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Fast-scan cyclic voltammetry with thiol-modified mercury electrodes distinguishes native from denatured BSA
Electrochemistry Communications 61 (2015) 114–116
Contents lists available at ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
Fast-scan cyclic voltammetry with thiol-modified mercury electrodes distinguishes native from denatured BSA Hana Černocká, Veronika Ostatná, Emil Paleček ⁎ Institute of Biophysics of the CAS, v. v. i., Královopolská 135, 612 65 Brno, Czech Republic
a r t i c l e
i n f o
Article history: Received 7 October 2015 Received in revised form 21 October 2015 Accepted 26 October 2015 Available online 3 November 2015 Keywords: Fast-scan cyclic voltammetry Protein denaturation Mercury electrode Chronopotentiometric stripping Bovine serum albumin Thiol-modified electrode
a b s t r a c t We studied native and denatured bovine serum albumin (BSA) at bare and dithiothreithol (DTT)-modified hanging mercury drop electrode (HMDE) by fast-scan cyclic voltammetry (fsCV) and chronopotentiometric stripping (CPS) to compare these methods for their ability to recognize changes in BSA structure. Using fsCV and bare HMDE, denatured BSA could be distinguished from its native form only between 10 and 20 V/s but at lower resolution than with CPS. At DTT-HMDE denatured BSA was recognized from native BSA in a wider range of scan rates suggesting new possibilities in development of voltammetric protein structure-sensitive sensing. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In the past decades label-free electrochemical analysis of proteins was predominantly limited to a small group of conjugated proteins, containing redox-active non-protein components [1–6]. We have found that at HMDE [7–9] all analyzed proteins produce CPS peaks at negative potentials allowing determination of proteins at nanomolar and subnanomolar concentrations [9,10]. We assigned these peaks to the catalytic hydrogen evolution reaction (CHER) [9,11,12] and denominated them as peaks H due to High sensitivity and Hydrogen evolution and in a tribute to J. Heyrovsky. Arginine, lysine, cysteine and histidine were shown as residues involved in CHER [13–16]. For decades it was believed that proteins denature when adsorbed at metal electrodes [9,17,18]. We found that proteins adsorbed at bare mercury electrodes at potentials close to zero charge are not denatured at the electrode surface. They can be, however, denatured due to prolonged exposure to negative potentials [7,19,20]. On the ground of this finding, we developed a CPS protein structure-sensitive method [9]. We showed that at high current densities this peak is sensitive to changes in protein structure, recognizing single amino exchange in mutant proteins [21] at bare and thiol-modified mercury electrodes [7,9]. The method was applied in the analysis of tumor suppressor p53 protein [21,22], α-synuclein [23,24] and peptides involved in neurodegenerative diseases [25], membrane proteins [26,27], protein glycation [28] ⁎ Corresponding author.
http://dx.doi.org/10.1016/j.elecom.2015.10.017 1388-2481/© 2015 Elsevier B.V. All rights reserved.
and for studies of sequence-specific p53-DNA binding [22]. Up to now it was believed that this type of protein structure-sensitive analysis is limited to CPS and cannot be performed by usual voltammetric methods [21]. In such analysis it is critical to control the rate of potential changes in CPS by choosing appropriate current densities (current intensities at constant electrode size) limiting the exposure of the surface-attached protein to negative potentials to ms time intervals. For example, using the polarizing stripping current intensity, Istr of −35 μA (corresponding current density of 8.75 mA cm−2), surface-attached BSA under the usual conditions did not show any sign of denaturation [7,9,29]. At this Istr, in the absence of the electrode process, the rates of potential changes corresponded to ~300 V/s (Table 1). Considering our data we have realized that fsCV offers similar or even higher rates [30,31]. This method was applied in studies of some metalloproteins, at carbon electrodes at scan rates up to 3000 V/s [32,33]. We were interested in whether fsCV stripping can replace or complement CPS in the non-conjugated protein structure analysis. In this paper we studied native and denatured BSA at bare and DTTmodified HMDE by fsCV and CPS to compare the ability of these methods to recognize changes in BSA structure. Our results showed that using CV at bare HMDE denatured BSA could be recognized from its native form in a relatively narrow scan rate range between 10 and 20 V/s. Under these conditions native BSA produced an appreciable CHER peak, suggesting significant denaturation of BSA at the electrode surface. At DTT-HMDE denatured BSA was recognized from its native
H. Černocká et al. / Electrochemistry Communications 61 (2015) 114–116
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Table 1 Rates of potential changes and ratios of denatured/native BSA peak heights in CV and CPS.a CPS
−Istr (μA)
CV
50 40 35 30 25 20 Scan rate (V/s) 50 100 310
Rate of potential changes (V/s) nat den 504 478 383 347 308 280 250 231 190 192 132 137
Time till peak H foot is reached (ms) nat den 3.5 3.6 4.5 4.8 5.6 6.1 6.8 7.3 8.8 8.7 12.6 12.1 Time till peak foot is reached (ms) nat den 39.5 38.8 21.9 21.8 8.2 ms till −2.5 V is reached 8.2 ms till −2.5 V is reached
Peak H duration (ms) nat den 16 107 34 173 56 228 367 312 557 462 887 746 Peak duration (ms) nat den 5.9 7.6 3.5 4.3 No peak No peak
Peak H heights ratio hden/hnat 17.96 8.81 5.32 0.91 0.95 0.95 Peak heights ratio Iden/Inat 1.25 1.24 −
a Time intervals related to different stages of CPS and CV measurements of BSA at bare HMDE at the respective Istr or scan rates. The rate of potential changes in CPS was calculated as the average rate from the initial potential of −0.1 V to the foot of the peak H.
form in a wider range of scan rates showing new potentiality of voltammetric methods in protein research. 2. Experimental All chemicals were of analytical grade, purchased from SigmaAldrich. Solutions were prepared in triply distilled water. Electrochemical measurements were performed on an Autolab analyzer PGSTAT30 (EcoChemie the Netherlands) with modules SCAN250, ADC10M for fsCV connected with VA-Stand 663 (Metrohm Switzerland). HMDE (0.4 mm2) as a working electrode, Ag|AgCl|3 M KCl as the reference and Pt wire as the auxiliary electrode were used in a standard thermostated cell. FsCV parameters: potential step 5 mV, initial potential −0.1 V, vertex potential −2.2 V. 300 nM protein was adsorbed at
the bare or DTT-HMDE [34] from 50 mM Na-phosphate pH 7 and 80 mM urea at the accumulation potential −0.1 V for 60 s accompanied by stirring 1500 rpm, followed by the chronopotentiometric (using GPES software) or voltammetric measurement (using NOVA software). All experiments were performed at room temperature, voltammetry in an argon atmosphere while CPS under air. BSA urea-denaturation was described [9,35]. 3. Results and discussion CV of native and denatured BSA at usual scan rates showed almost no differences between the electrocatalytic peaks of the two BSA forms, e.g. at 1 V/s almost identical peaks at − 1.89 V appeared for both forms of BSA (Fig. 1A). On the other hand, using CPS at Istr
Fig. 1. A., B., E., F. The cyclic voltammograms of 300 nM native (black, dashed) and denatured (red, solid) BSA at A., B., bare and E., F. DTT-modified HMDE at scan rates A. 1 V/s, B. 10 V/s. E. 2.5 V/s and F. 50 V/s. Inset: Chronopotentiograms of 300 nM native (black, dashed) and denatured (red, solid) BSA at B. bare HMDE at Istr −50 μA, F. DTT-HMDE at Istr −35 μA. C., D., G., H. Dependence of the peak C., G. potentials Ep, D., H. heights of native (black, dashed) and denatured (red, solid) BSA and dependence of the peak heights ratio Iden/Inat of BSA on scan rate ν at C., D., bare and G., H. DTT-modified HMDE.
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H. Černocká et al. / Electrochemistry Communications 61 (2015) 114–116
−50 μA, under otherwise the same conditions, denatured BSA produced a high peak at − 1.86 V, contrasting with the 18-fold smaller peak of native BSA (Fig. 1B Inset). Table 1 shows that at Istr −50 μA the rate of potential changes (before reaching the peak H foot) is about 500times higher than in CV at 1 V/s. We therefore decided to test fsCV, capable to reach scan rates equally high as potential changes obtained in protein structure-sensitive CPS analysis. 3.1. Fast-scan cyclic voltammetry 3.1.1. Bare HMDE With increasing scan rate peaks of native and denatured BSA grew almost linearly up to about 10 V/s and Ep shifted to more negative values (Fig. 1C,D). Up to ~ 5 V/s heights of native and denatured BSA peaks were almost identical. Between 5 and 20 V/s denatured BSA yielded higher peaks than native BSA, showing the largest differences between 10 and 20 V/s (Fig. 1B–D). At higher scan rates the peaks of both BSA forms as well as the ratio of Iden/Inat decreased. Such decrease of voltammetric peaks of biomacromolecules involving CHER was observed earlier [11,14] and can be explained by relatively slow electrocatalytic electrode process, not compatible with the fast scan rates. From about 50 V/s the peaks were rather small showing a tendency to merge with the background. Our results show that using fsCV at certain scan rates the denatured BSA can be distinguished from the native one (Fig. 1B–D) showing the highest Iden/Inat of ~ 1.5, which, compared to the CPS hden/hnat ~18 (Table 1), appears rather low suggesting substantial BSA denaturation during the fsCV potential scanning. Similar results were obtained also at solid amalgam electrodes (not shown), albeit with decreased reproducibility, usual with solid electrodes. The reasons for the difference between the fsCV and CPS responses can be deduced from Table 1; for example, in fsCV even at 100 V/s the time for reaching the potential of the peak (~ 22 ms) is longer than that in CPS (3.5 ms at − 50 μA). On the other hand, the time intervals for passing the potential range of the CPS peak H of native and denatured BSA are 16 ms and 107 ms respectively, while for the fsCV peak at 100 V/s ~ 4 ms for both BSA forms. These data suggest that under these conditions, in fsCV the surface-attached native BSA is exposed for a longer time to denaturation-inducing electric field effects at negative potentials [7] than in CPS, and that the fsCV peak of denatured BSA cannot fully develop because the time available for the electrode process is too short. These results (a) are in a good agreement with our previous papers [7,9,20] showing that the time of exposure of the surface-attached protein to negative potentials is critical for its denaturation (or intact state), and (b) lead us to the conclusion that, under the given conditions, increasing the scan rate does not turn the fsCV into a structure-sensitive method of protein analysis comparable to CPS. 3.1.2. DTT-modified HMDE Earlier we showed that modification of HMDE with DTT prevents surface-attached BSA from denaturation at higher ionic strengths [29] and that DTT-HMDE is particularly convenient for analysis of reduced proteins such as tumor suppressor protein p53 [21]. We supposed that with DTT-HMDE we might be able to get better resolution of native vs. denatured BSA using fsCV than with bare HMDE (Fig. 1A–D). Relatively high peak (Ep −1.93 V) of denatured BSA contrasting with a small peak of native BSA at 2.5 V/s is shown in Fig. 1E. At very low scan rates peak heights of native and denatured BSA differed only little (Fig. 1G,H), while starting from about 2.5 V/s, Ep's of native BSA were by ~ 50 mV less negative than those of the denatured BSA form (Fig. 1E–G). Other proteins, such as human serum albumin, ovalbumin and aldolase yielded similar differences in the peak heights of their native and denatured forms as BSA (Fig. 1E). Our results suggest that at DTT-modified HMDE native BSA is better protected against the denaturing electric field effects not only in CPS but also in voltammetry.
4. Conclusion We may conclude that CPS is the method of choice in the protein structure-sensitive analysis, but application of the fsCV in combination with DTT-HMDE opens the door also for voltammetric analysis of changes in protein structure. In addition to DTT, other thiols with different end groups [36] can be used for modification of mercury electrodes, in relation to properties of analyzed proteins. So far, CHER has been observed only at mercury-containing electrodes. On the other hand progress in studies of this reaction, related to energy problems [37–39], may result in finding conditions allowing structure-sensitive protein analysis at less negative potentials at different electrode materials. Acknowledgment This work was supported by Czech Science Foundation 15-15479S. References [1] F.A. Armstrong, H.A. Heering, J. Hirst, Chem. Soc. Rev. 26 (1997) 169. [2] G. Gilardi, A. Fantuzzi, S.J. Sadeghi, Curr. Opin. Struct. Biol. 11 (2001) 491. [3] P. Salvatore, D.D. Zeng, K.K. Karlsen, Q.J. Chi, J. Wengel, J. Ulstrup, ChemPhysChem 14 (2013) 2101. [4] D.E. Khoshtariya, T.D. Dolidze, M. Shushanyan, R. van Eldik, J. Phys. Chem. B 118 (2014) 692. [5] J.P. Layfield, S. Hammes-Schiffer, Chem. Rev. 114 (2014) 3466. [6] J.R. Winkler, H.B. Gray, Chem. Rev. 114 (2014) 3369. [7] H. Černocká, V. Ostatná, E. Paleček, Electrochim. Acta 174 (2015) 356. [8] V. Ostatna, B. Dogan, B. Uslu, S. Ozkan, E. Palecek, J. Electroanal. Chem. 593 (2006) 172. [9] E. Palecek, J. Tkac, M. Bartosik, T. Bertok, V. Ostatna, J. Palecek, Chem. Rev. 115 (2015) 2045. [10] M. Tomschik, L. Havran, E. Palecek, M. Heyrovsky, Electroanalysis 12 (2000) 274. [11] E. Palecek, M. Bartosik, V. Ostatna, M. Trefulka, Chem. Rec. 12 (2012) 27. [12] M. Heyrovsky, Electroanalysis 16 (2004) 1067. [13] V. Dorcak, V. Vargova, V. Ostatna, E. Palecek, Electroanalysis 27 (2015) 910. [14] V. Vargova, M. Zivanovic, V. Dorcak, E. Palecek, V. Ostatna, Electroanalysis 25 (2013) 2130. [15] M. Zivanovic, M. Aleksic, V. Ostatna, T. Doneux, E. Palecek, Electroanalysis 22 (2010) 2064. [16] T. Doneux, V. Dorcak, E. Palecek, Langmuir 26 (2010) 1347. [17] F.A. Armstrong, in: G.S. Wilson (Ed.)Bioelectrochemistry, vol. 9, Wiley-VCH, Weinheim 2002, p. 11. [18] F. Scheller, M. Jänchen, H.-J. Prümke, Biopolymers 14 (1975) 1553. [19] E. Palecek, V. Ostatna, Analyst 134 (2009) 2076. [20] H. Cernocka, V. Ostatna, E. Palecek, Anal. Chim. Acta 789 (2013) 41. [21] E. Palecek, V. Ostatna, H. Cernocka, A.C. Joerger, A.R. Fersht, J. Am. Chem. Soc. 133 (2011) 7190. [22] E. Palecek, H. Cernocka, V. Ostatna, L. Navratilova, M. Brazdova, Anal. Chim. Acta 828 (2014) 1. [23] C.D. Borsarelli, L.J. Falomir-Lockhart, V. Ostatna, J.A. Fauerbach, H.H. Hsiao, H. Urlaub, E. Palecek, E.A. Jares-Erijman, T.M. Jovin, Free Radic. Biol. Med. 53 (2012) 1004. [24] E. Palecek, V. Ostatna, M. Masarik, C.W. Bertoncini, T.M. Jovin, Analyst 133 (2008) 76. [25] K. Kurzatkowska, V. Ostatna, I.W. Hamley, T. Doneux, E. Palecek, Electrochim. Acta 106 (2013) 43. [26] M. Zatloukalova, E. Orolinova, M. Kubala, J. Hrbac, J. Vacek, Electroanalysis 24 (2012) 1758. [27] J. Vacek, M. Zatloukalova, M. Havlikova, J. Ulrichova, M. Kubala, Electrochem. Commun. 27 (2013) 104. [28] R. Vecerkova, L. Hernychova, P. Dobes, J. Vrba, B. Josypcuk, M. Bartosik, J. Vacek, Anal. Chim. Acta 830 (2014) 23. [29] E. Palecek, V. Ostatna, Chem. Commun. (2009) 1685. [30] D.O. Wipf, E.W. Kristensen, M.R. Deakin, R.M. Wightman, Anal. Chem. 60 (1988) 306. [31] R.G. Compton, C.E. Banks, Understanding Voltammetry 2nd Edition Preface, Imperial College Press, London, 2011. [32] J. Hirst, F.A. Armstrong, Anal. Chem. 70 (1998) 5062. [33] G. Munteanu, E. Dempsey, T. McCormac, C. Munteanu, J. Electroanal. Chem. 665 (2012) 12. [34] V. Ostatna, H. Cernocka, E. Palecek, Bioelectrochemistry 87 (2012) 84. [35] V. Ostatna, H. Cernocka, E. Palecek, J. Am. Chem. Soc. 132 (2010) 9408. [36] H.O. Finklea, in: R.A. Meyers (Ed.)Encyclopedia of Analytical Chemistry, vol. 11, Wiley, New York 2000, p. 10090. [37] E. Reisner, Eur. J. Inorg. Chem. (2011) 1005. [38] F. Safizadeh, E. Ghali, G. Houlachi, Int. J. Hydrog. Energy 40 (2015) 256. [39] Y. Yan, B. Xia, Z. Xu, X. Wang, ACS Catal. 4 (2014) 1693.
Contents lists available at ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
Fast-scan cyclic voltammetry with thiol-modified mercury electrodes distinguishes native from denatured BSA Hana Černocká, Veronika Ostatná, Emil Paleček ⁎ Institute of Biophysics of the CAS, v. v. i., Královopolská 135, 612 65 Brno, Czech Republic
a r t i c l e
i n f o
Article history: Received 7 October 2015 Received in revised form 21 October 2015 Accepted 26 October 2015 Available online 3 November 2015 Keywords: Fast-scan cyclic voltammetry Protein denaturation Mercury electrode Chronopotentiometric stripping Bovine serum albumin Thiol-modified electrode
a b s t r a c t We studied native and denatured bovine serum albumin (BSA) at bare and dithiothreithol (DTT)-modified hanging mercury drop electrode (HMDE) by fast-scan cyclic voltammetry (fsCV) and chronopotentiometric stripping (CPS) to compare these methods for their ability to recognize changes in BSA structure. Using fsCV and bare HMDE, denatured BSA could be distinguished from its native form only between 10 and 20 V/s but at lower resolution than with CPS. At DTT-HMDE denatured BSA was recognized from native BSA in a wider range of scan rates suggesting new possibilities in development of voltammetric protein structure-sensitive sensing. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In the past decades label-free electrochemical analysis of proteins was predominantly limited to a small group of conjugated proteins, containing redox-active non-protein components [1–6]. We have found that at HMDE [7–9] all analyzed proteins produce CPS peaks at negative potentials allowing determination of proteins at nanomolar and subnanomolar concentrations [9,10]. We assigned these peaks to the catalytic hydrogen evolution reaction (CHER) [9,11,12] and denominated them as peaks H due to High sensitivity and Hydrogen evolution and in a tribute to J. Heyrovsky. Arginine, lysine, cysteine and histidine were shown as residues involved in CHER [13–16]. For decades it was believed that proteins denature when adsorbed at metal electrodes [9,17,18]. We found that proteins adsorbed at bare mercury electrodes at potentials close to zero charge are not denatured at the electrode surface. They can be, however, denatured due to prolonged exposure to negative potentials [7,19,20]. On the ground of this finding, we developed a CPS protein structure-sensitive method [9]. We showed that at high current densities this peak is sensitive to changes in protein structure, recognizing single amino exchange in mutant proteins [21] at bare and thiol-modified mercury electrodes [7,9]. The method was applied in the analysis of tumor suppressor p53 protein [21,22], α-synuclein [23,24] and peptides involved in neurodegenerative diseases [25], membrane proteins [26,27], protein glycation [28] ⁎ Corresponding author.
http://dx.doi.org/10.1016/j.elecom.2015.10.017 1388-2481/© 2015 Elsevier B.V. All rights reserved.
and for studies of sequence-specific p53-DNA binding [22]. Up to now it was believed that this type of protein structure-sensitive analysis is limited to CPS and cannot be performed by usual voltammetric methods [21]. In such analysis it is critical to control the rate of potential changes in CPS by choosing appropriate current densities (current intensities at constant electrode size) limiting the exposure of the surface-attached protein to negative potentials to ms time intervals. For example, using the polarizing stripping current intensity, Istr of −35 μA (corresponding current density of 8.75 mA cm−2), surface-attached BSA under the usual conditions did not show any sign of denaturation [7,9,29]. At this Istr, in the absence of the electrode process, the rates of potential changes corresponded to ~300 V/s (Table 1). Considering our data we have realized that fsCV offers similar or even higher rates [30,31]. This method was applied in studies of some metalloproteins, at carbon electrodes at scan rates up to 3000 V/s [32,33]. We were interested in whether fsCV stripping can replace or complement CPS in the non-conjugated protein structure analysis. In this paper we studied native and denatured BSA at bare and DTTmodified HMDE by fsCV and CPS to compare the ability of these methods to recognize changes in BSA structure. Our results showed that using CV at bare HMDE denatured BSA could be recognized from its native form in a relatively narrow scan rate range between 10 and 20 V/s. Under these conditions native BSA produced an appreciable CHER peak, suggesting significant denaturation of BSA at the electrode surface. At DTT-HMDE denatured BSA was recognized from its native
H. Černocká et al. / Electrochemistry Communications 61 (2015) 114–116
115
Table 1 Rates of potential changes and ratios of denatured/native BSA peak heights in CV and CPS.a CPS
−Istr (μA)
CV
50 40 35 30 25 20 Scan rate (V/s) 50 100 310
Rate of potential changes (V/s) nat den 504 478 383 347 308 280 250 231 190 192 132 137
Time till peak H foot is reached (ms) nat den 3.5 3.6 4.5 4.8 5.6 6.1 6.8 7.3 8.8 8.7 12.6 12.1 Time till peak foot is reached (ms) nat den 39.5 38.8 21.9 21.8 8.2 ms till −2.5 V is reached 8.2 ms till −2.5 V is reached
Peak H duration (ms) nat den 16 107 34 173 56 228 367 312 557 462 887 746 Peak duration (ms) nat den 5.9 7.6 3.5 4.3 No peak No peak
Peak H heights ratio hden/hnat 17.96 8.81 5.32 0.91 0.95 0.95 Peak heights ratio Iden/Inat 1.25 1.24 −
a Time intervals related to different stages of CPS and CV measurements of BSA at bare HMDE at the respective Istr or scan rates. The rate of potential changes in CPS was calculated as the average rate from the initial potential of −0.1 V to the foot of the peak H.
form in a wider range of scan rates showing new potentiality of voltammetric methods in protein research. 2. Experimental All chemicals were of analytical grade, purchased from SigmaAldrich. Solutions were prepared in triply distilled water. Electrochemical measurements were performed on an Autolab analyzer PGSTAT30 (EcoChemie the Netherlands) with modules SCAN250, ADC10M for fsCV connected with VA-Stand 663 (Metrohm Switzerland). HMDE (0.4 mm2) as a working electrode, Ag|AgCl|3 M KCl as the reference and Pt wire as the auxiliary electrode were used in a standard thermostated cell. FsCV parameters: potential step 5 mV, initial potential −0.1 V, vertex potential −2.2 V. 300 nM protein was adsorbed at
the bare or DTT-HMDE [34] from 50 mM Na-phosphate pH 7 and 80 mM urea at the accumulation potential −0.1 V for 60 s accompanied by stirring 1500 rpm, followed by the chronopotentiometric (using GPES software) or voltammetric measurement (using NOVA software). All experiments were performed at room temperature, voltammetry in an argon atmosphere while CPS under air. BSA urea-denaturation was described [9,35]. 3. Results and discussion CV of native and denatured BSA at usual scan rates showed almost no differences between the electrocatalytic peaks of the two BSA forms, e.g. at 1 V/s almost identical peaks at − 1.89 V appeared for both forms of BSA (Fig. 1A). On the other hand, using CPS at Istr
Fig. 1. A., B., E., F. The cyclic voltammograms of 300 nM native (black, dashed) and denatured (red, solid) BSA at A., B., bare and E., F. DTT-modified HMDE at scan rates A. 1 V/s, B. 10 V/s. E. 2.5 V/s and F. 50 V/s. Inset: Chronopotentiograms of 300 nM native (black, dashed) and denatured (red, solid) BSA at B. bare HMDE at Istr −50 μA, F. DTT-HMDE at Istr −35 μA. C., D., G., H. Dependence of the peak C., G. potentials Ep, D., H. heights of native (black, dashed) and denatured (red, solid) BSA and dependence of the peak heights ratio Iden/Inat of BSA on scan rate ν at C., D., bare and G., H. DTT-modified HMDE.
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H. Černocká et al. / Electrochemistry Communications 61 (2015) 114–116
−50 μA, under otherwise the same conditions, denatured BSA produced a high peak at − 1.86 V, contrasting with the 18-fold smaller peak of native BSA (Fig. 1B Inset). Table 1 shows that at Istr −50 μA the rate of potential changes (before reaching the peak H foot) is about 500times higher than in CV at 1 V/s. We therefore decided to test fsCV, capable to reach scan rates equally high as potential changes obtained in protein structure-sensitive CPS analysis. 3.1. Fast-scan cyclic voltammetry 3.1.1. Bare HMDE With increasing scan rate peaks of native and denatured BSA grew almost linearly up to about 10 V/s and Ep shifted to more negative values (Fig. 1C,D). Up to ~ 5 V/s heights of native and denatured BSA peaks were almost identical. Between 5 and 20 V/s denatured BSA yielded higher peaks than native BSA, showing the largest differences between 10 and 20 V/s (Fig. 1B–D). At higher scan rates the peaks of both BSA forms as well as the ratio of Iden/Inat decreased. Such decrease of voltammetric peaks of biomacromolecules involving CHER was observed earlier [11,14] and can be explained by relatively slow electrocatalytic electrode process, not compatible with the fast scan rates. From about 50 V/s the peaks were rather small showing a tendency to merge with the background. Our results show that using fsCV at certain scan rates the denatured BSA can be distinguished from the native one (Fig. 1B–D) showing the highest Iden/Inat of ~ 1.5, which, compared to the CPS hden/hnat ~18 (Table 1), appears rather low suggesting substantial BSA denaturation during the fsCV potential scanning. Similar results were obtained also at solid amalgam electrodes (not shown), albeit with decreased reproducibility, usual with solid electrodes. The reasons for the difference between the fsCV and CPS responses can be deduced from Table 1; for example, in fsCV even at 100 V/s the time for reaching the potential of the peak (~ 22 ms) is longer than that in CPS (3.5 ms at − 50 μA). On the other hand, the time intervals for passing the potential range of the CPS peak H of native and denatured BSA are 16 ms and 107 ms respectively, while for the fsCV peak at 100 V/s ~ 4 ms for both BSA forms. These data suggest that under these conditions, in fsCV the surface-attached native BSA is exposed for a longer time to denaturation-inducing electric field effects at negative potentials [7] than in CPS, and that the fsCV peak of denatured BSA cannot fully develop because the time available for the electrode process is too short. These results (a) are in a good agreement with our previous papers [7,9,20] showing that the time of exposure of the surface-attached protein to negative potentials is critical for its denaturation (or intact state), and (b) lead us to the conclusion that, under the given conditions, increasing the scan rate does not turn the fsCV into a structure-sensitive method of protein analysis comparable to CPS. 3.1.2. DTT-modified HMDE Earlier we showed that modification of HMDE with DTT prevents surface-attached BSA from denaturation at higher ionic strengths [29] and that DTT-HMDE is particularly convenient for analysis of reduced proteins such as tumor suppressor protein p53 [21]. We supposed that with DTT-HMDE we might be able to get better resolution of native vs. denatured BSA using fsCV than with bare HMDE (Fig. 1A–D). Relatively high peak (Ep −1.93 V) of denatured BSA contrasting with a small peak of native BSA at 2.5 V/s is shown in Fig. 1E. At very low scan rates peak heights of native and denatured BSA differed only little (Fig. 1G,H), while starting from about 2.5 V/s, Ep's of native BSA were by ~ 50 mV less negative than those of the denatured BSA form (Fig. 1E–G). Other proteins, such as human serum albumin, ovalbumin and aldolase yielded similar differences in the peak heights of their native and denatured forms as BSA (Fig. 1E). Our results suggest that at DTT-modified HMDE native BSA is better protected against the denaturing electric field effects not only in CPS but also in voltammetry.
4. Conclusion We may conclude that CPS is the method of choice in the protein structure-sensitive analysis, but application of the fsCV in combination with DTT-HMDE opens the door also for voltammetric analysis of changes in protein structure. In addition to DTT, other thiols with different end groups [36] can be used for modification of mercury electrodes, in relation to properties of analyzed proteins. So far, CHER has been observed only at mercury-containing electrodes. On the other hand progress in studies of this reaction, related to energy problems [37–39], may result in finding conditions allowing structure-sensitive protein analysis at less negative potentials at different electrode materials. Acknowledgment This work was supported by Czech Science Foundation 15-15479S. References [1] F.A. Armstrong, H.A. Heering, J. Hirst, Chem. Soc. Rev. 26 (1997) 169. [2] G. Gilardi, A. Fantuzzi, S.J. Sadeghi, Curr. Opin. Struct. Biol. 11 (2001) 491. [3] P. Salvatore, D.D. Zeng, K.K. Karlsen, Q.J. Chi, J. Wengel, J. Ulstrup, ChemPhysChem 14 (2013) 2101. [4] D.E. Khoshtariya, T.D. Dolidze, M. Shushanyan, R. van Eldik, J. Phys. Chem. B 118 (2014) 692. [5] J.P. Layfield, S. Hammes-Schiffer, Chem. Rev. 114 (2014) 3466. [6] J.R. Winkler, H.B. Gray, Chem. Rev. 114 (2014) 3369. [7] H. Černocká, V. Ostatná, E. Paleček, Electrochim. Acta 174 (2015) 356. [8] V. Ostatna, B. Dogan, B. Uslu, S. Ozkan, E. Palecek, J. Electroanal. Chem. 593 (2006) 172. [9] E. Palecek, J. Tkac, M. Bartosik, T. Bertok, V. Ostatna, J. Palecek, Chem. Rev. 115 (2015) 2045. [10] M. Tomschik, L. Havran, E. Palecek, M. Heyrovsky, Electroanalysis 12 (2000) 274. [11] E. Palecek, M. Bartosik, V. Ostatna, M. Trefulka, Chem. Rec. 12 (2012) 27. [12] M. Heyrovsky, Electroanalysis 16 (2004) 1067. [13] V. Dorcak, V. Vargova, V. Ostatna, E. Palecek, Electroanalysis 27 (2015) 910. [14] V. Vargova, M. Zivanovic, V. Dorcak, E. Palecek, V. Ostatna, Electroanalysis 25 (2013) 2130. [15] M. Zivanovic, M. Aleksic, V. Ostatna, T. Doneux, E. Palecek, Electroanalysis 22 (2010) 2064. [16] T. Doneux, V. Dorcak, E. Palecek, Langmuir 26 (2010) 1347. [17] F.A. Armstrong, in: G.S. Wilson (Ed.)Bioelectrochemistry, vol. 9, Wiley-VCH, Weinheim 2002, p. 11. [18] F. Scheller, M. Jänchen, H.-J. Prümke, Biopolymers 14 (1975) 1553. [19] E. Palecek, V. Ostatna, Analyst 134 (2009) 2076. [20] H. Cernocka, V. Ostatna, E. Palecek, Anal. Chim. Acta 789 (2013) 41. [21] E. Palecek, V. Ostatna, H. Cernocka, A.C. Joerger, A.R. Fersht, J. Am. Chem. Soc. 133 (2011) 7190. [22] E. Palecek, H. Cernocka, V. Ostatna, L. Navratilova, M. Brazdova, Anal. Chim. Acta 828 (2014) 1. [23] C.D. Borsarelli, L.J. Falomir-Lockhart, V. Ostatna, J.A. Fauerbach, H.H. Hsiao, H. Urlaub, E. Palecek, E.A. Jares-Erijman, T.M. Jovin, Free Radic. Biol. Med. 53 (2012) 1004. [24] E. Palecek, V. Ostatna, M. Masarik, C.W. Bertoncini, T.M. Jovin, Analyst 133 (2008) 76. [25] K. Kurzatkowska, V. Ostatna, I.W. Hamley, T. Doneux, E. Palecek, Electrochim. Acta 106 (2013) 43. [26] M. Zatloukalova, E. Orolinova, M. Kubala, J. Hrbac, J. Vacek, Electroanalysis 24 (2012) 1758. [27] J. Vacek, M. Zatloukalova, M. Havlikova, J. Ulrichova, M. Kubala, Electrochem. Commun. 27 (2013) 104. [28] R. Vecerkova, L. Hernychova, P. Dobes, J. Vrba, B. Josypcuk, M. Bartosik, J. Vacek, Anal. Chim. Acta 830 (2014) 23. [29] E. Palecek, V. Ostatna, Chem. Commun. (2009) 1685. [30] D.O. Wipf, E.W. Kristensen, M.R. Deakin, R.M. Wightman, Anal. Chem. 60 (1988) 306. [31] R.G. Compton, C.E. Banks, Understanding Voltammetry 2nd Edition Preface, Imperial College Press, London, 2011. [32] J. Hirst, F.A. Armstrong, Anal. Chem. 70 (1998) 5062. [33] G. Munteanu, E. Dempsey, T. McCormac, C. Munteanu, J. Electroanal. Chem. 665 (2012) 12. [34] V. Ostatna, H. Cernocka, E. Palecek, Bioelectrochemistry 87 (2012) 84. [35] V. Ostatna, H. Cernocka, E. Palecek, J. Am. Chem. Soc. 132 (2010) 9408. [36] H.O. Finklea, in: R.A. Meyers (Ed.)Encyclopedia of Analytical Chemistry, vol. 11, Wiley, New York 2000, p. 10090. [37] E. Reisner, Eur. J. Inorg. Chem. (2011) 1005. [38] F. Safizadeh, E. Ghali, G. Houlachi, Int. J. Hydrog. Energy 40 (2015) 256. [39] Y. Yan, B. Xia, Z. Xu, X. Wang, ACS Catal. 4 (2014) 1693.