Enzymatic glyceride synthesis in a foam reactor

We report the results of our study on Rhizomucor miehei lipaseâ catalyzed lauric acidâ glycerol esterification in a foam reactor. A satisfactory yield of glyceride synthesis can be achieved with an unusually high initial water content (50% w/w). We found that product formation could be regulated by controlling foaming. Foaming was a function of the air flow rate, reaction temperature, pH value, ionic strength, and substrate molar ratio. Monolaurin and dilaurin, which constituted nearly 80% of the total yield, were the two dominant products in this reaction; trilaurin was also formed at the initial stages of the reaction. A study of pH and ionic strength effects on an independent basis revealed that they affect the interfacial mechanism in different manners. On varying the ratio of lauric acid and glycerol, only a slight change in the degree of conversion was detected and the consumption rate of fatty acid was approximately the same.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141443/1/aocs0643.pd

Structural And Functional Characterization Of Basic Pla2 Isolated From Crotalus Durissus Terrificus Venom

The venom of Crotalus durissus terrificus was fractionated by reverse-phase HPLC to obtain crotapotins (F5 and F7) and PLA2 (F15, F16, and F17) of high purity. The phospholipases A2 (PLA2s) and crotapotins showed antimicrobial activity against Xanthomonas axonopodis pv. passiflorae, although the unseparated crotoxin did not. The F17 of the PLA 2 also revealed significant anticoagulant activity, althrough for this to occur the presence of Glu 53 and Trp 61 is important. The F17 of the PLA2 showed allosteric behavior in the presence of a synthetic substrate. The amino acid sequence of this PLA2 isoform, determined by automatic sequencing, was HLLQFNKMLKFETRK NAVPFYAFGCYCGWGGQRRPKDATDRCCFVHDCCYEKVTKCNTKWDFYRYSLKSGY ITCGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPDSRCREPSETC. Analysis showed that the sequence of this PLA2 isoform differed slightly from the amino acid sequence of the basic crotoxin subunit reported in the literature. The homology with other crotalid PLA2 cited in the lit-erature varied from 60% to 90%. The pL was estimated to be 8.15, and the calculated molecular weight was 14664.14 as determined by Tricine SDS-PAGE, two-dimensional electrophoresis, and MALDI-TOFF. These results also suggested that the enzymatic activity plays an important role in the bactericidal effect of the F17 PLA2 as well as that of anticoagulation, although other regions of the molecule may also be involved in this biological activity. © 2002 Plenum Publishing Corporation.213161168Aird, S.D., Kaiser, I.I., (1985) Biochemistry, 24, pp. 7054-7058Aird, S.D., Kruggel, W.G., Kaiser, I.I., (1985) Toxicon, 28, pp. 669-673Anderson, N.L., Anderson, N.G., (1991) Electrophoresis, 12, pp. 883-906Beghini, D.G., Toyama, M.H., Hyslop, S., Sodek, L., Novello, J.C., Marangoni, S., (2000) J. Prot. Chem., 19, pp. 603-607Breithaupt, H., (1976) Toxicon, 14, pp. 221-233Carredano, B., Westerlind, B., Persson, M., Saareinen, S., Ramaswamy, D., Eaker, H., Eklund, M.W., (1998) Toxicon, 36, pp. 75-92Cho, W., Kezdy, F.J., (1991) Methods Enzymol., 23, pp. 75-79Faure, G., Choumet, V., Bouchier, C., Camoin, L., Guillaume, J.L., Monegier, B., Vuilhorgne, M., Bon, C., (1994) Eur. J. Biochem., 223, pp. 161-164Faure, G., Guillaume, J.L., Camoin, L., Saliou, B., Bon, C., (1991) Biochemistry, 30, pp. 8074-8083Gutierrez, J.M., Lomonte, B., (1995) Toxicon., 33, pp. 1405-1424Habermann, E., Breithaupt, H., (1978) Toxicon., 16, pp. 19-30Hendon, R.A., Fraenkel-Conrat, H., (1976) Toxicon., 14, pp. 283-289Holzer, M., Mackessy, S.P., (1996) Toxicon., 34, pp. 1149-1155Kini, R.M., Evans, H.J., (1989) Toxicon., 27, pp. 613-635Kini, R.M., Evans, H.J., (1987) J. Biol. Chem., 262, pp. 14402-14407Lambeau, G., Ancian, P., Nicolas, J.P., Cupillard, L., Zvaritch, E., Lazdunski, M., (1996) Seances Soc. Biol. Fil., 190, pp. 425-435Lomonte, B., Moreno, E., Tarkowski, A., Hanson, L.A., Maccarana, M., (1994) J. Biol. Chem., 269, pp. 29867-29873Paramo, L., Lomonte, B., Pizarro-Cerda, J., Bengoechea, J.A., Gorvel, J.P., Moreno, E., (1998) Eur. J. Biochem., 253, pp. 452-461Pieterson, W.A., Volwerk, J.J., Haas, G.H., (1974) Biochemistry, 13, pp. 1439-1445Rubsamen, K., Breithaupt, H., Habermann, E., (1971) Arch. Pharmacol., 270, pp. 274-288Schagger, H., Von Jagow, G., (1987) Anal. Biochem., 166, pp. 368-379Selistre De Araujo, H.S., White, S.P., Ownby, C.L., (1996) Arch. Biochem. Biophys., 326, pp. 21-30Shiomi, K.A., Kazama, A., Shimakura, K., Nagashima, Y., (1998) Toxicon, 36, pp. 589-599Soares, A.M., Andrião-Escaso, S.H., Bortoleto, R.K., Rodrigues-Simioni, L., Arni, R.K., Ward, R.J., Gutierrez, J.M., Giglio, J.R., (2001) Arch. Biochem. Biophys., 387, pp. 188-196Toyama, M.H., Soares, A.M., Wen-Hwa, L., Polikarpov, I., Giglio, J.R., Marangoni, S., (2000) Biochimie, 82, pp. 245-250Verheij, H.M., Boffa, M.C., Rothen, C., Bryckaert, M.C., Verger, R., De Hass, G.H., (1980) Eur. J. Biochem., 112, pp. 25-32Zhao, K., Zhou, Y., Lin, Z., (2000) Toxicon., 38, pp. 901-91

Enzymatic Characterization Of A Novel Phospholipase A2 From Crotalus Durissus Cascavella Rattlesnake (maracambóia) Venom

The PLA2 and crotapotin subunits of crotoxin from Crotalus durissus cascavella venom were purified by a combination of HPLC molecular exclusion (Protein Pack 300SW column) and reversephase HPLC (RP-HPLC). Tricine SDS-PAGE showed that the PLA2 and crotapotins migrated as single bands with estimated molecular masses of 15 and 9 kDa, respectively. The amino acid composition of the PLA2 showed the presence of 14 half-cysteines and a high content of basic residues (Lys, Arg, His), whereas the crotapotins were rich in hydrophobic, negatively charged residues and half-cysteines. The PLA2 showed allosteric behavior, with maximal activity at pH 8.3 and 35-40oC. The C. d. cascavella PLA2 required Ca2- for activity, but was inhibited by Cu2+ and Zn2+ and by Cu2 and Mg2 in the presence and absence of Ca2, respectively. Crotapotin (F3) and heparin inhibited the catalytic activity of the PLA2 by acting as allosteric inhibitors. © 2000 Plenum Publishing Corporation.197603607Aleksiev, B., Tchorbanov, B., (1976) Toxicon, 14, pp. 477-485Azevedo-Marques, M.M., Cupo, P., Coimbra, T.M., Hering, S.E., Rossi, M.A., Laure, C.J., (1982) Toxicon, 23, pp. 631-636Bon, C., Changeux, J.P., Jeng, T.W., Fraenkel-Conrat, H., (1979) Eur. J. Biochem., 99, pp. 471-1181Breithaupt, H., (1976) Toxicon, 14, pp. 221-233Cho, W., Kezdy, F.J., (1991) Meth. Enzymol., 197, pp. 75-79Cupo, P., Azevedo-Marques, M.M., Hering, S.E., (1988) Trans. R. Soc. Trop. Med. Hyg., 82, pp. 924-929Faure, G., Bon, C., (1988) Biochemistry, 27, pp. 730-738Faure, G., Guillaume, J.L., Camoin, L., Saliou, B., Bon, C., (1991) Biochemistry, 30, pp. 8074-8083Faure, G., Choumet, V., Bouchier, C., Camoin, L., Guillaume, J.L., Monegier, B., Vuilhorgne, M., Bon, C., (1994) Eur. J. Biochem., 223, pp. 161-164Habermann, E., Breithaupt, H., (1978) Toxicon, 16, pp. 19-30Hendon, R.A., Fraenkel-Conrat, H., (1971) Proc. Natl. Acad. Sei. USA, 68, pp. 1560-1563Holzer, M., Mackessy, S.P., (1996) Toxicon, 34, pp. 1149-1155Jarret, H.W., Cooksy, K.D., Ellis, B., Anderson, J.M., (1986) Anal. Biochem., 153, pp. 189-198Landucci, E.C.T., Condino-Neto, A., Ferez, A.C., Hyslop, S., Corrado, A.P., Novello, J.C., Marangoni, S., De Nucci, G., (1994) Toxicon, 32, pp. 217-226Landucci, E.C.T., Toyama, M., Marangoni, S., Oliveira, B., Cirino, G., Antunes, E., De Nucci, G., (2000) Toxicon, 38, pp. 199-208Martins, A.M.C., Monteiro, H.S.A., Junior, E.O.G., Menezes, D.B., Fonteies, M.C., (1988) Toxicon, 36, pp. 1441-1450Murakami, M., Hara, N., Kudo, I., Inoue, K., (1993) J. Immunol., 151, pp. 5675-5684Pieterson, W.A., Volwerck, J.J., De Haas, G.H., (1974) Biochemistry, 13, pp. 1439-1445Rosenfeld, G., (1971) Venomous Animals and Their Venoms, 2, pp. 345-362. , (Bucheri, W., and Buckley, E. E., eds.), Academic Press, New YorkRubsamen, K., Breithaupt, H., Habermann, E., (1971) NaunynSchmiedeberg's Arch. Pharmacol., 270, pp. 274-288Schagger, H., Von Jagow, G., (1987) Anal. Biochem., 166, pp. 368-379Toyama, M.H., Cameiro, E.M., Marangoni, S., Barbosa, R.L., Corso, G., Boschero, A.C., (2000) Biochim. Biophys. Acta, 1474, pp. 56-60Tu, A.T., Passey, R.B., Toom, P.M., (1970) Arch. Biochem. Biophys., 140, pp. 96-10

Anti-sera Raised In Rabbits Against Crotoxin And Phospholipase A 2 From Crotalus Durissus Cascavella Venom Neutralize The Neurotoxicity Of The Venom And Crotoxin

Crotoxin, the principal neurotoxin in venom of the South American rattlesnakes Crotalus durissus terrificus and Crotalus durissus cascavella, contains a basic phospholipase A2 (PLA2) and an acidic protein, crotapotin. In this work, we examined the ability of rabbit anti-sera against crotoxin and its PLA2 subunit to neutralize the neurotoxicity of venom and crotoxin from C. d. cascavella in mouse phrenic nerve-diaphragm and chick biventer cervicis preparations. Immunoblotting showed that the anti-sera recognized C. d. cascavella crotoxin and PLA2. This was confirmed by ELISA, with both anti-sera having end-point dilutions of 3×10-6. Anti-crotoxin serum neutralized the neuromuscular blockade in phrenic nerve-diaphragm muscle preparations at venom or crotoxin:anti-serum ratios of 1:2 and 1:3, respectively. Anti-PLA2 serum also neutralized this neuromuscular activity at a venom or crotoxin:anti-serum ratio of 1:1. In biventer cervicis preparations, the corresponding ratio for anti-crotoxin serum was 1:3 for venom and crotoxin, and 1:1 and 1:2 for anti-PLA2 serum. The neutralizing capacity of the sera in mouse preparations was comparable to that of commercial anti-serum raised against C. d. terrificus venom. These results show that anti-sera against crotoxin and PLA2 from C. d. cascavella venom neutralized the neuromuscular blockade induced by venom and crotoxin in both nerve-muscle preparations, with the anti-serum against crotoxin being slightly less potent than that against crotoxin. © 2004 Elsevier Ltd. All rights reserved.442141148Azevedo-Marques, M.M., Cupo, P., Coimbra, T.M., Hering, S.E., Rossi, M.A., Laure, C.J., Myonecrosis, myoglobinuria and acute renal failure induced by South-American rattlesnake (Crotalus durissus terrificus) envenomation in Brazil (1982) Toxicon, 23, pp. 631-636Beghini, D.G., Toyama, M.H., Hyslop, S., Sodek, L., Novello, J.C., Marangoni, S., Enzymatic characterization of a novel phospholipase A2 from Crotalus durissus cascavella rattlesnake (maracambóia) venom (2000) J. Protein Chem., 19, pp. 603-607Bon, C., Changeux, J.P., Jeng, T.W., Fraenkel-Conrat, H., Postsynaptic effects of crotoxin and of its isolated subunits (1979) Eur. J. Biochem., 99, pp. 471-481Breithaupt, H., Enzymatic characteristics of Crotalus phospholipase A2 and the crotoxin complex (1976) Toxicon, 14, pp. 221-233Bülbring, E., Observation on the isolated phrenic nerve diaphragm preparation of the rat (1946) Br. J. Pharmacol., 1, pp. 38-61Camara, P.R., Esquisatto, L.C., Camargo, E.A., Ribela, M.T., Toyama, M.H., Marangoni, S., De Nucci, G., Antunes, E., Inflammatory oedema induced by phospholipases A2 isolated from Crotalus durissus sp. in the rat dorsal skin: A role for mast cells and sensory C-fibers (2003) Toxicon, 41, pp. 823-829Chavez-Olórtegui, C., Silva Lopes, C., Drumond Cordeiro, F., Granier, C., Diniz, C.R., An enzyme linked immunosorbent assay (ELISA) that discriminates between Bothrops atrox and Lachesis muta muta venoms (1993) Toxicon, 31, pp. 417-425Choumet, V., Jiang, M.S., Radvanyi, F., Ownby, C., Bon, C., Neutralization of lethal potency and inhibition of enzymatic activity of a phospholipase A2 neurotoxin, crotoxin, by non-precipitating antibodies (Fab) (1989) FEBS Lett., 244, pp. 167-173Choumet, V., Faure, G., Robbe-Vincent, A., Saliou, B., Mazié, J.C., Bon, C., Immunochemical analysis of a snake venom phospholipase A2 neurotoxin, crotoxin, with monoclonal antibodies (1992) Mol. Immunol., 29, pp. 871-882Choumet, V., Lafaye, P., Mazié, J.C., Bon, C., A monoclonal antibody directed against the non-toxic subunit of a dimeric phospholipase A2 neurotoxin, crotoxin, neutralizes its toxicity (1998) J. Biol. Chem., 379, pp. 899-906Dos Santos, M.C., Diniz, C.R., Pacheco, M.A., Dias Da Silva, W., Phospholipase A2 injection in mice induces immunity against the lethal effects of Crotalus durissus terrificus venom (1988) Toxicon, 26, pp. 207-213Faure, G., Harvey, A.L., Thomson, E., Saliou, B., Radvani, F., Bon, C., Comparison of crotoxin isoforms reveals that stability of the complex plays a major role in its pharmacological action (1993) Eur. J. Biochem., 214, pp. 491-496Freitas, T.V., Fortes-Dias, C.L., Diniz, C.R., Protection against the lethal effects of Crotalus durissus terrificus (South American rattlesnake) venom in animals immunized with crotoxin (1990) Toxicon, 28, pp. 1491-1496Ginsborg, B.L., Warriner, J., The isolated chick biventer cervicis nerve muscle preparation (1960) Br. J. Pharmacol., 15, pp. 410-411Hendon, R.A., Fraenkel-Conrat, H., Biological roles of the two components of crotoxin (1971) Proc. Natl Acad. Sci. USA, 68, pp. 1560-1563Jeng, T.W., Hendon, R.A., Fraenkel-Conrat, H., Search for relationship among the hemolytic, phospholipolytic and neurotoxic activities of snake venoms (1978) Proc. Natl Acad. Sci. USA, 75, pp. 600-604Landucci, E.C., Condino-Neto, A., Perez, A.C., Hyslop, S., Corrado, A.P., Novello, J.C., Marangoni, S., De Nucci, G., Crotoxin induces aggregation of human washed platelets (1994) Toxicon, 32, pp. 217-226Lomonte, B., Gutiérrez, J.M., Moreno, E., Cerdas, L., Antibody neutralization of a myotoxin from the venom of Bothrops asper (terciopelo) (1987) Toxicon, 25, pp. 443-449Marlas, G., Purification and preliminary structure of a potent platelet aggregating glycoprotein isolated from the venom of Crotalus durissus cascavella (1982) Toxicon, 20, pp. 289-290Marlas, G., Isolation and characterization of the alpha and beta subunits of the platelet-activating glycoprotein from the venom of Crotalus durissus cascavella (1985) Biochimie, 67, pp. 1232-1239Marlas, G., Bon, C., Relationship between the pharmacological action of crotoxin and its phospholipase activity (1982) Eur. J. Biochem., 125, pp. 157-165Marlas, G., Joseph, D., Huet, C., I-isolation and electron microscope studies of a potent platelet-aggregating glycoprotein from the venom of Crotalus durissus cascavella (1983) Biochimie, 65, pp. 405-416Marlas, G., Joseph, D., Huet, C., Subunit structure of a potent platelet-activating glycoprotein isolated from the venom of Crotalus durissus cascavella (1983) Biochimie, 65, pp. 619-628Martins, A.M., Monteiro, H.S., Júnior, E.O., Menezes, D.B., Fonteles, M.C., Effects of Crotalus durissus cascavella venom in the isolated rat kidney (1998) Toxicon, 36, pp. 1441-1450Martins, A.M., Toyama, M.H., Havt, A., Novello, J.C., Marangoni, S., Fonteles, M.C., Monteiro, H.S., Determination of Crotalus durissus cascavella venom components that induce renal toxicity in isolated rat kidneys (2002) Toxicon, 40, pp. 1165-1171Martins, A.M., Lima, A.A., Toyama, M.H., Marangoni, S., Fonteles, M.C., Monteiro, H.S., Renal effects of supernatant from macrophages activated by Crotalus durissus cascavella venom: The role of phospholipase A2 and cyclooxygenase (2003) Pharmacol. Toxicol., 92, pp. 14-20Oliveira, D.G., Toyama, M.H., Martins, A.M., Havt, A., Nobre, A.C., Marangoni, S., Câmara, P.R., Monteiro, H.S., Structural and biological characterization of a crotapotin isoform isolated from Crotalus durissus cascavella venom (2003) Toxicon, 42, pp. 53-62Oshima-Franco, Y., Hyslop, S., Prado-Franceschi, J., Cruz-Höfling, M.A., Rodrigues-Simioni, L., Neutralizing capacity of antisera raised in horses and rabbits against Crotalus durissus terrificus (South American rattlesnake) venom and its main toxin, crotoxin (1999) Toxicon, 37, pp. 1341-1457Ouchterlony, O., Antigen-antibody reactions in gels (1949) Acta Path. Microbiol. Scand., 26, pp. 507-515Rosenfeld, G., Symptomatology, pathology and treatment of snake bites in South America (1971) Venomous Animals and Their Venoms, 2, pp. 345-384. , W. Bücherl, Buckley E.E. New York: Academic PressSantoro, M.L., Sousa-E-Silva, M.C.C., Gonçalvez, L.R.C., Almeida-Santos, S.M., Cardoso, D.F., Laporta-Ferreira, I.L., Saiki, M., Sano-Martins, I.S., Comparison of the biological activities in venoms from three subspecies of the South American rattlesnake (Crotalus durissus terrificus, C. durissus cascavella and C. durissus collilineatus) (1999) Comp. Biochem. Physiol. Part C, 122, pp. 61-73Slotta, K.H., Fraenkel-Conrat, H.L., Schlangengifte, III: Mitteilung Reiningung und Krystallization des Klapperschangengiftes (1938) Ber. Dich. Chem. Ges., 71, pp. 1076-1081Vital Brazil, O., Pharmacology of crystalline crotoxin. II. Neuromuscular blocking action (1966) Mem. Inst. Butantan, 33, pp. 981-99

Structural, Enzymatic And Biological Properties Of New Pla2 Isoform From Crotalus Durissus Terrificus Venom

We isolated a new PLA2 from the Crotalus durissus terrificus venom that designated F15, which showed allosteric behavior with a V max of 8.5nmol/min/mg and a Km of 38.5 mM. The incubated heparin salt of this isolated F15 act a positive allosteric effector by increasing the Vmax to 10.2nmol/min/mg, with decreasing the V max value to 20.5 mM. The crotapotin, on the other hand acts as a negative allosteric effector by increasing the Vmax values to 58.4 mM. F15also showed high calcium dependence for its catalysis similar to that found for other PLA2 enzymes isolated from these snake venoms. The replacement of calcium by other divalent ions such Mg2+, Mn 2+, Cd2+, Sn2+ and Cu2+ resulted in lower enzymatic activity. The optimum pH and temperature for the enzyme was 8.5 and 18 °C, respectively. F15 alone showed moderate neurotoxic activity in isolated mouse phrenic nerve diaphragm in comparison to other strong myotoxic PLA2 such as bothropstoxin-I (BThtx-I), but this activity was highly neurotoxic in a chick biventrer cervis preparation, whereas BthTx-I did not reveal this high neurotoxicity. This new protein showed a high bactericidal effect against both Gram-negative and Gram-positive bacterial strains. F15 contained 122 amino acid residues, with a primary structure of: HLLQFNKMIKFETRKNAVPFYAFYGCYCGWGGQRRPKDATDRCCFVHDCCYGKLTKCNTKWDIYRYSLKSGYIT CGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPKSRCRRPSETC. Its molecular mass and isoeletric point were 14.5 kDa and 8.85, both estimated by two dimensional electrophoresis. The amino acid sequence of the F15 revealed high sequence homology with F16 and F17. F15 and the other PLA2s revealed highly conserved amino acid sequences principally for calcium binding loop and active site helix. F15 also showed a high homology with the lysine-rich region of myotoxic PLA2. © 2003 Elsevier Science Ltd. All rights reserved.41810331038Beghini, D.G., Toyama, M.H., Hyslop, S., Sodek, L., Novello, J.C., Marangoni, S., Enzymatic characterization of a novel phospholipase A2 from Crotalus durissus cascavella rattlesnake (maracambóia) venom (2000) J. Protein Chem., 19, pp. 603-607Bon, C., Changeux, J.P., Jeng, T.W., Fraenkel-Conrat, H., Postsynaptic effects of crotoxin and of its isolated subunits (1979) Eur. J. Biochem., 99, pp. 471-481Breithaupt, H., Enzymatic characteristics of Crotalus phospholipase A2 and the crotoxin complex (1976) Toxicon, 14, pp. 221-233Bülbring, E., Observation on the isolated phrenic nerve diaphragm preparation of the rat (1946) Br. J. Pharmacol., 1, pp. 38-61Gambero, A., Landucci, E.C.T., Toyama, M.H., Marangoni, S., Giglio, J.R., Nader, H.B., Dietrich, C.P., Antunes, E., Human neutrophil migration in vitro induced by secretory Phospholipases A2: Role of cell surface glycosaminoglycans (2002) Biochem. Pharmacol., 63, pp. 65-72Ginsborg, B.L., Warriner, J., The isolated chick biventer nerve muscle preparation (1960) Br. J. Pharmacol., 15, pp. 410-421Habermann, E., Breithaupt, H., The crotoxin complex - An example of biochemical and pharmacological protein complementation (1978) Toxicon, 16, pp. 19-30Holzer, M., Mackessy, S.P., An aqueous endpoint assay of snake venom phospholipase A2 (1996) Toxicon, 34, pp. 1149-1155Lee, W.-H., Marangoni, S., Toyama, M.H., Da Silva Giotto, M.T., Polikarpov, I., Garrat, R.C., The structural basis for catalytic inactivity in Lys49 phospholipase A2 - A hypothesis: The crystal structure of Piratoxin II complexed to fatty acid (2001) J. Biochem., 40, pp. 28-36Oliveira, D.G., Toyama, M.H., Novello, J.C., Beliam, L.O.S., Marangoni, S., Biochemical and biological characterization of basic PLA2 isoform from Crotalus durissus terrificus venom (2001) J. Protein Chem., 21 (3), pp. 131-136Pieterson, W.A., Volwerk, J.J., De Haas, G.H., Interaction of phospholipase A2 and its zymogen with divalent metal ions (1974) Biochemistry, 13 (7), pp. 14-39Soares, A.M., Andrião-Escaso, S.H., Angulo, Y., Lomonte, B., Gutierrez, J.M., Marangoni, S., Toyama, M.H., Giglio, J.R., Structural and funtional characterization of myotoxin I, a Lys phospholipase A2 homologue form Bothrops moojeni (Caissaca) snake venom (2000) Arch. Biochem. Biophys., 373, pp. 7-15Toyama, M.H., Soares, A.M., Wen-Hwa, L., Polikarpov, I., Giglio, J.R., Marangoni, S., Amino acid sequence of piratoxin-II, a myotoxic lys49 phospholipase A2 homologue from Bothrops pirajai venom (2000) Biochimie, 82, pp. 245-250Toyama, M.H., Soares, A.M., Andrião-Escaso, S.H., Novello, J.C., Oliveira, B., Giglio, J.R., Fontes, M.R.M., Marangoni, S., Comparative biochemical studies of myotoxic phospholipase A2 from Bothrops venom (2001) Protein Peptide Lett., 8, pp. 179-78

Ability Of Rabbit Antiserum Against Crotapotin To Neutralize The Neurotoxic, Myotoxic And Phospholipase A2 Activities Of Crotoxin From Crotalus Durissus Cascavella Snake Venom

The toxicity of crotoxin, the major toxin of Crotalus durissus terrificus (South American rattlesnake) venom, is mediated by its basic phospholipase A2 (PLA2) subunit. This PLA2 is non-covalently associated with crotapotin, an acidic, enzymatically inactive subunit of the crotoxin complex. In this work, rabbit antiserum raised against crotapotin purified from Crotalus durissus cascavella venom was tested for its ability to neutralize the neurotoxicity of this venom and its crotoxin in vitro. The ability of this antiserum to inhibit the enzymatic activity of the crotoxin complex and PLA2 alone was also assessed, and its potency in preventing myotoxicity was compared with that of antisera raised against crotoxin and PLA2. Antiserum to crotapotin partially neutralized the neuromuscular blockade caused by venom and crotoxin in electrically stimulated mouse phrenic nerve-hemidiaphragm preparations and prevented the venom-induced myotoxicity, but did not inhibit the enzymatic activity of crotoxin and purified PLA2. In contrast, previous findings showed that antisera against crotoxin and PLA2 from C. d. cascavella effectively neutralized the neuromuscular blockade and PLA2 activity of this venom and its crotoxin. The partial neutralization of crotoxin-mediated neurotoxicity by antiserum to crotapotin probably reduced the binding of crotoxin to its receptor following interaction of the antiserum with the crotapotin moiety of the complex. © 2007 Elsevier Ltd. All rights reserved.221240248Aird, S.D., Kaiser, I.I., Lewis, R.V., Krugger, W.G., Rattlesnake pre-synaptic neurotoxins: primary structure and evolutionary origin of the acidic subunit (1985) Biochemistry, 24, pp. 7054-7058Azevedo-Marques, M.M., Cupo, P., Coimbra, T.M., Hering, S.E., Rossi, M.A., Laure, C.J., Myonecrosis, myoglobinuria and acute renal failure induced by South-American rattlesnake (Crotalus durissus terrificus) envenomation in Brazil (1985) Toxicon, 23, pp. 631-636Beghini, D.G., Toyama, M.H., Hyslop, S., Sodek, L., Novello, J.C., Marangoni, S., Enzymatic characterization of a novel phospholipase A2 from Crotalus durissus cascavella rattlesnake (maracambóia) venom (2000) J. Protein Chem., 19, pp. 603-607Beghini, D.G., Hernandez-Oliveira, S., Rodrigues-Simioni, L., Novello, J.C., Hyslop, S., Marangoni, S., Anti-sera raised in rabbits against crotoxin and phospholipase A2 from Crotalus durissus cascavella venom neutralize the neurotoxicity of the venom and crotoxin (2004) Toxicon, 44, pp. 141-148Beghini, D.G., Rodrigues-Simioni, L., Toyama, M.H., Novello, J.C., Cruz-Höfling, M.A., Marangoni, S., Neurotoxic and myotoxic actions of crotoxin-like and Crotalus durissus cascavella whole venom in the chick biventer cervicis preparation (2004) Toxicon, 43, pp. 255-261Bon, C., Changeux, J.P., Jeng, T.W., Fraenkel-Conrat, H., Postsynaptic effects of crotoxin and of its isolated subunits (1979) Eur. J. Biochem., 99, pp. 471-481Bradford, M.M., A rapid and sensitive method for the quantification of microgram quantities of protein using the principal of protein-dye binding (1976) Anal. 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Cross-neutralization Of The Neurotoxicity Of Crotalus Durissus Terrificus And Bothrops Jararacussu Venoms By Antisera Against Crotoxin And Phospholipase A2 From Crotalus Durissus Cascavella Venom

We have previously demonstrated that rabbit antisera raised against crotoxin from Crotalus durissus cascavella venom (cdc-crotoxin) and its PLA 2 (cdc-PLA2) neutralized the neurotoxicity of this venom and its crotoxin. In this study, we examined the ability of these antisera to neutralize the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms and their major toxins, cdt-crotoxin and bothropstoxin-I (BthTX-I), respectively, in mouse isolated phrenic nerve-diaphragm preparations. Immunoblotting showed that antiserum to cdc-crotoxin recognized cdt-crotoxin and BthTX-I, while antiserum to cdc-PLA2 recognized cdt-PLA 2 and BthTX-I. ELISA corroborated this cross-reactivity. Antiserum to cdc-crotoxin prevented the neuromuscular blockade caused by C. d. terrificus venom and its crotoxin at a venom/crotoxin:antiserum ratio of 1:3. Antiserum to cdc-PLA2 also neutralized the neuromuscular blockade caused by C. d. terrificus venom or its crotoxin at venom or toxin:antiserum ratios of 1:3 and 1:1, respectively. The neuromuscular blockade caused by B. jararacussu venom and BthTX-I was also neutralized by the antisera to cdc-crotoxin and cdc-PLA 2 at a venom/toxin:antiserum ratio of 1:10 for both. Commercial equine antivenom raised against C. d. terrificus venom was effective in preventing the neuromuscular blockade typical of B. jararacussu venom (venom:antivenom ratio of 1:2), whereas for BthTX-I the ratio was 1:10. These results show that antiserum produced against PLA2, the major toxin in C. durissus cascavella venom, efficiently neutralized the neurotoxicity of C. d. terrificus and B. jararacussu venoms and their PLA2 toxins.466604611Beghini, D.G., Toyama, M.H., Hyslop, S., Sodek, L., Novello, J.C., Marangoni, S., Enzymatic characterization of a novel phospholipase A2 from crotalus durissus cascavella rattlesnake (maracambóia) venom (2000) J. 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