1 Protocolo de purificación en dos etapas de fosfolipasas A2 a partir de la anémona marina Condylactis gigantea Two-steps phospholipases A2 purification protocol from the sea anemone Condylactis gigantea Protocolo de purificación en dos etapas de fosfolipasas A2 a partir de la anémona marina Condylactis gigantea Two-steps phospholipases A2 purification protocol from the sea anemone Condylactis gigantea Resumen: Numerosas investigaciones han demostrado similitud entre la composición del veneno de los celenterados marinos con los de serpiente en base a las propiedades farmacológicas que poseen. Estos constituyen mezclas complejas de varios componentes, muchos de naturaleza proteica, y para los cuales se ha descrito la actividad fosfolipásica A2. La cromatografía de afinidad constituye uno de los métodos de mayor utilidad para la purificación de proteínas debido a la especificidad de la interacción que se establece en este método, de ahí la importancia de seleccionar soportes adecuados para lograr este fin, por lo que se planteó como objetivo de la presente investigación purificar las fosfolipasas A2 (FLA2) procedentes de la anémona marina Condylactis gigantea a partir de un protocolo de dos etapas para su posterior caracterización. En el presente trabajo se muestra la purificación de FLA 2 en un soporte cromatográfico de afinidad con fosfatidilcolina de huevo inmovilizada covalentemente para la purificación de fosfolipasas A2, a los que se les comprobó cualitativamente la actividad fosfolipásica A2 por cromatografía en placa (TLC) utilizando sustrato marcado con fluorescencia. Dicho soporte cromatográfico permite que en un protocolo de purificación de solamente dos pasos se obtengan tres componentes proteicos de pesos moleculares entre 18000 y 14000, con al menos un componente que posee actividad fosfolipásica A2. Introduction: These enzymes are widely spread in nature (Berner & Hammond, 1970; Six & Dennis, 2000) and their functional versatility has conditioned their use in Specialized and Applied Biochemistry, Ecotoxicology and Fine Chemistry (Madoery & Minchiotti, 2006; Minchiotti et al., 2008) where is not always needed a highly purified enzyme. Marine organisms have been less explored than mammals; snakes, bees and scorpions poisons and microorganisms and there is also a higher probability for finding enzymes having different specificities towards non-natural substrates in those environments, that’s why the quest for phospholipase activity in marine invertebrates has been a major research goal in this field (Nevalainen et al., 2004; Knotz et al., 2006; Park et al., 2008; Perera et al., 2008) and they have demonstrated similarity with those from snakes based on their pharmacological properties and where PLA2 activity has been described in some species, where is a representative of this family is the anemone Condylactis gigantea.Berner & Hammond, 1970Six & Dennis, 2000Madoery & Minchiotti, 2006Minchiotti et al., 2008Nevalainen et al., 2004Knotz et al., 2006Park et al., 2008Perera et al., 2008 Materials and methods Sea anemone Condylactis gigantea was collected at the coast of Havana and extracts were obtained according to Romero et al., (1987). Chromatographic partitioning. - The initial step of chromatographic partitioning in Sephadex G-50 thin grade (Pharmacia Fine Chemicals) was the same used for isolation and purification of marine toxins from this coelenterate according to Romero (1997 ).1997 - Affinity chromatography in PC-MANA-Sepharose CL-4B was carried out at 25°C in a glass column (0.7x8 cm) with buffer TrisHCl 0.05 M pH 7.5 (fixation buffer containing CaCl 2 0.04 M). Elution was performed adding 0.05 M EDTA (without Ca 2+ ) at a flow rate of 90 cm/h and fractions of 3mL were collected. Phospholipase A2 activity was corroborated qualitatively by a TLC-based method after exposure to purified ePC and fluorogenic substrate 1-palmitoil–2-NBD-C12-PC. SDS-PAGE 12.5% was carried out according to Laemmli (1970), in a vertical chamber (30 min., 100 V, 50 mA and 12 watt at concentration gel; 1:07 h, 150 V, 100 mA and 12 watt for).1970 -4 different flow speeds (22-90 cm/h) was checked in order to verify the interactions of proteins fixed to the chromatographic support. As observed, reducing flow speed there is a better resolution of the fractions, which could be explained for differential hydrophobic interactions of proteins with the immobilized ligand in the chromatographic support.Results: - Due to complexities to determine and/or to quantify the specific enzymatic activity by continuous method for those enzymes, the proteins yield in each step is one of the useful parameters - C. gigantea, was purified as showed in SDS-PAGE. - Fluorescence fatty acid presence in TLC indicated that eluted fractions from affinity of C. gigantea contain PLA2 activity (Abe et al. 2010).Abe et al. 2010 Table 1. Total proteins recovered after each purification step in a continuous system (direct system). References: Abe, A. et al. 2010. J. Lipid Res. 51 : 2464-2470. Aiba, S. et al. 1973. Biochemical Engineering. 2nd edition ed. Academic Press Inc New York. Albert, D.H., and Snyder, F. 1983. The Journal of Biological Chemistry 258 : 97-102. Berner, D.L., and Hammond, E.G. 1970. Lipids 5 (6): 558-562. Knotz, S., Boersma, M., and Saborowski, R. 2006. Comparative biochemistry and physiology. Part A 145 (3): 406-411. Laemmli, U.K. 1970. Nature 227 : 680-685. Madoery, R., and Minchiotti, M. 2006. Enzyme and Microbial Technology 38 (7): 869-872. Minchiotti, M. et al. 2008. Enzyme and Microbial Technology 42 (5): 389-394. Nevalainen, T.J. et al. 2004. Comp. Biochem. Physiol. 139 Part B : 731-735. Park, J., Cho, S.Y., and Choi, S.J. 2008. BMB Reports 41 (3): 254-258. Perera, E. et al. 2008.. Comp Biochem Physiol B 150 (3): 247-254. Romero, L. et al. 2010. Biochimie 92 :1063-1071. Scope, R. 1984. Protein Purification. Principles and Practice. Second Edition, Ed. SpringerVerlag, New York. Silveira, L.B. et al. 2013. Journal of Pharmaceutical and Biomedical Analysis 73 (0): 35-43. Six, D.A., and Dennis, E.A. 2000. Biochim. Biophys. Acta 1488 : 1-19 Sun, M.Z. et al. 2009. Biochimie 91 (4): 558-567. Valentin, E., Lambeau, G. 2000. Biochimie 82 (9): 815- 831. Wang, Y. et al. 2008. Toxicon 51 (7): 1131-1139. Wurl, M., Kunze, H. 1985. Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism 834 (3): 411-418. Total proteins (mg)Yield (%) Crude extract 148,05 +/- 6,78100 Gel partitioning 77,22 +/- 8,7952,16 +/- 5,92 Affinity chromatography 28,83 +/- 4,7719,47 +/- 3,22 Frenkel Guisado-Bourzac a*, Dolores Lázara Romero del Sol b, Jorge González-Baceiro b, José Manuel Guisán-Seijas c, Joaquín Díaz-Brito b and Alberto del Monte-Martínez b a Universidad de Oriente, Ave Patricio Lumumba s/n esq. Ave de las Américas, Santiago de Cuba, Cuba. b Centro de Estudio de Proteínas, 25 # 455 e/ J e I, Facultad de Biología, Universidad de La Habana, Cuba. c Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Cantoblanco, 28049, Madrid, España. *Corresponding author: Frenkel Guisado-Bourzac E-mail address: [email protected]@uo.edu.cu Palabras claves: cromatografía de afinidad, fosfolipasa A2, anémona marina, Condylactis gigantea, purificación de proteínas Keywords: affinity, phospholipase A2, Condylactis gigantea, sea anemone, protein purification Abstract: Numerous investigations have demonstrated similarities between marine coelenterates poisons composition with those from snakes, based on pharmacological properties they possess. They constitute complex mixtures of several components, mainly of protein nature, and phospholipase A2 activity has been described. Affinity chromatography constitutes one of the most effective methods for proteins purification due to the specificity of interactions established in this method, hence the importance of selecting supports adapted to achieve this purpose. The objective of the present research is to test a two-steps purification protocol for phospholipases A2 (PLA2) that allows the purification of those enzymes for his later characterization. In the present work the purification of PLA2 from the sea anemone Condylactis gigantea is accomplished through a chromatographic affinity support MANA - Sepharose CL 4B with egg phosphatidylcholine covalent immobilized. The phospholipase activity A2 was corroborated by qualitative TLC using fluorogenic substrate. The above mentioned support allows the purification of three protein components, with molecular weights between 18000 and 14000, in a two steps protocol, with, at least, a component which possesses phospholipase activity A2 Numerous investigations have demonstrated similarities between marine coelenterates poisons composition with those from snakes, based on pharmacological properties they possess. They constitute complex mixtures of several components, mainly of protein nature, and phospholipase A2 activity has been described. Affinity chromatography constitutes one of the most effective methods for proteins purification due to the specificity of interactions established in this method, hence the importance of selecting supports adapted to achieve this purpose. The objective of the present research is to test a two-steps purification protocol for phospholipases A2 (PLA2) that allows the purification of those enzymes for his later characterization. In the present work the purification of PLA2 from the sea anemone Condylactis gigantea is accomplished through a chromatographic affinity support MANA - Sepharose CL 4B with egg phosphatidylcholine covalent immobilized. The phospholipase activity A2 was corroborated by qualitative TLC using fluorogenic substrate. The above mentioned support allows the purification of three protein components, with molecular weights between 18000 and 14000, in a two steps protocol, with, at least, a component which possesses phospholipase activity A2. Figure 2 Electrophoresis in polyacrylamide gels at 12.5 % (Laemmli, 1970) of collected fractions. From left to right: 1: Crude extract from C. gigantea ; 2: Peak 1 from gel filtration; 3: Eluted fraction from affinity (PLA2 fraction); 4: Standards Figure 3: Elution profiles from affinity chromatography of Peak 1 from Sephadex G-50 fractioning of crude extract of Condylactis gigantea after EDTA addition, tested at different flow rates: a) 90 cm/h; b) 60 cm/h; c) 30 cm/h and d) 22 cm/h. After affinity, three bands with closely molecular weights between 14000 – 20000 Da were observed, a clearly visible one and another two very tenuous bands, in correspondence with those informed for secreted PLA2 from mammals, snakes and vertebrates in general (Valentin & Lambeau, 2000; Wurl & Kunze, 1985; Sun et al., 2009; Romero et al., 2010).Valentin & Lambeau, 2000; Wurl & Kunze, 1985; Sun et al., 2009; Romero et al., 2010 This easy and not expensive purification protocol of PLA2 allows the purification of at least one PLA2 from Condylactis gigantea with an acceptable purity for such a variety of biotechnological and pharmacological uses. This affinity procedure avoid presence of many other peaks such as those obtained by DEAE Sephadex A-50 (Wang et al. 2008).Wang et al. 2008 Concluding remarks The two-steps protocol allows the purification of three protein components of molecular weight between 18000 and 14000 Da, which at least one of the components possessing phospholipase activity A2.