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XXI SICAT. Mlaga-Benalmdena-Costa.

Espaa, 2008

Purification of industrial enzymes: Synthesis of an affinity support for Phospholipase A2.

Lzara Romeroa, Frenkel Guisadoa, Jorge Gonzlez-Bacerioa, Clayton Zambellib, Sylvana Marcussib, Roberto Fernndez-Lafuentec, Jos Manuel Guisnc, Joaqun Daza, Andreimar Soaresb and Alberto del Montea* a Centro de Estudio de Protenas (CEP), Facultad de Biologa, Universidad de La Habana, calle 25 No. 455, J e I, Vedado, Ciudad Habana, Cuba.La Habana, CP 10400, Cuba. b Dep. Anlises Clnicas, Toxicolgicas e Bromatolgicas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Avenida do Cafe, s/n, Ribeirao Preto, Sao Paulo, 14040-903, Brasil. c Instituto de Catlisis y Petroleoqumica, CSIC, Campus Cantoblanco, Madrid, 28049, Espaa. * Alberto del Monte, Fax: 53 7 8321321, E-mail: Abstract. Affinity chromatography is a very useful method for the purification of proteins. For this reason, the obtainment of the appropriate supports to achieve this purpose is very important. The present paper shows the obtainment of an affinity support for the purification of phospholipases A2. As the initial step the Sepharose CL 4B was activated, oxidized and aminated. With the objective to obtain different degrees of amination and to obtain several supports with differences in the degree of immobilization of the phospholipids, three different concentrations of the ethylenediamine were rehearsed. The egg phosphatidylcholine was immobilized by covalent method. In order to know the immobilized phospholipid, the quantity of this in the departure material and in the laundries was determined by phosphorus (P) determination. In order to prove the validity of the support affinity chromatography with different samples were carried out: Extracts (with phospholipase A2 activity) from the sea anemones Condylactis gigantea and Stichodactyla helianthus, as well as of the snake venom from Crotalus durisus terrificus. The typical maximum corresponding in the elution was obtained. Esterase activity was determined by p-NPA and the phospholipase A2 activity using fluorescent substrate by qualitative TLC methods.

Key words: Affinity chromatography, egg yolk phosphatidylcholine, immobilization, phospholipase A2, sea anemone, snake venom.

Introduction. The phospholipases A2 (PLA2) are enzymes that participate in numerous important physiologic and metabolic processes [1, 2, 3]. The phospholipase A2 activity also, is associated to different pathologies for this reason the purification and later study of these enzymes are of great interest, in basic uses as well as in applied uses [4, 5]. Up to this moment, a variety of purification procedures for the PLA2 have been described, nevertheless, the final yield of many of this is low, due to the number of stages or steps in the case of complex sources [6, 7, 8]. Thus


more potent techniques are required for the purification of these enzymes, such as affinity chromatography. Affinity chromatography has been a very effective method for the purification of PLA2, and thats why the obtainment of supports for this purpose has a great importance and utility when working with these enzymes. The present work describes the obtainment of an affinity support for the purification of PLA2.

Material and Methods. The sea anemones Condylactis gigantea and Stichodactyla helianthus were collected at the coast of Havana by specialists from the Faculty of Biology, University of Havana. The sea anemone extracts were obtained by [9, 10] respectively. The Crotalus durrisus terrificus snake venom was kindly supplied by Dep. Anlises Clnicas, Toxicolgicas e Bromatolgicas, FCFRP, USP, Ribeirao Preto, Sao Paulo, Brasil. Extracts and snake venom were kept at -20 oC until use. Phosphatidylcholine oxidation. Egg yolk phosphatidylcholins was purified according to the protocol of [11] for obtaining of the ligand of the chromatographic support. The phospholipid was oxidized, previous to the immobilization, according to the protocol reported by [12]. Support synthesis for the affinity chromatography. Obtainment amine support. For the synthesis, the Sepharose CL 4B support was activated and oxidized according to the methodology reported by [13]. Sepharose CL 4B was activated with 1,2 epoxy 3-propanol (glycidol). The glicerylSepharose CL 4B obtained was oxidized then with NaIO4 in such way that a maximum degree oxidation is achieved (50 moles aldehyde /mL support). Once obtained the glyoxyl-Sepharose CL 4B, the support was aminated according to the method reported by [14]. In this manner the monoamimoethyl-N-aminoethyl-Sepharose CL 4B support (MANA-Sepharose CL 4B) was obtained. Different ethylendiamine concentrations were used with the objective of achieving different amination degrees of the support. Immobilization of oxidized phosphatidylcholine in MANA-Sepharose CL 4B support. The immobilization was carried out according to the method reported by [12] and egg yolk phosphatidylcholine oxidized was used it. In order to compare a commercial support AH Sepharose 4B (Pharmacia Biotech, Sweden) was submitted to the same procedure. Imobilization process characterization by ligand immobilized evaluation. The phosphatidylcholine immobilized was determined by the direct method, by means of the phosphates quantification according to the method reported by [15] previous support


degradation, including a control with the oxidized and aminated support without immobilizing. The sample concentration is informed in phosphorous mg/mL. Study of the proteins fixation capacity by affinity chromatographic support synthesized. The capacity of fixation of proteins for the synthesized and of AH - Sepharose 4B supports with binding PCo was determined by the indirect method. The Condylactis gigantea extract, that the PLA2 contains [16], in a relationship 0.47 mg Protein/g support, were incubated in batch during 24 hours. After the incubation the samples were centrifuged for to measure the protein quantity by [17]. The capacity was calculated subtracting the total protein applied of the protein not fixed. The support maximal adsorption capacity was determined by dynamic methods. For this, different quantities of proteins from the extract of C. gigantea were applied to the column containing the synthesized support. The absorbance of the binding protein was measured at 280 nm. We considered the maximal saturation the not increase of the absorbance. Affinity chromatography All samples were dialyzed with buffer Tris-HCl 0.05 mol/L CaCl2 0.02 mol/L pH 7.5 (fixation buffer) before of the application. Chromatography PC-MANA-Sepharose CL 4B was carried out at 25 C in a glass column (0.7 x 8 cm). Elution was performed with Tris-HCl 0.05 mol/L EDTA 0.04 mol/L pH 7.5, at a flow rate of 15 cm/h and fraction of 1mL were collected. The elution was monitored at 280 nm. In a Ultrospec 4000 spectrophotometer (Pharmacia Biotech. Sweden). Protein concentration Protein concentration was measured by the method of [17] and measured at 280 nm for the protein detection during the chromatographic procedures. For the last, was considering = 1 mg/mL cm-1 [18]. Enzimatic Activity Determination. The esterase activity was determined using pNPA [19]. The phospholipasic A2 activity was corroborated by the method of Len et al [20], using (1palmitoil2NBD-C12-PC (Avanti Polar Lipids). The presence of the labelled fatty acid in the plate corroborated this enzymatic activity.

Results. During the amination process different degrees of these were obtained (results dont show). To continue this work it was selected the aminated support more similar that the commercial support AH-Sepharose CL 4B described by the manufacturer (11-17 mol NH2/mL support).


In order to know the quantity of PCo binding to amine support, the determination of Phosphorus (P) (direct methods) was performed. The results appear in the Table I. Table I. Quantification of PCo binding to the supports by Fiske and Subarow phosphorus determination method. g P x mL Supp.-1 Synthesized support (MANA-Sepharose CL 4B) Commercial support (AH-Sepharose 4B) 4.78 0.31 6.05 2.07 moles FC x mL Supp.-1 0.31 0.010 0.40 0.010

Also, this determination was made for an affinity support synthesized by the same conditions and used for 27 months and the stability of this support was of the 92.34 7.61 % (Table II).

Table II. Ligand binding stability in the time to the synthesized support by Fiske and Subarow phosphorus determination method. g P x mL Supp.-1 Affnity support (time 0) Affnity support (27 months) 4.78 0.31 4.42 0.69

The comparative study of the protein capacity fixation between the AH-Sepharose 4B and MANA-Sepharose CL 4B is shown in Table III. This capacity measured for the indirect method is same for both supports.

Table III. Comparison of the protein capacity fixation to the supports by semi-batch system. Total protein (mg) C. gigantea extract 0.047 0.003 Yield (%) 100 32.38 2.98 32.86 2.02

MANA-Sepharose CL 4B 0.015 0.002 AH-Sepharose 4B 0.015 0.002

The results of the support dynamic capacity are showed in the Figure 1. A lineal increment of the fixed protein is observed to 100 mg of the applied extract after which the tendency is to stand constant.


Fixed Protein (mg) 25 20 15 10 5 0 0 50 100 150

y = 5,8995Ln(x) - 13,828 2 R = 0,9718




Applied Protein (mg)

Figure 1. Dynamic capacity of the support as a function of the applied protein. Different quantities of Condylactis gigantea extract (10-300 mg) were applied to the column and the concentration of the fixed protein measured at 280 nm. In order to know the functional characteristic of the synthesized support, affinity chromatography in column were performed. For it, the Crotalus durrisus terrificus venom (100mg) was applied. The typical profile for this chromatography was obtained (Figure 2a.), a first fraction that elutes with fixation buffer and a second fraction that elute by addition of the buffer with Calcium ion.

Furthermore, the Stichodactyla helianthus and Condylactis gigantea extracts were submitted to affinity chromatography with the same results (Figure 2b and 2c). The presence of the fluorescence fatty acid in the TLC analysis (results dont show) indicated that all applied extracts contain PLA2.

Discussion. The synthesis of a glioxil-Sepharose support from Sepharose CL 4B is functional because this support have the vantage of it can be chemically modified for the obtainment of the others affinity supports. In this work the amination was performed. The egg yolk

phosphatydilcholine was selected because the PLA2 show an elevated affinity for this phospholipid [2, 21]. After of the immobilization it is necessary to corroborate if the process was satisfactory. The determination of the Phosphorus binding to the support by direct method revelled that both, the synthesized support and the commercial binding a similar quantity of PCo. Also the indirect method demonstrated that the capacity for the fixation of proteins is similar to.


Figure 2. Typical elution profile from affinity chromatography of Crotalus durrisus terrificus snake venom (a), Condylactis gigantea extract (b) and Stychodactila helianthus extract (c). Samples (100 mg) were applied to the column (0.7 x 8 cm) in 0.05 mol/L TrisHCl, 0.02 mol/L CaCl2 pH 7.5 and eluted with 0.05 mol/L TrisHCl, EDTA 0.04 mol/L pH 7.5 at a flow rate of 15 cm/h. Fraction of 1 mL were collected. The binding stability in the time of the ligand to the synthesized support was also determined. It was proven that after 27 months, the quantity of coupled phosphatidylcholine was very similar to the initial. This result reveals a great stability, due to the character covalent of the binding between PCo and MANA-Sepharose CL 4B support. The study of the support dynamic capacity demonstrated that for the amination degree selected the maximum fixation was 100 mg protein. This result is achieved it allows us to optimize the affinity chromatographic process and this way not to lose enzyme during this practice.


Affinity chromatography in column was performed to corroborate the effectiveness of synthesized support. The application of the Crotalus durisus terrificus snake venom, grateful source of PLA2 enzyme, offered a typical profile for this chromatography. The application of Stichodactyla helianthus and Condylactis gigantea sea anemone extracts allows similar results. On the other hand the qualitative determination of the PLA2 activity, demonstrated that in all the cases the eluted protein have this activity type in effect.

Concluding remarks The synthesized support manifests a great stability in the time and permits the PLA2 purification from different sources like it is the snake venom Crotalus durrisus terrificus and the sea anemones Stichodactyla helianthus and Condylactis gigantea.

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The authors wish to thank BITaly S.r.l., Bergamo, Italy, for kindly provided financial support, and thanks to Havana University, Cuba and CNPq, Brasil for scientific grants.