Conformational Change Detection in Nonmetal Proteins by Direct

Anal. Chem. 2008, 80, 5783–5787

Conformational Change Detection in Nonmetal Proteins by Direct Electrochemical Oxidation Using Diamond Electrodes Masanobu Chiku,† Jin Nakamura,‡ Akira Fujishima,§ and Yasuaki Einaga*,† Department of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan, Department of Applied Physics and Chemistry, The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan, and Kanagawa Academy of Science and Technology, KSP 3-2-1 Sakado, Kawasaki 213-0012, Japan In this report, we established a new electrochemical method for the detection of conformational changes in large, non-metalloproteins such as bovine serum albumin, using flow injection analysis coupled with hydrogenterminated, boron-doped diamond electrodes. The oxidation current was used as a signal reporter in the monitoring of urea-induced BSA denaturation. In the denatured state at high urea concentrations, the electrochemical signal increased, and the amperometric responses for the oxidation potential at 1300 mV were consistent with the results of conventional methods of denaturation monitoring using fluorescence spectroscopy. The oxidation involved at least five redox-active species (cysteine, tryptophan, tyrosine, methionine, and disulfide bonds). Furthermore, the method also showed high sensitivity for quantitative analysis of protein. A linear dynamic in the concentration range 50-400 µg/mL (r2 ) 0.977) with a lower detection limit of 190 ng/mL was achieved for BSA. Direct electrochemical detection of conformation changes of proteins using BDD electrodes can be performed with advantages in terms of simplicity and sensitivity. Protein folding/unfolding is the most fundamental and universal example of biological self-assembly. This complex process has been studied for several decades to understand the relationship between protein sequence, structure, and function.1,2 The sequences of natural proteins have emerged through evolutionary processes such that their unique native states can be found very efficiently even in the complex environment inside a living cell. However, under some conditions, proteins fail to fold correctly in living systems, and this failure can result in a wide range of diseases, known as amyloidoses, which includes Alzheimer’s disease and Parkinson’s disease. Plaques that contain misfolded peptides called amyloid β are formed in the brain of Alzheimer’s * To whom correspondence should be addressed. E-mail: einaga@ chem.keio.ac.jp. † Keio University. ‡ The University of Electro-Communications. § Kanagawa Academy of Science and Technology. (1) Dobson, C. M. Nature 2003, 426 (6968), 884–890. (2) Go, N. Annu. Rev. Biophys. Bioeng. 1983, 12, 183–210. 10.1021/ac800394n CCC: $40.75  2008 American Chemical Society Published on Web 07/09/2008

disease patients over many years.3 This protein fold is shared by other peptides such as prions associated with protein misfolding diseases. All known prions induce the formation of an amyloid fold, in which the protein polymerizes into an aggregate consisting of tightly packed β sheets. Prions cause a number of diseases in a variety of mammals, including bovine spongiform encephalopathy (mad cow disease) in cattle and Creutzfeldt-Jakob disease in humans.4 Conformational changes of protein can be induced by a number of chemicals (such as acids, guanidine hydrochloride, urea, calcium chloride), or heat. Urea decreases hydrophobic interactions between nonpolar groups of a protein to destabilize its higher structures. This unfolds the natural three-dimensional structure of the protein into a disordered and randomly coiled form, bringing about changes in many of its physicochemical properties and a loss of its biological activities, such as enzymatic, informational, carrier, and immunological actions. Such a change in the natural three-dimensional form of a protein is called its denaturation. Denaturation involves changes in the native secondary, tertiary, and quaternary structures of a protein because of the disruption of the weak noncovalent bonds stabilizing those higher orders of protein structure. However, the primary structure, consisting of the amino acid sequence of the protein, is not changed because the covalent peptide bonds, joining the amino acids, are not cleaved during denaturation. Denaturation of proteins has been extensively investigated by a number of techniques including nuclear magnetic resonance, UV absorption, electron spin resonance, circular dichroism, birefringence, and fluorescence spectroscopy.5–8 In recent years, electrochemical detection has gained prominence as a sensitive and facile detection technique for electroactive compounds. Electrochemistry has also been applied to the study of protein folding. Metal-containing proteins, such as cytochrome c, exhibit reversible electron-transfer characteristics in their native (3) Emilien, G.; et al. Alzheimer Disease: Neuropsychology and Pharmacology; Birkhauser: Boston, MA, 2004; pp 19-26. (4) Brown, D. R., Ed. Neurodegeneration and Prion Disease; Springer Science: New York, 2005; p 21. (5) Khan, M. Y.; Agarwal, S. K.; Hangloo, S. J. Biochem. 1987, 102 (2), 313– 317. (6) Sanghamitra, N. J. M.; Mazumdar, S. Biochemistry 2008, 47 (5), 1309– 1318. (7) Moya, I.; Silvestri, M.; Vallon, O.; Cinque, G.; Bassi, R. Biochemistry 2001, 40 (42), 12552–12561. (8) Muralidharan, V.; Muir, T. M. Nat. Methods 2006, 3 (6), 429–438.

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state, and many researchers have studied the thermodynamics and kinetics of metalloproteins using electrochemical techniques.9–11 Moreover, applications of protein electrochemistry such as silicon nanotube array electrodes,12 alkanethiol-modified electrodes,13 and electrochemical methods in investigations of enzymes14 have greatly increased in the past few years. But, of course the utility of this approach is limited to proteins with redox-active cofactors. Reynaud et al. and Brabec et al. showed that tyrosine and tryptophan residues are electrochemically oxidizable at carbon electrodes.15,16 Non-metalloproteins have been detected by this electrochemical oxidation of tyrosine and tryptophan residues. Direct electrochemical oxidation of electroactive amino acid residues was utilized in quantitative and conformational analyses.17,18 Recently, electrochemical monitoring of urea-induced unfolding of bovine serum albumin (BSA) using the electron mediator Os(bpy)2dppz was performed.19 However, direct electrochemical detection of conformational changes in relatively large nonmetalloproteins (e.g., albumin) has rarely been reported. The limited number of reports is not only due to the complexity of protein structure but also the strong adsorption and polymerization of proteins on the electrode surface,20 which can lead to signal reduction, unpredictability, and irreproducibility. Boron-doped diamond (BDD) electrodes have attracted much interest due to their superior properties, including low background currents, a wide working potential window, favorable electrontransfer kinetics, and surface inertness, which result in high resistance to deactivation.21,22 Our group has recently reported the electrochemical detection of BSA by direct electrochemical oxidation of tyrosine, tryptophan, and cysteine residues using H-terminated BDD (H-BDD) electrodes.23 H-terminated BDD electrodes are well-faceted, hydrophobic, and have low surface energy, so can be a viable material for the direct oxidation of proteins.24 Herein, we report the results of an investigation into the detection of conformational changes in a non-metalloprotein (albumin) by direct electrochemical oxidation using BDD electrodes. A protein changes its conformation after the addition of 8 (9) Bixler, J.; Bakker, G.; McLendon, G. J. Am. Chem. Soc. 1992, 114 (17), 6938–6939. (10) Yeh, P.; Kuwana, T. Chem. Lett. 1977, 1145–1148. (11) Armstrong, F. A.; Hill, H. A. O.; Walton, N. J. Acc. Chem. Res. 1988, 21 (11), 407–413. (12) Mu, C.; Zhao, Q.; Xu, D. S.; Zhuang, Q. K.; Shao, Y. H. J. Phys. Chem. B 2007, 111 (6), 1491–1495. (13) Ferapontova, E. E.; Ruzgas, T.; Gorton, L. Anal. Chem. 2003, 75 (18), 4841– 4850. (14) Armstrong, F. A. Curr. Opin. Chem. Biol. 2005, 9 (2), 110–117. (15) Reynaud, J. A.; Malfoy, B.; Bere, A. J. Electroanal. Chem. 1980, 116, 595– 606. (16) Brabec, V. J. Electroanal. Chem. 1980, 116, 69–82. (17) Vestergaard, M.; Kerman, K.; Saito, M.; Nagatani, N.; Takamura, Y.; Tamiya, E. J. Am. Chem. Soc. 2005, 127, 11892–11893. (18) Zhang, M. G.; Mullens, C.; Gorski, W. Anal. Chem. 2005, 77 (19), 6396– 6401. (19) Guo, L. H.; Qu, N. Anal. Chem. 2006, 78 (17), 6275–6278. (20) Lehrer, S. S.; Fasman, G. D. Biochemistry 1967, 6, 757–767. (21) Watanabe, T.; Ivandini, T. A.; Makide, Y.; Fujishima, A.; Einaga, Y. Anal. Chem. 2006, 78 (22), 7857–7860. (22) Suzuki, A.; Ivandini, T. A.; Yoshimi, K.; Fujishima, A.; Oyama, G.; Nakazato, T.; Hattori, N.; Kitazawa, S.; Einaga, Y. Anal. Chem. 2007, 79 (22), 8608– 8615. (23) Chiku, M.; Ivandini, T. A.; Kamiya, A.; Fujishima, A.; Einaga, Y. J. Electroanal. Chem. 2008, 612, 201–207. (24) Shin, D.; Tryk, D. A.; Fujishima, A.; Merkoci, A.; Wang, J. Electroanalysis 2005, 17 (4), 305–311.

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M urea in such a way that the number of its electrochemically active amino acid residues accessible for reaction with a BDD electrode is significantly increased. However, electrochemical polymerization of tyrosine and tryptophan residues in proteins may be the most serious damage resulting from direct electrochemical oxidation of proteins.25 Bruins et al. demonstrated electrochemical cleavage of peptides using relatively high potential electrochemical oxidation.26,27 Therefore, we applied high potential oxidation with flow injection analysis (FIA) preventing the deactivation of electrodes for the detection of conformational changes of BSA. EXPERIMENTAL SECTION Chemicals. BSA (MW, 66 000) and other compounds were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). BSA was purified three times by a dialysis method (total 24 h) using Slide-A-Lyzer Dialysis Products (MWCO 10 k, 0.5-3 mL capacity) purchased from Pierce (Rockford, IL). Protein concentrations were determined by measuring the optical density of their solutions at 278 nm. The water used in the experiments was purified using a simple laboratory water system (Millipore) to a specific resistivity of 18.2 M. Methods. The BDD electrodes were deposited on Si(100) wafers in a microwave plasma-assisted chemical vapor deposition system (ASTeX Corp.). The procedure has been described in detail previously.28 A mixed acetone/methanol (9:1, v/v) solution of B2O3 with a B/C atomic concentration ratio of 10 000 ppm was used as the carbon source. The typical grain size of the resulting BDD thin films was up to ∼5 µm with a thickness of ∼20 µm for a deposition time of 7 h using 5-kW plasma power. The electrochemical measurements were conducted using a potentiostat (HZ-100 and HZ-5000, Hokuto Denko) with a standard three-electrode configuration and a single-compartment glass cell. An Ag/AgCl electrode was used as reference electrode, and a Pt wire was used as the counter electrode. The FIA system used in the present study consisted of a thin-layer flow cell (GL Science, Inc.), an amperometric detector (Bioanalytical system LC-4C), a degassing unit (DG660B, GL Science, Inc.), and a data acquisition system (EZ Chrom Elite, Scientific Software). The pump flow rate was 1 mL/min. The interval between each measurement was 40 min. The wall-jet-type flow cell consisted of an Ag/AgCl reference electrode and a stainless steel tube counter electrode, which also served as the tube for the solution outlet. UV-visible absorption spectra were recorded on a V-560 spectrophotometer (Jasco), and fluorescence emission spectra were measured on a FP-6300 luminescence spectrometer (Jasco). RESULTS AND DISCUSSION Figure. 1 shows linear sweep voltammograms (LSV)s using H-BDD electrodes (background current subtracted) of 50 µg/ mL BSA in 0.1 M phosphate buffer solution (PBS) with and without 8 M urea as the denaturating agent. Three separated peaks were observed in each urea solution. Our previous work (25) Marx, K. A.; Zhou, T. J. Electroanal. Chem. 2002, 521 (1-2), 53–60. (26) Permentier, H. P.; Bruins, A. P. J. Am. Soc. Mass Spectrom. 2004, 15 (12), 1707–1716. (27) Permentier, H. P.; Jurva, U.; Barroso, B.; Bruins, A. P. Rapid Commun. Mass Spectrom. 2003, 17 (14), 1585–1592. (28) Ivandini, T. A.; Sarada, B. V.; Terashima, C.; Rao, T. N.; Tryk, D. A.; Ishiguro, H.; Kubota, Y.; Fujishima, A. J. Electroanal. Chem. 2002, 521 (1-2), 117– 126.

Figure 1. Cyclic voltammetry responses of 50 µg/mL BSA in 0.1 M phosphate buffer solution (pH 7.4) at a scan rate of 100 mV s-1 in the absence and presence of 8 M urea at BDD electrode (background current subtracted).

with BSA oxidation showed that the three oxidation peaks of cysteine, tyrosine, and tryptophan were observed at an almost identical potential, 0.75 V in BSA oxidation. Cysteine oxidation can be written as the coupling of disulfied bonds and it is widely accepted.29 Tyrosine oxidation was suggested an irreversible oneelectron and one-proton process.30 Tryptophan oxidation was suggested an irreversible two-electrons and two-protons process.31 In order to confirm the identities of the other redox-active sites of BSA at the BDD electrode, comparison was made between the LSVs of BSA and methionine and glutathione disulfide (GSSG). CV signals were recorded for 1 mM standard solutions of glutathione disulfide (Figure 2a) and methionine (Figure 2b) in 0.1 M PBS, pH 7.4 using BDD electrodes. The tripeptide glutathione contains the amino acid residues of cysteine, glutamine, and glycine. It is found in vivo as both glutathione (GSH) and GSSG. Under oxidative stress, GSH is known to be oxidized to GSSG in living cells. As shown in Figure 2a, GSSG produced two oxidation peak at 0.75 and 1.1 V. The oxidation poteintal at 0.75 V is similar to the oxidation of thiol (cysteine), so it seemed that GSSG contained GSH as an impurity. The oxidation potential at 1.1 V seems to be the oxidation of disulfide. The oxidation current was very low despite the high concentration (1 mM) of GSSG. Direct electrochemical oxidation of GSSG was performed by Fujishima et al. using oxidized BDD electrodes, and H-BDD showed relatively low sensitivity for GSSG.32 They explained it on the basis of the ion-dipole interactions at the electrode surface. As mentioned in our previous reports, we believe that the oxygen functional groups, such as carbonyl or hydroxyl groups, formed on the facets of the diamond microcrystals form a negative dipolar field, which electrostatically attracts the positively charged GSSG molecule. On the other hand, H-termination is required to promote the oxidation reaction of BSA at BDD electrodes. The behavior is in agreement with the properties of BSA in PBS pH 7.4 since the pI of BSA is ∼4.7; the BSA is negatively charged at pH 7.4. Previously, our group has discussed the advantages of H-terminated BDD over the Oterminated one for oxidation of negatively charged molecules. (29) Spataru, N.; Sarada, B. V.; Popa, E.; Tryk, D. A.; Fujishima, A. Anal. Chem. 2001, 73 (3), 514–519. (30) Ivandini, T. A.; Sarada, B. V.; Terashima, C.; Rao, T. N.; Tryk, D. A.; Ishiguro, H.; Kubota, Y.; Fujishima, A. J. Chromatogr., B 2003, 791 (1-2), 63–72. (31) Jin, G. P.; Lin, X. Q. Electrochem. Commun. 2004, 6 (5), 454–460. (32) Terashima, C.; Tata, N.; Rao, B. V.; Sarada, Y.; Kubota, Fujishima, A. Anal. Chem. 2003, 75 (7), 1564–1572.

Figure 2. (a) Cyclic voltammetry responses of a 0.1 M phosphate buffer solution (pH 7.4) at a scan rate of 100 mV s-1 in the absence (dashed line) and presence (solid line) of 1 mM glutathione disulfide at BDD electrode. (b) Cyclic voltammetry responses of a 0.1 M phosphate buffer solution (pH 7.4) at a scan rate of 100 mV s-1 in the absence (dashed line) and presence (solid line) of 1 mM methionine at BDD electrode.

Methionine is known to be an electrochemicaly oxidizable amino acid at high potential.33 As shown in Figure 2b, methionine produced an oxidation potential at 1.2 V. Oxidation of methionine has been speculated to occur with cleavage of the C-S bond to yield methanesulfonic acid and an adsorbed amino acid fragment.34 In conclusion, it was confirmed that the electroactive sites of BSA at the BDD electrode were amino acid residues (cysteine, tryptophan, tyrosine, methionine) and disulfide bonds. After BSA was denatured with 8 M urea, its oxidation signal increased relative to the native protein. This increase in oxidation current likely results from a higher affinity of the denatured structure of the BSA molecule in the bulk solution to the surface, possibly resulting in multilayer adsorption.35 Linear current dependence on the scan rate confirmed that the BSA is surface attached (Figure 3). It indicates that the utility of cyclic voltammetry for the detection of conformational change of BSA is impractical. Given this factor, FIA was used for further experiments to minimize the adsorbing effects of the protein. Urea-induced unfolding of BSA has been studied previously by monitoring the change of its intrinsic fluorescence. As the protein unfolds, the emission intensity of tryptophan residues decreases. To validate our new method, urea-induced denaturation of BSA was monitored by both fluorescence and FIA. Unlike (33) Metal ions in biological systems; Sigel, H., Sigel, A., Eds.; CRC Press: New York, 1991; Vol. 27, p 36. (34) Reynaud, J. A.; Malfoy, B.; Canesson, P. J. Electroanal. Chem. 1980, 114 (2), 195–211. (35) Jackson, D. R.; Omanovic, S.; Roscoe, S. G. Langmuir 2000, 16 (12), 5449– 5457.


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Figure 3. Peak currents for 50 µg/mL BSA using CVs at 0.75 V in 0.1 M phosphate buffer solution (pH 7.4) as a function of varying potential sweep rate.

Figure 4. (O) Intrinsic fluorescence emission intensity of 50 µg/mL BSA in 0.1 M phosphate buffer solution (pH 7.4) as a function of urea concentration. Excitation wavelength, 280 nm; emission wavelength, 290-500 nm. (0) Reciprocal of the oxidation current of 50 µg/mL BSA in 0.1 M phosphate buffer solution (pH 7.4) as a function of urea concentration. The current is corrected by FIA using BDD electrodes. Oxidation potential is 1300 mV vs Ag/AgCl. Flow rate is 1 mL/min.

fluorescence, the electrochemical signal increased at high urea concentrations. Because BSA changes its conformation, the number of its electrochemicaly oxidizable amino acid residues accessible for the reaction with BDD electrode is significantly increased. To make a better comparison between fluorescence and electrochemistry, the reciprocal of the oxidation current is used as an indicator for the state of protein unfolding and is plotted. Figures 4 and 5 show the changes of fluorescence intensities and reciprocals of oxidation currents by FIA using BDD electrodes at several oxidation potentials as a function of urea concentration. The background current was subtracted before the inverse of the current was calculated. When developing an assay involving an electrochemical sensor, it is necessary to select an operating potential that is high enough to drive the redox reaction of interest and decrease adsorption of proteins, but low enough to avoid oxidization of the BDD surface. Three denaturation curves for oxidation potential at 1100, 1200, and 1400 mV showed limited similarity between the electrochemical and fluorescence traces, but the oxidation potential at 1300 mV showed strong similarity. Surface fouling on the BDD electrode due to a buildup of adsorbed reaction products was observed during BSA oxidation at 1100 and 1200 mV, and the oxidation current decreased during the oxidation of denatured BSA. The oxidation of the BDD surface itself was caused because of the higher applied potential at 1400 mV versus 5786


Figure 5. (a-c) (O) Intrinsic fluorescence emission intensity of 50 µg/mL BSA in 0.1 M phosphate buffer solution (pH 7.4) as a function of urea concentration. Excitation wavelength, 280 nm; emission wavelength, 290-500 nm. (0) Reciprocal of the oxidation current of 50 µg/mL BSA in 0.1 M phosphate buffer solution (pH 7.4) as a function of urea concentration. The current is corrected by FIA using BDD electrodes. Oxidation potentials are (a) 1100, (b)1200, and (c)1400 mV vs Ag/AgCl.

Ag/AgCl.36 It means the hydrogen-terminated surfaces were kept below 1300 mV. In contrast, oxidation of BSA at 1300 mV using BDD electrodes indicated its inertness to adsorption of oxidation products and kept the hydrogen-terminated surface during FIA at least for 20 h. Bruins et al. suggested that oxidation of tyrosine and tryptophan can give rise to peptide bond cleavage at their C-terminal side, and the presence of disulfide bonds was expected to be a major cause of lack of cleavage products for some proteins.26,27 Disulfide bonds were oxidized at 1.1 V on H-BDD electrodes (shown in Figures 1 and 2). For this reason, oxidation of BSA at higher potential avoids the adsorption of BSA on BDD electrodes. The similarity of the pattern displayed in the two (36) Yagi, I.; Notsu, H.; Kondo, T.; Tryk, D. A.; Fujishima, A. J. Electroanal. Chem. 1999, 473 (1-2), 173–178.

Figure 6. Signal responses of BSA using flow injection analysis with a BDD electrode as the detector in the concentration range 50-400 µg/mL. (O) The applied potential was 800 mV without urea. (b) The applied potential was 800 mV with 8 M urea. (0) The applied potential was 1300 mV without urea, (9) The applied potential was 1300 mV with 8 M urea. The mobile phase was a 0.1 M phosphate buffer solution at pH 7.4. The flow rate was 1 mL/min. Table 1. Slopes for the Calibration Curves in Figure 6

urea 0 M urea 8 M

800 mV

1300 mV

(2.73 ± 0.13) × 10-3 (2.43 ± 0.32) × 10-3

1.17 ± 0.11 3.18 ± 0.24

denaturation curves suggests that direct oxidation of proteins using BDD electrodes at high (1300 mV) potential under FIA conditions can be employed as a new tool for the study of the unfolding processes of cofactor-free proteins. Quantitative analysis of BSA was carried out using an oxidation potential of 800 mV. Figure 6 shows a comparison of the amperometric responses for BSA with FIA using BDD electrodes at different oxidation potentials, 800 and 1300 mV, and different urea concentrations, 0 and 8 M. The amperometric responses at 800 mV show a linear function with almost the same slopes for the native and denatured states in the concentration range 50-400 µg/mL of BSA (Table. 1). It means that protein denaturation was

not detected with an oxidation potential of 800 mV. On the other hand, the oxidation current using an oxidation potential of 1300 mV shows a linear function in the same concentration range but shows different slopes for the native and denatured states of BSA (Table 1). These phenomena suggest that FIA using BDD electrodes with an oxidation potential at 800 mV can determine the quantity of proteins independent of native or denatured state, and FIA with oxidation potential of 1300 mV can detect the conformational change of proteins. Furthermore, the oxidation current at 1300 mV was 1000-fold higher than that at 800 mV. High-potential oxidation of BSA using BDD electrodes kept the electrode surface active and clean, so the sensitivity for proteins was higher than for low-potential oxidation methods. The detection limit at oxidation potential 1300 mV was ∼190 ng/mL (2.89 nM) BSA (S/N ) 3), and it is ∼1000fold smaller than previous work. CONCLUSION To the best of our knowledge, BSA conformational change detection using direct electrochemical oxidation was performed successfully for the first time using a BDD electrode at high potential. The analysis of such solutions, in conjunction with voltammetric data, suggests that the electrode process involves the oxidation of BSA’s tyrosine, tryptophan, cysteine, methionine, and disulfides. The analytical performance of FIA using H-BDD electrodes indicates that this can be useful as a sensitive amperometric detector for relatively large, nonmetalloproteins such as BSA. Furthermore, the detection limit of quantitative analysis was also improved to 190 ng/mL, 1000-fold lower than at the low oxidation potential (800 mV). Furthermore, the BDD electrodes have the potential to simplify the analysis of other proteins that contain at least one redox-active amino acid such as tyrosine, tryptophan, cystein, methionine, and disulfide. Received for review February 26, 2008. Accepted June 4, 2008. AC800394N


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Conformational Change Detection in Nonmetal Proteins by Direct

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