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Variations in the composition of echis venoms
8th World Conlp,'ess on
AMnud, Plant and Microbial Toxins
ISOLATION /LIE) PARTIAL ~ R I Z A T I O N OF A TIlROHBIII-LII(EENZ~ FROH VEMOHOF ~ SOUTHERNPACIFIC RATrLESIMIOE (CROTALUSVIRIDIS IIELLERI) By R.C. Schaeffer, J r . , J. Resk, D. Marsh, S-M. C'h'T~-on'-an~"l~11~-.Car--rl-s'6"n~. Dept. Med., Mayne State Univ. Sch. Med., Detroit, MI 48201, USA The thrombtn-ltke enzyme from C. vtridts heller4 venom was tsolated by two methods. Method A: Fracttonatton (200 ullf'rac~.~l'on,.1JS~Ium acetate, pH 7.4, 1 ml/mtn) of crude venom (50 mg/ml, 25 ul/inJectton), on a calibrated gel permeation htgh performance liquid chromatography (HPLC) SM 3000 (7.5X30 cm)-2OO0 (7.5X60 cm) series column produced 6 Low~y protein peaks. The f t r s t peak (0.01 NIH thrombtn unIts/ug Lowry protein; 0.5 O.D./mtn/ug protein, using the synthetic substrate; PPAN, Stgmn Chemical Co.) was fracttonated tnto 4 peaks by hydrophobtc HPLC (TSK Phenyl-5P~ column, 15 mtn linear gradient of decreasing ammonium sulfate 1.7 to OM, pH 7.0). The 2nd of these peaks contained the clotting a c t i v i t y (<.01 NIH untt/ug Lowry protein). Thts fraction showed no Coomasste blue stained bands on a non-reduced discontinuous SDS-lOY~oolyacrylamtde electrephorettc gel (SDS-IO%PAGE; Laenmlt, 1970). Method B: Fraction 1 (50 mg/ml,lO0 ulltnJection, from Sephedex G-IOO chromatography, Schaeffer eL e l . , 1984) was separated into 5 peaks by DEAE ton-exchange HPLC (B buffer .05H sodium-ph~phate ,pH 8.0; A buffer-8 buffer +.5M NaCl;gredtent condttion:15 mtn lOOm B, 1 mtn pulse elution of 45% k, 2 mtn 100% 8, and a 10 mtn ltnear gradient 100% 8 to 100~ k). Peak 4 (45% pulse elution) was separated tnto 4 peaks by hydrophobtc HPLC. SDS-IO%PAGEof the 2nd peak (.04 NZH untts/ug protein and .010 O.D./mtn/ug protein of PPAN a c t i v i t y ) again dtd not stain with Coomasste blue but showed a single yellow-stained band (ca.53-61ktloDalton) after Intense stlver staining. Preliminary reverse-phase HPLC analysis of the reaction products of this enzyme's degradation of purified human flbrlnogen suggests that flbrlnopeptlde k Is the main product released after 5-30 mtn of Incubation. These data show that the thrombtn-11ke enzyme from C. vtrtdts hellert venom ts a r e l a t i v e l y large molecular weight protease that appears to a~tac-'k'-~cl~'~tn-'~tn a manner stmtlar to other throabtn-11ke enzymes dertved from crotalId venoms. KEFEKEIIOES Laemmlt, U.K. (1970) Nature (Lmnd) 227,680. Schaeffer, R.C., J r . , Brtston, C., ChilLon, S-M., Carlson, R.M. (1984b) J. Pharmncol. Exp. Thera. 230,393. KEY MOR9S C. vtrtdts heller] thrombtn-11ke enzyme, HPLC, coagulation
VARIATIONS IN TIIECOIIPOSITIOIIOFECHISVENONS By R.C. Schaeffer, J r . , S~. ChilLon, R.]~'T~nktewicz and R.W. Carlson. Dept. Med., Wayne State Unlv. Sch. Med., Detroit, M1 48201, USA Different lots of Echts cartnatus venom (ECV) and Echts coloratus venom (ECOV)were analyzed for the per ce--e'nE-dryw--~ (% prectpttatedm----~-e'rta-'l--a'~'e~-crude venom dtluted tn detontzed water, 1 mg/ml) ,clotting acttvtty (thrembtn equivalent units, control dog plasma; Schaeffer elm1., 1984a), % Lowry proteln (% protetn of crude venom, 1 aqllml) and esterolytlc actlvl~y(N-benzoyl-L-prolyl-L-phenylalanyl-L-arglnlne-p-nltroanlllde HCI,PPAN, Stgma Chemtcal Co.; a synthetic enzyme substrate; Schaeffer et e l . , 1984b and Kluft et e l . , 1978.; units are O.D.ImtnilO ug crude weight; Table 1)7 " m T'aSle'-l. Conq)osttton of Echts cartnatus and Echts colorata venoms. Smple 1 (ECV A) and 2 (ECV C) were g i f t s f r o m ~ ~ a l t k an--n'd'TJt~ebs. Samples 3-5 were Sigma lots 31F, 39C and 103F of ECV, respectively. Sample 6 (ECOV) was a g i f t from Dr. E. Kochva and 7-9 were Latoxan lot C008 (ECOV) and lots A144 and C0039 (ECV), respectively. Sample t % Dry Wt Clotttng Protein PPAN (AanK) 1 34 2.6 67.7 0.131 ~ " 2 16 <0.1 49.6 0.475 3 18 4.0 73.0 0.015 4 25.5 4.0 78.7 0.008 5 18.4 <0.1 101.1 0.038 6 10.2 2.7 85.3 0.015 7 23.5 1.5 78.7 0.045 8 29.8 0.7 114.7 0.005 9 28.3 4.0 98.7 0.660 These data suggest that variation between venom lots may affect the production of potent antivenin to the clotting and PPANa c t i v i t i e s . REFERENCES Schaeffer, R.C., J r . , ChilLon, S-M., Hadden, T.J., Carlson, R.W. (1984a) J. Appl. Physiol.:Resptret. Envtren. Exercise Phystol. 57,1824. Schaeffer, R.C., J r . , 8rlston, C., ChilLon, S-M., Carlson, R.W. (1984b) J. Pharmacol. Exp. There. 230,393. Kluft C. (1978) J. lab. Cltn. lied. 91.83. KEY MORDS Echts cartnatus and Echts colorata venom, clotttng and esterelyttc a c t i v i t i e s
AMnud, Plant and Microbial Toxins
ISOLATION /LIE) PARTIAL ~ R I Z A T I O N OF A TIlROHBIII-LII(EENZ~ FROH VEMOHOF ~ SOUTHERNPACIFIC RATrLESIMIOE (CROTALUSVIRIDIS IIELLERI) By R.C. Schaeffer, J r . , J. Resk, D. Marsh, S-M. C'h'T~-on'-an~"l~11~-.Car--rl-s'6"n~. Dept. Med., Mayne State Univ. Sch. Med., Detroit, MI 48201, USA The thrombtn-ltke enzyme from C. vtridts heller4 venom was tsolated by two methods. Method A: Fracttonatton (200 ullf'rac~.~l'on,.1JS~Ium acetate, pH 7.4, 1 ml/mtn) of crude venom (50 mg/ml, 25 ul/inJectton), on a calibrated gel permeation htgh performance liquid chromatography (HPLC) SM 3000 (7.5X30 cm)-2OO0 (7.5X60 cm) series column produced 6 Low~y protein peaks. The f t r s t peak (0.01 NIH thrombtn unIts/ug Lowry protein; 0.5 O.D./mtn/ug protein, using the synthetic substrate; PPAN, Stgmn Chemical Co.) was fracttonated tnto 4 peaks by hydrophobtc HPLC (TSK Phenyl-5P~ column, 15 mtn linear gradient of decreasing ammonium sulfate 1.7 to OM, pH 7.0). The 2nd of these peaks contained the clotting a c t i v i t y (<.01 NIH untt/ug Lowry protein). Thts fraction showed no Coomasste blue stained bands on a non-reduced discontinuous SDS-lOY~oolyacrylamtde electrephorettc gel (SDS-IO%PAGE; Laenmlt, 1970). Method B: Fraction 1 (50 mg/ml,lO0 ulltnJection, from Sephedex G-IOO chromatography, Schaeffer eL e l . , 1984) was separated into 5 peaks by DEAE ton-exchange HPLC (B buffer .05H sodium-ph~phate ,pH 8.0; A buffer-8 buffer +.5M NaCl;gredtent condttion:15 mtn lOOm B, 1 mtn pulse elution of 45% k, 2 mtn 100% 8, and a 10 mtn ltnear gradient 100% 8 to 100~ k). Peak 4 (45% pulse elution) was separated tnto 4 peaks by hydrophobtc HPLC. SDS-IO%PAGEof the 2nd peak (.04 NZH untts/ug protein and .010 O.D./mtn/ug protein of PPAN a c t i v i t y ) again dtd not stain with Coomasste blue but showed a single yellow-stained band (ca.53-61ktloDalton) after Intense stlver staining. Preliminary reverse-phase HPLC analysis of the reaction products of this enzyme's degradation of purified human flbrlnogen suggests that flbrlnopeptlde k Is the main product released after 5-30 mtn of Incubation. These data show that the thrombtn-11ke enzyme from C. vtrtdts hellert venom ts a r e l a t i v e l y large molecular weight protease that appears to a~tac-'k'-~cl~'~tn-'~tn a manner stmtlar to other throabtn-11ke enzymes dertved from crotalId venoms. KEFEKEIIOES Laemmlt, U.K. (1970) Nature (Lmnd) 227,680. Schaeffer, R.C., J r . , Brtston, C., ChilLon, S-M., Carlson, R.M. (1984b) J. Pharmncol. Exp. Thera. 230,393. KEY MOR9S C. vtrtdts heller] thrombtn-11ke enzyme, HPLC, coagulation
VARIATIONS IN TIIECOIIPOSITIOIIOFECHISVENONS By R.C. Schaeffer, J r . , S~. ChilLon, R.]~'T~nktewicz and R.W. Carlson. Dept. Med., Wayne State Unlv. Sch. Med., Detroit, M1 48201, USA Different lots of Echts cartnatus venom (ECV) and Echts coloratus venom (ECOV)were analyzed for the per ce--e'nE-dryw--~ (% prectpttatedm----~-e'rta-'l--a'~'e~-crude venom dtluted tn detontzed water, 1 mg/ml) ,clotting acttvtty (thrembtn equivalent units, control dog plasma; Schaeffer elm1., 1984a), % Lowry proteln (% protetn of crude venom, 1 aqllml) and esterolytlc actlvl~y(N-benzoyl-L-prolyl-L-phenylalanyl-L-arglnlne-p-nltroanlllde HCI,PPAN, Stgma Chemtcal Co.; a synthetic enzyme substrate; Schaeffer et e l . , 1984b and Kluft et e l . , 1978.; units are O.D.ImtnilO ug crude weight; Table 1)7 " m T'aSle'-l. Conq)osttton of Echts cartnatus and Echts colorata venoms. Smple 1 (ECV A) and 2 (ECV C) were g i f t s f r o m ~ ~ a l t k an--n'd'TJt~ebs. Samples 3-5 were Sigma lots 31F, 39C and 103F of ECV, respectively. Sample 6 (ECOV) was a g i f t from Dr. E. Kochva and 7-9 were Latoxan lot C008 (ECOV) and lots A144 and C0039 (ECV), respectively. Sample t % Dry Wt Clotttng Protein PPAN (AanK) 1 34 2.6 67.7 0.131 ~ " 2 16 <0.1 49.6 0.475 3 18 4.0 73.0 0.015 4 25.5 4.0 78.7 0.008 5 18.4 <0.1 101.1 0.038 6 10.2 2.7 85.3 0.015 7 23.5 1.5 78.7 0.045 8 29.8 0.7 114.7 0.005 9 28.3 4.0 98.7 0.660 These data suggest that variation between venom lots may affect the production of potent antivenin to the clotting and PPANa c t i v i t i e s . REFERENCES Schaeffer, R.C., J r . , ChilLon, S-M., Hadden, T.J., Carlson, R.W. (1984a) J. Appl. Physiol.:Resptret. Envtren. Exercise Phystol. 57,1824. Schaeffer, R.C., J r . , 8rlston, C., ChilLon, S-M., Carlson, R.W. (1984b) J. Pharmacol. Exp. There. 230,393. Kluft C. (1978) J. lab. Cltn. lied. 91.83. KEY MORDS Echts cartnatus and Echts colorata venom, clotttng and esterelyttc a c t i v i t i e s