roxlcon Vol . 30, No. 3, pp. 331-338, 1992 . Printed in Great Britain.
00"1-0101/92 tá.00+ .00 ® 1992 Perpmon Pus plc
DERMONECROTIC AND LETHAL COMPONENTS OF LOXOSCELES GAUCHO SPIDER VENOM K. C.
BARBARO,'w J . L .
C.
CARD~
V. R. D.
EicxsTEDT3
and I.
MoTA'
'Centro de Pesquisa e Formagio em Imunologia, =Hospital Vital Brazil, and 3Sc9io de Artrópodos Peçonhentos, Instituto Butantan, Slïo Paulo, Brazil (Received 30 Judy 1991 ; accepted 15 October 1991)
K. C. BARBARO, J . L. C. CARDOSO, V. R. D. EICKSTEDT and I . MOTH . Dermonecrotic and lethal components of Loxosceles gaucho spider venom. Toxicon 30, 331-338, 1992. Loxosceles gaucho spider venom causes a typical dermonecrotic lesion in bitten patients and rarely causes lethal systemic effects. Gel filtration on Sephadex G 100 of L. gaucho spider venom resulted in three fractions: fraction A, containing the higher mol. wt components (approximately 35,000); fraction B, containing lower mol. wt components (approximately 15,000); and fraction C, containing very low mol. wt components (probably small peptides). The dermonecrotic and lethal activities were detected exclusively in fraction A. The venom and fraction A produced large dermonecrotic lesions in rabbits with necrosis spreading by gravity to the skin of the lateral body wall . Analysis by SDS-PAGE showed that the proteins contained in fraction A are approximately 35,000 and 33,000 mol. wt. Immunoblotting analysis showed that the proteins responsible for the dermonecrotic and lethal activity are very immunogenic and the first to be detected by antibodies during the course of immunization .
INTRODUCTION
has been recognized as a health problem in Brazil (ROSENFEL.D et al., 1957). Most accidents are caused by Loxosceles gaucho spider, and the most common finding after the spider's bite is a dermonecrotic lesion; very rarely it causes systemic effects that may be lethal (CARDOSO et al., 1988). Very little is known of the immunochemical and biological properties of the spider venom, except for a report by CICAREL.Li et al. (1983), who studied by immunodiffusion and immunoelectrophoresis the venom of L. gaucho . In this paper we report some of the immunochemical and biological properties of L. gaucho spider venom, attempting to characterize the components responsible for its dermonecrotic and lethal activities . LoxoscELIsm
Author to whom correspondence should be addressed at: Centro de Pesquisa e Formagio em Imunologia, Instituto Butantan, AV . Vital Brazil, 1500, 05504SAo Paulo-SP, Brazil . 331
33 2
K. C. BARBARO et al. MATERIALS AND METHODS
Animals and venom
Inbred A/Sn mice weighing 20-25 g and adult white rabbits weighing 3-4 kg were provided by the Instituto Butantan animal house. Specimens of L. gaucho were collected in the state of Süo Paulo, Brazil. After capture the spiders were kept in quarantine for a week without food before venom collection. The venom was obtained by electrostimulation (Bucmm., 1969), with slight modifications . Briefly, 15 V electrostimulation was applied repeatedly to the spider until release of the venom, which was collected with a micropipette, dried under vacuum and stored at -20°C. The pure venom is a transparent liquid that becomes cloudy when contaminated with stomach content. Contaminated venom was always disregarded. A pool of venom collected from approximately 1000 spiders was used . The protein content of the venom was determined according to the method of Lownv et al. (1951). Preparation of antiserum against the venom
Antiserum against the venom was prepared in rabbits by injecting intradermally (i .d .) 0.2 ml of 0.15 M NaCI containing 10 hg of venom. Animals were boosted 45 days later with the same dose of the antigen. Blood was collected from the marginal ear vein weekly after the first immunizing dose and after the booster. Serum was obtained by centrifugation (10 min, 4°C, ß00g). Antibody assays were performed by ELISA according to the method of THCAK.4TON et al. (1977), using L. gaucho venom (20 yg/ml) to coat the plates . The level of IgG antibodies was assayed after each bleeding. Normal rabbit serum obtained before immunization was used as a control. Gel filtration
A column (1 .8 x 70.0 cm) of Sephadex G 100 (Pharmacia, Fine Chemicals Ltd, Sweden) equilibrated with ammonium bicarbonate buffer (0.02 M pH 8.0) was used . Six milligrams of venom were applied to the column and 1.5 ml samples were collected with a flow rate of 20 ml/hr. Elution was monitored by absorbance at 280 nm . Fractions corresponding to each protein peak were then pooled, lyophilized and stored at -20°C. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)
The venom and its fractions were analysed by SDS-PAGE (8-1ß°/a acrylamide resolution gels) under nonreducing conditions, using the method of LAommr (1970) . Prior to electrophoresis, protein samples (30 pg) were mixed 1 : 1 (v/v) with sample buffer (125 mM Tris-HCI, pH 6 .8), SDS (sodium dodecyl sulfate) 2.5%, glycerol 20%, 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM TLCK (N-alfa-p-tosyl-r,-lysine chloromethyl ketone), 4 mM EDTA (ethylenediaminetetraacetic acid) and bromophenol blue (0 .05°/.), boiled for 3 min and placed on the gel. Gel electrophoresis was carried out at 25 mA for 4 hr, and then the gels were stained with 0. I% amido black. Mouse IgG (150,000), BSA (67,000), hen egg albumin (43,000), trypsinogen (24,000) and lysozyme (14,000) (Sigma Chemical Company, St . Louis, MO, U.S .A.) were used as mol. wt markers. Immunoblotting
Venom (300 hg) was first fractionated by SDS-PAGE, as previously described. The gel was placed in the electroblot apparatus adjacent to nitrocellulose paper in buffer, as described by TowBIN et al. (1979), and transferred overnight at 180 mA. Nitrocellulose paper was then incubated with immune rabbit serum at a dilution of 1 : 20 and the immunoreactive proteins identified using peroxidase labeled goat anti-rabbit IgG revealed with 0.05% 4-chloro-I-naphthol in 15% methanol (v/v), in the presence of 0.03% H202 (v/v). Normal rabbit serum was used as a control . Dermonecrotic activity of the venom and its fractions
Since preliminary observations showed that the venom does not induce dermonecrosis in mice, this activity was studied in rabbits. Dermonecrotic activity was assayed by i.d . injections, into a shaved area in the rabbit dorsum of 0.2 ml of 0.15 M NaCI containing 3 pg of venom or its fractions. The area of skin necrosis was inspected 6, 24, 48 and 168 hr after injection. Determination of the minimal lethal dose of the venom and its fractions
Lethality assay was performed using A/Sn mice which received i.d . injections of 0.2 ml of 0.15 M NaCl containing increasing concentrations (0.25; 0.50; 1 .00; 1.50; 2.00 and 2.50 mg/kg weight) of total venom protein, and the lethal activity was determined 6, 12, 24 and 48 hr after injection. Data were analysed using groups of five
Loxosce%s gaucho Venom
333
t1 0.8
á d
0.7 Q4 Q3 A 20
B
40 TUBE
FIG.
I.
FRACTIONATION
OF
L.
60 Bo NUMBER
gaucho
spiDER VENOM CHROMATOGRAPHY .
100
BY
120
SEPHADEx
G 100
COLUMN
A sample containing 6 Ing venom was loaded onto the column and eluted with 0 .02 M NH 4 HCO3 at a flow rate of 20 ml/hr . Samples of 1 .5 ml/tube were collected, and their protein content monitored by absorbance at 280 nm. mice for each dose (WORLD HEALTH ORGANIzAmoN, 1981) . To determine the lethal activity of the venom fraction, groups of mice were injected with a dose of each fraction equal to the minimal lethal dose of the total venom .
RESULTS
Figure 1 shows that in a typical elution profile of L. gaucho venom on a Sephadex G 100 column three major fractions A, B and C were obtained. These fractions were analysed for detmonecrotic and lethal activities. Preliminary experiments showed that 3 Rg of venom per site was required to produce a definite dermonecrotic lesion . To locate the dermonecrotic activity, 3 yg protein of each fraction was injected i.d. in rabbits. Fraction A, containing the higher mol. wt components showed dermonecrotic activity, while fractions B and C had no activity (Fig. 2). The venom and fraction A produced large dermonecrotic lesions in rabbits, with necrosis spreading by gravity to the skin of the lateral body wall . The minimal dose of the total venom to kill 100% of the mice within 48 hr was 1 .5 mg/kg body weight (Table 1). Lethal activity of the venom fractions was also located exclusively in fraction A (Table 1). The incubation of venom with antivenom produced in rabbits (57 days after immunization) abolished the lethal activity of the venom in mice (results not shown). The results of SDS-PAGE analysis of the venom and its fractions are shown in Fig. 3. At least nine components were revealed in the total venom. The major component was localized between 36,000 and 32,000 mol. wt. Fraction A was resolved into two major bands with mol. wts around 35,000 and 33,000, plus two minor bands of 30,000 and 28,000 . Fraction B showed a major component at 15,000 slightly contaminated with 35,000 and 33,000 proteins . No protein band was detected in fraction C. Some com-
K . C . BARBARO et at.
334
V
A
C
B
6 hr
24 hr
48 hr
168 hr
FIG. 2 .
DERMONECAOTnC LESIONS INDUCE BY TOTAL VENOM Arm FeAcnoNs L . gaucho VENOM.
A, B
AND C of
The venom and its fractions were prepared as described in the text. Three micrograms of each one were injected i .d. into the rabbit's dorsal skin . The lesions were inspected and photographed 6, 24, 48 and 168 hr after inoculation . Left to right: (V) venom ; (A) fraction A; (B) fraction B; and (C) fraction C .
Loxosceks gaucho Venom TASu
1.
DemunmATtoN of Tm? LuniAL Dosa of
Dose of venom (mg/kg of weight) Whole venom
Fractions A B C
6
L. gaucho
335 mENoat
erra
rrs
FRACTIONS
Mortality Hours after injection 12 24
48
0.25 0.50 1 .00 1 .50 2.00 2.50
0/5 " 0/5 0/5 0/5 0/5 0/5
0/5 0/5 0/5 0/5 5/5 5/5
0/5 0/5 1/5 5/5 -
0/5 0/5 1/5 -
1 .50 1 .50 1 .50
0/5 0/5 0/5
3/5 0/5 0/5
5/5 0/5 0/5
0/5 0/5
To determine the minimal lethal dose, groups of five mice were injected i .d. with whole venom or its fractions, and death recorded within 48 hr after injection . *Dead animals/injected animals .
-150 kDa -67 kDa -43 k Da -24 kDa -14kDa
1
2
3
4
FiG. 3 . SDS POLYAeaevr "urne ELEcrxotHoxEnc SEPARATION of L. gaucho TOTAL vENoat AND ris A, B AND C FRACTIONS. The fractions were prepared as described in the text. (1) Fraction A; (2) fraction B; (3) fraction C ; (4) whole venom. Numbers at right correspond to position of mol . wt markers .
33 6
K. C. BARBARO et al.
FIG .
4. Loxosceles gaucho
VENOM wAs FRACnoNATED BY SDS-PAGE AND REVEALED By WtsTERN BLOTTING USING RABBIT ANTI-TOTAL VENOM SERUM .
Normal sera ; (2) antiserum 7 days post-immunizatioin (d.p .i .) ; (3) 14d.p .i .; (4) 21 d.p.i.; (5) 29 d.p.i .; (6) 36 d.p.i .; (7) 43 d.p.i .; (8) 57 d.p.i .; (9) 64 d.p.i.; (10) 72 d.p .i. The booster (10 pg) was administered 45 days after the first dose . Numbers at the right correspond to position of mol. wt markers. (1)
ponents of the venom present in very low amount were not included in the fractions and so their bands do not appear in the electrophoresis . Immunization of rabbits with L. gaucho venom induced an antibody response which increased after the booster dose. As shown in Fig. 4, immunoblotting analysis of serum samples collected during the course of immunization showed that the first antibodies to appear reacted with two components of 35,000 and 33,000 mol. wt . After the booster, these components stained more strongly and an additional band of 15,000 was also detected . DISCUSSION
Separation of L. gaucho venom by Sephadex G 100 gel filtration produced three major fractions. The dermonecrotic and lethal activities were found only in the first peak containing the higher mol. wt components (fraction A); the other fractions (B and Q were devoid of these activities .
Loxoscelej gaucho Venom
337
Analysis of the L. gaucho venom by SDS-PAGE showed at least nine components . The proteins responsible for the dermonecrotic and lethal effects present in fraction A have higher mol. wts of approximately 35,000 and 33,000 . This is in agreement with results of GEREN et al. (1976) and REES et al. (1984), who verified that L. reclusa venom has two toxins of approximately 34,000 mol. wt responsible for the dermonecrotic and lethal activities of the venom. The observation that the venom produces necrotic lesions that tend to spread to the skin of the lateral body wall has been reported by DILLAHA et al. (1964) working with L. reclusa, and attributed to the presence of spreading factors and other enzymes in the venom (WRIGHT et al., 1973 ; FORRESTER et al., 1978; NORmwT et al., 1979). The L. gaucho venom was also able to produce this phenomenom . Thus, a similar spreading factor may also be present in this venom. Antivenom antibodies have been demonstrated by immunodiffusion in the serum of dogs and rabbits surviving several spider bites but not a single bite (DENNY et al., 1964). As shown in this paper, rabbits immunized with total venom produced high levels of IgG antibodies. It was also shown that these antibodies react strongly with the proteins responsible for the dermonecrotic and lethal activities of the venom. Human antiLoxosceles serum collected from patients bitten by Loxosceles sp. also has antibodies that recognize the L. gaucho venom components responsible for dermonecrotic and lethal activities of the venom (to be published). There are many reports describing the multiple components of Loxosceles venom and their various biological properties. However, it is difficult to make meaningful comparisons or relate specific biological effects to one venom component, because there are large differences in the venom preparation, scheme of purification and methods used to study the mechanisms of the venom components action . However, it seems that dermonecrotic and lethal activities are characteristic of the Loxosceles genus, and that the active components from all species have similar mol. wts between 30,000 and 37,000. A definite therapy for the lesion caused by the venom of Loxosceles spider has not yet been established. Surgical excision of the bitten site (REES et al., 1985), specific serum therapy (CARDOSO et al., 1990), diamine-diphenyl sulfone (dapsone) (KING and REFS, 1983) and steroids treatment (REES et al., 1981 ; ScHENONE et al., 1989) have been tried but their efficacy is uncertain. Since the dermonecrotic and lethal factors were the most immunogenic components of the venom, and are also the components recognized by horse serum anti-Loxosceles gaucho venom (not shown), the use of serum therapy as treatment of loxoscelism may be indicated in the cases when the patients can be treated early after the bite. Before immunoprophylaxis and immunotherapy of loxoscelism can become useful, further immunological and biochemical understanding of the venom is required . Acknowledgements-This paper was supported by Fundaçiio de Amparo a Pesquisa do Estado de Sio Paulo
(FÀPESP grant 90/1879-2). K. C . BARBARo was recipient of a FAPESP fellowship (89/1189-9). The authors are indebted to Mr CARL.Os A . JARm for excellent technical assistance . REFERENCES and venoms of the most important South American spiders of the genera Phoneutria, Loxosceks, Lycosa and Latrodectus . Am. Zoologist 9, 157-159 . CARnaeo, J . L. C., FRAN9A, F . O. S ., EICICSIEDT, V . R . D ., BoRGEs, I . and Noo~, M . T. (1988) Loxoscelismo : Estudo de 242 trios (1980-1984). Rev. Soc. bras. Toxicol. 1, 58-60. BücHERL,
W . (1969)
Biology
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CARDoso, J. L. C., WEN, F. H., FRANç~ F. O. S., WARRELL, D. A. and 1HEAKsToN, R. D. G. (1990) Detection by enzyme immunoassay of Loxosoeks gaucho venom in necrotic skin lesions caused by spider bites in Brazil. Trans. R. Soc. trop. Med Hyg. 84, 608-609. CScARELLI, R. M. B., Sum Vn t ARRoIa., M., GuinoInv, R and CynivELLARo, O. (1983) Estudo immunoquímico dos venenos aranbioos das espdcies Loxosceks gaucho, Phoneutria nigriventer e Lycosa erythrognatha. Mem. Inst . Butantan 47, 33-43. DENNY, W. F., DII-LAHA, C. J. and MoRGAN, P. N. (1964) Hemotoxic effect of Loxosceks rechisa venom: in vivo and in vitro studies . J. Lab. clin . Med 64, 291-298. DiLLAxA, C. J., JANSEN, G. T., HONEYcuTr, W. M. and HAYDEN, C. R. (1964) North American loxoscelism. J. Am . med. Ass. 188, 153-156. FoRRESrER, L. J., BARRm, J. T. and CAMPBELL, B. J. (1978) Red blood cell lysis induced by the venom of the brown recluse spider. The role of sphingomyelinase D. Archs Biochem. Biophys. 187, 355-365. GEREN, C. R., CHAN, T. K., HOwELL, D. E. and ODELL, G. V. (1976) Isolation and characterization of toxins from brown recluse spider venom (Loxosceles reclusa) . Archs Biochem. Biophys. 174, 90-99. KING, L. E. and REPS, R. S. (1983) Dapsone treatment of a brown recluse bite. J. Am . med. Ass. 250, 648. LAan_I, U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 68085. LowRY, O. H., RomRouGH, N. J., FARR, A. C. and RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. NORMENT, B. R., JONG, Y. S. and HErrz, J. R. (1979) Separation and characterization of venom components in Loxosceks reclusa-III. Hydrolytic enzyme activity . Toxicon 17, 539-548. REEs, R. S., SHACK, R. B., WrrHE s, E., MADDEN, J., FRANKLIN, J. and LYNCH, J. B. (1981) Management of the brown recluse spider bite . Plastic reconstr. Surg. 68, 768-773. REEs, R. S., NANNEY, L. B., YATES, R. A. and KING, L. E. (1984) Interaction of brown recluse spider venom on cell membranes: the inciting mechanism? J. invest. Derm . 83, 270-275. REEs, R. S., ALTENBERN, P., LYNCH, J. B. and KING, L. E. (1985) Brown recluse spider bites. A comparison of early surgical excision versus dapsone and delayed surgical excision . Ann. Surg. 202, 65963. RosENPELD, G., NAHAs, L., CiLLo, D. M. and FLEIray, C. T. (1957) Envenenam entos por serpentes, aranhas e escorpi6es. In : Atualizaçdo Terapeutica, pp . 931-944 (CINTRA Do PRADo, F. et al., Eds) . Silo Paulo, Brazil: Livraria luso espanhola e brasileira. ScHmIomE, H., SAAvEDRA, T., Roms, A. and Vn r ARRm, F. (1989) Loxoscelismo en Chile. Estudios epidemiol6gioos, cliníoos y experimentales . Rev. Inst . Med. Trop. Sd o Paulo 31, 403-415. 1HEAKsroN, R. D. G., LLoYD-JONES, M. J. and REID, H. A. (1977) Micro-ELISA for detecting and assaying snare venom and anti-venom antibody. Lancet ü, 639-641. TowBna, H., STAEHELnv, T. and GoRDoN, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc . natn. Acad. Sci. U.S.A . 76, 4350-4354. WoRLD HEALTH ORGANIZATION (1981) Progress in the characterization of venoms and standardization of antivenoms . WHO Offset Public. 58. WRIGHT, R. P., ELGERT, K. D., CAMPBELL, B. J. and BARRETT, J. T. (1973) Hyaluronidase and esterase activities of the venom of the poisonous brown recluse spider. Archs Biochem. Biophys. 159, 415-426.
00"1-0101/92 tá.00+ .00 ® 1992 Perpmon Pus plc
DERMONECROTIC AND LETHAL COMPONENTS OF LOXOSCELES GAUCHO SPIDER VENOM K. C.
BARBARO,'w J . L .
C.
CARD~
V. R. D.
EicxsTEDT3
and I.
MoTA'
'Centro de Pesquisa e Formagio em Imunologia, =Hospital Vital Brazil, and 3Sc9io de Artrópodos Peçonhentos, Instituto Butantan, Slïo Paulo, Brazil (Received 30 Judy 1991 ; accepted 15 October 1991)
K. C. BARBARO, J . L. C. CARDOSO, V. R. D. EICKSTEDT and I . MOTH . Dermonecrotic and lethal components of Loxosceles gaucho spider venom. Toxicon 30, 331-338, 1992. Loxosceles gaucho spider venom causes a typical dermonecrotic lesion in bitten patients and rarely causes lethal systemic effects. Gel filtration on Sephadex G 100 of L. gaucho spider venom resulted in three fractions: fraction A, containing the higher mol. wt components (approximately 35,000); fraction B, containing lower mol. wt components (approximately 15,000); and fraction C, containing very low mol. wt components (probably small peptides). The dermonecrotic and lethal activities were detected exclusively in fraction A. The venom and fraction A produced large dermonecrotic lesions in rabbits with necrosis spreading by gravity to the skin of the lateral body wall . Analysis by SDS-PAGE showed that the proteins contained in fraction A are approximately 35,000 and 33,000 mol. wt. Immunoblotting analysis showed that the proteins responsible for the dermonecrotic and lethal activity are very immunogenic and the first to be detected by antibodies during the course of immunization .
INTRODUCTION
has been recognized as a health problem in Brazil (ROSENFEL.D et al., 1957). Most accidents are caused by Loxosceles gaucho spider, and the most common finding after the spider's bite is a dermonecrotic lesion; very rarely it causes systemic effects that may be lethal (CARDOSO et al., 1988). Very little is known of the immunochemical and biological properties of the spider venom, except for a report by CICAREL.Li et al. (1983), who studied by immunodiffusion and immunoelectrophoresis the venom of L. gaucho . In this paper we report some of the immunochemical and biological properties of L. gaucho spider venom, attempting to characterize the components responsible for its dermonecrotic and lethal activities . LoxoscELIsm
Author to whom correspondence should be addressed at: Centro de Pesquisa e Formagio em Imunologia, Instituto Butantan, AV . Vital Brazil, 1500, 05504SAo Paulo-SP, Brazil . 331
33 2
K. C. BARBARO et al. MATERIALS AND METHODS
Animals and venom
Inbred A/Sn mice weighing 20-25 g and adult white rabbits weighing 3-4 kg were provided by the Instituto Butantan animal house. Specimens of L. gaucho were collected in the state of Süo Paulo, Brazil. After capture the spiders were kept in quarantine for a week without food before venom collection. The venom was obtained by electrostimulation (Bucmm., 1969), with slight modifications . Briefly, 15 V electrostimulation was applied repeatedly to the spider until release of the venom, which was collected with a micropipette, dried under vacuum and stored at -20°C. The pure venom is a transparent liquid that becomes cloudy when contaminated with stomach content. Contaminated venom was always disregarded. A pool of venom collected from approximately 1000 spiders was used . The protein content of the venom was determined according to the method of Lownv et al. (1951). Preparation of antiserum against the venom
Antiserum against the venom was prepared in rabbits by injecting intradermally (i .d .) 0.2 ml of 0.15 M NaCI containing 10 hg of venom. Animals were boosted 45 days later with the same dose of the antigen. Blood was collected from the marginal ear vein weekly after the first immunizing dose and after the booster. Serum was obtained by centrifugation (10 min, 4°C, ß00g). Antibody assays were performed by ELISA according to the method of THCAK.4TON et al. (1977), using L. gaucho venom (20 yg/ml) to coat the plates . The level of IgG antibodies was assayed after each bleeding. Normal rabbit serum obtained before immunization was used as a control. Gel filtration
A column (1 .8 x 70.0 cm) of Sephadex G 100 (Pharmacia, Fine Chemicals Ltd, Sweden) equilibrated with ammonium bicarbonate buffer (0.02 M pH 8.0) was used . Six milligrams of venom were applied to the column and 1.5 ml samples were collected with a flow rate of 20 ml/hr. Elution was monitored by absorbance at 280 nm . Fractions corresponding to each protein peak were then pooled, lyophilized and stored at -20°C. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)
The venom and its fractions were analysed by SDS-PAGE (8-1ß°/a acrylamide resolution gels) under nonreducing conditions, using the method of LAommr (1970) . Prior to electrophoresis, protein samples (30 pg) were mixed 1 : 1 (v/v) with sample buffer (125 mM Tris-HCI, pH 6 .8), SDS (sodium dodecyl sulfate) 2.5%, glycerol 20%, 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM TLCK (N-alfa-p-tosyl-r,-lysine chloromethyl ketone), 4 mM EDTA (ethylenediaminetetraacetic acid) and bromophenol blue (0 .05°/.), boiled for 3 min and placed on the gel. Gel electrophoresis was carried out at 25 mA for 4 hr, and then the gels were stained with 0. I% amido black. Mouse IgG (150,000), BSA (67,000), hen egg albumin (43,000), trypsinogen (24,000) and lysozyme (14,000) (Sigma Chemical Company, St . Louis, MO, U.S .A.) were used as mol. wt markers. Immunoblotting
Venom (300 hg) was first fractionated by SDS-PAGE, as previously described. The gel was placed in the electroblot apparatus adjacent to nitrocellulose paper in buffer, as described by TowBIN et al. (1979), and transferred overnight at 180 mA. Nitrocellulose paper was then incubated with immune rabbit serum at a dilution of 1 : 20 and the immunoreactive proteins identified using peroxidase labeled goat anti-rabbit IgG revealed with 0.05% 4-chloro-I-naphthol in 15% methanol (v/v), in the presence of 0.03% H202 (v/v). Normal rabbit serum was used as a control . Dermonecrotic activity of the venom and its fractions
Since preliminary observations showed that the venom does not induce dermonecrosis in mice, this activity was studied in rabbits. Dermonecrotic activity was assayed by i.d . injections, into a shaved area in the rabbit dorsum of 0.2 ml of 0.15 M NaCI containing 3 pg of venom or its fractions. The area of skin necrosis was inspected 6, 24, 48 and 168 hr after injection. Determination of the minimal lethal dose of the venom and its fractions
Lethality assay was performed using A/Sn mice which received i.d . injections of 0.2 ml of 0.15 M NaCl containing increasing concentrations (0.25; 0.50; 1 .00; 1.50; 2.00 and 2.50 mg/kg weight) of total venom protein, and the lethal activity was determined 6, 12, 24 and 48 hr after injection. Data were analysed using groups of five
Loxosce%s gaucho Venom
333
t1 0.8
á d
0.7 Q4 Q3 A 20
B
40 TUBE
FIG.
I.
FRACTIONATION
OF
L.
60 Bo NUMBER
gaucho
spiDER VENOM CHROMATOGRAPHY .
100
BY
120
SEPHADEx
G 100
COLUMN
A sample containing 6 Ing venom was loaded onto the column and eluted with 0 .02 M NH 4 HCO3 at a flow rate of 20 ml/hr . Samples of 1 .5 ml/tube were collected, and their protein content monitored by absorbance at 280 nm. mice for each dose (WORLD HEALTH ORGANIzAmoN, 1981) . To determine the lethal activity of the venom fraction, groups of mice were injected with a dose of each fraction equal to the minimal lethal dose of the total venom .
RESULTS
Figure 1 shows that in a typical elution profile of L. gaucho venom on a Sephadex G 100 column three major fractions A, B and C were obtained. These fractions were analysed for detmonecrotic and lethal activities. Preliminary experiments showed that 3 Rg of venom per site was required to produce a definite dermonecrotic lesion . To locate the dermonecrotic activity, 3 yg protein of each fraction was injected i.d. in rabbits. Fraction A, containing the higher mol. wt components showed dermonecrotic activity, while fractions B and C had no activity (Fig. 2). The venom and fraction A produced large dermonecrotic lesions in rabbits, with necrosis spreading by gravity to the skin of the lateral body wall . The minimal dose of the total venom to kill 100% of the mice within 48 hr was 1 .5 mg/kg body weight (Table 1). Lethal activity of the venom fractions was also located exclusively in fraction A (Table 1). The incubation of venom with antivenom produced in rabbits (57 days after immunization) abolished the lethal activity of the venom in mice (results not shown). The results of SDS-PAGE analysis of the venom and its fractions are shown in Fig. 3. At least nine components were revealed in the total venom. The major component was localized between 36,000 and 32,000 mol. wt. Fraction A was resolved into two major bands with mol. wts around 35,000 and 33,000, plus two minor bands of 30,000 and 28,000 . Fraction B showed a major component at 15,000 slightly contaminated with 35,000 and 33,000 proteins . No protein band was detected in fraction C. Some com-
K . C . BARBARO et at.
334
V
A
C
B
6 hr
24 hr
48 hr
168 hr
FIG. 2 .
DERMONECAOTnC LESIONS INDUCE BY TOTAL VENOM Arm FeAcnoNs L . gaucho VENOM.
A, B
AND C of
The venom and its fractions were prepared as described in the text. Three micrograms of each one were injected i .d. into the rabbit's dorsal skin . The lesions were inspected and photographed 6, 24, 48 and 168 hr after inoculation . Left to right: (V) venom ; (A) fraction A; (B) fraction B; and (C) fraction C .
Loxosceks gaucho Venom TASu
1.
DemunmATtoN of Tm? LuniAL Dosa of
Dose of venom (mg/kg of weight) Whole venom
Fractions A B C
6
L. gaucho
335 mENoat
erra
rrs
FRACTIONS
Mortality Hours after injection 12 24
48
0.25 0.50 1 .00 1 .50 2.00 2.50
0/5 " 0/5 0/5 0/5 0/5 0/5
0/5 0/5 0/5 0/5 5/5 5/5
0/5 0/5 1/5 5/5 -
0/5 0/5 1/5 -
1 .50 1 .50 1 .50
0/5 0/5 0/5
3/5 0/5 0/5
5/5 0/5 0/5
0/5 0/5
To determine the minimal lethal dose, groups of five mice were injected i .d. with whole venom or its fractions, and death recorded within 48 hr after injection . *Dead animals/injected animals .
-150 kDa -67 kDa -43 k Da -24 kDa -14kDa
1
2
3
4
FiG. 3 . SDS POLYAeaevr "urne ELEcrxotHoxEnc SEPARATION of L. gaucho TOTAL vENoat AND ris A, B AND C FRACTIONS. The fractions were prepared as described in the text. (1) Fraction A; (2) fraction B; (3) fraction C ; (4) whole venom. Numbers at right correspond to position of mol . wt markers .
33 6
K. C. BARBARO et al.
FIG .
4. Loxosceles gaucho
VENOM wAs FRACnoNATED BY SDS-PAGE AND REVEALED By WtsTERN BLOTTING USING RABBIT ANTI-TOTAL VENOM SERUM .
Normal sera ; (2) antiserum 7 days post-immunizatioin (d.p .i .) ; (3) 14d.p .i .; (4) 21 d.p.i.; (5) 29 d.p.i .; (6) 36 d.p.i .; (7) 43 d.p.i .; (8) 57 d.p.i .; (9) 64 d.p.i.; (10) 72 d.p .i. The booster (10 pg) was administered 45 days after the first dose . Numbers at the right correspond to position of mol. wt markers. (1)
ponents of the venom present in very low amount were not included in the fractions and so their bands do not appear in the electrophoresis . Immunization of rabbits with L. gaucho venom induced an antibody response which increased after the booster dose. As shown in Fig. 4, immunoblotting analysis of serum samples collected during the course of immunization showed that the first antibodies to appear reacted with two components of 35,000 and 33,000 mol. wt . After the booster, these components stained more strongly and an additional band of 15,000 was also detected . DISCUSSION
Separation of L. gaucho venom by Sephadex G 100 gel filtration produced three major fractions. The dermonecrotic and lethal activities were found only in the first peak containing the higher mol. wt components (fraction A); the other fractions (B and Q were devoid of these activities .
Loxoscelej gaucho Venom
337
Analysis of the L. gaucho venom by SDS-PAGE showed at least nine components . The proteins responsible for the dermonecrotic and lethal effects present in fraction A have higher mol. wts of approximately 35,000 and 33,000 . This is in agreement with results of GEREN et al. (1976) and REES et al. (1984), who verified that L. reclusa venom has two toxins of approximately 34,000 mol. wt responsible for the dermonecrotic and lethal activities of the venom. The observation that the venom produces necrotic lesions that tend to spread to the skin of the lateral body wall has been reported by DILLAHA et al. (1964) working with L. reclusa, and attributed to the presence of spreading factors and other enzymes in the venom (WRIGHT et al., 1973 ; FORRESTER et al., 1978; NORmwT et al., 1979). The L. gaucho venom was also able to produce this phenomenom . Thus, a similar spreading factor may also be present in this venom. Antivenom antibodies have been demonstrated by immunodiffusion in the serum of dogs and rabbits surviving several spider bites but not a single bite (DENNY et al., 1964). As shown in this paper, rabbits immunized with total venom produced high levels of IgG antibodies. It was also shown that these antibodies react strongly with the proteins responsible for the dermonecrotic and lethal activities of the venom. Human antiLoxosceles serum collected from patients bitten by Loxosceles sp. also has antibodies that recognize the L. gaucho venom components responsible for dermonecrotic and lethal activities of the venom (to be published). There are many reports describing the multiple components of Loxosceles venom and their various biological properties. However, it is difficult to make meaningful comparisons or relate specific biological effects to one venom component, because there are large differences in the venom preparation, scheme of purification and methods used to study the mechanisms of the venom components action . However, it seems that dermonecrotic and lethal activities are characteristic of the Loxosceles genus, and that the active components from all species have similar mol. wts between 30,000 and 37,000. A definite therapy for the lesion caused by the venom of Loxosceles spider has not yet been established. Surgical excision of the bitten site (REES et al., 1985), specific serum therapy (CARDOSO et al., 1990), diamine-diphenyl sulfone (dapsone) (KING and REFS, 1983) and steroids treatment (REES et al., 1981 ; ScHENONE et al., 1989) have been tried but their efficacy is uncertain. Since the dermonecrotic and lethal factors were the most immunogenic components of the venom, and are also the components recognized by horse serum anti-Loxosceles gaucho venom (not shown), the use of serum therapy as treatment of loxoscelism may be indicated in the cases when the patients can be treated early after the bite. Before immunoprophylaxis and immunotherapy of loxoscelism can become useful, further immunological and biochemical understanding of the venom is required . Acknowledgements-This paper was supported by Fundaçiio de Amparo a Pesquisa do Estado de Sio Paulo
(FÀPESP grant 90/1879-2). K. C . BARBARo was recipient of a FAPESP fellowship (89/1189-9). The authors are indebted to Mr CARL.Os A . JARm for excellent technical assistance . REFERENCES and venoms of the most important South American spiders of the genera Phoneutria, Loxosceks, Lycosa and Latrodectus . Am. Zoologist 9, 157-159 . CARnaeo, J . L. C., FRAN9A, F . O. S ., EICICSIEDT, V . R . D ., BoRGEs, I . and Noo~, M . T. (1988) Loxoscelismo : Estudo de 242 trios (1980-1984). Rev. Soc. bras. Toxicol. 1, 58-60. BücHERL,
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