Enzymatic And Structural Characterization Of New Pla2 Isoform Isolated From White Venom Of Crotalus Durissus Ruruima

This work reports the structural and enzymatic characterization of a new sPLA2 from the white venom of Crotalus durissus ruruima, nominated PLA2A. The homogeneity of the PLA2A fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14,299.34 Da. Structural investigation, through circular dichroism spectroscopy, revealed that PLA2A has a high content of alpha helix and beta-turn structures, 45.7% and 35.6% respectively. Its amino acid sequence, determined by Edman degradation and "de novo amino acid sequencing", exhibited high identity to PLA2 Cdt F15 from Crotalus durissus terrificus. The enzymatic investigation, conducted using the synthetic substrate 4-nitro-3-(octanoyloxy)-benzoic acid, determined its Vmax (7.56 nmoles/min) and KM (2.76 mM). Moreover, PLA2A showed an allosteric behavior and its enzymatic activity was dependent on Ca2+. Intrinsic fluorescence measurements suggested that Ca2+ induced a significant increase of PLA2A fluorescence, whereas its replacement for Mg2+, Mn2+, Sn2+ and Cd2+ apparently induced no structural modifications. The optimal pH and temperature for the enzymatic activity of PLA2A were 8.4 and 40 °C, respectively, and the minimal concentration of p-BPB and crotapotin that significantly inhibited such activity was 0.75 mM and 0.4 μM, respectively. In addition, PLA2A showed a significant antibacterial effect that was not strictly dependent on the enzymatic activity of such sPLA2. © 2008 Elsevier Ltd. All rights reserved.531104114Bercovici, D., Chudzinski, A.M., Dias, W.O., Esteves, M.I., Hiraichi, E., Oishi, N.Y., Picarelli, Z.P., Raw, I., A systematic fractionation of Crotalus durissus terrificus venom (1987) Mem. Inst. Butantan (São Paulo), 49, pp. 69-78Barrio, A., Estudio electroforético del veneno de serpiente de cascabel (1954) Cienc. Investig., 10, pp. 368-370Bon, C., Bouchier, C., Choumet, V., Faure, G., Jiang, M.S., Lambezat, M.P., Radvanyi, F., Saliou, B., Crotoxin, half-century of investigations on a phospholipase A2 neurotoxin (1989) Acta Physiol. Pharmacol. Latinoam., 39, pp. 439-448Chen, Y.H., Wang, Y.M., Hseu, M.J., Tsai, I.H., Molecular evolution and structure-function relationships of crotoxin-like and asparagine-6-containing phospholipases A2 in pit viper venoms (2004) Biochem. J., 381 (PART 1), pp. 25-34Chang, L.S., Lin, S.R., Chang, C.C., Probing calcium ion-induced conformational changes of Taiwan cobra phospholipase A2 by trinitrophenylation of lysine residues (1994) J. Protein Chem., 16, pp. 51-57Chang, L.S., Wu, P.F., Liou, J.C., Chiang-Lin, W.H., Yang, C.C., Chemical modification of arginine residues of Notechis scutatus scutatus notexin (2004) Toxicon, 44, pp. 491-497Chu, Y.P., Cheng, Y.C., Yang, C.C., Chang, L.S., The structural events associated with the binding of divalent cations to beta-bungarotoxin (2005) Toxicon, 45, pp. 139-145Dal Belo, C.A., Toyama, M.H., Toyama Dde, O., Marangoni, S., Moreno, F.B., Cavada, B.S., Fontana, M.D., Boschero, A.C., Determination of the amino acid sequence of a new phospholipase A(2) (MIDCA1) isolated from Micrurus dumerilii carinicauda venom (2005) Protein J., 24 (3), pp. 147-153Dos Santos, M.C., Ferreira, L.C., Da Silva, W.D., Furtado, M.F., Characterization of the biological activities of the 'yellow' and 'white' venoms from Crotalus durissus ruruima compared with the Crotalus durissus terrificus venom. Neutralizing activity of Crotalus durissus ruruima antivenins (1993) Toxicon, 31, pp. 1459-1469Dos Santos, M.C., Assis, E.B., Moreira, T.D., Pinheiro, J., Fortes-Dias, C.L., Individual venom variability in Crotalus durissus ruruima snakes, a subspecies of Crotalus durissus from the Amazonian region (2005) Toxicon, 46, pp. 958-961Faure, G., Choumet, V., Bouchier, C., Camoin, L., Guillaume, J.L., Monegier, B., Vuilhorgne, M., Bon, C., The origin of the diversity of crotoxin isoforms in the venom of Crotalus durissus terrificus (1994) Eur. J. Biochem., 223, pp. 161-164Faure, G., Bon, C., Crotoxin, a phospholipase A2 neurotoxin from the South American rattlesnake Crotalus durissus terrificus: purification of several isoforms and comparison of their molecular structure and of their biological activities (1988) Biochemistry, 27, pp. 730-738Fuly, A.L., de Miranda, A.L., Zingali, R.B., Guimarães, J.A., Purification and characterization of a phospholipase A2 isoenzyme isolated from Lachesis muta snake venom (2002) Biochem. Pharmacol., 63, pp. 1589-1597Gonçalves, J.M., Vieira, L.G., Estudos sobre venenos de serpentes brasileiras. I. Análise eletroforética (1950) An. Acad. Bras. Ciênc., 22, pp. 141-150Garcia, F., Toyama, M.H., Castro, F.R., Proença, P.L., Marangoni, S., Santos, L.M., Crotapotin induced modification of T lymphocyte proliferative response through interference with PGE2 synthesis (2003) Toxicon, 42, pp. 433-437Georgieva, D.N., Betzel, C., Aleksiev, B., Genov, N., Spectroscopic investigation of calcium binding sites in the neurotoxin vipoxin and its components-relation with the X-ray structure (2000) Spectrochim. Acta A Mol. Biomol. Spectrosc., 56, pp. 2811-2816Gibbs, H.L., Prior, K.A., Weatherhead, P.J., Johnson, G., Genetic structure of populations of the threatened eastern massasauga rattlesnake, Sistrurus c. catenatus: evidence from microsatellite DNA markers (1997) Mol. Ecol., 6 (12), pp. 1123-1132John, T.R., Smith, L.A., Kaiser, I.I., Genomic sequences encoding the acidic and basic subunits of Mojave toxin: unusually high sequence identity of non-coding regions (1994) Gene, 139 (2), pp. 229-234Kini, R.M., Chan, Y.M., Accelerated evolution and molecular surface of venom phospholipase A2 enzymes (1999) J. Mol. Evol., 48, pp. 125-132Kini, R.M., Evans, H.J., A model to explain the pharmacological effects of snake venom phospholipases A2 (1989) Toxicon, 27, pp. 613-635Landucci, E.C., Antunes, E., Donato, J.L., Faro, R., Hyslop, S., Marangoni, S., Oliveira, B., de Nucci, G., Inhibition of carrageenin-induced rat paw oedema by crotapotin, a polypeptide complexed with phospholipase A2 (1995) Br. J. Pharmacol., 114, pp. 578-583Landucci, E.C., Toyama, M., Marangoni, S., Oliveira, B., Cirino, G., Antunes, E., de Nucci, G., Effect of crotapotin and heparin on the rat paw oedema induced by different secretory phospholipases A2 (2000) Toxicon, 38, pp. 199-208Lee, W.H., Toyama, M.H., Soares, A.M., Giglio, J.R., Marangoni, S., Polikarpov, I., Crystallization and preliminary X-ray diffraction studies of piratoxin III, a D-49 phospholipase A2 from the venom of Bothrops pirajai (1999) Acta Crystallogr. D Biol. Crystallogr., 55, pp. 1229-1230Liu, X., Zhu, H., Huang, B., Rogers, J., Yu, B.Z., Kumar, A., Jain, M.K., Tsai, M.D., Phospholipase A2 engineering. Probing the structural and functional roles of N-terminal residues with site-directed mutagenesis, X-ray, and NMR (1995) Biochemistry, 34, pp. 7322-7334Ladokhin, A.S., Jayasinghe, S., White, S.H., How to measure and analyze tryptophan fluorescence in membranes properly, and why bother? (2000) Anal. Biochem., 285, pp. 235-245Nakashima, K., Nobuhisa, I., Deshimaru, M., Nakai, M., Ogawa, T., Shimohigashi, Y., Fukumaki, Y., Ohno, M., Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes (1995) Proc. Natl. Acad. Sci. U.S.A., 92, pp. 5605-5609Oliveira, 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. 53-62Oliveira, D.G., Toyama, M.H., Novello, J.C., Beriam, L.O., Marangoni, S., Structural and functional characterization of basic PLA2 isolated from Crotalus durissus terrificus venom (2002) J. Protein Chem., 21, pp. 161-168Ogawa, T., Oda, N., Nakashima, K., Sasaki, H., Hattori, M., Sakaki, Y., Kihara, H., Ohno, M., Unusually high conservation of untranslated sequences in cDNAs for Trimeresurus flavoviridis phospholipase A2 isozymes (1992) Proc. Natl. Acad. Sci. U.S.A., 89, pp. 8557-8561Ohno, M., Chijiwa, T., Oda-Ueda, N., Ogawa, T., Hattori, S., Molecular evolution of myotoxic phospholipases A2 from snake venom (2003) Toxicon, 42, pp. 841-854Ogawa, T., Nakashima, K., Nobuhisa, I., Deshimaru, M., Shimohigashi, Y., Fukumaki, Y., Sakaki, Y., Ohno, M., Accelerated evolution of snake venom phospholipase A2 isozymes for acquisition of diverse physiological functions (1996) Toxicon, 34, pp. 1229-1236Oda, N., Yoshida, M., Tanaka, S., Kihara, H., Ohno, M., Interactions of Trimeresurus flavoviridis phospholipase A2 and its N-terminal octapeptide-removed and p-bromophenacylated derivatives with acridine and anilinonaphthalene dyes (1986) J. Biochem., 100, pp. 1551-1560Ponce-Soto, L.A., Baldasso, P.A., Romero-Vargas, F.F., Winck, F.V., Novello, J.C., Marangoni, S., Biochemical, pharmacological and structural characterization of two PLA2 isoforms Cdr-12 and Cdr-13 from Crotalus durissus ruruima snake venom (2007) Protein J., 26, pp. 39-49Passero, L.F., Tomokane, T.Y., Corbett, C.E., Laurenti, M.D., Toyama, M.H., Comparative studies of the anti-leishmanial activity of three Crotalus durissus ssp. Venoms (2007) Parasitol. Res., 101, pp. 1365-1371Santoro, M.L., Sousa-e-Silva, M.C., Gonçalves, L.R., 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. C Pharmacol. Toxicol. Endocrinol., 122, pp. 61-73Schägger, H., von Jagow, G., Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa (1987) Anal. Biochem., 166, pp. 368-379Scott, D.L., Sigler, P.B., Structure and catalytic mechanism of secretory phospholipases A2 (1994) Adv. Protein Chem., 45, pp. 53-88Toyama, M.H., de Oliveira, D.G., Beriam, L.O., Novello, J.C., Rodrigues-Simioni, L., Marangoni, S., Structural, enzymatic and biological properties of new PLA(2) isoform from Crotalus durissus terrificus venom (2003) Toxicon, 41, pp. 1033-1038Toyama, M.H., Toyama, D.O., Joazeiro, P.P., Carneiro, E.M., Beriam, L.O., Marangoni, L.S., Boschero, A.C., Biological and structural characterization of a new PLA2 from the Crotalus durissus collilineatus venom (2005) Protein J., 24, pp. 103-112Toyama, M.H., Carneiro, E.M., Marangoni, S., Barbosa, R.L., Corso, G., Boschero, A.C., Biochemical characterization of two crotamine isoforms isolated by a single step RP-HPLC from Crotalus durissus terrificus (South American rattlesnake) venom and their action on insulin secretion by pancreatic islets (2000) Biochim. Biophys. Acta, 1474, pp. 56-60Toyama, M.H., Carneiro, E.M., Marangoni, S., Amaral, M.E.C., Velloso, L.A., Boschero, A.C., Isolation and characterization of a convulxin-like protein from Crotalus durissus collilineatus venom (2001) J. Protein Chem., 20, pp. 585-591Valiente, C., Moreno, E., Sittenfeld, A., Lomonte, B., Gutiérrez, J.M., An electrophoretic study on phospholipase A2 isoenzymes in the venoms of Central American crotaline snakes (1992) Toxicon, 30, pp. 815-823Zhao, H., Tang, L., Wang, X., Zhou, Y., Lin, Z., Structure of a snake venom phospholipase A2 modified by p-bromo-phenacyl-bromide (1998) Toxicon, 36, pp. 875-88