Thiol-Reactive Support to Immobilize Thiol

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Novel Bifunctional Epoxy/Thiol-Reactive Support to Immobilize Thiol Containing Proteins by the Epoxy Chemistry Valeria Grazu´ ,† Olga Abian,‡ Cesar Mateo,‡ Franciso Batista-Viera,†,§ Roberto Ferna´ ndez-Lafuente,*,‡ and Jose´ Manuel Guisa´ n*,‡ Department of Biocatalysis, Instituto de Cata´ lisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spain, Unidad Asociada de Bioquı´mica (Fac. Quı´mica)-I.Q.B (Fac. Ciencias), Montevideo, Uruguay, and Ca´ tedra de Bioquı´mica, Fac. de Quı´mica, Montevideo, Uruguay Received July 29, 2003; Revised Manuscript Received September 19, 2003

In this manuscript, we present a new bifunctional support containing epoxide and thiol-reactive groups for its use in protein immobilization. In a first step, the proteins are reversibly immobilized by reaction of its thiol groups with the thiol-reactive groups of the support under mild experimental conditions (pH 7.0, 24 °C). Then, the remaining epoxides of the support can form irreversible bonds with nucleophile surface groups of the already immobilized protein in a rapid way. The partial derivatization of EP-Sepabeads (a commercial matrix containing 120 µmol epoxy groups/g drained support) was optimized, using dithiotreitol (DTT) as thiolating agent. It was possible to achieve a partial thiolation of the support proportional to the concentration of DTT used (3, 8, and 15 µmol SH groups/g wet support). The remaining epoxide content after the thiolation treatment was high (e.g., nearly 70% for the highest thiolation degree). High immobilization yields were obtained for the three model enzymes selected (60% for Penicillin G acylase, 65% and 100% for K. lactis and E. coli β-galactosidase, respectively). In all cases, no significant immobilization onto an unmodified epoxy support was found, thus demonstrating that the first step of attachment takes place through thiol-disulfide exchange reactions. In the case of the bifunctional support, progressive formation of enzymesupport attachments involving the epoxy groups was showed by the irreversible covalent attachment of the proteins on the support. The promotion of this multipoint covalent immobilization required long incubation periods at basic pH values. Introduction Epoxy activated supports have been proposed as very efficient materials for the immobilization of proteins at the industrial scale for different reasons: e.g., high stability of the groups, high stability of the enzyme-support bonds, possibility of performing a final blocking of the remaining groups, and possibility of achieving stabilization of the enzymes via multipoint covalent attachment.1-9 The immobilization on these activated supports follows a two-step mechanism: a rapid physical adsorption followed by an “intramolecular” covalent reaction. Thus, conventional epoxy supports have a fairly hydrophobic nature, and immobilization must proceed at high ionic strength.10-13 Recently, it has been shown that the modification of some epoxy groups with different groups which are able to promote the physical adsorption of the proteins may be enough to promote this first physical adsorption.9,14-18 The immobilization does not require neither high ionic strength nor a hydrophobic support. Thus, it is possible to immobilize one protein via different regions (those having the highest * To whom correspondence should be addressed. Fax: 34-91-5854760. Phone: 91-585-4809. E-mail: [email protected] (R.F.L.); [email protected] (J.M.G.). † Unidad Asociada de Bioquı´mica. ‡ Instituto de Cata ´ lisis. § Ca ´ tedra de Bioquı´mica.

negative or positive charges, the highest density of His groups, etc). However, it is very likely that each protein may have more than one area with these properties, promoting the existence of different subpopulations, perhaps with different properties. Moreover, the immobilization needs to be performed under conditions where the protein is adsorbed on the support. In the present study, we now report the development of a new bifunctional epoxy support containing: (i) a small proportion of thiol-reactive groups and (ii) a high number of remaining epoxy groups. The enzymes are first covalently immobilized via a thiol-disulfide exchange between the surface thiol groups of the protein and the thiol-reactive moieties of the support or vice versa (Scheme 1). Thiol groups are the most reactive nucleophiles found in proteins, and they react with disulfide groups at a relatively high rate under a wide range of pH. One possible disadvantage of this idea is that the frequency of the sulfhydryl groups’ occurrence in proteins is usually low.19 However, this can be used as a new and very efficient tool to drive the attachment of the protein to the support via a well-defined area within the protein. Therefore, the development of this bifunctional support could permit: (i) a great increase of the range of conditions where the immobilization can be performed (no requirements of ionic

10.1021/bm034262f CCC: $25.00 © 2003 American Chemical Society Published on Web 10/10/2003

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Scheme 1. Partial Thiolation of Sepabeads with DTT as Thiolating Agent and Alternative Ways of Immobilizing Proteins onto the Obtained Bifunctional Support

strength or presence of organic solvents or detergents, at pH over 6); (ii) the site specific immobilization (via the Cys contained in the native protein, chemically introduced thiol groups, or genetically engineered cysteine residues of the protein) via a disulfide covalent attachment; (iii) the subsequent immobilization via secondary amine, thioether, or ether bonds, fully stable under any reaction conditions; (iv) the rigidification of the protein structure via the formation of multipoint covalent linkages between its very different surface nucleophiles (Lys, His, Tyr, Ser, etc.) and the epoxy groups of the support. In this paper, we report the optimization of the preparation of this mixed support using dithiothreitol (DTT) as the thiolating agent. DTT was selected because it contains two sulfydryl groups per molecule; this allowed the covalent reaction of one of the thiol groups with an epoxy group of the support while the other thiol group remained free for its use in protein immobilization or subsequent activation. Materials Epoxy-Sepabeads (EP-HG-15) was kindly supplied by Resindion S.R.L. (Mitsubishi Chem. Corp, Milan, Italy). 2,2Dipyridyl disulfide (2-PDS), o-nitrophenyl-β-D-galactopyranoside (ONPG), 6-nitro-3-(phenylacetamido)benzoic acid (NIPAB), dithiothreitol (DTT), and Escherichia coli β-galactosidase were from Sigma Chemical Co (St. Louis, Mo,USA). KluyVeromices lactis β-galactosidase (Maxilact LX 5000) was a gift from Gist-Brocades (Cedex, France). Penicillin G acylase (PGA) from Escherichia coli was generously supplied by Antibio´ticos S.A (Burgos, Spain). PD-10 columns (Sephadex G-25) and N-succinimidyl-3-(2pyridyldithio)propionate (SPDP) were purchased from Phar-

macia BTG-LKB (Uppsala, Sweden). Other chemicals were of reagent grade. Methods All experiments were performed at least in triplicate, and the results are given as a mean. Experimental error was never over 7%. Synthesis of Bifunctional Sepabeads by Partial Thiolation with DTT [Epoxy/SH(DTT)-Sepabeads]. Aliquots (10 g) of Epoxy-Sepabeads prewashed with deionized water were incubated at room temperature with 200 mL of 0.2 M potassium bicarbonate pH 8.5 and 200 mL of 1 mM EDTA containing different DTT concentrations (5.5, 11, and 22 mM). After 1 h, the reaction was stopped by washing the gel on a sintered glass filter with 0.1 M potassium bicarbonate pH 8.5, deionized water, and finally 0.2 M sodium acetate pH 5.0. The partially thiolated support derivatives were stored in 50 mM sodium phosphate pH 7.0 at 4 °C until used. Activation of Partially Thiolated Supports with 2-PDS [Epoxy/PyS2(DTT)-Sepabeads]. This was performed essentially as described by Carlsson et al.20 Partially thiolated Sepabeads with DTT (10 g) were added to a mixture of 40 mL of acetone-deionized water (60:40, v/v) and 60 mL of 0.3 M 2-PDS dissolved in acetone-0.05 M sodium bicarbonate (60:40, v/v). The solution was mixed by end-overend rotation during the reaction, which was allowed to proceed for 1 h at room temperature. The product was washed with acetone-water (60-40, v/v) and finally with 1 mM EDTA. The activated supports were stored in 50 mM sodium phosphate pH 7.0 at 4 °C. Thiol Group Analysis. The thiol content of both soluble and insoluble material was determined spectrophotometrically by titration with 2-PDS (saturated solution; 1.5 mM) dissolved in 0.1 M sodium phosphate buffer, pH 8.0 as described by Brocklehurst et al.21

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Determination of the Total Amount of Remaining Epoxy Groups. By Binding of Iminodiacetic Acid (IDA) and Titration with Copper. First, the epoxy groups of the synthesized bifunctional supports were substituted with IDA by the following protocol: 5 g aliquots of each support were agitated by end-over-end rotation with 5.5 mL of 2 M IDA dissolved in 0.1 M sodium bicarbonate pH 11, for 16 h at room temperature. Then, 2 g aliquots of each support were incubated with 0.17 M copper sulfate for 2 h at room temperature. After thoroughly washing with deinozed water, the binded copper was desorbed with 0.1 M EDTA in 0.1 M sodium phosphate pH 7.0, and titrated at 730 nm. EpoxySepabeads without activation was used as blank sample. Thiol Enrichment of PGA. This was performed essentially as described by Brena et al.22 Aliquots (4 mL) of PGA (565 EU/ml, 22.5 mg/mL) in 50 mM sodium phosphate pH 6.8 were incubated for 30 min with aliquots of 25 mM SPDP in ethanol to give a SPDP/protein molar ratio of 1. After the incubation period, a 1.5 molar excess of DTT over the starting amount of SPDP was added and incubated for 1 h with stirring. The thiolated enzyme (thiol-PGA) was separated from excess DTT and other low molecular weight products on a Sephadex G-25 column (PD-10 column). The thiolated enzyme was purified from the nonthiolated one by immobilization onto PyS2-Sepabeads containing the maximum substitution degree (100 µmol thiol-reactive groups/g of wet support). Then, the thiolated enzyme was eluted from the support by reduction with DTT, and the excess of reducing agent was removed by gel filtration. Reduction of K. lactis β-Galactosidase. Aliquots (1.0 mL) of β-galactosidase (60 mg/mL, 1400 EU/mL) were incubated for 30 min with 1 mL of 200 mM DTT in 0.1 M potassium phosphate pH 8.0.23 The excess of reducing agent and other low molecular weight molecules were removed by gel filtration. Immobilization of Enzymes onto Sepabeads Supports. (i) Onto Bifunctional Epoxy/PyS2(DTT)-Sepabeads Support. 10 mL aliquots of each thiol-containing enzyme (17 EU) in the corresponding activity buffer were applied to 1 g of epoxy/PyS2(DTT)-Sepabeads support containing 3 µmol of thiol-reactive groups/g of wet support. The mixture was gently agitated by end-over-end rotation at room temperature. Periodically, samples of the supernatant fluids were taken at different time intervals and analyzed for enzyme activity. The insoluble derivatives were then thoroughly washed with activity buffer. (ii) Onto Monofunctional, Glycine Blocked Support. The immobilization was performed as described above but using the mixed support with its epoxy groups previously blocked with glycine. We used the term monofunctional glycineblocked deriVatiVes to refer to these enzyme derivatives. (iii) Onto Sepabeads without DeriVatization (Monofunctional Epoxy-Support). This was performed as described above, using Epoxy-Sepabeads prewashed with deionized water as support. Enzyme-Support Irreversible Covalent Attachment. After immobilization, the insoluble PGA and K. lactis β-galactosidase derivatives obtained were incubated under different pHs and time periods to allow the reaction between

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the epoxy groups still present on the support surface and the reactive groups of the enzyme. In the case of PGA, when using alkaline pH values, 100 mM phenyl acetic acid and 20% glycerine were added to the suspension to prevent enzyme inactivation.24 Blocking Process of Epoxy Groups. (i) To completely block epoxy groups in the mixed (bifunctional) support previous to its incubation with the enzymes, 1 g aliquots of epoxy/PyS2(DTT)-Sepabeads support containing 3 µmol of thiol-reactive groups/g of wet support were incubated with 4 mL of 3 M glycine at pH 8.5. The mixtures were gently agitated for 24 h at room temperature. The blocked support, monofunctional glycine-blocked support, was then thoroughly washed with 50 mM sodium phosphate pH 7.0. The supports were stored in 50 mM sodium phosphate pH 7.0 at 4 °C until its use in enzyme immobilization. (ii) End Point to the Support Enzyme Reaction. To block completely the remaining epoxy groups in the enzyme derivatives obtained by incubation of the mixed (bifunctional) support with the enzymes under different conditions, 1 g aliquots of the obtained enzyme derivatives were incubated in 4 mL of 3 M glycine at pH 8.5. The mixtures were gently agitated for 24 h at room temperature. The blocked derivatives were then thoroughly washed with 50 mM sodium phosphate pH 7.0. Elution with DTT of the Proteins Covalently Attached Only by Disulfide Bonds. The insoluble enzymes derivatives obtained (1 g) were treated with 10 mL of 400 mM DTT in 50 mM sodium phosphate pH 8.0 at room temperature for 1 h. Afterward, protein and enzyme activity content of the supernatants were assayed. This was performed to evaluate if the covalent reaction between surface amino groups of the protein and the remaining epoxy groups of the partially thiolated supports took place. Proteins attached to the support only by disulfide bonds are eluted by its reduction with DTT. The elution yield was defined as the activity elution yield: (eluted EU/g of wet support)/(immobilized EU/g of wet support). The elution yields of the enzymes were confirmed by protein determination and represent averages of at least three experiments (the expanded uncertainty was under 10%). Enzyme Activity Assays. (i) In Solution. the assay was performed at room temperature with 10 mM ONPG or 28 mM ONPG for E. coli and K. lactis β-galactosidase respectively, in the corresponding activity buffer, and the rate of formation of free o-nitrophenol (ONP) was determined at 405 nm.25 PGA activity was measured by following the increase of absorbance at 405 nm, which accompanies the hydrolysis of the synthetic substrate NIPAB.26 (ii) Immobilized Enzyme DeriVatiVes. The activity was measured under identical conditions in spectrophotometric cells provided with magnetic stirring and expressed as the intrinsic activity (percentage of activity exhibited by the immobilized enzyme when compared to the soluble form). Activity buffers for (i) K. lactis β-galactosidase: 20 mM potassium phosphate pH 7.0 containing 0.1 M KCl and 2 mM MgCl2; (ii) E. coli β-galactosidase: 50 mM sodium phosphate pH 7.0 containing 2 mM MgCl2; and (iii) E. coli PGA: 25 mM sodium phosphate pH 7.0.

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Table 1. Effect of the Concentration of DTT on the Modification of Epoxy Groups of EP-Sepabeadsa DTT (mM)

substitution degree (µmol SH/g wet support)

percentage of remaining epoxides (%)

11 5.5 2.75

15 ( 2 8(1 3(1

65 ( 8 80 ( 6 90 ( 8

a The initial epoxy group content of EP-Sepabeads is 120 µmol epoxy groups/g of wet support.

One unit of enzyme (EU) was defined as the amount of enzyme catalyzing the hydrolysis of 1 µmol of substrate per minute under the specified conditions Results and Discussion Control of the Thiolation of the Epoxy Groups. Table 1 shows that it was possible to produce bifunctional supports containing (i) a small proportion of thiol-reactive groups and (ii) epoxy groups that enable further multipoint covalent immobilization of the protein. The achieved partial thiolation of the support was proportional to the concentration of DTT used. Moreover, the remaining content of epoxy groups after the thiolation treatment was high. Immobilization of Thiolated Proteins on Epoxy-Disulfide Supports: Thiol/Disulfide Interchange. The essential condition to promote the first step of the immobilization process (via thiol-disulfide exchange) is the presence of surfacel thiol groups in the protein. Bearing in mind this point, we have immobilized three different enzymes having very different starting situation: (i) Naturally Containing Surface Thiol Groups. β-Galactosidase from E. coli has a high content of cysteins (64 residues per tetramer, one-fourth of which react with sulfydryl reagents without affecting enzymatic activity);27,28 thus, its immobilization does not require previous reduction or thiolation steps. (ii) Enzymes Whose Thiol Groups Need to Be Generated from Reduction of Indigenous Disulfides. β-Galactosidase from K. lactis exhibits a low level of free thiol groups (detected by titration with thiol reagents), but because of their lack of reactivity toward thiol-reactive adsorbents, the enzyme must be reduced with DTT for its immobilization onto these supports.23 (iii) Enzymes Whose Thiol Groups Need to Be Created by Thiolation with SPDP. PGA from E. coli does not contain accessible thiol groups or disulfide groups susceptible of reduction;29 therefore, its thiolation with SPDP19,22 for its immobilization onto this bifunctional support was necessary. The modification of the epoxy groups of the support with the thiol reactive group must reach a compromise between a high degree of substitution for a rapid immobilization of the proteins and a sufficiently high amount of reactive epoxy groups in order to achieve a intense multipoint covalent immobilization via others nucleophiles present in the protein.14,15 For this reason, the immobilization of proteins was carried out using the less substituted bifunctional support we have obtained (3 µmol of thiol-reactive groups/g of wet support) (Figure 1).

Figure 1. Immobilization course of different enzymes onto EpoxySepabeads support (rhombus), and derivatized thiol-reactive/epoxy Sepabeads bifunctional support (triangles) containing 3 µmol thiolreactive groups/g of wet support. (a) E. coli β-galactosidase; (b) thiolated and reduced E. coli penicillin G acylase from Escherichia coli; (c) reduced K. lactis β-galactosidase. The activity of soluble enzyme incubated under similar conditions are represented by (squares).

High immobilization yields were achieved for the three proteins studied when partially thiolated Sepabeads was used as a support (nearly 60% for PGA, 65% and 100% for β-galactosidases from K. lactis and E. coli, respectively). Moreover, the intrinsic activities (that is, the ratio between immobilized activity and observed activity) of the obtained insoluble enzyme derivatives were 40% for PGA and 65% and 80% for β-galactosidases from K. lactis and E. coli, respectively. The more rapid rate of immobilization for E. coli β-galactosidase is probably due to its high content of surface thiol groups. When Epoxy-Sepabeads without de-

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Scheme 2. Strategy Used to Prove the Enzyme-Support Irreversible Covalent Attachment Obtained in Enzyme Derivatives: (a) Bifunctional Support; (b) DTT-modified and Glycine-Blocked Support

Figure 2. Percentage of activity eluted with DTT from enzyme derivatives immobilized on bifunctional Thiol-Sepabeads after 24 h of incubation at pH 7. Elution yield (% EU) ) (eluted EU/g of wet derivative)/(bond EU/g of wet derivative).

rivatization was used (monofunctional support), immobilization was found to be negligible in the case of PGA and K. lactis β-galactosidase protein. Only the β-galactosidase from E. coli was significantly immobilized (20% after 20 h of enzyme-support interaction), although at a much slower rate than when using the heterofunctional support, very likely because of its high thiol content.27,28 It is also important to point out that the rate of immobilization of each enzyme was similar both on the bifunctional support as on the bifunctional support with its epoxy groups previously blocked with glycine (monofunctional glycine-blocked support). This reinforces the idea that the reaction with the thiol reactive groups is the first step in the immobilization on the new heterofunctional supports. Reaction of the Thiol Interchange Immobilized Enzyme with Epoxy Groups of the Support. Protein molecules that were attached on the support only via a disulfide bond are eluted away from the support by incubation with a reducing agent (e.g., DTT), whereas the reaction with epoxy groups implies that this protein cannot be eluted by DTT incubation (Scheme 2). Figure 2 shows that, even after 24 h of incubation at pH 7, a large percentage of the thiolated PGA and β-galactosidase from K. lactis could be desorbed from the support.

In the case of E. coli β-galactosidase derivatives, immobilization via epoxy groups was very rapid (less than 10% of the bound enzyme was eluted with DTT after 24 h at pH 7). This could be due to the high content of very reactive Cys residues on the surface of this enzyme. However, for the PGA and K. lactis β-galactosidase derivatives, it was necessary to further incubate the already immobilized enzyme at basic pHs values and long incubation times to accelerate this second step of the immobilization process (Figure 3). The size of the spacer arm having the thiol group coupled to the size of the artificially introduced thiol groups in the proteins may have some impact in these difficulties for reacting the protein residues with the epoxy groups in the support. The use of Cys introduced via genetic manipulation of the protein and the optimization of the thiol reactive group may greatly improve this reaction rate even at neutral pH values, like in any other heterofunctional supports.16-18 In fact, such variables (pH and time) were previously found to greatly increase the formation of multipoint covalent linkages when using glyoxyl-agarose gels30-37 and other epoxy supports (8, 9, 14, and 15). When monofunctional glycine-blocked support immobilized proteins were treated with DTT, more than 90% of enzyme elution was observed in all cases. These results together demonstrate that the first step in the immobilization process onto the bifunctional support proceeds via thiol-disulfide interchange reactions. General Discussion We could confirm the two-step immobilization mechanism proposed for this new mixed support. Several results suggested that the first step of immobilization proceeds via thiol-disulfide interchange. (i) In the case of PGA and K. lactis β-galactosidase, it is necessary to previously thiolate or reduce the native enzymes to achieve their immobilization onto the bifunctional support, respectively. (ii) The rate of immobilization of each enzyme was similar either on the bifunctional support or on the monofunctional glycine-blocked support.

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References and Notes

Figure 3. Elution of the proteins immobilized on bifunctional supports by reduction with DTT: (a) PGA and (b) K. lactis β-galactosidase derivatives with 48 and 56 h, respectively, of incubation postimmobilization. Both derivatives were incubated respectively in sodium or potassium phosphate buffer 25 mM pH 7, 8, or 10 K. lactis β-galactosidase derivative was not incubated at pH 10 due to the low stability of the enzyme at basic pH values. Elution yield (% EU) ) (eluted EU/g of wet derivative)/(bond EU/g of wet derivative).

(iii) Most of the proteins immobilized on the new bifunctional support could be released from it just by incubation with DTT, suggesting that only this disulfide bond had been produced. (iv) Further incubation under proper conditions permits the reaction of the epoxy groups with nearby nucleophile groups of the already immobilized proteins. These new supports open the possibility of a fully sitedirected immobilization of proteins via the epoxy chemistry by coupling the use of this support with protein engineering techniques, by preparing enzymes with only one surface Cys in different places of the protein surface. Acknowledgment. This project has been funded by the Spanish CICYT (BIO2000-0747-C05-02 and BIO20012259), Resindion S.R.L (Mitsubishi Chemical Corporation), “Program for the Development of Basic Sciences” (PEDECIBA, Uruguay) and IPICS (Uppsala University, Sweden). A Ph.D. fellowship from CAM for O.A. is gratefully recognized. We also thank Antibio´ticos S.A. and Gist Brocades for the kind donation of E. coli penicillin G acylase and K. lactis β-galactosidase, respectively. MSc. A Ä ngel Berenguer (Departamento de Quı´mica Inorga´nica, Universidad de Alicante) is gratefully acknowledged for his help during the writing of this paper

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