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Electrochemical oxidation of proteins using ionic liquids as solubilizers, adsorption solvents and electrolytes
Electrochimica Acta 126 (2014) 31–36
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Electrochemical oxidation of proteins using ionic liquids as solubilizers, adsorption solvents and electrolytes Jan Vacek a,∗ , Jiˇrí Vrba a , Martina Zatloukalová a , Martin Kubala b a Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic b Department of Biophysics, Faculty of Science, Palacky University, 17. listopadu 12, 771 46 Olomouc, Czech Republic
a r t i c l e
i n f o
Article history: Received 3 May 2013 Received in revised form 19 June 2013 Accepted 20 June 2013 Available online 4 July 2013 Keywords: Protein Ionic liquid Electrochemical sensing Protein stability Electrolytes
a b s t r a c t This study focuses on application of room temperature ionic liquids (RTILs) as solubilizers, adsorption solvents and supporting electrolytes for electrochemical analysis of human and bovine serum albumins, HSA and BSA. The proteins were analyzed by ex situ, adsorptive transfer, square-wave voltammetry (SWV) at a basal-plane pyrolytic graphite electrode after solubilization using imidazolium- and ammoniumbased RTILs. The application of RTILs enabled SWV scan from 0 to +1.5 V (vs. Ag/AgCl/3 M KCl) without interference with the anodic response of the proteins. Concretely, Tyr (Y) and Trp (W) oxidation currents of HSA and BSA, peak Y&W around +0.85 V, were observed and characterized under different RTILs and RTIL/water conditions. The electrochemical data were supported by electrophoresis under denaturing and native conditions. These provided evidence for the structural changes and stability of the studied proteins in the presence of RTILs. The data acquired using BSA and HSA model proteins, could be used in further applications of RTILs in protein electrochemistry and for developing new protein sensing strategies. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Room temperature ionic liquids (RTILs) are organic salts with low melting points, low vapor pressure, good stability and high conductivity. RTILs exist in the liquid state under ambient laboratory conditions which enable their use in many chemistry and biochemistry fields, including protein research. They also have a broad spectrum of electrochemical applications [1,2]. This generation of solvents provides not only an environment for protein solubilization but also a highly efficient reaction medium for enzyme catalysis and stabilization, and novel protein crystallization tool [1,3–6]. In this paper, we focus on the application of RTILs for protein solubilization, adsorption and application as electrolytes in electrochemical measurements using ex situ (adsorptive transfer [7]) voltammetry. For this purpose, human serum and bovine serum albumins, HSA and BSA, were used as model proteins. Their primary structures are composed of a single chain of about 585 amino acid residues, and tertiary structures of both proteins were well-characterized using X-ray spectroscopy and NMR [8,9]. Both proteins were characterized using several spectral and/or
∗ Corresponding author. Tel.: +420 585 632 303; fax: +420 585 632 302. E-mail address: [email protected] (J. Vacek). 0013-4686/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.electacta.2013.06.115
electrochemical methods, based on intrinsic electroactivity measurement in aqueous buffered solutions [10]. One way of analyzing proteins is through the anodic reactions of aromatic amino acids in polypeptide chain, namely Tyr (Y) and Trp (W) residues, at carbon electrodes [11–13]. This approach was previously used for electrooxidation and analysis of HSA and BSA in an aqueous environment (reviewed in [10,14,15]). This study aimed at i) investigating the electrochemical oxidation of serum albumins after solubilization and adsorption using RTILs, ii) analyzing and discussing the stability of the proteins after solubilization in RTILs and their aqueous mixtures, and iii) testing RTILs as supporting electrolytes in the anodic voltammetry of proteins.
2. Experimental 2.1. Chemicals HSA, BSA and buffer components were obtained from Sigma–Aldrich (St. Louis, USA). All solutions were prepared using reverse-osmosis deionized water (Ultrapur, Watrex, CZ). The sample preparation and analyses were performed under aerobic conditions. The pH of buffer solutions and RTILs was measured by pH/ORP Meter (HI2211) equipped with HT 1131 electrode (HANNA Instruments, USA).
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2.2. Tested RTILs The proteins were solubilized using the following RTILs with the specification of [purity in %]. Ammonium-based RTILs: ethylammonium nitrate [>97%] and 2-hydroxyethylammonium formate [>97%]. Imidazolium-based RTILs: 1-ethyl-3-methylimidazolium ethylsulfate [99%]; 1-butyl-3-methylimidazolium dicyanamide [98%]; 1-ethyl-3-methylimidazolium tetrafluoroborate [98%]; 1ethyl-3-methylimidazolium acetate [>95%]. RTILs were donated by Ionic Liquids Technologies GmbH (Heilbronn, DE). 2.3. Electrochemical measurement The proteins were analyzed using ex situ (adsorptive transfer) voltammetric analysis with a basal-plane pyrolytic graphite working electrode, PGE (3 × 3 mm, source of PG: Momentive Performance Materials, USA). PGE was first dipped into 5-L aliquot of the studied sample. After an accumulation period, the electrode was washed by deionized water and placed in an electrochemical cell containing supporting electrolyte. Square-wave voltammetry (SWV) was performed at room temperature with a Autolab III analyzer (Metrohm Autolab, NL) in a three-electrode setup with Ag/AgCl/3 M KCl electrode as a reference and platinum wire as an auxiliary electrode. Other parameters: time of accumulation, tA = 30 s, supporting electrolyte: acetate buffer (pH 5.0), frequency: 200 Hz, if not stated otherwise. 2.4. Gel electrophoresis of HSA and BSA Integrity of albumins was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by nondenaturing (native) polyacrylamide gel electrophoresis (PAGE). SDS-PAGE was performed according to Laemmli [16]. In brief, a 10% resolving gel contained 375 mM Tris–Cl, 0.1% (w/v) SDS, pH 8.8, and a 5.2% stacking gel contained 125 mM Tris–Cl, 0.1% (w/v) SDS, pH 6.8. Samples of albumins were mixed 1:1 with sample buffer (125 mM Tris–Cl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 200 mM dithiothreitol, 0.02% (w/v) bromophenol blue) and boiled for 5 min at 95 ◦ C. The samples (10 g of protein per lane) were subjected to electrophoresis using an electrode buffer containing 25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS, pH 8.3. The gels were subsequently incubated for 1 h in staining solution containing 0.1% (w/v) Coomassie brilliant blue G-250, 40% (v/v) methanol, 10% (v/v) glacial acetic acid, and 50% (v/v) water. The stained gels were agitated in destaining solution (40% (v/v) methanol, 10% (v/v) glacial acetic acid, 50% (v/v) water) until the background became clear. Nondenaturing PAGE employed a system developed by Ornstein [17] and Davis [18]. A 10% resolving gel contained 375 mM Tris–Cl, pH 8.8, and a 4% stacking gel contained 125 mM Tris–Cl, pH 6.8. Samples of albumins were mixed 1:1 with sample buffer (125 mM Tris–Cl, pH 6.8, 20% (v/v) glycerol, 0.02% (w/v) bromophenol blue) and subjected to electrophoresis (10 g of protein per lane) using an electrode buffer consisting of 25 mM Tris, 192 mM glycine, pH 8.3. Proteins in the gels were visualized by Coomassie blue staining as described above. 3. Results and discussion BSA and HSA, used as model proteins, were electrochemically examined after their adsorption (adsorptive transfer [7]) onto PGE surface using the ex situ SWV method. The RTILs were used either as a medium for protein solubilization and adsorption or as a supporting electrolyte. First, we tested the two ammonium- and four imidazolium-based RTILs given in Section 2.2, using the ex situ technique, where RTILs without the protein were adsorbed (tA = 30 s)
Table 1 Tyrosine and tryptophan residues in HSA and BSA. Protein HSA BSA
Total Tyr (Y)
Trp (W)
Surface exposed Tyr (Y) Trp (W)
18 20
1 2
9 12
0 0
onto the PGE surface. We then monitored interferences to electrochemical analysis of HSA and BSA (not shown). No interfering currents were found for the tested solvents. 3.1. Electrooxidation of proteins adsorbed from ionic liquids We focused on the usability of RTILs for HSA and BSA solubilization and adsorption for ex situ SWV measurement. The dissolved proteins (final conc. 10 M) were adsorbed onto the PGE surface (tA = 30 s). After formation of the adsorbed layer, the electrode was washed with distilled water, dried and placed in an electrochemical cell, where SWV (200 Hz) was performed in 0.2 M acetate buffer, pH 5.0. The scan was performed from 0 V to +1.5 V as described previously [19]. Using these conditions for sample adsorption, the PGE surface was always fully covered by the analyzed proteins (not shown). The proteins dissolved and adsorbed from the Britton–Robinson buffer (pH 7.4), in the absence RTILs, exhibited SWV oxidation peaks Y&W at potential around +0.8 V (Fig. 1A). In proteins, this oxidation peak is assigned to the Tyr (Y) and Trp (W) oxidations [11–13]. HSA and BSA contain in total 18 Y and 1 W and 20 Y and 2 W, respectively (Table 1). Generally, it is considered that intrinsic protein electroactivity is caused by the electroactive amino acid residues that are, after protein adsorption, in direct contact with an electrode surface. Hence, it may be assumed that only the residues localized on a protein surface can be oxidized. The possible oxidation centers are displayed in Scheme 1. It is very likely that only Tyr residues contribute to the current response because the Trp residues are localized in the protein interior or in the cavities that are accessible from the solvent only by a narrow tunnel. Based on the high-resolution structures, we can identify 9 Tyr in HSA and 12 Tyr in BSA on the protein surface [20,21] (Table 1). The contribution of Trp residues to anodic response cannot be strictly excluded, especially in cases where experimental conditions involve significant structural changes of the proteins. Further, both proteins were solubilized and adsorbed from the RTILs/water mixture under the same conditions as described in the first paragraph of this section. All six examined RTILs were used for this purpose; however, RTILs competed for the electrode surface with the analyzed proteins. Hence, we observed the peak Y&W decrease (see below) due to co-adsorption processes. Some RTILs (e.g. ethylammonium nitrate) also suppress protein–protein interactions (aggregation) [3,22], and hence, RTILs can find use as a co-adsorbing species for formation of an adsorbed layer with lower density of adsorbed protein molecules due to molecular repulsions at the electrode surface. In order to minimize the denaturizing changes of proteins, a defined RTIL/water ratio was used [3,5]. SW voltammograms of HSA and BSA solubilized and adsorbed in 1-ethyl-3-methylimidazolium acetate/water or ethylammonium nitrate/water (80:20, v/v) mixtures are shown in Fig. 1B and C. The co-adsorption effect of RTIL vs. studied protein was examined with various RTIL/water ratios. With increasing amounts of RTILs, we observed a gradual decrease in Y&W peak for both HSA and BSA (Fig. 2A). From the view point of co-adsorption processes, there is almost no interference of ethylammonium nitrate and only small decrease in the Y&W peak was observed in the presence of 2-hydroxyethylammonium formate. A stronger
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Scheme 1. The aromatic residues Tyr (blue) and Trp (magenta) exposed to the surface of HSA or BSA are highlighted. The left and right images of the same molecule are mutually rotated by 180◦ along the vertical axis. The images for HSA and BSA were created based on the high-resolution structures deposited in RCSB Protein Data Bank 1AO6 [21] and 4F5S [20], respectively.
Fig. 1. Ex situ SW voltammograms of HSA and BSA after solubilization in: BrittonRobinson buffer at pH 7.4 (A), 1-ethyl-3-methylimidazolium acetate-water (B) and ethylammonium nitrate-water (C) solutions (80:20, v/v); concentration of proteins: 10 M; supporting electrolyte: 0.2 M acetate buffer, pH 5.0. Potential vs. Ag/AgCl/3 M KCl.
co-adsorption effect was observed for imidazolium RTILs, 1-ethyl3-methylimidazolium acetate and 1-buthyl-3-methylimidazolium dicyanamide. In the case of protein solubilization using 100% RTILs, we observed a 50% decrease in current response (Fig. 2A). The observed co-adsorption effects for imidazolium RTILs are in agreement with previously published studies where these RTILs were used to intentionally suppress protein adsorption in electrophoretic capillaries [23]. In addition to the quantitative effects connected with the abovedescribed co-adsorption processes, we also examined changes in the potential of oxidation peaks of HSA and BSA. With increasing amounts of RTILs in the sample, we observed a small but proportional shift of the Y&W peak toward less positive values (Fig. 2B). This observation indicates that the presence of RTILs facilitates the protein electrooxidation, which may be due to the good conductive properties of RTILs [3,24]. An alternative explanation could be some partial RTIL-modification of the PGE surface or modification
Fig. 2. Effect of RTILs/water percentage representation (v/v) used for HSA solubilization on peak Y&W height (A) and potential (B). The following RTILs were used for solubilization of HSA: (1) ethylammonium nitrate, (2) 2hydroxyethylammonium formate, (3) 1-ethyl-3-methylimidazolium acetate, (4) 1-buthyl-3-methylimidazolium dicyanamide. Ex situ SWV, supporting electrolyte: 0.2 M acetate buffer (pH 5.0). Concentration of HSA was 10 M. 100% peak heights = water without RTILs in samples. Similar effects can be observed for BSA. Potential vs. Ag/AgCl/3 M KCl.
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Fig. 3. Denaturing (A) and native (B) gel electrophoreograms of HSA after its solubilization using RTILs-water (80:20, v/v) solutions. Very similar results were obtained for BSA. Lanes: (ctrl) control–no RTIL, (a) 1-ethyl-3-methylimidazolium ethylsulfate, (b) 1-ethyl-3-methylimidazolium acetate, (c) 1-buthyl-3-methylimidazolium dicyanamide, (d) ethylammonium nitrate, (e) 2-hydroxyethylammonium formate, and (f) 1-ethyl-3-methylimidazolium tetrafluoroborate.
of the protein structure, i.e. stabilization of adsorbed film that may facilitate the anodic reaction. For interpretation of these results and also to check for protein integrity, we analyzed the HSA and BSA after their incubation with the RTILs using gel electrophoresis.
3.2. Protein stability after ionic liquids solubilization Both studied proteins were analyzed by polyacrylamide gel electrophoresis (PAGE) [16–18]. For this experiment, HSA and BSA were dissolved at a concentration of 4 mg/ml in Britton–Robinson buffer pH 7.4 (control) or in RTIL/water (80:20, v/v) mixtures and incubated for 10 min at laboratory temperature. The samples were then diluted and prepared for PAGE analysis as given in Section 2.4. Electrophoretic separation was performed under both denaturing (sodium dodecyl sulfate, SDS) or non-denaturing (native) conditions. In the case of SDS-PAGE, we could observe well-separated bands of HSA and BSA, exhibiting the same electrophoretic mobility for RTIL-treated samples as for the control (Fig. 3A). In the case of native PAGE, we observed the typical electrophoretic profiles of HSA and BSA without any qualitative difference between control and RTIL-containing samples (Fig. 3B). The results by native PAGE also revealed that RTILs under given conditions probably do not influence the intermolecular interactions in HSA and BSA (in solution) and this is indicated by bands of dimers and oligomers (Fig. 3B). Interaction of imidazolium RTILs with BSA has already been examined by other groups [25,26]. Based on these findings, we can assume that the RTILs used in this study interact with the studied proteins primarily through weak non-covalent hydrophilic interactions and H-bonds. Inevitable unfolding effects were observed for imidazolium RTILs only in the case when their structure was modified by a sufficiently long hydrophobic hydrocarbon chain. In this case, denaturation can be caused by the interaction of hydrophobic chains with the hydrophobic protein interior [25]. The results of PAGE confirmed that RTILs application does not cause aggregation, fragmentation or structural changes of the studied proteins, which could affect their electrophoretic mobility.
Fig. 4. Ex situ SW voltammograms of HSA and BSA solubilized in Britton-Robinson buffer at pH 7.4. The SWV was performed using supporting electrolytes: 1-ethyl-3methylimidazolium acetate (A) and ethylammonium nitrate (B). Concentration of proteins was 10 M. Potential vs. Ag/AgCl/3 M KCl. Table 2 The potentials of SWV peaks Y&W of HSA and BSA in 0.2 M acetate buffer (pH 5.0) and selected RTILs. Average values of potentials (n = 6) are expressed vs. Ag/AgCl/3 M KCl. The standard deviations (S.D.) were less than ±3 mV for all potentials. Electrolytes
pH
Peak Y&W potential (V) HSA BSA
Acetate buffer, 0.2 M 1-Ethyl-3-methylimidazolium ethylsulfate 1-Ethyl-3-methylimidazolium tetrafluoroborate 1-Ethyl-3-methylimidazolium acetate 1-Buthyl-3-methylimidazolium dicyanamide Ethylammonium nitrate 2-Hydroxyethylammonium formate
5.0 7.6 3.9 12.1 8.5 4.5 7.0
0.908 0.772 0.943 0.518 0.645 0.864 0.843
0.925 0.786 0.896 0.552 0.694 0.874 0.850
3.3. Ionic liquids as electrolytes for protein electrooxidation Finally, we studied the possible use of RTILs as a supporting electrolyte for the SWV of proteins. HSA and BSA were first adsorbed onto the PGE surface from Britton–Robinson buffer (pH 7.4) for tA = 30 s, subsequently washed, dried and placed in an electrochemical cell containing RTIL electrolyte. All tested RTIL electrolytes were useful for electrochemical oxidation of both proteins (Fig. 4 and Table 2). Due to substantially different pH and other physicochemical parameters of examined RTILs, the Y&W peak of both HSA and BSA was observed at different potentials (Table 2). Our results suggested that the pH of tested RTILs is the driving factor affecting the peak Y&W potential for both proteins. Increasing pH of the supporting electrolyte, shifts the potential peak Y&W toward less positive values, by −55 mV per pH unit, which is in good agreement with pH effects recently reported for peptide methionine sulfoxide reductase A [27]. The pH effects presented here were obtained in a series of experiments with Britton-Robinson buffer in the pH
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References
Fig. 5. Effect of pH on potential of peak Y&W for HSA and BSA in aqueous BrittonRobinson buffer (full circles and squares) and in RTILs (open circles and squares) as supporting electrolytes for SWV. (a) 1-ethyl-3-methylimidazolium ethylsulfate, (b) 1-ethyl-3-methylimidazolium acetate, (c) 1-buthyl-3-methylimidazolium dicyanamide, (d) ethylammonium nitrate, (e) 2-hydroxyethylammonium formate, (f) 1-ethyl-3-methylimidazolium tetrafluoroborate. Potential vs. Ag/AgCl/3 M KCl.
range 3–12. Shifts of peak Y&W potentials recorded for the aqueous buffered electrolyte correspond very well to the results obtained using RTILs electrolytes (Fig. 5). In contrast to ammonium-based RTILs, SWV recorded in imidazolium RTIL electrolytes also revealed currents from the electrolyte itself but not the same as the potential for peak Y&W of HSA or BSA (compare Fig. 4A and 4B). The measurement can also be done in RTIL/water mixtures, or in RTILs whose pH is modified using an aqueous buffer. We found the optimal aqueous buffer was the Britton–Robinson buffer as it did not result in precipitate formation in the solution (not shown). The need to control the pH of supporting electrolytes on the basis of RTILs still presents a challenge. Currently developed ionic liquid buffers [28,29] could be a solution to this problem but they have not been examined electrochemically and their applicability to protein analysis will need further research. 4. Conclusions This study focused on the applicability of selected RTILs with the imidazolium or ammonium cation for solubilization of model proteins HSA and BSA. We examined the adsorption of these proteins from RTILs onto PGE surface and also electroooxidation of the adsorbed protein layer in the RTIL environment. Our results show that 1-ethyl-3-methylimidazolium ethylsulfate, 1-ethyl-3methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium acetate, 1-buthyl-3-methylimidazolium dicyanamide, ethylammonium nitrate and 2-hydroxyethylammonium formate could be used as solubilizers and adsorption solvents in ex situ voltammetry and as a supporting electrolyte for SWV analysis of both proteins. The results could be applied in electrochemical examination of other proteins and in developing new approaches, e.g. protein extracting electrodes [30]. Further, RTILs properly modified by hydrophobic functional groups could be useful for solubilization of water-insoluble proteins and their subsequent electroanalysis [19,31,32]. Acknowledgement The authors thank Dr. Alexander Oulton for language correction and Ms. Alˇzbˇeta Balharová for excellent technical support. This work was supported by the Institutional Financial Support of Palacky University (Faculty of Medicine and Dentistry) and by the grant No. CZ.1.07/2.3.00/20.0057–Operational Programme
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Electrochemical oxidation of proteins using ionic liquids as solubilizers, adsorption solvents and electrolytes Jan Vacek a,∗ , Jiˇrí Vrba a , Martina Zatloukalová a , Martin Kubala b a Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic b Department of Biophysics, Faculty of Science, Palacky University, 17. listopadu 12, 771 46 Olomouc, Czech Republic
a r t i c l e
i n f o
Article history: Received 3 May 2013 Received in revised form 19 June 2013 Accepted 20 June 2013 Available online 4 July 2013 Keywords: Protein Ionic liquid Electrochemical sensing Protein stability Electrolytes
a b s t r a c t This study focuses on application of room temperature ionic liquids (RTILs) as solubilizers, adsorption solvents and supporting electrolytes for electrochemical analysis of human and bovine serum albumins, HSA and BSA. The proteins were analyzed by ex situ, adsorptive transfer, square-wave voltammetry (SWV) at a basal-plane pyrolytic graphite electrode after solubilization using imidazolium- and ammoniumbased RTILs. The application of RTILs enabled SWV scan from 0 to +1.5 V (vs. Ag/AgCl/3 M KCl) without interference with the anodic response of the proteins. Concretely, Tyr (Y) and Trp (W) oxidation currents of HSA and BSA, peak Y&W around +0.85 V, were observed and characterized under different RTILs and RTIL/water conditions. The electrochemical data were supported by electrophoresis under denaturing and native conditions. These provided evidence for the structural changes and stability of the studied proteins in the presence of RTILs. The data acquired using BSA and HSA model proteins, could be used in further applications of RTILs in protein electrochemistry and for developing new protein sensing strategies. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Room temperature ionic liquids (RTILs) are organic salts with low melting points, low vapor pressure, good stability and high conductivity. RTILs exist in the liquid state under ambient laboratory conditions which enable their use in many chemistry and biochemistry fields, including protein research. They also have a broad spectrum of electrochemical applications [1,2]. This generation of solvents provides not only an environment for protein solubilization but also a highly efficient reaction medium for enzyme catalysis and stabilization, and novel protein crystallization tool [1,3–6]. In this paper, we focus on the application of RTILs for protein solubilization, adsorption and application as electrolytes in electrochemical measurements using ex situ (adsorptive transfer [7]) voltammetry. For this purpose, human serum and bovine serum albumins, HSA and BSA, were used as model proteins. Their primary structures are composed of a single chain of about 585 amino acid residues, and tertiary structures of both proteins were well-characterized using X-ray spectroscopy and NMR [8,9]. Both proteins were characterized using several spectral and/or
∗ Corresponding author. Tel.: +420 585 632 303; fax: +420 585 632 302. E-mail address: [email protected] (J. Vacek). 0013-4686/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.electacta.2013.06.115
electrochemical methods, based on intrinsic electroactivity measurement in aqueous buffered solutions [10]. One way of analyzing proteins is through the anodic reactions of aromatic amino acids in polypeptide chain, namely Tyr (Y) and Trp (W) residues, at carbon electrodes [11–13]. This approach was previously used for electrooxidation and analysis of HSA and BSA in an aqueous environment (reviewed in [10,14,15]). This study aimed at i) investigating the electrochemical oxidation of serum albumins after solubilization and adsorption using RTILs, ii) analyzing and discussing the stability of the proteins after solubilization in RTILs and their aqueous mixtures, and iii) testing RTILs as supporting electrolytes in the anodic voltammetry of proteins.
2. Experimental 2.1. Chemicals HSA, BSA and buffer components were obtained from Sigma–Aldrich (St. Louis, USA). All solutions were prepared using reverse-osmosis deionized water (Ultrapur, Watrex, CZ). The sample preparation and analyses were performed under aerobic conditions. The pH of buffer solutions and RTILs was measured by pH/ORP Meter (HI2211) equipped with HT 1131 electrode (HANNA Instruments, USA).
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2.2. Tested RTILs The proteins were solubilized using the following RTILs with the specification of [purity in %]. Ammonium-based RTILs: ethylammonium nitrate [>97%] and 2-hydroxyethylammonium formate [>97%]. Imidazolium-based RTILs: 1-ethyl-3-methylimidazolium ethylsulfate [99%]; 1-butyl-3-methylimidazolium dicyanamide [98%]; 1-ethyl-3-methylimidazolium tetrafluoroborate [98%]; 1ethyl-3-methylimidazolium acetate [>95%]. RTILs were donated by Ionic Liquids Technologies GmbH (Heilbronn, DE). 2.3. Electrochemical measurement The proteins were analyzed using ex situ (adsorptive transfer) voltammetric analysis with a basal-plane pyrolytic graphite working electrode, PGE (3 × 3 mm, source of PG: Momentive Performance Materials, USA). PGE was first dipped into 5-L aliquot of the studied sample. After an accumulation period, the electrode was washed by deionized water and placed in an electrochemical cell containing supporting electrolyte. Square-wave voltammetry (SWV) was performed at room temperature with a Autolab III analyzer (Metrohm Autolab, NL) in a three-electrode setup with Ag/AgCl/3 M KCl electrode as a reference and platinum wire as an auxiliary electrode. Other parameters: time of accumulation, tA = 30 s, supporting electrolyte: acetate buffer (pH 5.0), frequency: 200 Hz, if not stated otherwise. 2.4. Gel electrophoresis of HSA and BSA Integrity of albumins was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by nondenaturing (native) polyacrylamide gel electrophoresis (PAGE). SDS-PAGE was performed according to Laemmli [16]. In brief, a 10% resolving gel contained 375 mM Tris–Cl, 0.1% (w/v) SDS, pH 8.8, and a 5.2% stacking gel contained 125 mM Tris–Cl, 0.1% (w/v) SDS, pH 6.8. Samples of albumins were mixed 1:1 with sample buffer (125 mM Tris–Cl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 200 mM dithiothreitol, 0.02% (w/v) bromophenol blue) and boiled for 5 min at 95 ◦ C. The samples (10 g of protein per lane) were subjected to electrophoresis using an electrode buffer containing 25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS, pH 8.3. The gels were subsequently incubated for 1 h in staining solution containing 0.1% (w/v) Coomassie brilliant blue G-250, 40% (v/v) methanol, 10% (v/v) glacial acetic acid, and 50% (v/v) water. The stained gels were agitated in destaining solution (40% (v/v) methanol, 10% (v/v) glacial acetic acid, 50% (v/v) water) until the background became clear. Nondenaturing PAGE employed a system developed by Ornstein [17] and Davis [18]. A 10% resolving gel contained 375 mM Tris–Cl, pH 8.8, and a 4% stacking gel contained 125 mM Tris–Cl, pH 6.8. Samples of albumins were mixed 1:1 with sample buffer (125 mM Tris–Cl, pH 6.8, 20% (v/v) glycerol, 0.02% (w/v) bromophenol blue) and subjected to electrophoresis (10 g of protein per lane) using an electrode buffer consisting of 25 mM Tris, 192 mM glycine, pH 8.3. Proteins in the gels were visualized by Coomassie blue staining as described above. 3. Results and discussion BSA and HSA, used as model proteins, were electrochemically examined after their adsorption (adsorptive transfer [7]) onto PGE surface using the ex situ SWV method. The RTILs were used either as a medium for protein solubilization and adsorption or as a supporting electrolyte. First, we tested the two ammonium- and four imidazolium-based RTILs given in Section 2.2, using the ex situ technique, where RTILs without the protein were adsorbed (tA = 30 s)
Table 1 Tyrosine and tryptophan residues in HSA and BSA. Protein HSA BSA
Total Tyr (Y)
Trp (W)
Surface exposed Tyr (Y) Trp (W)
18 20
1 2
9 12
0 0
onto the PGE surface. We then monitored interferences to electrochemical analysis of HSA and BSA (not shown). No interfering currents were found for the tested solvents. 3.1. Electrooxidation of proteins adsorbed from ionic liquids We focused on the usability of RTILs for HSA and BSA solubilization and adsorption for ex situ SWV measurement. The dissolved proteins (final conc. 10 M) were adsorbed onto the PGE surface (tA = 30 s). After formation of the adsorbed layer, the electrode was washed with distilled water, dried and placed in an electrochemical cell, where SWV (200 Hz) was performed in 0.2 M acetate buffer, pH 5.0. The scan was performed from 0 V to +1.5 V as described previously [19]. Using these conditions for sample adsorption, the PGE surface was always fully covered by the analyzed proteins (not shown). The proteins dissolved and adsorbed from the Britton–Robinson buffer (pH 7.4), in the absence RTILs, exhibited SWV oxidation peaks Y&W at potential around +0.8 V (Fig. 1A). In proteins, this oxidation peak is assigned to the Tyr (Y) and Trp (W) oxidations [11–13]. HSA and BSA contain in total 18 Y and 1 W and 20 Y and 2 W, respectively (Table 1). Generally, it is considered that intrinsic protein electroactivity is caused by the electroactive amino acid residues that are, after protein adsorption, in direct contact with an electrode surface. Hence, it may be assumed that only the residues localized on a protein surface can be oxidized. The possible oxidation centers are displayed in Scheme 1. It is very likely that only Tyr residues contribute to the current response because the Trp residues are localized in the protein interior or in the cavities that are accessible from the solvent only by a narrow tunnel. Based on the high-resolution structures, we can identify 9 Tyr in HSA and 12 Tyr in BSA on the protein surface [20,21] (Table 1). The contribution of Trp residues to anodic response cannot be strictly excluded, especially in cases where experimental conditions involve significant structural changes of the proteins. Further, both proteins were solubilized and adsorbed from the RTILs/water mixture under the same conditions as described in the first paragraph of this section. All six examined RTILs were used for this purpose; however, RTILs competed for the electrode surface with the analyzed proteins. Hence, we observed the peak Y&W decrease (see below) due to co-adsorption processes. Some RTILs (e.g. ethylammonium nitrate) also suppress protein–protein interactions (aggregation) [3,22], and hence, RTILs can find use as a co-adsorbing species for formation of an adsorbed layer with lower density of adsorbed protein molecules due to molecular repulsions at the electrode surface. In order to minimize the denaturizing changes of proteins, a defined RTIL/water ratio was used [3,5]. SW voltammograms of HSA and BSA solubilized and adsorbed in 1-ethyl-3-methylimidazolium acetate/water or ethylammonium nitrate/water (80:20, v/v) mixtures are shown in Fig. 1B and C. The co-adsorption effect of RTIL vs. studied protein was examined with various RTIL/water ratios. With increasing amounts of RTILs, we observed a gradual decrease in Y&W peak for both HSA and BSA (Fig. 2A). From the view point of co-adsorption processes, there is almost no interference of ethylammonium nitrate and only small decrease in the Y&W peak was observed in the presence of 2-hydroxyethylammonium formate. A stronger
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Scheme 1. The aromatic residues Tyr (blue) and Trp (magenta) exposed to the surface of HSA or BSA are highlighted. The left and right images of the same molecule are mutually rotated by 180◦ along the vertical axis. The images for HSA and BSA were created based on the high-resolution structures deposited in RCSB Protein Data Bank 1AO6 [21] and 4F5S [20], respectively.
Fig. 1. Ex situ SW voltammograms of HSA and BSA after solubilization in: BrittonRobinson buffer at pH 7.4 (A), 1-ethyl-3-methylimidazolium acetate-water (B) and ethylammonium nitrate-water (C) solutions (80:20, v/v); concentration of proteins: 10 M; supporting electrolyte: 0.2 M acetate buffer, pH 5.0. Potential vs. Ag/AgCl/3 M KCl.
co-adsorption effect was observed for imidazolium RTILs, 1-ethyl3-methylimidazolium acetate and 1-buthyl-3-methylimidazolium dicyanamide. In the case of protein solubilization using 100% RTILs, we observed a 50% decrease in current response (Fig. 2A). The observed co-adsorption effects for imidazolium RTILs are in agreement with previously published studies where these RTILs were used to intentionally suppress protein adsorption in electrophoretic capillaries [23]. In addition to the quantitative effects connected with the abovedescribed co-adsorption processes, we also examined changes in the potential of oxidation peaks of HSA and BSA. With increasing amounts of RTILs in the sample, we observed a small but proportional shift of the Y&W peak toward less positive values (Fig. 2B). This observation indicates that the presence of RTILs facilitates the protein electrooxidation, which may be due to the good conductive properties of RTILs [3,24]. An alternative explanation could be some partial RTIL-modification of the PGE surface or modification
Fig. 2. Effect of RTILs/water percentage representation (v/v) used for HSA solubilization on peak Y&W height (A) and potential (B). The following RTILs were used for solubilization of HSA: (1) ethylammonium nitrate, (2) 2hydroxyethylammonium formate, (3) 1-ethyl-3-methylimidazolium acetate, (4) 1-buthyl-3-methylimidazolium dicyanamide. Ex situ SWV, supporting electrolyte: 0.2 M acetate buffer (pH 5.0). Concentration of HSA was 10 M. 100% peak heights = water without RTILs in samples. Similar effects can be observed for BSA. Potential vs. Ag/AgCl/3 M KCl.
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Fig. 3. Denaturing (A) and native (B) gel electrophoreograms of HSA after its solubilization using RTILs-water (80:20, v/v) solutions. Very similar results were obtained for BSA. Lanes: (ctrl) control–no RTIL, (a) 1-ethyl-3-methylimidazolium ethylsulfate, (b) 1-ethyl-3-methylimidazolium acetate, (c) 1-buthyl-3-methylimidazolium dicyanamide, (d) ethylammonium nitrate, (e) 2-hydroxyethylammonium formate, and (f) 1-ethyl-3-methylimidazolium tetrafluoroborate.
of the protein structure, i.e. stabilization of adsorbed film that may facilitate the anodic reaction. For interpretation of these results and also to check for protein integrity, we analyzed the HSA and BSA after their incubation with the RTILs using gel electrophoresis.
3.2. Protein stability after ionic liquids solubilization Both studied proteins were analyzed by polyacrylamide gel electrophoresis (PAGE) [16–18]. For this experiment, HSA and BSA were dissolved at a concentration of 4 mg/ml in Britton–Robinson buffer pH 7.4 (control) or in RTIL/water (80:20, v/v) mixtures and incubated for 10 min at laboratory temperature. The samples were then diluted and prepared for PAGE analysis as given in Section 2.4. Electrophoretic separation was performed under both denaturing (sodium dodecyl sulfate, SDS) or non-denaturing (native) conditions. In the case of SDS-PAGE, we could observe well-separated bands of HSA and BSA, exhibiting the same electrophoretic mobility for RTIL-treated samples as for the control (Fig. 3A). In the case of native PAGE, we observed the typical electrophoretic profiles of HSA and BSA without any qualitative difference between control and RTIL-containing samples (Fig. 3B). The results by native PAGE also revealed that RTILs under given conditions probably do not influence the intermolecular interactions in HSA and BSA (in solution) and this is indicated by bands of dimers and oligomers (Fig. 3B). Interaction of imidazolium RTILs with BSA has already been examined by other groups [25,26]. Based on these findings, we can assume that the RTILs used in this study interact with the studied proteins primarily through weak non-covalent hydrophilic interactions and H-bonds. Inevitable unfolding effects were observed for imidazolium RTILs only in the case when their structure was modified by a sufficiently long hydrophobic hydrocarbon chain. In this case, denaturation can be caused by the interaction of hydrophobic chains with the hydrophobic protein interior [25]. The results of PAGE confirmed that RTILs application does not cause aggregation, fragmentation or structural changes of the studied proteins, which could affect their electrophoretic mobility.
Fig. 4. Ex situ SW voltammograms of HSA and BSA solubilized in Britton-Robinson buffer at pH 7.4. The SWV was performed using supporting electrolytes: 1-ethyl-3methylimidazolium acetate (A) and ethylammonium nitrate (B). Concentration of proteins was 10 M. Potential vs. Ag/AgCl/3 M KCl. Table 2 The potentials of SWV peaks Y&W of HSA and BSA in 0.2 M acetate buffer (pH 5.0) and selected RTILs. Average values of potentials (n = 6) are expressed vs. Ag/AgCl/3 M KCl. The standard deviations (S.D.) were less than ±3 mV for all potentials. Electrolytes
pH
Peak Y&W potential (V) HSA BSA
Acetate buffer, 0.2 M 1-Ethyl-3-methylimidazolium ethylsulfate 1-Ethyl-3-methylimidazolium tetrafluoroborate 1-Ethyl-3-methylimidazolium acetate 1-Buthyl-3-methylimidazolium dicyanamide Ethylammonium nitrate 2-Hydroxyethylammonium formate
5.0 7.6 3.9 12.1 8.5 4.5 7.0
0.908 0.772 0.943 0.518 0.645 0.864 0.843
0.925 0.786 0.896 0.552 0.694 0.874 0.850
3.3. Ionic liquids as electrolytes for protein electrooxidation Finally, we studied the possible use of RTILs as a supporting electrolyte for the SWV of proteins. HSA and BSA were first adsorbed onto the PGE surface from Britton–Robinson buffer (pH 7.4) for tA = 30 s, subsequently washed, dried and placed in an electrochemical cell containing RTIL electrolyte. All tested RTIL electrolytes were useful for electrochemical oxidation of both proteins (Fig. 4 and Table 2). Due to substantially different pH and other physicochemical parameters of examined RTILs, the Y&W peak of both HSA and BSA was observed at different potentials (Table 2). Our results suggested that the pH of tested RTILs is the driving factor affecting the peak Y&W potential for both proteins. Increasing pH of the supporting electrolyte, shifts the potential peak Y&W toward less positive values, by −55 mV per pH unit, which is in good agreement with pH effects recently reported for peptide methionine sulfoxide reductase A [27]. The pH effects presented here were obtained in a series of experiments with Britton-Robinson buffer in the pH
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Education for Competitiveness from the Ministry of Education, Youth and Sports, Czech Republic.
References
Fig. 5. Effect of pH on potential of peak Y&W for HSA and BSA in aqueous BrittonRobinson buffer (full circles and squares) and in RTILs (open circles and squares) as supporting electrolytes for SWV. (a) 1-ethyl-3-methylimidazolium ethylsulfate, (b) 1-ethyl-3-methylimidazolium acetate, (c) 1-buthyl-3-methylimidazolium dicyanamide, (d) ethylammonium nitrate, (e) 2-hydroxyethylammonium formate, (f) 1-ethyl-3-methylimidazolium tetrafluoroborate. Potential vs. Ag/AgCl/3 M KCl.
range 3–12. Shifts of peak Y&W potentials recorded for the aqueous buffered electrolyte correspond very well to the results obtained using RTILs electrolytes (Fig. 5). In contrast to ammonium-based RTILs, SWV recorded in imidazolium RTIL electrolytes also revealed currents from the electrolyte itself but not the same as the potential for peak Y&W of HSA or BSA (compare Fig. 4A and 4B). The measurement can also be done in RTIL/water mixtures, or in RTILs whose pH is modified using an aqueous buffer. We found the optimal aqueous buffer was the Britton–Robinson buffer as it did not result in precipitate formation in the solution (not shown). The need to control the pH of supporting electrolytes on the basis of RTILs still presents a challenge. Currently developed ionic liquid buffers [28,29] could be a solution to this problem but they have not been examined electrochemically and their applicability to protein analysis will need further research. 4. Conclusions This study focused on the applicability of selected RTILs with the imidazolium or ammonium cation for solubilization of model proteins HSA and BSA. We examined the adsorption of these proteins from RTILs onto PGE surface and also electroooxidation of the adsorbed protein layer in the RTIL environment. Our results show that 1-ethyl-3-methylimidazolium ethylsulfate, 1-ethyl-3methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium acetate, 1-buthyl-3-methylimidazolium dicyanamide, ethylammonium nitrate and 2-hydroxyethylammonium formate could be used as solubilizers and adsorption solvents in ex situ voltammetry and as a supporting electrolyte for SWV analysis of both proteins. The results could be applied in electrochemical examination of other proteins and in developing new approaches, e.g. protein extracting electrodes [30]. Further, RTILs properly modified by hydrophobic functional groups could be useful for solubilization of water-insoluble proteins and their subsequent electroanalysis [19,31,32]. Acknowledgement The authors thank Dr. Alexander Oulton for language correction and Ms. Alˇzbˇeta Balharová for excellent technical support. This work was supported by the Institutional Financial Support of Palacky University (Faculty of Medicine and Dentistry) and by the grant No. CZ.1.07/2.3.00/20.0057–Operational Programme
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