toxicon 6, 45 . B~xrx~e, E. H. and Mnxn, A. d. M. (1969) Sea wasp (Chironcx flecked) venom: lethal, haemolytic and dermonecrotic properties. Taxiton 7, 195 . B~, E. H., M~ex, A. (I . M. and Lnx$, W. R. (1970) Sea wasp (Chironex Jleckcd) toxin-Experimental immunity. 2nd Int. Syrrlp . Animal and Plant Toxins, Israel, 1970. (Abstract in Toxicon 8, 121). Du~ecco, R. and VoaT, M. (1954) Plaque formation and isolation of pun lines with poliomyelitis viruses. J. exp . Med. 99, 167. Erro~v, R and Hsnro~esox, L. (1969) Furtherstudies of toxic material from nematocysts of thecubomedusan Chironex decked Southcott. Toxicon 7, 303. Sou~xarrr, R V. (1956) Studies on Australian cubomedusae including a new genus and species apparently harmful to man. Aunt. J, mar. Freshwat . Res. 7, 254.
TOXICON 1971 Yo1. 9.