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Study of variables involved in horseradish and soybean peroxidase purification by affinity chromatography on concanavalin A-Agarose
Process Biochemistry 38 (2002) 537 /543 www.elsevier.com/locate/procbio
Study of variables involved in horseradish and soybean peroxidase purification by affinity chromatography on concanavalin A-Agarose M.V. Miranda, M.L. Magri, A.A. Navarro del Can˜izo, O. Cascone * Ca´tedra de Microbiologı´a Industrial y Biotecnologı´a, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junı´n 956, 1113 Buenos Aires, Argentina Received 8 January 2002; received in revised form 15 April 2002; accepted 16 May 2002
Abstract Variables involved in adsorption and elution of horseradish and soybean seed peroxidases from a concanavalin A-Agarose matrix were studied. The effect of pH, ion strength and Ca2 /Mg2 concentration on maximum capacity and dissociation constant was assessed through adsorption isotherms. The effect of flow rate and peroxidase concentration on dynamic capacity was assessed through breakthrough curves. For the elution step, NaCl and a-D-methylmannopyranoside concentrations were optimised. The best conditions for soybean peroxidase adsorption were pH 5.0 and 1 mM Ca2 /Mg2 in the absence of salt, at a flow rate up to 3.2 cm/ min and 3 mg/ml peroxidase concentration. Elution required the addition of 0.48 M a-D-methylmannopyranoside and 0.97 M NaCl. Under these conditions, the dynamic capacity was 16.9 mg/ml matrix and purification yield, 84.3%. In the case of horseradish peroxidase, the best adsorption conditions were pH 7.0, 5 mM ions and 0.75 M NaCl, at a flow rate up to 1.5 cm/min and a 4 mg/ml peroxidase concentration. Dynamic capacity was 9.6 mg/ml matrix, and elution required 0.36 M a-D-methylmannopyranoside to yield 75% of enzyme. The dynamic-to-static capacity ratios were 0.71 and 0.62 for horseradish and soybean peroxidases, respectively. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Peroxidase; Horseradish; Soybean; Adsorption; Concanavalin A-Agarose; Chromatography
1. Introduction Peroxidases (EC 1.11.1.7) are ubiquitous oxidoreductases that utilise hydrogen peroxide or organic hydroperoxides as oxidants. Most peroxidases are glycoproteins containing N-linked oligosaccharide chains [1,2]. Horseradish peroxidase (HRP), from Armoracia rusticana roots, is a commercially important enzyme that occurs as a large family of isoenzymes [3]. More recently, a peroxidase extracted from soybean hulls (SBP), an inexpensive food industry by-product, has been described. This peroxidase was obtained as a single anionic isoenzyme with an isoelectric point of 4.1 [4]. Its unusually high thermostability even at low pHs and its activity in organic solvents, make it useful for the synthetic organic chemist and for different industrial applications [5]. * Corresponding author. Tel./fax: /54-11-4901-6284 E-mail address: [email protected] (O. Cascone).
Many methods for plant peroxidase purification have been reported. After homogenising the crude material with a buffer or water, the homogenate is filtered, concentrated by precipitation, centrifuged, and the enzyme purified by applying different chromatographic steps, depending on its intended utilisation. When the aim is to obtain a high purity enzyme, every purification method includes an affinity chromatographic step. In this way, lectin chromatography is used in most methods reported so far taking into account that HRP and SBP are glycoproteins [4,6 /8]. In order to reduce the purification process costs, we have reported other possibilities based on partitioning in aqueous two-phase systems, but every proposed scheme had to include an affinity chromatography step to obtain high purity products [9]. Plant lectins bind monosaccharides with relatively low affinity (KA /1 /103 M), and oligosaccharides far more tightly (KA /1 /106 M) [10]. Concanavalin A (Con A), from the jack bean Cannavalia ensiformis , is the most
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M.V. Miranda et al. / Process Biochemistry 38 (2002) 537 /543
extensively studied member of the lectin family. It consists of 26.5 kDa subunits that readily form tetramers. The association of subunits is pH-dependent. Each subunit has binding sites for one Mg2 or Mn2, one Ca2 and one saccharide. Mg2 or Mn2 must be bound before Ca2 binding, and both metal ions must be present for saccharide binding [11]. Con A binds molecules containing a-D-mannopyranosyl and aD-glucopyranosyl residues, and has been extensively utilised for isolation, fractionation, structural characterisation and immobilisation of glycoproteins carrying these kinds of residues. Peroxidase is usually desorbed with a-methyl-D-mannopyranoside, a sugar that competes with the enzyme for the binding sites. The aim of this work was to study the variables involved in HRP and SBP chromatographic purification on Con A-Agarose. Ion strength, pH and Ca2/Mg2 concentrations were chosen as the variables in the adsorption step, and sodium chloride and a-methyl-Dmannopyranoside concentrations for the elution step.
2. Materials and methods 2.1. Materials HRP (P-8000), SBP (P-1432), Con A-Agarose type III-ASCL (C-7911) and a-methyl-D-mannopyranoside were from Sigma-Aldrich, St. Louis, MO. Both enzymes were used without further purification. Guaiacol was from Mallinkrodt Chemical Works, St. Louis, MO. All other reagents were AR grade. 2.2. Peroxidase assay HRP: The assay mixture contained 105 mM guaiacol and 250 mM hydrogen peroxide in 1 ml of 100 mM potassium phosphate buffer, pH 7.0. SBP: The assay mixture contained 28 mM guaiacol and 500 mM hydrogen peroxide in 1 ml of 100 mM potassium phosphate buffer, pH 5.5. After addition of a 10 ml sample, absorbance at 470 nm was recorded within 1 min. Activity calculations were made as per Tijssen [8]. 2.3. Adsorption isotherms They were measured in batch systems, basically as described by Chase [12]. Samples of 0.1, 0.25, 0.5, 1.0, 2.5 and 3.5 mg/ml enzyme in 20 mM sodium acetate buffer, pH 5.0, or 20 mM sodium phosphate buffer, pH 6.0 and 7.0, containing Ca2/Mg2 at 1 or 5 mM, were used. NaCl was added when required. 200 ml of a 50% (v/v) suspension of Con A-Agarose in buffer was then added to each flask. The suspensions, in a final volume of 1 ml, were gently agitated for 15/20 h at 20 8C to
allow the system to reach its equilibrium. Then, peroxidase concentration in the supernatant (c*) was measured spectrophotometrically at 403 nm (absorbance of the prosthetic heme group). The equilibrium concentration of peroxidase bound to the adsorbent (q *) was calculated as the total amount of enzyme present at the beginning of the experiment less the amount still in the soluble phase at equilibrium. The binding data were fitted to a Langmuir-type isotherm in which q* varies with c* as follows: q+
qm c + c+ K D
qm (maximum bound peroxidase concentration at equilibrium) and KD (equilibrium dissociation constant) were calculated from this formula as per Chase [12]. 2.4. Adsorption breakthrough curves for binding of SBP and HRP to Con A-Agarose They were measured to assess the effect of changes in flow rate and feed concentration on matrix dynamic capacity. Adsorption breakthrough curves were obtained by pumping SBP or HRP solutions to a column (0.5 /4.0 cm) of Con A-Agarose equilibrated with the adsorption buffer selected for each enzyme and collecting 1 ml fractions. Enzyme activity was measured in each collected fraction. 2.5. Fast protein liquid chromatography A column (0.5 /4.0 cm) of Con A-Agarose was equilibrated with the optimised adsorption buffer (20 mM sodium acetate buffer, pH 5.0, 1 mM CaCl2 and MgCl2 for SBP, or 20 mM sodium phosphate buffer, pH 7.0, 5 mM CaCl2 and MgCl2, 0.75 M NaCl, for HRP). After loading 50 ml of 12.4 mg/ml peroxidase, 1-ml fractions were collected at a linear flow rate of 1.5 cm/ min for HRP or 3.2 cm/min for SBP. Fractions eluted were monitored by their absorbance at 280 and 403 nm, and enzyme activity. HRP was eluted by a 0 /1 M amethyl-D-mannopyranoside gradient and SBP by a simultaneous dual gradient (0 /1 M a-methyl-D-mannopyranoside and 0 /0.5, 0 /1 or 0/2 M NaCl).
3. Results and discussion 3.1. Peroxidase adsorption optimisation To assess the effect of different variables (pH, ion strength and Ca2/Mg2 concentration) on peroxidase binding to Con A-Agarose, adsorption isotherms were developed, and KD and qm values obtained were
M.V. Miranda et al. / Process Biochemistry 38 (2002) 537 /543
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compared to find the best adsorption conditions for SBP and HRP.
3.1.1. Soybean peroxidase Fig. 1 shows the isotherms for SBP adsorption on Con A-Agarose, at different pH values (A) and NaCl concentrations (B), and Fig. 2 is a 3D-graphic that allows finding the best conditions by combining the variables involved in SBP adsorption on Con A-Agarose (for an improved presentation, KA /1/KD was plotted instead of KD). At pH 6.0 and 7.0, ion strength had little influence on KD. In contrast, at pH 5.0 ion strength had a strong influence, as KD increased from 2.4 /10 6 to 7.1 / 10 6 M when NaCl concentration increased from 0 to 0.75 M (Fig. 1).
Fig. 2. 3-D plot showing the influence of pH and NaCl concentration on the KA( /1/KD) value obtained from the adsorption isotherms for SBP binding to Con A-Agarose. Ca2 /Mg2 concentration /1 mM.
Both KD and qm did not show significant differences when working at 1 or 5 mM cation concentration (not shown). Therefore, 1 mM cation was chosen for further experiments. From these results, it is evident that pH 5.0 in the absence of salt and 1 mM cation is the condition that minimises KD and maximises qm and, therefore, it is the best for SBP adsorption.
Fig. 1. (A) Adsorption isotherms for SBP binding to Con A-Agarose, at pH 5.0 (j), 6.0 (%) and 7.0 (m). (B) Adsorption isotherms for SBP binding to Con A-Agarose at pH 5.0, and 0.25 (j), 0.50 (m), 0.75 M NaCl ('), and absence of salt (%). Buffer: 20 mM sodium acetate, pH 5.0, or 20 mM sodium phosphate, pH 6.0 or 7.0. Ca2 /Mg2 concentration /1 mM. Details in Section 2.
3.1.2. Horseradish peroxidase Fig. 3 shows the isotherms for HRP adsorption on Con A-Agarose, at different pH values (A) and different NaCl concentrations (B), and Fig. 4 integrates the results in a 3D-graphic. Data indicate that pH had some influence on KD but, in contrast with SBP, at a higher NaCl concentration. In the presence of 0.75 M NaCl and 5 mM cation, KD value decreased from 2.0 /10 5 to 9.5 /10 6 M when the pH was raised from 5.0 to 7.0. This effect was not evident at 1 mM cation concentration (not shown). When the effect of NaCl concentration on KD was assessed, results indicated a strong influence of salt on KD at pH 7.0 and 5 mM cation concentration. No significant influence of salt, at pH 5.0 or 6.0 was evidenced. At pH 7 in the presence of 0.75 M NaCl, cation concentration had a great influence on KD: the higher the cation concentration, the better enzyme adsorption to the matrix (KD dropped from 3 /10 5 at 1 mM cation to 2.6 /10 6 at 5 mM cation).
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Fig. 4. 3-D plot showing the influence of pH and NaCl concentration on the KA( /1/KD) value obtained from the adsorption isotherms for HRP binding to Con A-Agarose. Cation concentration /5 mM.
Fig. 3. (A) Adsorption isotherms for HRP binding to Con A-Agarose, at pH 5.0 (j), 6.0 (%) and 7.0 (m). (B) Adsorption isotherms for HRP binding to Con A-Agarose at pH 7.0, and 0.25 (j), 0.50 (m), 0.75 M NaCl ('), and absence of salt (%). Buffer: 20 mM sodium acetate, pH 5.0, or 20 mM sodium phosphate, pH 6.0 or 7.0. Ca2 /Mg2 concentration /5 mM. Details in Section 2.
All the above results indicate that the best conditions for HRP adsorption to Con A-Agarose are pH 7.0, 5 mM Ca2/Mg2 and 0.75 M NaCl. 3.2. Adsorption breakthrough curves Breakthrough curves were measured to assess the influence of peroxidase concentration feed and flow rate on the dynamic capacity of the matrix, under the conditions selected in static experiments. Figs. 5 and 6 show adsorption breakthrough curves for the binding of SBP and HRP at different feed concentrations (A) and flow rates (B), respectively. For SBP, dynamic capacity increased with the increase in
feed concentration between 0.5 and 3 mg/ml peroxidase. At concentrations higher than 3 mg/ml, breakthrough curves overlapped thus indicating that the dynamic capacity remained constant at approximately 17 mg/ml matrix, and 3 mg/ml is the lower peroxidase concentration allowing an effective utilisation of the chromatographic matrix (Fig. 5A). On the other hand, when flow rate was the variable under study, dynamic capacity remained unchanged between 1.0 and 3.2 cm/min, but decreased by 76% when the linear flow rate was raised to 5.1 cm/min. This result indicates that for SBP it is possible to operate the chromatographic purification up to 3.2 cm/min without any loss in dynamic capacity (Fig. 5B). For HRP, dynamic capacity also increased with the rise in feed peroxidase concentration, between 1 and 4 mg/ml HRP. Over 4 mg/ml, dynamic capacity remained constant at approximately 10 mg peroxidase/ml matrix (Fig. 6A). With regard to flow rate, dynamic capacity increased as flow rate decreased from 4.0 to 1.5 cm/min (Fig. 6B). From the above results, conditions selected for SBP were 3 mg/ml feed concentration at a flow rate of 3.2 cm/min. Under these conditions, the dynamic capacity was 62% of the maximum capacity (qm /27.1 mg/ml matrix). For HRP, a feed concentration of 4 mg/ml was required for an effective utilisation of the chromatographic matrix, at a flow rate of 1.5 cm/min. Dynamic capacity corresponds to 71% of the maximum capacity of the chromatographic matrix (qm /13.5 mg/ml matrix).
M.V. Miranda et al. / Process Biochemistry 38 (2002) 537 /543
Fig. 5. (A) Breakthrough curves for SBP binding to Con A-Agarose at 0.5 ("), 2.0 (j), 3.0 (') and 5.0 (m) mg/ml SBP feed concentration. (B) Breakthrough curves for SBP binding to Con A-Agarose at 1.0 ("), 1.5 (j), 3.2 (') and 5.1 (m) cm/min feed velocity. Buffer: 20 mM sodium acetate, pH 5.0. Ca2 /Mg2 concentration /1 mM. C , SBP concentration in the outlet stream; C0, SBP concentration in the feedstock; f , volumetric flux; t , time elapsed; C0.f.t, SBP amount loaded on the column. Details in Section 2.
3.3. Selecting conditions for the elution step
3.3.1. Soybean peroxidase Under the binding conditions selected, a sample of SBP was loaded on a Con A-Agarose column and, after a wash step with adsorption buffer, a simultaneous dual gradient of a-methyl-D-mannopyranoside (0 /1 M) and NaCl (0 /0.5 M or 0/1 M or 0 /2 M) was developed. With the 0/0.5 M NaCl/0/1 M sugar gradient, the enzyme eluted at 0.45 M sugar and 0.22 M NaCl. When the 0 /1 M NaCl/0 /1 M sugar gradient was applied, peroxidase eluted at 0.30 M sugar and 0.30 M NaCl. With the 0 /2 M NaCl/0 /1 M sugar gradient, 0.25 M sugar and 0.50 M NaCl were required for enzyme elution. These results indicate that NaCl in the elution buffer allows reduction of the sugar amount required to elute the peroxidase. Fig. 7A shows the chromato-
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Fig. 6. (A) Breakthrough curves for HRP binding to Con A-Agarose at 1.0 ("), 2.0 (j), 4.0 (') and 6.0 (m) mg/ml HRP feed concentration. (B) Breakthrough curves for HRP binding to Con AAgarose at 1.0 ("), 1.5 (j), 2.9 (') and 4.0 (m) cm/min feed velocity. Buffer: 20 mM sodium phosphate, pH 7.0. Ca2 /Mg2 concentration /5 mM. C , HRP concentration in the outlet stream; C0, HRP concentration in the feedstock; f , volumetric flux; t , time elapsed; C0.f.t, HRP amount loaded on the column. Details in Section 2.
graphic profile. Based on these results, a step-elution was designed: after the wash step with adsorption buffer, peroxidase was eluted by changing the mobile phase to 0.48 M a-methyl-D-mannopyranoside and 0.97 M NaCl (these concentrations are those corresponding to the end of the peek). Under these conditions, 84.3% SBP recovery was obtained without any enzyme loss during the wash step. Gillikin and Graham [4], working at pH 6.8, recovered less than 50% SBP by eluting the enzyme with 0.75 /1 M mannose in the presence of 2.5 M NaCl. Our results suggest that the different working pH could account for this difference. 3.3.2. Horseradish peroxidase As a high NaCl concentration helps HRP binding to Con A-Agarose (see adsorption experiments), the elution was performed only with a gradient of 0/1 M amethyl-D-mannopyranoside. The enzyme eluted at
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Fig. 7. (A) Chromatographic profile of SBP purification on Con A-Agarose under the conditions selected. A column (0.5 /4.0 cm) of Con AAgarose was equilibrated with a 20 mM sodium acetate buffer, pH 5.0, 1 mM CaCl2 and MgCl2. After loading 50 ml of 12.4 mg/ml SBP, 1-ml fractions were collected at a linear flow rate of 3.2 cm/min and monitored by their absorbance at 280 nm (j) and 403 nm ('), and enzyme activity (m). After a wash step with 5 ml of adsorption buffer, SBP elution was carried out with a simultaneous 0 /1 M a-methyl-D-mannopyranoside/0 /2 M NaCl gradient. (B) Chromatographic profile of HRP purification on Con A-Agarose under the conditions selected. A column (0.5 /4.0 cm) of Con A-Agarose was equilibrated with a 20 mM sodium phosphate buffer, pH 7.0, 5 mM CaCl2 and MgCl2, 0.75 M NaCl. After loading 50 ml of 12.4 mg/ ml HRP, 1-ml fractions were collected at a linear flow rate of 1.5 cm/min and their absorbance at 280 nm (j) and 403 nm ('), and enzyme activity (m) were monitored. After a wash step with 5 ml of adsorption buffer, HRP elution was carried out with a 0 /1 M a-methyl-D-mannopyranoside eluent.
0.16 /0.36 M sugar (Fig. 7B); therefore, a step elution with 0.36 M a-methyl-D-mannopyranoside was developed. Recovery was 75.0% and, in contrast with SBP, 20 /25% of peroxidase activity passed through during the wash step. The most active part of a glycan structure towards Con A is not that involving the a-mannose residues in a terminal position but the trisaccharide a-Man-(1-3)-[aMan-(1-6)]Man [13]. On the other hand, it has been stated that binding of glycoproteins to immobilised lectins is of a multivalent nature, thus meaning that more than one lectin molecule should be involved in the binding of one glycocompound molecule [14]. SBP contains 18.2% carbohydrate, at five or six glycosylation sites [15]. The site at the N-terminus, Asn 16, has been clearly defined as being substituted mainly for high mannose-type glycans (Manx GlcNAc2, x /4, 5, 6 or 7) though there is sufficient evidence that this site may also be substituted with (Xyl)Man3(Fuc)GlcNAc2 in different batches of SBP [2]. In contrast to SBP, HRPc (the best-studied HRP isoenzyme) has only traces of high mannose-type glycans, where a significant part ( /16%) of the total glycan pool is composed of high-mannose species confined to a single glycosylation site [16]. There are no references as regards glycosylation patterns of the various HRP isoenzymes. It is important to point out that while peroxidases are multivalent, the lectin used in this work is tetravalent.
In the case of SBP, it was not easy to dissociate the affinity complex. A possible explanation is that the high mannose-type glycans present in this peroxidase bind to Con A-Agarose very tightly and, as a consequence of this resulting polyvalent interaction, dissociation of the complex by adding a monovalent sugar like methylmannoside is very difficult. NaCl addition was useful to decrease the sugar concentration for peroxidase elution but even so, it was not enough to bring out a quantitative recovery of the enzyme. For HRP, the loss in the wash step could be due to difference in the glycosylation pattern of the various isoenzymes. Results obtained in this work evidence the usefulness of static and dynamic chromatographic parameter determination to select the best conditions for SBP and HRP peroxidase purification.
Acknowledgements This work was supported by grants from the Universidad de Buenos Aires, the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas de la Repu´blica Argentina and the Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica de la Repu´blica Argentina. M.V. Miranda and O. Cascone are career researchers of the CONICET.
M.V. Miranda et al. / Process Biochemistry 38 (2002) 537 /543
References [1] Dunford HB, Stillman JS. On the function and mechanism of action of peroxidases. Coordination Chem Rev 1976;19:187 /251. [2] Gray JS, Montgomery R. The N -glycosylation sites of soybean seed coat peroxidase. Glycobiology 1997;7:679 /85. [3] Kay E, Shannon LM, Lew JY. Peroxidase isozymes from horseradish roots. J Biol Chem 1967;242:2470 /3. [4] Gillikin JW, Graham JS. Purification and developmental analysis of the major anionic peroxidase from the seed coat of Glycine max . Plant Physiol 1991;96:214 /20. [5] MacEldoon JP, Dordick JS. Unusual thermal stability of soybean peroxidase. Biotechnol Prog 1996;12:555 /8. [6] Paradkar VM, Dordick JS. Affinity-based reverse micellar extraction and separation (ARMES): a facile technique for the purification of peroxidase from soybean hulls. Biotechnol Prog 1993;9:199 /203. [7] Casl MT, Kostrencic C. Isolation and purification of horseradish peroxidase. Ann Clin Biochem 1987;24:271. [8] Tijssen P. Enzymes for immunoassays. In: Burdon RH, van Knippenberg PH, editors. Practice and theory of enzyme immunoassays (10). Amsterdam, New York, Oxford: Elsevier, 1985:173 /220.
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[9] Miranda MV, Ferna´ndez-Lahore HM, Dobrecky J, Cascone O. The extractive purification of peroxidase from plant raw materials in aqueous two-phase systems. Acta Biotechnologica 1998;18:179 /88. [10] Sanders DAR, Moothoo DN, Raftery J, Howard AJ, Helliwell JR, Naismith JH. The 1.2 A structure of the Con A-dimannose complex. J Mol Biol 2001;310:875 /84. [11] Reeke GN, Becker JW, Cunningham BA, Gunther FR, Wang JC, Edelman GN. Relationships between the structure and activities of concanavalin A. Ann NY Acad Sci 1974;234:369 /82. [12] Chase H. Prediction of the performance of preparative affinity chromatography. J Chromatog 1984;297:179 /202. [13] Naismith JH, Field RA. Structural basis of trimannoside recognition by concanavalin A. J Biol Chem 1996;271:972 /6. [14] Franco-Fraguas L, Carlsson J, Batista-Viera F. The adsorption/ desorption behaviour of horseradish peroxidase and porcine thyroglobulin on concanavalin A-Sepharose with different ligand densities. LIBC 2000;5:1 /11. [15] Gray JS, Yung Yang B, Hull SR, Venzke DP, Montgomery R. The glycans of soybean peroxidase. Gycobiology 1996;6:23 /32. [16] Gray JS, Yun Yang B, Montgomery R. Heterogeneity of glycans at each N -glycosylation site of horseradish peroxidase. Carbohydrate Res 1998;311:61 /9.
Study of variables involved in horseradish and soybean peroxidase purification by affinity chromatography on concanavalin A-Agarose M.V. Miranda, M.L. Magri, A.A. Navarro del Can˜izo, O. Cascone * Ca´tedra de Microbiologı´a Industrial y Biotecnologı´a, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junı´n 956, 1113 Buenos Aires, Argentina Received 8 January 2002; received in revised form 15 April 2002; accepted 16 May 2002
Abstract Variables involved in adsorption and elution of horseradish and soybean seed peroxidases from a concanavalin A-Agarose matrix were studied. The effect of pH, ion strength and Ca2 /Mg2 concentration on maximum capacity and dissociation constant was assessed through adsorption isotherms. The effect of flow rate and peroxidase concentration on dynamic capacity was assessed through breakthrough curves. For the elution step, NaCl and a-D-methylmannopyranoside concentrations were optimised. The best conditions for soybean peroxidase adsorption were pH 5.0 and 1 mM Ca2 /Mg2 in the absence of salt, at a flow rate up to 3.2 cm/ min and 3 mg/ml peroxidase concentration. Elution required the addition of 0.48 M a-D-methylmannopyranoside and 0.97 M NaCl. Under these conditions, the dynamic capacity was 16.9 mg/ml matrix and purification yield, 84.3%. In the case of horseradish peroxidase, the best adsorption conditions were pH 7.0, 5 mM ions and 0.75 M NaCl, at a flow rate up to 1.5 cm/min and a 4 mg/ml peroxidase concentration. Dynamic capacity was 9.6 mg/ml matrix, and elution required 0.36 M a-D-methylmannopyranoside to yield 75% of enzyme. The dynamic-to-static capacity ratios were 0.71 and 0.62 for horseradish and soybean peroxidases, respectively. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Peroxidase; Horseradish; Soybean; Adsorption; Concanavalin A-Agarose; Chromatography
1. Introduction Peroxidases (EC 1.11.1.7) are ubiquitous oxidoreductases that utilise hydrogen peroxide or organic hydroperoxides as oxidants. Most peroxidases are glycoproteins containing N-linked oligosaccharide chains [1,2]. Horseradish peroxidase (HRP), from Armoracia rusticana roots, is a commercially important enzyme that occurs as a large family of isoenzymes [3]. More recently, a peroxidase extracted from soybean hulls (SBP), an inexpensive food industry by-product, has been described. This peroxidase was obtained as a single anionic isoenzyme with an isoelectric point of 4.1 [4]. Its unusually high thermostability even at low pHs and its activity in organic solvents, make it useful for the synthetic organic chemist and for different industrial applications [5]. * Corresponding author. Tel./fax: /54-11-4901-6284 E-mail address: [email protected] (O. Cascone).
Many methods for plant peroxidase purification have been reported. After homogenising the crude material with a buffer or water, the homogenate is filtered, concentrated by precipitation, centrifuged, and the enzyme purified by applying different chromatographic steps, depending on its intended utilisation. When the aim is to obtain a high purity enzyme, every purification method includes an affinity chromatographic step. In this way, lectin chromatography is used in most methods reported so far taking into account that HRP and SBP are glycoproteins [4,6 /8]. In order to reduce the purification process costs, we have reported other possibilities based on partitioning in aqueous two-phase systems, but every proposed scheme had to include an affinity chromatography step to obtain high purity products [9]. Plant lectins bind monosaccharides with relatively low affinity (KA /1 /103 M), and oligosaccharides far more tightly (KA /1 /106 M) [10]. Concanavalin A (Con A), from the jack bean Cannavalia ensiformis , is the most
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 1 6 6 - 8
538
M.V. Miranda et al. / Process Biochemistry 38 (2002) 537 /543
extensively studied member of the lectin family. It consists of 26.5 kDa subunits that readily form tetramers. The association of subunits is pH-dependent. Each subunit has binding sites for one Mg2 or Mn2, one Ca2 and one saccharide. Mg2 or Mn2 must be bound before Ca2 binding, and both metal ions must be present for saccharide binding [11]. Con A binds molecules containing a-D-mannopyranosyl and aD-glucopyranosyl residues, and has been extensively utilised for isolation, fractionation, structural characterisation and immobilisation of glycoproteins carrying these kinds of residues. Peroxidase is usually desorbed with a-methyl-D-mannopyranoside, a sugar that competes with the enzyme for the binding sites. The aim of this work was to study the variables involved in HRP and SBP chromatographic purification on Con A-Agarose. Ion strength, pH and Ca2/Mg2 concentrations were chosen as the variables in the adsorption step, and sodium chloride and a-methyl-Dmannopyranoside concentrations for the elution step.
2. Materials and methods 2.1. Materials HRP (P-8000), SBP (P-1432), Con A-Agarose type III-ASCL (C-7911) and a-methyl-D-mannopyranoside were from Sigma-Aldrich, St. Louis, MO. Both enzymes were used without further purification. Guaiacol was from Mallinkrodt Chemical Works, St. Louis, MO. All other reagents were AR grade. 2.2. Peroxidase assay HRP: The assay mixture contained 105 mM guaiacol and 250 mM hydrogen peroxide in 1 ml of 100 mM potassium phosphate buffer, pH 7.0. SBP: The assay mixture contained 28 mM guaiacol and 500 mM hydrogen peroxide in 1 ml of 100 mM potassium phosphate buffer, pH 5.5. After addition of a 10 ml sample, absorbance at 470 nm was recorded within 1 min. Activity calculations were made as per Tijssen [8]. 2.3. Adsorption isotherms They were measured in batch systems, basically as described by Chase [12]. Samples of 0.1, 0.25, 0.5, 1.0, 2.5 and 3.5 mg/ml enzyme in 20 mM sodium acetate buffer, pH 5.0, or 20 mM sodium phosphate buffer, pH 6.0 and 7.0, containing Ca2/Mg2 at 1 or 5 mM, were used. NaCl was added when required. 200 ml of a 50% (v/v) suspension of Con A-Agarose in buffer was then added to each flask. The suspensions, in a final volume of 1 ml, were gently agitated for 15/20 h at 20 8C to
allow the system to reach its equilibrium. Then, peroxidase concentration in the supernatant (c*) was measured spectrophotometrically at 403 nm (absorbance of the prosthetic heme group). The equilibrium concentration of peroxidase bound to the adsorbent (q *) was calculated as the total amount of enzyme present at the beginning of the experiment less the amount still in the soluble phase at equilibrium. The binding data were fitted to a Langmuir-type isotherm in which q* varies with c* as follows: q+
qm c + c+ K D
qm (maximum bound peroxidase concentration at equilibrium) and KD (equilibrium dissociation constant) were calculated from this formula as per Chase [12]. 2.4. Adsorption breakthrough curves for binding of SBP and HRP to Con A-Agarose They were measured to assess the effect of changes in flow rate and feed concentration on matrix dynamic capacity. Adsorption breakthrough curves were obtained by pumping SBP or HRP solutions to a column (0.5 /4.0 cm) of Con A-Agarose equilibrated with the adsorption buffer selected for each enzyme and collecting 1 ml fractions. Enzyme activity was measured in each collected fraction. 2.5. Fast protein liquid chromatography A column (0.5 /4.0 cm) of Con A-Agarose was equilibrated with the optimised adsorption buffer (20 mM sodium acetate buffer, pH 5.0, 1 mM CaCl2 and MgCl2 for SBP, or 20 mM sodium phosphate buffer, pH 7.0, 5 mM CaCl2 and MgCl2, 0.75 M NaCl, for HRP). After loading 50 ml of 12.4 mg/ml peroxidase, 1-ml fractions were collected at a linear flow rate of 1.5 cm/ min for HRP or 3.2 cm/min for SBP. Fractions eluted were monitored by their absorbance at 280 and 403 nm, and enzyme activity. HRP was eluted by a 0 /1 M amethyl-D-mannopyranoside gradient and SBP by a simultaneous dual gradient (0 /1 M a-methyl-D-mannopyranoside and 0 /0.5, 0 /1 or 0/2 M NaCl).
3. Results and discussion 3.1. Peroxidase adsorption optimisation To assess the effect of different variables (pH, ion strength and Ca2/Mg2 concentration) on peroxidase binding to Con A-Agarose, adsorption isotherms were developed, and KD and qm values obtained were
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compared to find the best adsorption conditions for SBP and HRP.
3.1.1. Soybean peroxidase Fig. 1 shows the isotherms for SBP adsorption on Con A-Agarose, at different pH values (A) and NaCl concentrations (B), and Fig. 2 is a 3D-graphic that allows finding the best conditions by combining the variables involved in SBP adsorption on Con A-Agarose (for an improved presentation, KA /1/KD was plotted instead of KD). At pH 6.0 and 7.0, ion strength had little influence on KD. In contrast, at pH 5.0 ion strength had a strong influence, as KD increased from 2.4 /10 6 to 7.1 / 10 6 M when NaCl concentration increased from 0 to 0.75 M (Fig. 1).
Fig. 2. 3-D plot showing the influence of pH and NaCl concentration on the KA( /1/KD) value obtained from the adsorption isotherms for SBP binding to Con A-Agarose. Ca2 /Mg2 concentration /1 mM.
Both KD and qm did not show significant differences when working at 1 or 5 mM cation concentration (not shown). Therefore, 1 mM cation was chosen for further experiments. From these results, it is evident that pH 5.0 in the absence of salt and 1 mM cation is the condition that minimises KD and maximises qm and, therefore, it is the best for SBP adsorption.
Fig. 1. (A) Adsorption isotherms for SBP binding to Con A-Agarose, at pH 5.0 (j), 6.0 (%) and 7.0 (m). (B) Adsorption isotherms for SBP binding to Con A-Agarose at pH 5.0, and 0.25 (j), 0.50 (m), 0.75 M NaCl ('), and absence of salt (%). Buffer: 20 mM sodium acetate, pH 5.0, or 20 mM sodium phosphate, pH 6.0 or 7.0. Ca2 /Mg2 concentration /1 mM. Details in Section 2.
3.1.2. Horseradish peroxidase Fig. 3 shows the isotherms for HRP adsorption on Con A-Agarose, at different pH values (A) and different NaCl concentrations (B), and Fig. 4 integrates the results in a 3D-graphic. Data indicate that pH had some influence on KD but, in contrast with SBP, at a higher NaCl concentration. In the presence of 0.75 M NaCl and 5 mM cation, KD value decreased from 2.0 /10 5 to 9.5 /10 6 M when the pH was raised from 5.0 to 7.0. This effect was not evident at 1 mM cation concentration (not shown). When the effect of NaCl concentration on KD was assessed, results indicated a strong influence of salt on KD at pH 7.0 and 5 mM cation concentration. No significant influence of salt, at pH 5.0 or 6.0 was evidenced. At pH 7 in the presence of 0.75 M NaCl, cation concentration had a great influence on KD: the higher the cation concentration, the better enzyme adsorption to the matrix (KD dropped from 3 /10 5 at 1 mM cation to 2.6 /10 6 at 5 mM cation).
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Fig. 4. 3-D plot showing the influence of pH and NaCl concentration on the KA( /1/KD) value obtained from the adsorption isotherms for HRP binding to Con A-Agarose. Cation concentration /5 mM.
Fig. 3. (A) Adsorption isotherms for HRP binding to Con A-Agarose, at pH 5.0 (j), 6.0 (%) and 7.0 (m). (B) Adsorption isotherms for HRP binding to Con A-Agarose at pH 7.0, and 0.25 (j), 0.50 (m), 0.75 M NaCl ('), and absence of salt (%). Buffer: 20 mM sodium acetate, pH 5.0, or 20 mM sodium phosphate, pH 6.0 or 7.0. Ca2 /Mg2 concentration /5 mM. Details in Section 2.
All the above results indicate that the best conditions for HRP adsorption to Con A-Agarose are pH 7.0, 5 mM Ca2/Mg2 and 0.75 M NaCl. 3.2. Adsorption breakthrough curves Breakthrough curves were measured to assess the influence of peroxidase concentration feed and flow rate on the dynamic capacity of the matrix, under the conditions selected in static experiments. Figs. 5 and 6 show adsorption breakthrough curves for the binding of SBP and HRP at different feed concentrations (A) and flow rates (B), respectively. For SBP, dynamic capacity increased with the increase in
feed concentration between 0.5 and 3 mg/ml peroxidase. At concentrations higher than 3 mg/ml, breakthrough curves overlapped thus indicating that the dynamic capacity remained constant at approximately 17 mg/ml matrix, and 3 mg/ml is the lower peroxidase concentration allowing an effective utilisation of the chromatographic matrix (Fig. 5A). On the other hand, when flow rate was the variable under study, dynamic capacity remained unchanged between 1.0 and 3.2 cm/min, but decreased by 76% when the linear flow rate was raised to 5.1 cm/min. This result indicates that for SBP it is possible to operate the chromatographic purification up to 3.2 cm/min without any loss in dynamic capacity (Fig. 5B). For HRP, dynamic capacity also increased with the rise in feed peroxidase concentration, between 1 and 4 mg/ml HRP. Over 4 mg/ml, dynamic capacity remained constant at approximately 10 mg peroxidase/ml matrix (Fig. 6A). With regard to flow rate, dynamic capacity increased as flow rate decreased from 4.0 to 1.5 cm/min (Fig. 6B). From the above results, conditions selected for SBP were 3 mg/ml feed concentration at a flow rate of 3.2 cm/min. Under these conditions, the dynamic capacity was 62% of the maximum capacity (qm /27.1 mg/ml matrix). For HRP, a feed concentration of 4 mg/ml was required for an effective utilisation of the chromatographic matrix, at a flow rate of 1.5 cm/min. Dynamic capacity corresponds to 71% of the maximum capacity of the chromatographic matrix (qm /13.5 mg/ml matrix).
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Fig. 5. (A) Breakthrough curves for SBP binding to Con A-Agarose at 0.5 ("), 2.0 (j), 3.0 (') and 5.0 (m) mg/ml SBP feed concentration. (B) Breakthrough curves for SBP binding to Con A-Agarose at 1.0 ("), 1.5 (j), 3.2 (') and 5.1 (m) cm/min feed velocity. Buffer: 20 mM sodium acetate, pH 5.0. Ca2 /Mg2 concentration /1 mM. C , SBP concentration in the outlet stream; C0, SBP concentration in the feedstock; f , volumetric flux; t , time elapsed; C0.f.t, SBP amount loaded on the column. Details in Section 2.
3.3. Selecting conditions for the elution step
3.3.1. Soybean peroxidase Under the binding conditions selected, a sample of SBP was loaded on a Con A-Agarose column and, after a wash step with adsorption buffer, a simultaneous dual gradient of a-methyl-D-mannopyranoside (0 /1 M) and NaCl (0 /0.5 M or 0/1 M or 0 /2 M) was developed. With the 0/0.5 M NaCl/0/1 M sugar gradient, the enzyme eluted at 0.45 M sugar and 0.22 M NaCl. When the 0 /1 M NaCl/0 /1 M sugar gradient was applied, peroxidase eluted at 0.30 M sugar and 0.30 M NaCl. With the 0 /2 M NaCl/0 /1 M sugar gradient, 0.25 M sugar and 0.50 M NaCl were required for enzyme elution. These results indicate that NaCl in the elution buffer allows reduction of the sugar amount required to elute the peroxidase. Fig. 7A shows the chromato-
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Fig. 6. (A) Breakthrough curves for HRP binding to Con A-Agarose at 1.0 ("), 2.0 (j), 4.0 (') and 6.0 (m) mg/ml HRP feed concentration. (B) Breakthrough curves for HRP binding to Con AAgarose at 1.0 ("), 1.5 (j), 2.9 (') and 4.0 (m) cm/min feed velocity. Buffer: 20 mM sodium phosphate, pH 7.0. Ca2 /Mg2 concentration /5 mM. C , HRP concentration in the outlet stream; C0, HRP concentration in the feedstock; f , volumetric flux; t , time elapsed; C0.f.t, HRP amount loaded on the column. Details in Section 2.
graphic profile. Based on these results, a step-elution was designed: after the wash step with adsorption buffer, peroxidase was eluted by changing the mobile phase to 0.48 M a-methyl-D-mannopyranoside and 0.97 M NaCl (these concentrations are those corresponding to the end of the peek). Under these conditions, 84.3% SBP recovery was obtained without any enzyme loss during the wash step. Gillikin and Graham [4], working at pH 6.8, recovered less than 50% SBP by eluting the enzyme with 0.75 /1 M mannose in the presence of 2.5 M NaCl. Our results suggest that the different working pH could account for this difference. 3.3.2. Horseradish peroxidase As a high NaCl concentration helps HRP binding to Con A-Agarose (see adsorption experiments), the elution was performed only with a gradient of 0/1 M amethyl-D-mannopyranoside. The enzyme eluted at
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Fig. 7. (A) Chromatographic profile of SBP purification on Con A-Agarose under the conditions selected. A column (0.5 /4.0 cm) of Con AAgarose was equilibrated with a 20 mM sodium acetate buffer, pH 5.0, 1 mM CaCl2 and MgCl2. After loading 50 ml of 12.4 mg/ml SBP, 1-ml fractions were collected at a linear flow rate of 3.2 cm/min and monitored by their absorbance at 280 nm (j) and 403 nm ('), and enzyme activity (m). After a wash step with 5 ml of adsorption buffer, SBP elution was carried out with a simultaneous 0 /1 M a-methyl-D-mannopyranoside/0 /2 M NaCl gradient. (B) Chromatographic profile of HRP purification on Con A-Agarose under the conditions selected. A column (0.5 /4.0 cm) of Con A-Agarose was equilibrated with a 20 mM sodium phosphate buffer, pH 7.0, 5 mM CaCl2 and MgCl2, 0.75 M NaCl. After loading 50 ml of 12.4 mg/ ml HRP, 1-ml fractions were collected at a linear flow rate of 1.5 cm/min and their absorbance at 280 nm (j) and 403 nm ('), and enzyme activity (m) were monitored. After a wash step with 5 ml of adsorption buffer, HRP elution was carried out with a 0 /1 M a-methyl-D-mannopyranoside eluent.
0.16 /0.36 M sugar (Fig. 7B); therefore, a step elution with 0.36 M a-methyl-D-mannopyranoside was developed. Recovery was 75.0% and, in contrast with SBP, 20 /25% of peroxidase activity passed through during the wash step. The most active part of a glycan structure towards Con A is not that involving the a-mannose residues in a terminal position but the trisaccharide a-Man-(1-3)-[aMan-(1-6)]Man [13]. On the other hand, it has been stated that binding of glycoproteins to immobilised lectins is of a multivalent nature, thus meaning that more than one lectin molecule should be involved in the binding of one glycocompound molecule [14]. SBP contains 18.2% carbohydrate, at five or six glycosylation sites [15]. The site at the N-terminus, Asn 16, has been clearly defined as being substituted mainly for high mannose-type glycans (Manx GlcNAc2, x /4, 5, 6 or 7) though there is sufficient evidence that this site may also be substituted with (Xyl)Man3(Fuc)GlcNAc2 in different batches of SBP [2]. In contrast to SBP, HRPc (the best-studied HRP isoenzyme) has only traces of high mannose-type glycans, where a significant part ( /16%) of the total glycan pool is composed of high-mannose species confined to a single glycosylation site [16]. There are no references as regards glycosylation patterns of the various HRP isoenzymes. It is important to point out that while peroxidases are multivalent, the lectin used in this work is tetravalent.
In the case of SBP, it was not easy to dissociate the affinity complex. A possible explanation is that the high mannose-type glycans present in this peroxidase bind to Con A-Agarose very tightly and, as a consequence of this resulting polyvalent interaction, dissociation of the complex by adding a monovalent sugar like methylmannoside is very difficult. NaCl addition was useful to decrease the sugar concentration for peroxidase elution but even so, it was not enough to bring out a quantitative recovery of the enzyme. For HRP, the loss in the wash step could be due to difference in the glycosylation pattern of the various isoenzymes. Results obtained in this work evidence the usefulness of static and dynamic chromatographic parameter determination to select the best conditions for SBP and HRP peroxidase purification.
Acknowledgements This work was supported by grants from the Universidad de Buenos Aires, the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas de la Repu´blica Argentina and the Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica de la Repu´blica Argentina. M.V. Miranda and O. Cascone are career researchers of the CONICET.
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