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Purification of chymotrypsin from bovine pancreas using precipitation with a strong anionic polyelectrolyte
Process Biochemistry 44 (2009) 588–592
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Purification of chymotrypsin from bovine pancreas using precipitation with a strong anionic polyelectrolyte Valeria Boeris, Diana Romanini, Beatriz Farruggia, Guillemo Pico´ * Bioseparation Lab., Physic Chemical Department, Faculty of Biochemical and Pharmaceutical Sciences, CONICET, FonCyT and CIUNR, National University of Rosario, Suipacha 570, (S2002RLK) Rosario, Argentina
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 July 2008 Received in revised form 4 December 2008 Accepted 16 February 2009
The separation of chymotrypsin from a crude filtrate of bovine pancreas homogenate was carried out using precipitation with a commercially available negatively charged strong polyelectrolyte: polyvinyl sulfonate. The zymogen form of chymotrypsin was activated by addition of trypsin (0.01 mg/g homogenate), then, the enzyme was precipitated by polyelectrolyte addition at pH 2.5 in the pancreas homogenate. A stoichiometric ratio of 670 bound molecules of chymotrypsin per polyelectrolyte molecule was found in the non-soluble form of the enzyme–polyelectrolyte complex. The non-soluble complex was separated by simple centrifugation and re-dissolved by a pH change to 8.0. The recovery of chymotrypsin biological activity was 61% of the initial activity in the homogenate with 4.7-fold increase in its specific activity. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Chymotrypsin Polyvinyl sulfonate Protein precipitation
1. Introduction Precipitation is a common approach to obtain enzymes and other macromolecules. This technique offers the possibility of concentrating and purifying the target macromolecule at a low cost. Polyelectrolyte precipitation uses a poly-charged macromolecule of opposite electrical charge to the target macromolecule, forming a soluble protein–polyelectrolyte complex. Under desired experimental conditions, these complexes interact among each other, producing insoluble macroaggregates. This is a suitable method for protein isolation because very low polyelectrolyte concentrations are used (up to 0.1%, w/w). This method sometimes offers a high selectivity and the insoluble complex can be redissolved by a pH change or adding a salt [1]. Production of proteins is a prime biotechnological application which includes upstream and downstream processing steps to obtain the final product in the desired purified form, the downstream processing being often the most expensive one. Bioseparation steps for the recovery of the final product can account for 50–80% of overall production costs. Most purification technologies use precipitation of proteins as one of the initial operations aimed at concentrating the product for further downstream steps. Precipitation using salts, organic solvents, non-ionic polymers and polyelectrolytes is a well known and simple
technique for protein concentration. Attempts are usually made to derive some degree of purification of target products in the precipitation step [2]. One protease widely used in food and pharmaceutical industry is chymotrypsin which has a single polypeptidic chain of 324 amino acid residues and a molecular weight of 25.7 kDa. It is one of the proteolytic enzymes of vertebrate pancreas juice, being its optimum activity pH 8.2 and its isoelectric point 9.1 [3]. A great amount of this enzyme is required for different industrial purposes, which makes it necessary to develop scaling up methodologies. In the area where our laboratory is located meat industries are very important, therefore, great amounts of meat waste are produced. One of these products is the bovine pancreas, which is very rich in enzymes such as different types of protease, amylase and lipase of wide application in numerous biotechnological processes. In a previous paper [4] we described the molecular mechanism of interaction between polyvinil sulfonate (CH2–CH–O–SO32 )x, a strong anionic polyelectrolyte (pKa 1, molecular mass: 220 kDa) and determined the medium variable values at which the insoluble protein–polyelectrolyte formation is optimal. In this paper, these conditions are used as a method designed for the isolation and purification of this enzyme from bovine pancreas homogenate. 2. Materials and methods 2.1. Chemicals
* Corresponding author. E-mail address: pico@ifise.gov.ar (G. Pico´). Abbreviations: ChTRP, chymotrypsin; PVS, polyvinyl sulfonate sodium salt. 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.02.009
Chymotrypsin (ChTRP) and N-benzoyl-l-tyrosine ethyl ester (BTEE) were purchased from sigma Chem. Co., polyvinyl sulfonate (PVS), sodium salt in aqueous solution at 25% (w/w), from Aldrich and used without further purification.
V. Boeris et al. / Process Biochemistry 44 (2009) 588–592
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Buffer solutions formed by a mixture of sodium citrate (50 mM) and Tris–HCl (50 mM) of different pH were prepared, the pH was adjusted with NaOH or HCl in each case. 2.2. Methodology 2.2.1. Bovine pancreas homogenate preparation The pancreas was removed from a recently killed bovine, washed with isotonical saline solution and cut in small pieces mixed with Tris–HCl–sodium citrate buffer, pH 2.5 (ratio 1:3) and homogenized for 5 min in a Minipimmer homogenizer at a rate of 4000 rpm. The resulting homogenate was divided in aliquots and frozen at 30 8C. 2.2.2. Chymotrypsin activation from freshly homogenate pancreas Chymotrypsinogen is an inactive precursor of ChTRP, therefore, a previous activation step was required. The zymogen activation was initiated by adding a small aliquot of trypsin (0.0001%, w/w) in buffer Tris–HCl 90 mM pH 8.2 and CaCl2 45 mM. The time required to complete the activation process was also determined by measuring the ChTRP activity at different intervals until a maximal value was reached. 2.2.3. Determination of the ChTRP activity The chymotrypsin assay is based on the hydrolysis of BTEE [5]. The reaction rate was determined by measuring the absorbance increase at 256 nm, at 25 8C, which results from the hydrolysis of the substrate at 0.6 mM concentration in 100 mM buffer Tris–HCl pH 8.2 with 100 mM CaCl2. The ChTRP enzymatic activity is the slope of the plot absorbance vs. time. 2.2.4. ChTRP turbidimetric titration curves with polymer The formation of the insoluble polymer–protein complex was followed by means of turbidimetric titration [6]. Buffer Tris–HCl–sodium citrate solutions (10 mL) with a fixed protein concentration were titrated at 25 8C in a glass cell with the polymer solution as the titrant. To avoid changes in pH during titration, both protein and polyelectrolyte solutions were adjusted to the same pH value. The absorbance of solution at 420 nm was used to follow the protein– polyelectrolyte complex formation and plotted vs. the total concentration of polymer in the tube. The results were fitted with a 4-parameters sigmoidal function in order to determine the value of the PVS minimal concentration required to precipitate ChTRP. This parameter was calculated as the intersection of the tangent at the inflection point with the plateau of the plot. The [ChTRP]/[PVS] molar ratio can be calculated as the rate between the ChTRP total concentration and the [PVS] calculated [4]. Absorbance solutions were measured using a Jasco 520 spectrophotometer with a thermostatized cell of 1 cm of path length.
Fig. 1. Turbidimetric titration of ChTRP (4 mM) with PVS. Medium buffer sodium citrate 50 mM–Tris–HCl pH 2.5. Temperature 25 8C. The activity was measured at pH 8.2.
electrical charges, each one can interact with one ChTRP molecule by electrostatic interaction, and as a result, very low PVS concentration is necessary (in the nano-molar order) to precipitate the ChTRP in solution. However, the low stoichiometry found for the lysozyme–PVS interaction in a previous work [7] as opposed to the present results, shows that the ChTRP favors the interaction between the soluble complex particles to form macroaggregates. This finding, in which the same polyelectrolyte was used to precipitate different proteins obtaining different stoichiometry ratios, suggests that the non-soluble complex formation depends not only on the number of opposite electrical charges both in the protein and the polyelectrolyte, but also on the nature of protein superficial area exposed to the polyelectrolyte contact. The ratio protein–polymer value found is important because it enables us to calculate the minimal polymer concentration necessary to precipitate the enzyme. 3.2. PVS effect on the ChTRP enzymatic activity
2.2.5. Evaluation of the purification process by SDS-PAGE Aliquots of pancreas homogenate and re-dissolved precipitate were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) using a vertical system. The running time was about 45 min and the constant intensity was 23 mA for the resolving gel. Proteins were stained with Coomassie brilliant blue and destained with 10% (v/v) methanol–10% (v/v) acetic acid. The proteins bands were identified using horse serum albumin and ovalbumin as molecular weight markers (Sigma Chemical Co.).
3. Results 3.1. Titration of ChTRP with PVS Fig. 1 shows the absorbance dependence at 420 nm when ChTRP is titrated at a constant concentration with PVS in pH 2.5 media. This pH was selected because ChTRP is stable at such pH, lower pH values were not assayed due to the possibility of enzyme denaturalization. A higher pH was not assayed because ChTRP forms aggregates and acquires biological activity from pH 6, inducing the auto-proteolysis of the enzyme. Fig. 1 also shows the residual activity in the supernatant solution before the precipitate was separated. A decrease in the activity can be seen as the precipitate is formed, which confirms that PVS forms a non-soluble complex with ChTRP. The stoichiometric protein/polymer ratio which corresponds to the case in which most ChTRP has been precipitated as an insoluble complex was calculated from Fig. 1. A high value was obtained: 670 enzyme molecules per polymer molecule. This value is explained by the fact that one PVS molecule has a great number of negative
In a previous report [8], we have demonstrated that polyelectrolytes influence the enzymatic activity because they induce a modification of the secondary and tertiary protein structure. ChTRP at constant concentration (4 mM) was incubated for 30 min in media of increasing PVS concentrations at the pH of the enzyme maximal activity (8.2). Under these conditions, the ChTRP–PVS complex remains in the soluble form, then the enzyme activity of the ChTRP was determined with respect to a medium reference in the absence of PVS. The polyelectrolyte induced a slight increase in the enzyme activity (data not shown) which reached a plateau, suggesting that the change in the activity is closely associated with the enzyme–PVS complex formation in agreement with a previous report where PVS was found to stabilize the secondary structure of the ChTRP [8]. The effect of formation of the insoluble complex ChTRP–PVS on the enzyme activity of ChTRP was also tested. At a constant concentration, the enzyme was precipitated with a constant PVS concentration at pH 2.5. At different times, the precipitate was dissolved by changing the pH to 8.2 and the enzyme activity was determined. Fig. 2 shows the recovered ChTRP biological activity against time. A loss of activity with respect to the control (taken at zero time) with a maximal value of 35% of the initial value could be seen. After 30 min, the enzyme activity remained constant over time. 3.3. Isolation of ChTRP from an artificial protein mixture The precipitation of ChTRP was carried out from an artificial mixture of ChTRP and BSA in a mass ratio 1:5. The experiment was
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Fig. 2. Incubation time effect on the biological activity of ChTRP (4 mM). Medium sodium citrate–Tris–HCl 50 mM pH 8.2.
Fig. 3. ChTRP activity from bovine pancreas homogenate vs. the incubation time. Medium sodium citrate–Tris–HCl 50 mM pH 8.2.
performed to simulate the ChTRP in the presence of other proteins as is the case of pancreas homogenate. Bovine albumin simulates the other proteins present in the pancreas homogenate. This enables us to check whether the best conditions found in the previous experiments can be applied to a mixture of proteins. Aliquots of increasing amounts of PVS were added to tubes containing the mixture. The precipitate obtained was separated by centrifugation (4 min at 500 g) and then dissolved by addition of 4 ml Tris–HCl buffer, pH 8.2. The recovered ChTRP activity and total protein concentration were determined in the original mixture, in the supernatant and in the re-dissolved precipitate. The values of recovery and purification factor for this process are shown in Table 1. A decrease in the PVS–enzyme ratio is seen to induce an increase in the ChTRP precipitation and therefore in its recovery. However, the purification factor decreased in a significant way. This was expected to happen, since the increase in ChTRP concentration induced a greater number of molecules of this enzyme which is available to interact with PVS forming a larger amount of precipitate. Serum albumin molecules might have mixed themselves with the ChTRP–PVS complex when the precipitate was formed, thus decreasing the enzyme purity.
these conditions were set to determine the best experimental conditions for the chymotrypsinogen activation in the pancreas homogenate. It was found that the presence of trypsin increased the activation process significantly in less than 1 h (see Fig. 3), while the presence of PEI did not modify the activation rate in a significant way (data not shown). However, a decrease in the homogenate viscosity solution was observed, which facilitated its handling. No significant difference in the activation time was observed between 8 and 25 8C (data not shown). The control solution was a homogenate without trypsin addition. Fig. 3 also shows that the homogenate control activity remained constant for 24 h while in the trypsin-treated homogenate, the activity remained constant for the first 10 h. Then a significant decrease was observed which can be attributed to the destruction of the ChTRP due to the trypsin which was self-generated from the non-active form of trypsin present in the homogenate. Therefore, the following experiments were performed with a homogenate which was previously incubated with trypsin for 30 min. 3.5. ChTRP precipitation from the activated homogenate of bovine pancreas
3.4. Zymogen activation from the bovine pancreas homogenate ChTRP is present in the pancreas in a biological inactive form, therefore, it was activated in the following way: a mass of 10 g of bovine pancreas homogenate was pre-treated under different conditions. The following effects of the kinetics on the activation from zymogen to ChTRP were assayed: (1) the presence of small concentration of trypsin (0.01 mg/g of homogenate)—it is known that trypsin increases the rate of zymogen activation [3], (2) the temperature and (3) the effect of a previous elimination of the nucleic acid with 0.2% (w/w) of polyethylene imine (PEI) to decrease the medium viscosity. All
Table 1 Results of the precipitation process with PVS from an artificial mixture of ChTRP and serum albumin (1:5). [PVS]/[ChTRP]
Recovery (%)
Purification factor
1:1000 1:1500 1:2000 1:2500
61.2 0.2 72.6 0.2 79.1 0.2 83.9 0.2
4.4 0.5 3.4 0.5 3.0 0.5 2.3 0.5
Aliquots with a 10-g mass of activated homogenate in the absence and presence of 0.2% (w/w) of PEI (a synthetic basic polyelectrolyte used to precipitate nucleic acids) were titrated with increasing concentration of PVS solution (10–400 nM) by adding small aliquots of PVS concentrated solution. The precipitate obtained was separated by centrifugation (4 min at 500 g) and then dissolved by addition of 4 ml citrate–Tris–HCl buffer, pH 8.2. The ChTRP activity recovered and the total protein concentration were determined in the supernatant Fig. 4 shows that an increase in the PVS total concentration favors the ChTRP recovery, reaching a maximum yield of 61%, around a PVS concentration of 250 nM. The presence of 0.2% PEI induced a significant decrease in the ChTRP recovery. Fig. 4 also shows the PVS total concentration effect on the purification factor, where the PEI presence can be seen to induce poor purification factor values, while the absence of this polymer yielded high purification factor with values between 4.5 and 5.1 in the PVS concentration range between 0 and 250 nM. The SDS-PAGE patterns obtained from the re-dissolved precipitate (Fig. 5) yielded a single band corresponding to ChTRP.
V. Boeris et al. / Process Biochemistry 44 (2009) 588–592
Fig. 4. Recovery of the ChTRP activity and purification factor from the solubilization of the ChTRP–PVS precipitated at different initial concentration of precipitant agent (PVS). Medium sodium citrate 50 mM–Tris–HCl 50 mM pH 2.5. The activity was measured at pH 8.2.
4. Discussion There is a great interest in scaling up method for enzyme obtention using synthetic [4] and natural polyelectrolytes [9]. Chymotrypsinogen is activated to chymotrypsin by porcine enterokinase and the final step in the purification process is an ion exchange chromatography using NaCl gradient. All these operations are time-consuming, and cause a loss in the enzyme activity, thus making the standard methods expensive and difficult to scale up. Below pH 6.0, ChTRP interacts with the anionic polymer by means of electrostatic forces between the sulfonic negatively charged groups of the polymer and the positively charged histidine residues of ChTRP. However, at pH below 6, the positively charged histidine groups of the free remaining ChTRP do not interact with
591
negatively charged groups of the polymer. These positive groups of ChTRP molecules are repelled by similar groups of other ChTRP molecules inducing repulsion between the molecules of the complex. It is well known that an insoluble complex can be solubilized by breaking the coulombic interaction in two ways: either by changing the pH or by adding salt. Our finding suggests that the prototropic groups of ChTRP lose their positive electrical change around 6. Therefore, the increase in the pH induces a solubilization of the precipitate. The low purification factor and recovery of ChTRP observed in the polyethylene imine presence is due to the interaction between the cationic polymer and the nucleic acid with the anionic PVS. However, our finding suggests that the presence of nucleic acid in the homogenate does not seem to cause any difficulties in the handling of the solution and in the protein precipitation and recovery. The 61% of ChTRP recovered seems to be unable to increase due to the loss of the enzyme activity that occurs when ChTRP is in the precipitate form at acid pH (as shown in Fig. 3). This might be due to the strong interaction of PVS with the positively charged groups of ChTRP, which could induce an irreversible conformational change in the tertiary structure of ChTRP near its catalytic site which results in a partial loss of the enzyme biological activity. On the other hand, the increase in the PVS concentration induces an increase in the recovery from the pancreas homogenate, because a greater number of molecules of ChTRP can interact with PVS forming a larger amount of precipitate. The presence of molecules of other proteins (impurities) which might take part in the formation of the complex, which eventually precipitates, favors a decrease in the enzyme purity. There are several reports about the interaction between the strong anionic polyelectrolyte PVS and basic proteins [7,10]. The results show that PVS binds the basic proteins to different stoichiometric ratio, which suggests that the nature of the protein surface area together with the number of electrical charges present in the polyelectrolyte and the protein, condition their interaction. However, steric hindrance between the negative charge of the PVS and positive charge of the protein significantly conditions the number of protein molecules bound per PVS molecule. Due to the fact that the process is driven only by coulombic forces, it should be considered as an ion exchange process and not an affinity interaction one [2]. The methodology proved to be very useful and simple compared with long and tedious classical laboratory methodologies to isolate this enzyme. Another advantage is that the precipitation method can be potentially applied for scaling up. Acknowledgements This work was supported by a grant from FoNCyT PICT0612476/02 and CONICET PIP5053. We thank Marı´a Robson, Geraldine Raimundo, Mariana De Sanctis and Marcela Culasso for the language correction of the manuscript. References
Fig. 5. SDS-polyacrylamide (13%) gel electrophoresis (Coomassie blue staining) of the proteins present in the pancreas homogenate and in the re-dissolved precipitate. The molecular mass markers were electrophoresed in a parallel lane and consisted of horse serum albumin (79 kDa) and ovalbumin (42.7 kDa). The second lane is commercial ChTRP (25.7 kDa).
[1] Matsudo T, Ogawa K, Kokufuta E. Complex formation of protein with different water-soluble synthetic polymers. Biomacromolecules 2003;4:1794–9. [2] Chatterjee S, Chatterjee S, Chatterjee BP, Guha AK. Clarification of fruit juice with chitosan. Process Biochem 2004;39:2229–32. [3] Sharmat SK, Hopkins TR. Activation of bovine chymotrypsinogen A. Isolation and characterization of chymotrypsin. Biochemistry 1979;18:1008–13. [4] Boeris V, Spelzini D, Peleteiro Salgado J, Pico´ G, Romanini D, Farruggia B. Chymotrypsin–poly vinyl sulfonate interaction studied by dynamic light scattering and turbidimetric approaches. Biochim Biophys A 2008;1780:1032–7. [5] Guyonneta V, C¸uscikb FT, Longa PL, Polanowskib A, Travis J. Purification and partial characterization of the pancreatic proteolytic enzymes trypsin, chymotrypsin and elastase from the chicken. J Chromatogr A 1999;852:217–25. [6] Patrickios CS, Sharma LR, Armes SP, Billingham NC. Precipitation of a watersoluble ABC triblock methacrylic polyampholyte: effects of time, pH, polymer
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concentration, salt type and concentration, and presence of a protein. Langmuir 1999;15:1613–20. [7] Romanini D, Braia M, Giatte Angarten R, Loh W, Pico´ G. Interaction of lysozyme with negatively charged flexible chain polymers. J Chromatogr B 2007;857: 25–31. [8] Boeris V, Farruggia B, Nerli B, Romanini D, Pico´ G. Protein-flexible chain polymer interactions to explain protein partition in aqueous two-phase sys-
tems and the protein–polyelectrolyte complex formation. I. J Biol Macromol 2007;41:286–94. [9] Chatterjee S, Adhya M, Guha AK, Chatterjee BP. Chitosan from Mucor rouxii: production and physico-chemical characterization. Process Biochem 2005;40: 395–400. [10] Izumrudov VA, Galaev IY, Mattiasson B. Polycomplexes–potential for bioseparation. Bioseparation 1999;7:207–20.
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Purification of chymotrypsin from bovine pancreas using precipitation with a strong anionic polyelectrolyte Valeria Boeris, Diana Romanini, Beatriz Farruggia, Guillemo Pico´ * Bioseparation Lab., Physic Chemical Department, Faculty of Biochemical and Pharmaceutical Sciences, CONICET, FonCyT and CIUNR, National University of Rosario, Suipacha 570, (S2002RLK) Rosario, Argentina
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 July 2008 Received in revised form 4 December 2008 Accepted 16 February 2009
The separation of chymotrypsin from a crude filtrate of bovine pancreas homogenate was carried out using precipitation with a commercially available negatively charged strong polyelectrolyte: polyvinyl sulfonate. The zymogen form of chymotrypsin was activated by addition of trypsin (0.01 mg/g homogenate), then, the enzyme was precipitated by polyelectrolyte addition at pH 2.5 in the pancreas homogenate. A stoichiometric ratio of 670 bound molecules of chymotrypsin per polyelectrolyte molecule was found in the non-soluble form of the enzyme–polyelectrolyte complex. The non-soluble complex was separated by simple centrifugation and re-dissolved by a pH change to 8.0. The recovery of chymotrypsin biological activity was 61% of the initial activity in the homogenate with 4.7-fold increase in its specific activity. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Chymotrypsin Polyvinyl sulfonate Protein precipitation
1. Introduction Precipitation is a common approach to obtain enzymes and other macromolecules. This technique offers the possibility of concentrating and purifying the target macromolecule at a low cost. Polyelectrolyte precipitation uses a poly-charged macromolecule of opposite electrical charge to the target macromolecule, forming a soluble protein–polyelectrolyte complex. Under desired experimental conditions, these complexes interact among each other, producing insoluble macroaggregates. This is a suitable method for protein isolation because very low polyelectrolyte concentrations are used (up to 0.1%, w/w). This method sometimes offers a high selectivity and the insoluble complex can be redissolved by a pH change or adding a salt [1]. Production of proteins is a prime biotechnological application which includes upstream and downstream processing steps to obtain the final product in the desired purified form, the downstream processing being often the most expensive one. Bioseparation steps for the recovery of the final product can account for 50–80% of overall production costs. Most purification technologies use precipitation of proteins as one of the initial operations aimed at concentrating the product for further downstream steps. Precipitation using salts, organic solvents, non-ionic polymers and polyelectrolytes is a well known and simple
technique for protein concentration. Attempts are usually made to derive some degree of purification of target products in the precipitation step [2]. One protease widely used in food and pharmaceutical industry is chymotrypsin which has a single polypeptidic chain of 324 amino acid residues and a molecular weight of 25.7 kDa. It is one of the proteolytic enzymes of vertebrate pancreas juice, being its optimum activity pH 8.2 and its isoelectric point 9.1 [3]. A great amount of this enzyme is required for different industrial purposes, which makes it necessary to develop scaling up methodologies. In the area where our laboratory is located meat industries are very important, therefore, great amounts of meat waste are produced. One of these products is the bovine pancreas, which is very rich in enzymes such as different types of protease, amylase and lipase of wide application in numerous biotechnological processes. In a previous paper [4] we described the molecular mechanism of interaction between polyvinil sulfonate (CH2–CH–O–SO32 )x, a strong anionic polyelectrolyte (pKa 1, molecular mass: 220 kDa) and determined the medium variable values at which the insoluble protein–polyelectrolyte formation is optimal. In this paper, these conditions are used as a method designed for the isolation and purification of this enzyme from bovine pancreas homogenate. 2. Materials and methods 2.1. Chemicals
* Corresponding author. E-mail address: pico@ifise.gov.ar (G. Pico´). Abbreviations: ChTRP, chymotrypsin; PVS, polyvinyl sulfonate sodium salt. 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.02.009
Chymotrypsin (ChTRP) and N-benzoyl-l-tyrosine ethyl ester (BTEE) were purchased from sigma Chem. Co., polyvinyl sulfonate (PVS), sodium salt in aqueous solution at 25% (w/w), from Aldrich and used without further purification.
V. Boeris et al. / Process Biochemistry 44 (2009) 588–592
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Buffer solutions formed by a mixture of sodium citrate (50 mM) and Tris–HCl (50 mM) of different pH were prepared, the pH was adjusted with NaOH or HCl in each case. 2.2. Methodology 2.2.1. Bovine pancreas homogenate preparation The pancreas was removed from a recently killed bovine, washed with isotonical saline solution and cut in small pieces mixed with Tris–HCl–sodium citrate buffer, pH 2.5 (ratio 1:3) and homogenized for 5 min in a Minipimmer homogenizer at a rate of 4000 rpm. The resulting homogenate was divided in aliquots and frozen at 30 8C. 2.2.2. Chymotrypsin activation from freshly homogenate pancreas Chymotrypsinogen is an inactive precursor of ChTRP, therefore, a previous activation step was required. The zymogen activation was initiated by adding a small aliquot of trypsin (0.0001%, w/w) in buffer Tris–HCl 90 mM pH 8.2 and CaCl2 45 mM. The time required to complete the activation process was also determined by measuring the ChTRP activity at different intervals until a maximal value was reached. 2.2.3. Determination of the ChTRP activity The chymotrypsin assay is based on the hydrolysis of BTEE [5]. The reaction rate was determined by measuring the absorbance increase at 256 nm, at 25 8C, which results from the hydrolysis of the substrate at 0.6 mM concentration in 100 mM buffer Tris–HCl pH 8.2 with 100 mM CaCl2. The ChTRP enzymatic activity is the slope of the plot absorbance vs. time. 2.2.4. ChTRP turbidimetric titration curves with polymer The formation of the insoluble polymer–protein complex was followed by means of turbidimetric titration [6]. Buffer Tris–HCl–sodium citrate solutions (10 mL) with a fixed protein concentration were titrated at 25 8C in a glass cell with the polymer solution as the titrant. To avoid changes in pH during titration, both protein and polyelectrolyte solutions were adjusted to the same pH value. The absorbance of solution at 420 nm was used to follow the protein– polyelectrolyte complex formation and plotted vs. the total concentration of polymer in the tube. The results were fitted with a 4-parameters sigmoidal function in order to determine the value of the PVS minimal concentration required to precipitate ChTRP. This parameter was calculated as the intersection of the tangent at the inflection point with the plateau of the plot. The [ChTRP]/[PVS] molar ratio can be calculated as the rate between the ChTRP total concentration and the [PVS] calculated [4]. Absorbance solutions were measured using a Jasco 520 spectrophotometer with a thermostatized cell of 1 cm of path length.
Fig. 1. Turbidimetric titration of ChTRP (4 mM) with PVS. Medium buffer sodium citrate 50 mM–Tris–HCl pH 2.5. Temperature 25 8C. The activity was measured at pH 8.2.
electrical charges, each one can interact with one ChTRP molecule by electrostatic interaction, and as a result, very low PVS concentration is necessary (in the nano-molar order) to precipitate the ChTRP in solution. However, the low stoichiometry found for the lysozyme–PVS interaction in a previous work [7] as opposed to the present results, shows that the ChTRP favors the interaction between the soluble complex particles to form macroaggregates. This finding, in which the same polyelectrolyte was used to precipitate different proteins obtaining different stoichiometry ratios, suggests that the non-soluble complex formation depends not only on the number of opposite electrical charges both in the protein and the polyelectrolyte, but also on the nature of protein superficial area exposed to the polyelectrolyte contact. The ratio protein–polymer value found is important because it enables us to calculate the minimal polymer concentration necessary to precipitate the enzyme. 3.2. PVS effect on the ChTRP enzymatic activity
2.2.5. Evaluation of the purification process by SDS-PAGE Aliquots of pancreas homogenate and re-dissolved precipitate were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) using a vertical system. The running time was about 45 min and the constant intensity was 23 mA for the resolving gel. Proteins were stained with Coomassie brilliant blue and destained with 10% (v/v) methanol–10% (v/v) acetic acid. The proteins bands were identified using horse serum albumin and ovalbumin as molecular weight markers (Sigma Chemical Co.).
3. Results 3.1. Titration of ChTRP with PVS Fig. 1 shows the absorbance dependence at 420 nm when ChTRP is titrated at a constant concentration with PVS in pH 2.5 media. This pH was selected because ChTRP is stable at such pH, lower pH values were not assayed due to the possibility of enzyme denaturalization. A higher pH was not assayed because ChTRP forms aggregates and acquires biological activity from pH 6, inducing the auto-proteolysis of the enzyme. Fig. 1 also shows the residual activity in the supernatant solution before the precipitate was separated. A decrease in the activity can be seen as the precipitate is formed, which confirms that PVS forms a non-soluble complex with ChTRP. The stoichiometric protein/polymer ratio which corresponds to the case in which most ChTRP has been precipitated as an insoluble complex was calculated from Fig. 1. A high value was obtained: 670 enzyme molecules per polymer molecule. This value is explained by the fact that one PVS molecule has a great number of negative
In a previous report [8], we have demonstrated that polyelectrolytes influence the enzymatic activity because they induce a modification of the secondary and tertiary protein structure. ChTRP at constant concentration (4 mM) was incubated for 30 min in media of increasing PVS concentrations at the pH of the enzyme maximal activity (8.2). Under these conditions, the ChTRP–PVS complex remains in the soluble form, then the enzyme activity of the ChTRP was determined with respect to a medium reference in the absence of PVS. The polyelectrolyte induced a slight increase in the enzyme activity (data not shown) which reached a plateau, suggesting that the change in the activity is closely associated with the enzyme–PVS complex formation in agreement with a previous report where PVS was found to stabilize the secondary structure of the ChTRP [8]. The effect of formation of the insoluble complex ChTRP–PVS on the enzyme activity of ChTRP was also tested. At a constant concentration, the enzyme was precipitated with a constant PVS concentration at pH 2.5. At different times, the precipitate was dissolved by changing the pH to 8.2 and the enzyme activity was determined. Fig. 2 shows the recovered ChTRP biological activity against time. A loss of activity with respect to the control (taken at zero time) with a maximal value of 35% of the initial value could be seen. After 30 min, the enzyme activity remained constant over time. 3.3. Isolation of ChTRP from an artificial protein mixture The precipitation of ChTRP was carried out from an artificial mixture of ChTRP and BSA in a mass ratio 1:5. The experiment was
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Fig. 2. Incubation time effect on the biological activity of ChTRP (4 mM). Medium sodium citrate–Tris–HCl 50 mM pH 8.2.
Fig. 3. ChTRP activity from bovine pancreas homogenate vs. the incubation time. Medium sodium citrate–Tris–HCl 50 mM pH 8.2.
performed to simulate the ChTRP in the presence of other proteins as is the case of pancreas homogenate. Bovine albumin simulates the other proteins present in the pancreas homogenate. This enables us to check whether the best conditions found in the previous experiments can be applied to a mixture of proteins. Aliquots of increasing amounts of PVS were added to tubes containing the mixture. The precipitate obtained was separated by centrifugation (4 min at 500 g) and then dissolved by addition of 4 ml Tris–HCl buffer, pH 8.2. The recovered ChTRP activity and total protein concentration were determined in the original mixture, in the supernatant and in the re-dissolved precipitate. The values of recovery and purification factor for this process are shown in Table 1. A decrease in the PVS–enzyme ratio is seen to induce an increase in the ChTRP precipitation and therefore in its recovery. However, the purification factor decreased in a significant way. This was expected to happen, since the increase in ChTRP concentration induced a greater number of molecules of this enzyme which is available to interact with PVS forming a larger amount of precipitate. Serum albumin molecules might have mixed themselves with the ChTRP–PVS complex when the precipitate was formed, thus decreasing the enzyme purity.
these conditions were set to determine the best experimental conditions for the chymotrypsinogen activation in the pancreas homogenate. It was found that the presence of trypsin increased the activation process significantly in less than 1 h (see Fig. 3), while the presence of PEI did not modify the activation rate in a significant way (data not shown). However, a decrease in the homogenate viscosity solution was observed, which facilitated its handling. No significant difference in the activation time was observed between 8 and 25 8C (data not shown). The control solution was a homogenate without trypsin addition. Fig. 3 also shows that the homogenate control activity remained constant for 24 h while in the trypsin-treated homogenate, the activity remained constant for the first 10 h. Then a significant decrease was observed which can be attributed to the destruction of the ChTRP due to the trypsin which was self-generated from the non-active form of trypsin present in the homogenate. Therefore, the following experiments were performed with a homogenate which was previously incubated with trypsin for 30 min. 3.5. ChTRP precipitation from the activated homogenate of bovine pancreas
3.4. Zymogen activation from the bovine pancreas homogenate ChTRP is present in the pancreas in a biological inactive form, therefore, it was activated in the following way: a mass of 10 g of bovine pancreas homogenate was pre-treated under different conditions. The following effects of the kinetics on the activation from zymogen to ChTRP were assayed: (1) the presence of small concentration of trypsin (0.01 mg/g of homogenate)—it is known that trypsin increases the rate of zymogen activation [3], (2) the temperature and (3) the effect of a previous elimination of the nucleic acid with 0.2% (w/w) of polyethylene imine (PEI) to decrease the medium viscosity. All
Table 1 Results of the precipitation process with PVS from an artificial mixture of ChTRP and serum albumin (1:5). [PVS]/[ChTRP]
Recovery (%)
Purification factor
1:1000 1:1500 1:2000 1:2500
61.2 0.2 72.6 0.2 79.1 0.2 83.9 0.2
4.4 0.5 3.4 0.5 3.0 0.5 2.3 0.5
Aliquots with a 10-g mass of activated homogenate in the absence and presence of 0.2% (w/w) of PEI (a synthetic basic polyelectrolyte used to precipitate nucleic acids) were titrated with increasing concentration of PVS solution (10–400 nM) by adding small aliquots of PVS concentrated solution. The precipitate obtained was separated by centrifugation (4 min at 500 g) and then dissolved by addition of 4 ml citrate–Tris–HCl buffer, pH 8.2. The ChTRP activity recovered and the total protein concentration were determined in the supernatant Fig. 4 shows that an increase in the PVS total concentration favors the ChTRP recovery, reaching a maximum yield of 61%, around a PVS concentration of 250 nM. The presence of 0.2% PEI induced a significant decrease in the ChTRP recovery. Fig. 4 also shows the PVS total concentration effect on the purification factor, where the PEI presence can be seen to induce poor purification factor values, while the absence of this polymer yielded high purification factor with values between 4.5 and 5.1 in the PVS concentration range between 0 and 250 nM. The SDS-PAGE patterns obtained from the re-dissolved precipitate (Fig. 5) yielded a single band corresponding to ChTRP.
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Fig. 4. Recovery of the ChTRP activity and purification factor from the solubilization of the ChTRP–PVS precipitated at different initial concentration of precipitant agent (PVS). Medium sodium citrate 50 mM–Tris–HCl 50 mM pH 2.5. The activity was measured at pH 8.2.
4. Discussion There is a great interest in scaling up method for enzyme obtention using synthetic [4] and natural polyelectrolytes [9]. Chymotrypsinogen is activated to chymotrypsin by porcine enterokinase and the final step in the purification process is an ion exchange chromatography using NaCl gradient. All these operations are time-consuming, and cause a loss in the enzyme activity, thus making the standard methods expensive and difficult to scale up. Below pH 6.0, ChTRP interacts with the anionic polymer by means of electrostatic forces between the sulfonic negatively charged groups of the polymer and the positively charged histidine residues of ChTRP. However, at pH below 6, the positively charged histidine groups of the free remaining ChTRP do not interact with
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negatively charged groups of the polymer. These positive groups of ChTRP molecules are repelled by similar groups of other ChTRP molecules inducing repulsion between the molecules of the complex. It is well known that an insoluble complex can be solubilized by breaking the coulombic interaction in two ways: either by changing the pH or by adding salt. Our finding suggests that the prototropic groups of ChTRP lose their positive electrical change around 6. Therefore, the increase in the pH induces a solubilization of the precipitate. The low purification factor and recovery of ChTRP observed in the polyethylene imine presence is due to the interaction between the cationic polymer and the nucleic acid with the anionic PVS. However, our finding suggests that the presence of nucleic acid in the homogenate does not seem to cause any difficulties in the handling of the solution and in the protein precipitation and recovery. The 61% of ChTRP recovered seems to be unable to increase due to the loss of the enzyme activity that occurs when ChTRP is in the precipitate form at acid pH (as shown in Fig. 3). This might be due to the strong interaction of PVS with the positively charged groups of ChTRP, which could induce an irreversible conformational change in the tertiary structure of ChTRP near its catalytic site which results in a partial loss of the enzyme biological activity. On the other hand, the increase in the PVS concentration induces an increase in the recovery from the pancreas homogenate, because a greater number of molecules of ChTRP can interact with PVS forming a larger amount of precipitate. The presence of molecules of other proteins (impurities) which might take part in the formation of the complex, which eventually precipitates, favors a decrease in the enzyme purity. There are several reports about the interaction between the strong anionic polyelectrolyte PVS and basic proteins [7,10]. The results show that PVS binds the basic proteins to different stoichiometric ratio, which suggests that the nature of the protein surface area together with the number of electrical charges present in the polyelectrolyte and the protein, condition their interaction. However, steric hindrance between the negative charge of the PVS and positive charge of the protein significantly conditions the number of protein molecules bound per PVS molecule. Due to the fact that the process is driven only by coulombic forces, it should be considered as an ion exchange process and not an affinity interaction one [2]. The methodology proved to be very useful and simple compared with long and tedious classical laboratory methodologies to isolate this enzyme. Another advantage is that the precipitation method can be potentially applied for scaling up. Acknowledgements This work was supported by a grant from FoNCyT PICT0612476/02 and CONICET PIP5053. We thank Marı´a Robson, Geraldine Raimundo, Mariana De Sanctis and Marcela Culasso for the language correction of the manuscript. References
Fig. 5. SDS-polyacrylamide (13%) gel electrophoresis (Coomassie blue staining) of the proteins present in the pancreas homogenate and in the re-dissolved precipitate. The molecular mass markers were electrophoresed in a parallel lane and consisted of horse serum albumin (79 kDa) and ovalbumin (42.7 kDa). The second lane is commercial ChTRP (25.7 kDa).
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