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

Abstract

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