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Electrophoretic variants of mojave rattlesnake (Crotalus scutulatus scutulatus) venoms and migration differences of mojave toxin
Toxic+w. Vol. 22, No. 6, pp. 990-985, 1984 . Prlated in atert Maim
0041-0101/94 S3 .00+ .00 C 1994 Papmoa Pre= Ltd.
ELECTROPHORETIC VARIANTS OF MOJAVE RATTLESNAKE (CROTALUS SCUTULATUS SCUTULATUS) VENOMS AND MIGRATION DIFFERENCES OF MOJAVE TOXIN EPP>E D. RAEL, 1 R. ALEC KNIGHT' and HECTOR ZEPEDA' 'Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968, U .S .A., and 'Department of Biology, Sul Ross State University, Alpine, TX 79832, U .S .A . (Accepted for publkation 2 May 1984) E . D . RAEL, R. A . KNraHT and H . ZEPMA. Electrophoretic variants of Mojave rattlesnake (Crotales scutulatus scutulatus) venoms and migration differences of Mojave toxin . Taxiton 22, 980-985, 1984. - Mojave toxin was found in comparable quantities in venoms from Mojave rattlesnakes captured in the Big Bend region of Texas and southeastern Arizona. Taxicities in mice were also comparable . Electrophoretic profiles of venom differed significantly between the two groups, suggesting two genetic divergent groups. Immunotransfer revealed several electrophoretic variants of Mojave toxin among the Texas snake venoms, all of which migrated slower than Mojave toxin of venoms from the Arizona snakes .
THE PRESENCE or absence of Mojave toxin in the venom of the Mojave rattlesnake (Crotalu,s scutulatus scutulatus) is the basis for distinguishing two groups of venoms, type A and type B (GLENN and STRAIGHT, 1978; GLENN et al., 1983) . The mouse i.p. LDso for type A venoms, which have high concentrations of Mojave toxin, is 0.28 mg/kg, and for type B venoms, in which Mojave toxin is absent or in low concentrations, is 3 .33 mg/kg. The presence or absence of the toxin is important in snake bite treatment and may be important in determining phylogenetic relationships. We therefore studied venoms from snakes of this species captured in Arizona and the Big Bend region of Texas. Lyophilized venom (Sigma Chemical Company, St. Louis, MO) was fractionated by DEAF-Sephadex A-50 column chromatography (RAEL and JONES, 1983). Subfractionation was conducted by preparative disc polyacrylamide gel electrophoresis in a Canalco 'Prep-Disc' apparatus to isolate Mojave toxin . Ten milliliters of separation gel and 5 ml of stacking gel were used. Isolation was carried out at a running pH of 9.5, a stacking pH of 8.3, a constant current of 6 mA, a flow rate of 1 ml/min and at a constant temperature of 5°C. Isolated Mojave toxin was injected into rabbits to raise antibodies. Double immunodiffugion analysis with the antibodies showed that all the venoms formed one precipitin line of identity with isolated Mojave toxin . Venoms from Mojave rattlesnakes, three from snakes captured in southeastern Arizona (venoms 1- 3) and six from snakes captured in the Big Bend region of Texas (venoms 4-9), were injected i.p. into 15 g CD-1 mice . The LDso , calculated 48 hr after injection 980
98 1
FIG . 1 . ELECTROPHORETIC SEPARATION OF MOJAVE RATTLESNAKE vENOMB IN 7 .5% POLYACRYLAMIDE GEL. A running pH of 9 .3 and a stacking pH of 8 .3 were used . The numbers at the top of the
electrophoretogram indicate that the venoms were from snakes captured in southeastern Arizona (1-3) or from snakes captured in the Big Bend region of Texas (4-9) . S indicates venom purchased from Sigma Chemical Company . The electrophoretogram was assigned zones for reference .
98 2
FIo . 2 .
MOIAvE To?CIId IN vENoms FROM SNAKES CAPTURED IN souTHEABTERN ARIZONA TEXAS (4-9).
(1 -3)
AND
The numbers correspond to those shown in Fig . 1 . After electrophoresis in 7 .5% polyacrylamide, the venom proteins were transferred to nitrocellulose paper and Mojave toxin developed with anti Mojave toxin antibodies. S indicates venom purchased from Sigma Chemical Company and M is isolated Mojave toxin .
Short Communications
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(REED and WENCH, 1938), ranged from 0.07 to 0 .14 mg/kg, which is slightly higher than previously reported for this species (MwToN, 1959; GLENN et al., 1983). However, the mice were small and may have influenced this result . Quantities of Mojave toxin in these venoms, determined by dot-immunobinding titration (HAWKES et al., 1982), were within one 2-fold dilution of venom. Differences in toxicity were also not found according to geographical distribution, as has been reported for Mojave rattlesnakes found in Arizona (GLENN and STRAIGHT, 1978; GLENN et al., 1983). Electrophoresis of the venoms was in 7.5% polyacrylamide gel slabs with a running pH of 9.3 and a stacking pH of 8.3 (DAvis, 1964). The gel slabs were stained with Coomassie blue R-250 or transferred electrophoretically to nitrocellulose sheets using a trans-blot cell (Bio-Rad Laboratories) containing 0.7% acetic acid (TOwBIN et al., 1979). Unreacted sites on the nitrocellulose paper were blocked with 0.05 M Tris buffer, pH 7.4, containing 0.2 M NaCl, 3% bovine serum albumin (w/v) and 1% goat serum (v/v). The transferred proteins were reacted for 2 hr with rabbit anti-Mojave toxin diluted 1/16000, washed, then incubated for 2 hr with a peroxidase conjugated, affinity purified goat anti-rabbit IgG (Miles Laboratories, Elkhart, IN) diluted 1/2000. Specific bands were developed with 4-cloro-l-napthol and hydrogen peroxide (HARES et al., 1982). Figure 1 shows that the venoms were distinguishable into two groups corresponding to the two geographical areas where the specimens were captured. Immunotransfer (Fig. 2) shows that the antiserum recognized one major band and two minor bands in venoms 1, 2 and 3 and in purchased venom. The major bands migrated the same distance as isolated Mojave toxin and correspond to the major bands in zone III of the electrophoretogram (Fig. 1). Two to three prominent bands were recognized by the antiserum in each of the venoms from the Texas group, which correspond to the prominent bands in zone II (Fig . 1). These migrated more slowly and, apparently, are isotoxins. Mojave isotoxins have been previously isolated from pooled Mojave rattlesnake venom (HENDON and BIEBER, 1982). The bands recognized by the antiserum appear to be very closely related, and may vary perhaps in only a few amino acid residues . These differences are probably in the basic protein of the toxin complex, because the acidic protein, thought to migrate with the dye front (GLENN et al., 1983), and which migrated identically in all the venoms we studied (Fig. 1), was not recognized by the antiserum. The results show that the venoms were very similar within each group, but differed significantly between the two localities, indicating two divergent genetic populations. These results suggest geographical separation of Mojave rattlesnakes in Texas from those in Arizona, and might be the basis for recognizing the two populations as distinct subspecies. Although more work is required, venom differences may also indicate different treatment and management of bite for the two groups . The first concern is, of course, to neutralize Mojave toxin, since all had approximately the same concentration. The high toxicity indicates that Mojave rattlesnakes from the Big Bend produce type A venom (GLENN et al., 1983). work was supported by NIH grant Sob RR 8012-12 and by the Chihuahan Desert Research institute, Alpine, TX 79832. Thanks are also extended to Dr JAm s F. SCUDDAY from the Biology Department of Sul Ross State University, Alpine, Texas, for assistance.
Acknowledgements - This
DAVrs,
REFERENCES B. J. (1964) Disc eleárophoresis II . Method and applicationto human serum pr~. Ann. N. Y. Acad.
sct. 121, 404. (h~, J. L. and STxAtatrr,
R. C. (1978) Mojave rattlesnake Crotdm scutulalus scututatus venom: variation in
984
Short Communications
toxicity with geographical origin . Toxiton 16, 81 . GLENN, J. L., STRAtotrr, R. C., WOLt7E, M. C. and HARDY, D. L. (1983) Geographical variation in Crotales scutelatus scetelates (Mojave rattlesnake) venom properties . Toxicon 21, 119. HAwKEs, R., NiDAY, E. and GORDON, J. (1982) A dot-immunobinding assay for monoclonal and other antibodies . Analyt. Biochem . 119, 142. HENDON, R. A. and BnRBER, A. L. (1982) Presynaptic toxins from rattlesnake venoms . In Rattlesnake Venoms., Their Actions and Treatment, p. 211 ('Iii, A. T., Ed.) . New York : Marcel Dekker . MimroN, S. A. JR. (1958) Observations on the amphibians and reptiles of Big Bend region of Texas. SWest. Nat. 3,28 . RAEL, E. D. and JONES, L. P. (1983) Isolation of an anticomplement factor from the venom of the Mojave rattlesnake (Crotales scutulatus scutulatus). Toxlron 21, 57 . REED, L. J. and MuENcH, H. (1938) A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 625 . TOWBnv, H., STAEHELIN, T. and GORDON, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : procedure and some applications . Proc. natn. Acad. Scl. U.S.A . 76, 4350.
0041-0101/94 S3 .00+ .00 C 1994 Papmoa Pre= Ltd.
ELECTROPHORETIC VARIANTS OF MOJAVE RATTLESNAKE (CROTALUS SCUTULATUS SCUTULATUS) VENOMS AND MIGRATION DIFFERENCES OF MOJAVE TOXIN EPP>E D. RAEL, 1 R. ALEC KNIGHT' and HECTOR ZEPEDA' 'Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968, U .S .A., and 'Department of Biology, Sul Ross State University, Alpine, TX 79832, U .S .A . (Accepted for publkation 2 May 1984) E . D . RAEL, R. A . KNraHT and H . ZEPMA. Electrophoretic variants of Mojave rattlesnake (Crotales scutulatus scutulatus) venoms and migration differences of Mojave toxin . Taxiton 22, 980-985, 1984. - Mojave toxin was found in comparable quantities in venoms from Mojave rattlesnakes captured in the Big Bend region of Texas and southeastern Arizona. Taxicities in mice were also comparable . Electrophoretic profiles of venom differed significantly between the two groups, suggesting two genetic divergent groups. Immunotransfer revealed several electrophoretic variants of Mojave toxin among the Texas snake venoms, all of which migrated slower than Mojave toxin of venoms from the Arizona snakes .
THE PRESENCE or absence of Mojave toxin in the venom of the Mojave rattlesnake (Crotalu,s scutulatus scutulatus) is the basis for distinguishing two groups of venoms, type A and type B (GLENN and STRAIGHT, 1978; GLENN et al., 1983) . The mouse i.p. LDso for type A venoms, which have high concentrations of Mojave toxin, is 0.28 mg/kg, and for type B venoms, in which Mojave toxin is absent or in low concentrations, is 3 .33 mg/kg. The presence or absence of the toxin is important in snake bite treatment and may be important in determining phylogenetic relationships. We therefore studied venoms from snakes of this species captured in Arizona and the Big Bend region of Texas. Lyophilized venom (Sigma Chemical Company, St. Louis, MO) was fractionated by DEAF-Sephadex A-50 column chromatography (RAEL and JONES, 1983). Subfractionation was conducted by preparative disc polyacrylamide gel electrophoresis in a Canalco 'Prep-Disc' apparatus to isolate Mojave toxin . Ten milliliters of separation gel and 5 ml of stacking gel were used. Isolation was carried out at a running pH of 9.5, a stacking pH of 8.3, a constant current of 6 mA, a flow rate of 1 ml/min and at a constant temperature of 5°C. Isolated Mojave toxin was injected into rabbits to raise antibodies. Double immunodiffugion analysis with the antibodies showed that all the venoms formed one precipitin line of identity with isolated Mojave toxin . Venoms from Mojave rattlesnakes, three from snakes captured in southeastern Arizona (venoms 1- 3) and six from snakes captured in the Big Bend region of Texas (venoms 4-9), were injected i.p. into 15 g CD-1 mice . The LDso , calculated 48 hr after injection 980
98 1
FIG . 1 . ELECTROPHORETIC SEPARATION OF MOJAVE RATTLESNAKE vENOMB IN 7 .5% POLYACRYLAMIDE GEL. A running pH of 9 .3 and a stacking pH of 8 .3 were used . The numbers at the top of the
electrophoretogram indicate that the venoms were from snakes captured in southeastern Arizona (1-3) or from snakes captured in the Big Bend region of Texas (4-9) . S indicates venom purchased from Sigma Chemical Company . The electrophoretogram was assigned zones for reference .
98 2
FIo . 2 .
MOIAvE To?CIId IN vENoms FROM SNAKES CAPTURED IN souTHEABTERN ARIZONA TEXAS (4-9).
(1 -3)
AND
The numbers correspond to those shown in Fig . 1 . After electrophoresis in 7 .5% polyacrylamide, the venom proteins were transferred to nitrocellulose paper and Mojave toxin developed with anti Mojave toxin antibodies. S indicates venom purchased from Sigma Chemical Company and M is isolated Mojave toxin .
Short Communications
98 3
(REED and WENCH, 1938), ranged from 0.07 to 0 .14 mg/kg, which is slightly higher than previously reported for this species (MwToN, 1959; GLENN et al., 1983). However, the mice were small and may have influenced this result . Quantities of Mojave toxin in these venoms, determined by dot-immunobinding titration (HAWKES et al., 1982), were within one 2-fold dilution of venom. Differences in toxicity were also not found according to geographical distribution, as has been reported for Mojave rattlesnakes found in Arizona (GLENN and STRAIGHT, 1978; GLENN et al., 1983). Electrophoresis of the venoms was in 7.5% polyacrylamide gel slabs with a running pH of 9.3 and a stacking pH of 8.3 (DAvis, 1964). The gel slabs were stained with Coomassie blue R-250 or transferred electrophoretically to nitrocellulose sheets using a trans-blot cell (Bio-Rad Laboratories) containing 0.7% acetic acid (TOwBIN et al., 1979). Unreacted sites on the nitrocellulose paper were blocked with 0.05 M Tris buffer, pH 7.4, containing 0.2 M NaCl, 3% bovine serum albumin (w/v) and 1% goat serum (v/v). The transferred proteins were reacted for 2 hr with rabbit anti-Mojave toxin diluted 1/16000, washed, then incubated for 2 hr with a peroxidase conjugated, affinity purified goat anti-rabbit IgG (Miles Laboratories, Elkhart, IN) diluted 1/2000. Specific bands were developed with 4-cloro-l-napthol and hydrogen peroxide (HARES et al., 1982). Figure 1 shows that the venoms were distinguishable into two groups corresponding to the two geographical areas where the specimens were captured. Immunotransfer (Fig. 2) shows that the antiserum recognized one major band and two minor bands in venoms 1, 2 and 3 and in purchased venom. The major bands migrated the same distance as isolated Mojave toxin and correspond to the major bands in zone III of the electrophoretogram (Fig. 1). Two to three prominent bands were recognized by the antiserum in each of the venoms from the Texas group, which correspond to the prominent bands in zone II (Fig . 1). These migrated more slowly and, apparently, are isotoxins. Mojave isotoxins have been previously isolated from pooled Mojave rattlesnake venom (HENDON and BIEBER, 1982). The bands recognized by the antiserum appear to be very closely related, and may vary perhaps in only a few amino acid residues . These differences are probably in the basic protein of the toxin complex, because the acidic protein, thought to migrate with the dye front (GLENN et al., 1983), and which migrated identically in all the venoms we studied (Fig. 1), was not recognized by the antiserum. The results show that the venoms were very similar within each group, but differed significantly between the two localities, indicating two divergent genetic populations. These results suggest geographical separation of Mojave rattlesnakes in Texas from those in Arizona, and might be the basis for recognizing the two populations as distinct subspecies. Although more work is required, venom differences may also indicate different treatment and management of bite for the two groups . The first concern is, of course, to neutralize Mojave toxin, since all had approximately the same concentration. The high toxicity indicates that Mojave rattlesnakes from the Big Bend produce type A venom (GLENN et al., 1983). work was supported by NIH grant Sob RR 8012-12 and by the Chihuahan Desert Research institute, Alpine, TX 79832. Thanks are also extended to Dr JAm s F. SCUDDAY from the Biology Department of Sul Ross State University, Alpine, Texas, for assistance.
Acknowledgements - This
DAVrs,
REFERENCES B. J. (1964) Disc eleárophoresis II . Method and applicationto human serum pr~. Ann. N. Y. Acad.
sct. 121, 404. (h~, J. L. and STxAtatrr,
R. C. (1978) Mojave rattlesnake Crotdm scutulalus scututatus venom: variation in
984
Short Communications
toxicity with geographical origin . Toxiton 16, 81 . GLENN, J. L., STRAtotrr, R. C., WOLt7E, M. C. and HARDY, D. L. (1983) Geographical variation in Crotales scutelatus scetelates (Mojave rattlesnake) venom properties . Toxicon 21, 119. HAwKEs, R., NiDAY, E. and GORDON, J. (1982) A dot-immunobinding assay for monoclonal and other antibodies . Analyt. Biochem . 119, 142. HENDON, R. A. and BnRBER, A. L. (1982) Presynaptic toxins from rattlesnake venoms . In Rattlesnake Venoms., Their Actions and Treatment, p. 211 ('Iii, A. T., Ed.) . New York : Marcel Dekker . MimroN, S. A. JR. (1958) Observations on the amphibians and reptiles of Big Bend region of Texas. SWest. Nat. 3,28 . RAEL, E. D. and JONES, L. P. (1983) Isolation of an anticomplement factor from the venom of the Mojave rattlesnake (Crotales scutulatus scutulatus). Toxlron 21, 57 . REED, L. J. and MuENcH, H. (1938) A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 625 . TOWBnv, H., STAEHELIN, T. and GORDON, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : procedure and some applications . Proc. natn. Acad. Scl. U.S.A . 76, 4350.