- Email: [email protected]
Some methodological aspects on ion-exchange chromatography of toxins
146
8th European Symposium-Abstracts
signal are regular findings at 0.1 picomolar concentrations . ECG records show a rapid onset of bradycardia and arrythmia often expressed as AV block. This might indicate that the bundle of Hiss could have been affected, too. Results show that EqT II is a potent cardiotoxic protein which produces effects on isolated guinea-pig heart which are comparable to the effects observed in whole animal after i.v . injection of Equinatoxin (Stct:r et al., 1974). REFERENCES FEat,uv I and LÉSEZ, D. (1974) Toxicon 12, 57. Ho, C. L., Ko, J. L., LuE, H. M., LEE, C. Y. and FEiu .nx, I. (1987) Toxicon 25, 659. LEE, C. Y., LEE, S. Y., Ct~av, Y. M. and FER.LAN, I. (1983) Toxic Lett . 31, Suppl., 121. McL~tc, P. and LÉSEZ, D. (1988) Toxicon 26, 441. Sir, D., DRASLAR, K., FERLAN, I. and LÉSEZ, D. (1974) Toxicon 12, 63 . ~UPUT, D. (1986) Period. Biol. 88, 210. ~`ur~rr, D. (1988) Toxicon 26, 40 . ~upur, D., Rusw, N. and MEVES, H. (1987) In Progress in Venom and Toxin Research (GOPALAKRISHNAKONE, P. and Tax, C. K., eds.) pp. 46769. Information management by hypertext: aninwl venoms andpoisons. Irr~ Cuus, T. LnxaELt~ooErcE and D. MESS (Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, Frankfurt-70, F.R.G .) . HYrES~xr (OWL International Inc.) is a versatile data management software . Informations are organized by connecting files (text or graphic) or documents according to the actual request made by the user. During writing a text, document words and phrases can be marked (by buttons) and additional informations related to the specific subject can be added; they are hidden and are to be activated when necessary. This system has been used in constructing a data-base model which includes informations about poisoning and envenomations by animals and plants, the symptomatology, therapy as well as chemistry and mode of action of the toxins involved . The functional and morphological changes of rat's myocardium produced by Vipera betas snake venom. M. Cti+erxowE, D. Pstlcovt~, M. Pevr.ov~~, D. M~ctrr and K. ZDJELAR (Institute of Biochemistry and Institute of Physiology, Medical faculty, Vilegradska 26, 11000 Belgrade, Yugoslavia) . Vrr>Exe SExus belongs to the Viperidae snake family and in Europe stands as one of the most poisonous representatives. The present study was carried out with the aim to fractionate the venom into as many fractions as possible, to examine homogeneity of these obtained fractions and the effect of the whole venom and its fractions on the isolated rat heart. We obtained eight fractions on the Sephadex G-100 column. The first two of them are with proteolytic and the others with toxic activity . Efficiency of this separation was checked out by the method of PAAG electrophoresis in tubes. This method didn't show enough homogeneity of all fractions . Toxic effects of the whole venom and fractions were investigated on the isolated right ventricle of rat's heart. The whole venom stopped heart action in systole after 40 min. Toxic fractions IV and V produced decrease of the frequency and the amplitude of the heart contractions . They stopped heart action aRer 9 and IS min, respectively. These physiological results were checked out by histological investigations . Both the whole venom and fractions IV and V showed oedematous myofibrils, appearance of small granulations, cariolysis, haemorrhage and absence of cross-striation in some places . These changes are more significant after application of fractions IV and V. It seems that snake venom contains toxic substances which produce effects on the myocardium cells by destroying normal cell membranes. We suppose that there is a connection between toxic and proteolytic effects which could be due to the unhomogeneity of fractions IV and V found by electrophoresis. Some methodological aspects on ion-exchange chromatography of toxins. Cexr.os ~:RVEIANSKf~, Aurt L. H~revEY' and Evsrtr K~tu.ssoN3 ('Instituto Investigaciones BiolSgicas Clemente Estable, Avenida Italia 3318, Montevideo, Uruguay, =Strathclyde Institute for Drug Research, 204 George Street, Glasgow GL 1XW, U.K . and 3Department of Biochemistry, Biomedical Center, Box 576, 751 23 Uppsala, Sweden). (1) Aea~ornuM acetate is volatile and has been used with excellent results at neutral pH when it has a very low buffer capacity . High capacity buffers are usually recommended, but many of them such as Tris and phosphate are non-volatile . A desalting step is necessary before freeze-drying and besides extra work, some material is lost. (2) Cation exchangers Bio-Rex 70 and TSK SP-SPW: besides differences in charge, hydrophobic interactions and hydrogen bonding can also influence the separation, especially on Bio-Rex 70 (polymerized methacrylic acid
8th European Symposium-Abstracts
147
cross-linked with divinylbenzene). These interactions are much weaker on TSK SP-SPW (cross-linked vinyl polymer with hydroxyl groups, ion~xehange group sulphopropyl. ~HZ~Hz~H 2SO;). On both ion exchangers excellent resolution of very similar isotoxins have been obtained with ammonium acetate buffers. (3) Separation depends both on differences in net charge and on different localization of charges. Monoacetyl derivatives can be separated although they have the same net charge (one positive charge less than the parent molecule), but they have a different distribution of charged groups . Isotoxins differing only by a single amino acid replacement such as Ile/Ser, Pro/Ser and Tyr/Asn have been separated. (4) Different ions influence the elution of proteins to a different degree, and it is possible to change both resolution and elution order by changing buffer ions . Acetate gives in several cases separation compared with chloride . Isotoùns differing by a Pro/Ser mutation, co-eluted on Bio-Rex 70 in sodium phosphate buffers but were separated in ammonium acetate. Monoclonal antibodies and site-specific polyclonal antibodies help to reveal the site of toxicity ojammodytoxirts. V. ~uxrx-$trraec and F. Gtn3ex9etc (Dept. Biochem., J. Stefan Institute, Ljubljana, Yugoslavia). Try e~flrlo acid sequences of ammodytoùns A, B and C, and presynaptically toxic phospholipases A 2 from Vipers ammodytes ammodytes venom were determined . The differences in their primary structures provided first evidence for the region which is probably a part of the toùc site in ammodytoxins. Two marine monoclonal antibodies against a native ammodytoxin A have been raised . One of them, K321, was of the IgG, subclass with pI at pH 6,55 and the second one was of the IgG~ with pI at 5,85 . Monoclonal antibody K326 totally blocked the lethal activity of ammodytoùn A. Lethality of the complexes between ammodytoùn A and monoclonal antibodies was further compared to lethality of completes of ammodytoxin A and polyclonal site-specific antibodies raised in rabbits against four synthetic peptides (L,: 113-121, Lz: 106-113, L3: 70-78, L~: 12134) chosen from the primary structure of ammodytoxin A. In addition, monoclonal antibodies were also prepared against two synthetic peptides (L~ and L~ covalently linked to a protein carrier. Monoclonal antibodies against peptide Lz (IgG,) and Lj (IgM) were purified by the affinity chromatography on protein A-Sepharose 4B and by the gel filtration on Sephacryl 5-300, respectively . Anti-LZ monoclonal antibodies proved to be pure in the isoelectric focusing experiment as a single family of bands near the pH 6,85. This monoclonal antibody also completely blocked the lethality of ammodytoxin A. The results agree with those obtained with site-specific polyclonal antibodies and indicate the position of the binding site of the ammodytoùn A neutralizing monoclonal antibody raised against the native protein. In ammodytoxins, the tonic site probably comprises a part of the primary structure between residues covered by peptides L, and Lz. Scorpion toxins active on the sodi~an charutels of the cockroach Periplaneta americana. MARIA Et.etvA DE Llnu,' M~RIe Flulvce MARTIN-EAUCL.AIRE,' BeRIV~Rn Hue, 2 ERWAN LoRSr,' CnRws RlliE1R0 Dlxlz' and H>:Itv~ Roctur' ('CNRS UA 1179, INSERM U 172, Laboratoire de Biochimie, Faculté de Médecine Nord, Bd . Pierre Dramard, 13.326 Marseille Cedex l5, France, CNRS UA 611, Laboratoire de Neurophysiologie, Faculté de Médecine, Rue Haute de Reculée, 49 .045 Angers, France, and ~ Departamento de Bioquimica e Imunologia, Instituto de Ciências Biological, Universidade Federal de Minas Gerais, 30 .000 Belo Horizonte, M.G ., Brasil). SCORPION tOXin3 form a family of miniproteins of 6(1-70 amino acid residues cross-linked by four disulfide bridges that give the toxins a highly organized conformation, including a-helix and ß-sheet structures (FONIECILLA-CAMPS et al., 1980, 1988). These toùns have been divided into "mammal toùns", "insect toùns" and "crustacean toùns" according to their toxicity on mouse, blowfly larva or isopod . They generally act on the voltage-dependent sodium channels of excitable cells. '~I-iododerivatives of one insect toxin (AaH IT,) from Androctorrus australis Hector and of one toxin (Ts VII) from Tityus serrulatus were purified by HPLC and used in binding experiments on a nerve cord synaptosomal fraction of cockroach (P . arnericarra) . '~`I-AaH TI',_ n binds specifically and reversibly to a single class of non-interacting binding sites of high affinity (Kd' = 0.14f0.06 nM, n = 5) and low capacity (l .7 f 0.6 pmoles/mg protein) . The association (k,) and dissociation (k_,) constants are 3 ~ l(W M- 's -' (n = 2) and 7 ~ 10 -' s- ' (n = 2), respectively and are in good agreement with the equilibrium constant . The interaction of "SI-Ts VII-d was irreversible and the association kinetics wnstant (k) was 2-5 ~ 10~ M -' s-' (n = 4) . As already shown Ts VII and AaH IT, compete to the same binding site (LIMA et al., 1986, 1988). The binding site capacity of'uI-Ts VIII (1 .6 f 0.12 pmoles/mg of protein, n = 2) was similar to that obtained with'~I-AaH TT,_n in the present experiments and with '~I-AaH IT, (GoRnoly et al., 1984) on an insect synaptosomal fraction of central nervous system, and to that of toxin y from Tityus serrulatus, a toxin very similar, if not identical to Ts VII, on rat brain synaptosomes (BARxAxlx et al ., 1982). The injection of Ts VII in the 6th abdominal ganglion of the cockroach induces a progressive decrease in excitatory postsynaptic potentials and action potential.
8th European Symposium-Abstracts
signal are regular findings at 0.1 picomolar concentrations . ECG records show a rapid onset of bradycardia and arrythmia often expressed as AV block. This might indicate that the bundle of Hiss could have been affected, too. Results show that EqT II is a potent cardiotoxic protein which produces effects on isolated guinea-pig heart which are comparable to the effects observed in whole animal after i.v . injection of Equinatoxin (Stct:r et al., 1974). REFERENCES FEat,uv I and LÉSEZ, D. (1974) Toxicon 12, 57. Ho, C. L., Ko, J. L., LuE, H. M., LEE, C. Y. and FEiu .nx, I. (1987) Toxicon 25, 659. LEE, C. Y., LEE, S. Y., Ct~av, Y. M. and FER.LAN, I. (1983) Toxic Lett . 31, Suppl., 121. McL~tc, P. and LÉSEZ, D. (1988) Toxicon 26, 441. Sir, D., DRASLAR, K., FERLAN, I. and LÉSEZ, D. (1974) Toxicon 12, 63 . ~UPUT, D. (1986) Period. Biol. 88, 210. ~`ur~rr, D. (1988) Toxicon 26, 40 . ~upur, D., Rusw, N. and MEVES, H. (1987) In Progress in Venom and Toxin Research (GOPALAKRISHNAKONE, P. and Tax, C. K., eds.) pp. 46769. Information management by hypertext: aninwl venoms andpoisons. Irr~ Cuus, T. LnxaELt~ooErcE and D. MESS (Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, Frankfurt-70, F.R.G .) . HYrES~xr (OWL International Inc.) is a versatile data management software . Informations are organized by connecting files (text or graphic) or documents according to the actual request made by the user. During writing a text, document words and phrases can be marked (by buttons) and additional informations related to the specific subject can be added; they are hidden and are to be activated when necessary. This system has been used in constructing a data-base model which includes informations about poisoning and envenomations by animals and plants, the symptomatology, therapy as well as chemistry and mode of action of the toxins involved . The functional and morphological changes of rat's myocardium produced by Vipera betas snake venom. M. Cti+erxowE, D. Pstlcovt~, M. Pevr.ov~~, D. M~ctrr and K. ZDJELAR (Institute of Biochemistry and Institute of Physiology, Medical faculty, Vilegradska 26, 11000 Belgrade, Yugoslavia) . Vrr>Exe SExus belongs to the Viperidae snake family and in Europe stands as one of the most poisonous representatives. The present study was carried out with the aim to fractionate the venom into as many fractions as possible, to examine homogeneity of these obtained fractions and the effect of the whole venom and its fractions on the isolated rat heart. We obtained eight fractions on the Sephadex G-100 column. The first two of them are with proteolytic and the others with toxic activity . Efficiency of this separation was checked out by the method of PAAG electrophoresis in tubes. This method didn't show enough homogeneity of all fractions . Toxic effects of the whole venom and fractions were investigated on the isolated right ventricle of rat's heart. The whole venom stopped heart action in systole after 40 min. Toxic fractions IV and V produced decrease of the frequency and the amplitude of the heart contractions . They stopped heart action aRer 9 and IS min, respectively. These physiological results were checked out by histological investigations . Both the whole venom and fractions IV and V showed oedematous myofibrils, appearance of small granulations, cariolysis, haemorrhage and absence of cross-striation in some places . These changes are more significant after application of fractions IV and V. It seems that snake venom contains toxic substances which produce effects on the myocardium cells by destroying normal cell membranes. We suppose that there is a connection between toxic and proteolytic effects which could be due to the unhomogeneity of fractions IV and V found by electrophoresis. Some methodological aspects on ion-exchange chromatography of toxins. Cexr.os ~:RVEIANSKf~, Aurt L. H~revEY' and Evsrtr K~tu.ssoN3 ('Instituto Investigaciones BiolSgicas Clemente Estable, Avenida Italia 3318, Montevideo, Uruguay, =Strathclyde Institute for Drug Research, 204 George Street, Glasgow GL 1XW, U.K . and 3Department of Biochemistry, Biomedical Center, Box 576, 751 23 Uppsala, Sweden). (1) Aea~ornuM acetate is volatile and has been used with excellent results at neutral pH when it has a very low buffer capacity . High capacity buffers are usually recommended, but many of them such as Tris and phosphate are non-volatile . A desalting step is necessary before freeze-drying and besides extra work, some material is lost. (2) Cation exchangers Bio-Rex 70 and TSK SP-SPW: besides differences in charge, hydrophobic interactions and hydrogen bonding can also influence the separation, especially on Bio-Rex 70 (polymerized methacrylic acid
8th European Symposium-Abstracts
147
cross-linked with divinylbenzene). These interactions are much weaker on TSK SP-SPW (cross-linked vinyl polymer with hydroxyl groups, ion~xehange group sulphopropyl. ~HZ~Hz~H 2SO;). On both ion exchangers excellent resolution of very similar isotoxins have been obtained with ammonium acetate buffers. (3) Separation depends both on differences in net charge and on different localization of charges. Monoacetyl derivatives can be separated although they have the same net charge (one positive charge less than the parent molecule), but they have a different distribution of charged groups . Isotoxins differing only by a single amino acid replacement such as Ile/Ser, Pro/Ser and Tyr/Asn have been separated. (4) Different ions influence the elution of proteins to a different degree, and it is possible to change both resolution and elution order by changing buffer ions . Acetate gives in several cases separation compared with chloride . Isotoùns differing by a Pro/Ser mutation, co-eluted on Bio-Rex 70 in sodium phosphate buffers but were separated in ammonium acetate. Monoclonal antibodies and site-specific polyclonal antibodies help to reveal the site of toxicity ojammodytoxirts. V. ~uxrx-$trraec and F. Gtn3ex9etc (Dept. Biochem., J. Stefan Institute, Ljubljana, Yugoslavia). Try e~flrlo acid sequences of ammodytoùns A, B and C, and presynaptically toxic phospholipases A 2 from Vipers ammodytes ammodytes venom were determined . The differences in their primary structures provided first evidence for the region which is probably a part of the toùc site in ammodytoxins. Two marine monoclonal antibodies against a native ammodytoxin A have been raised . One of them, K321, was of the IgG, subclass with pI at pH 6,55 and the second one was of the IgG~ with pI at 5,85 . Monoclonal antibody K326 totally blocked the lethal activity of ammodytoùn A. Lethality of the complexes between ammodytoùn A and monoclonal antibodies was further compared to lethality of completes of ammodytoxin A and polyclonal site-specific antibodies raised in rabbits against four synthetic peptides (L,: 113-121, Lz: 106-113, L3: 70-78, L~: 12134) chosen from the primary structure of ammodytoxin A. In addition, monoclonal antibodies were also prepared against two synthetic peptides (L~ and L~ covalently linked to a protein carrier. Monoclonal antibodies against peptide Lz (IgG,) and Lj (IgM) were purified by the affinity chromatography on protein A-Sepharose 4B and by the gel filtration on Sephacryl 5-300, respectively . Anti-LZ monoclonal antibodies proved to be pure in the isoelectric focusing experiment as a single family of bands near the pH 6,85. This monoclonal antibody also completely blocked the lethality of ammodytoxin A. The results agree with those obtained with site-specific polyclonal antibodies and indicate the position of the binding site of the ammodytoùn A neutralizing monoclonal antibody raised against the native protein. In ammodytoxins, the tonic site probably comprises a part of the primary structure between residues covered by peptides L, and Lz. Scorpion toxins active on the sodi~an charutels of the cockroach Periplaneta americana. MARIA Et.etvA DE Llnu,' M~RIe Flulvce MARTIN-EAUCL.AIRE,' BeRIV~Rn Hue, 2 ERWAN LoRSr,' CnRws RlliE1R0 Dlxlz' and H>:Itv~ Roctur' ('CNRS UA 1179, INSERM U 172, Laboratoire de Biochimie, Faculté de Médecine Nord, Bd . Pierre Dramard, 13.326 Marseille Cedex l5, France, CNRS UA 611, Laboratoire de Neurophysiologie, Faculté de Médecine, Rue Haute de Reculée, 49 .045 Angers, France, and ~ Departamento de Bioquimica e Imunologia, Instituto de Ciências Biological, Universidade Federal de Minas Gerais, 30 .000 Belo Horizonte, M.G ., Brasil). SCORPION tOXin3 form a family of miniproteins of 6(1-70 amino acid residues cross-linked by four disulfide bridges that give the toxins a highly organized conformation, including a-helix and ß-sheet structures (FONIECILLA-CAMPS et al., 1980, 1988). These toùns have been divided into "mammal toùns", "insect toùns" and "crustacean toùns" according to their toxicity on mouse, blowfly larva or isopod . They generally act on the voltage-dependent sodium channels of excitable cells. '~I-iododerivatives of one insect toxin (AaH IT,) from Androctorrus australis Hector and of one toxin (Ts VII) from Tityus serrulatus were purified by HPLC and used in binding experiments on a nerve cord synaptosomal fraction of cockroach (P . arnericarra) . '~`I-AaH TI',_ n binds specifically and reversibly to a single class of non-interacting binding sites of high affinity (Kd' = 0.14f0.06 nM, n = 5) and low capacity (l .7 f 0.6 pmoles/mg protein) . The association (k,) and dissociation (k_,) constants are 3 ~ l(W M- 's -' (n = 2) and 7 ~ 10 -' s- ' (n = 2), respectively and are in good agreement with the equilibrium constant . The interaction of "SI-Ts VII-d was irreversible and the association kinetics wnstant (k) was 2-5 ~ 10~ M -' s-' (n = 4) . As already shown Ts VII and AaH IT, compete to the same binding site (LIMA et al., 1986, 1988). The binding site capacity of'uI-Ts VIII (1 .6 f 0.12 pmoles/mg of protein, n = 2) was similar to that obtained with'~I-AaH TT,_n in the present experiments and with '~I-AaH IT, (GoRnoly et al., 1984) on an insect synaptosomal fraction of central nervous system, and to that of toxin y from Tityus serrulatus, a toxin very similar, if not identical to Ts VII, on rat brain synaptosomes (BARxAxlx et al ., 1982). The injection of Ts VII in the 6th abdominal ganglion of the cockroach induces a progressive decrease in excitatory postsynaptic potentials and action potential.