Studies of Electrochemically Transformed Ferritin Adsorbed at Tin

These data reveal that ferritin adsorbs onto ITO from solution at controlled electrode ..... the i-E signature of ITO/ferritin would be similar to tha...
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Langmuir 2003, 19, 5808-5812

Studies of Electrochemically Transformed Ferritin Adsorbed at Tin-Doped Indium Oxide Electrodes Using X-ray Photoelectron Spectroscopy Kevin C. Martin,† Stephanie M. Villano,† Patrick R. McCurdy,‡ and Donald C. Zapien*,† Department of Chemistry, University of Colorado at Denver, Denver, Colorado 80217, and Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523 Received January 29, 2002. In Final Form: April 30, 2003 X-ray photoelectron spectroscopy (XPS) was used to (1) confirm the presence of ferritin on tin-doped indium oxide (ITO) as suggested by voltammetry, (2) determine whether ferritin desorbs upon reduction, (3) verify whether iron is released when ferritin is reduced in the presence of an iron-chelating agent, and (4) ascertain whether the protein-iron complex could be electrochemically reconstituted. The N 1s signal increases dramatically after the ITO electrode is exposed to ferritin. Substantial decreases in the Fe/N peak ratio support the hypothesis that iron release can be electrochemically induced by reducing ferritin in the presence of an iron-chelating agent. These data reveal that ferritin adsorbs onto ITO from solution at controlled electrode potential and that ferritin can be electrochemically induced to release iron without the need for homogeneous reducing agents. However, following the exposure of an ITO/ferritin (“emptied”) to ferrous ion at positive electrode potential, the XPS spectrum reveals a substantial increase in the iron signal and a decrease in substrate signals. These results indicate that iron deposits directly onto the ITO substrate, casting doubt on the previous conclusion that adsorbed ferritin can be electrochemically reconstituted.

Introduction Ferritin is a protein found in most organisms and functions principally to sequester excess iron from the cell. Ferritin has a diameter of 12.0 nm and is composed of a 2.0 nm thick protein sheath surrounding a “ferrihydrite” mineral core.1 The protein sheath is composed of 24 individual subunits assembled to give a roughly spherical quaternary structure. A schematic representation is found in ref 1. The 3-fold sites of contact form the hydrophilic channels through which iron enters the protein.2 The mechanism of iron uptake is not wellunderstood; however it is known that the process involves an oxidation step.3 In vitro experiments have suggested that once the core iron is reduced, iron can exit the protein shell.4 Thus, an understanding of ferritin’s redox reactions is an important component to elucidating the mechanisms of iron loading and unloading. In previous work, the voltammetry of ferritin adsorbed on tin-doped indium oxide (ITO) electrodes revealed that ferritin is electroactive in the adsorbed state.5 The experimental results suggested that iron can be removed from the ferritin core by reducing ferritin in the presence of an iron-chelating agent and that the iron-protein complex can be electrochemically reconstituted by holding an ITO/ferritin electrode at an oxidizing potential in the presence of ferrous ion.6 Whereas these results are * To whom correspondence should be addressed. † University of Colorado at Denver. ‡ Colorado State University. (1) Ford, G. C.; Harrison, P. M.; Rice, D. W.; Smith, J. M. A.; Treffery, A.; White, J. L.; Yariv, J. J. Philos. Trans. R. Soc. London, Ser. B 1984, 304, 551-565. (2) Thiel, E. C. Annu. Rev. Biochem. 1987, 56, 289-315. (3) Harrison, P. M.; Hoy, T. G.; Macara, I. G.; Hoare, R. J. Biochem. J. 1974, 143, 445-451. (4) Topham, R.; Goger, M.; Pearce, K.; Schultz, P. Biochem. J. 1989, 261, 137-143. (5) Cherry, R. J.; Bjornsen, A. J.; Zapien, D. C. Langmuir 1998, 14, 1971-1973.

promising, it is necessary to corroborate these findings by employing a technique that can provide direct evidence for the presence of both protein and iron on the ITO surface. X-ray photoelectron spectroscopy (XPS) has been a useful technique to study a wide variety of adsorbed proteins including collagen,7 fibronectin,8 albumin,9 lysozyme,10 γ-globulins,11,12 legumin,13 cytochrome c,14-16 azurin,17 hemoglobin,18 and horseradish peroxidase.19 The capability of XPS to yield information about the elemental composition of a surface has made the technique useful for studying proteins on conducting and nonconducting surfaces with minimal beam damage. XPS has been used to monitor protein concentration as a function of time,20 (6) Pyon, M.-S.; Cherry, R. J.; Bjornsen, A. J.; Zapien, D. C. Langmuir 1999, 15, 7040-7046. (7) Defreˆne, Y. F.; Marchal, T. G.; Rouxhet, P. G. Appl. Surf. Sci. 1999, 144-145, 638-643. (8) Amedeu Do Serro, A. P. V.; Fernandes, A. C.; De Jesus Vieira Saramago, B. Biomed. Mater. Res. 2000, 49, 349-352. (9) Gama, F. M.; Mota, M. Biocatal. Biotransform. 1997, 15, 237250. (10) Garrett, Q.; Chatelier, R. C.; Griesser, H. J.; Milthorpe, B. K. Biomaterials 1998, 19, 2175-2186. (11) McArthur, S. A.; McLean, K. M.; Kingshott, P.; St. John, H.; Chatelier, R. C.; Griesser, H. J. Colloids Surf., B 2000, 17, 37-48. (12) Wagner, M. S.; Horbett, T. A.; Castner, D. G. Langmuir 2003, 19, 1708-1715. (13) Lebugle, A; Subirade, M.; Gueguen, J. Biochim. Biophys. Acta 1995, 1248, 107-114. (14) Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564-6572. (15) Jiang, L.; Glidle, A.; Griffith, A.; McNeil, C. J.; Cooper, J. M. Bioelectrochem. Bioenerg. 1997, 42, 15-23. (16) Sophia, S. J.; Premnath, V.; Merrill, E. W. Macromolecules 1998, 31, 5059-5070. (17) Chi, Q.; Zhang, J.; Nielsen, J. U.; Friis, E. P.; Chorkindorff, F.; Canters, G. W.; Anderson, E. T.; Ulstrup, J. J. Am. Chem. Soc. 2000, 122, 4047-4055. (18) Paynter, R. W.; Ratner, B. D.; Horbett, T. A.; Thomas, H. R. J. Colloid Interface Sci. 1984, 101, 233-245. (19) Fitspatrick, H.; Luckham, P. F.; Eriksen, S.; Hammond, K. J. Colloid Interface Sci. 1992, 149, 1-9. (20) Kuboki, Y.; Teroka, K; Okada, S. J. Dent. Res. 1987, 66, 10161019.

10.1021/la020098q CCC: $25.00 © 2003 American Chemical Society Published on Web 06/12/2003

Ferritin Adsorbed on ITO Electrodes

surface type,16,21-22 the presence of competing proteins23 and denaturing agents,24 and dissolved protein concentration.25 The organization,7,12,14,17 the type of bonding to the surface,14-15,17 and the thickness of protein films19 have been studied to shed light on the nature of protein surface chemistry. Of particular note, XPS, atomic force microscopy, and contact angle measurements were used by Caruso and co-workers to characterize ferritin layers on bare gold.26 Previous studies have shown that ITO is a sufficiently conductive material for XPS analysis.27,28 The studies mentioned above support the suitability of XPS to probe the composition of the ITO/ferritin surface. In this work, high-resolution XPS spectra of ITO/ferritin electrodes were collected before and after various electrochemical transformations to determine whether ferritin adsorbs on the ITO surface and whether the processes of iron release and uptake are induced electrochemically. The N 1s signal was measured as the most reliable indication of the relative coverage of protein on the surface.26 The Fe 3p signal was used to measure the relative amount of iron. The 3p transition was monitored, instead of the more commonly used Fe 2p transition, due to the significant and varying interference of both the In 3p1/2 (703 eV) and Sn 3p3/2 (715 eV) with the Fe 2p (711, 724 eV) peaks. In addition, the In 3d5/2 signal was monitored to detect relative changes in exposed ITO substrate. Experimental Section Cyclic voltammetry was performed with a Cypress model Omni 90 potentiostat (Lawrence, KS) and recorded on a BioAnalytical systems model RXY recorder (West Lafayette, IN). The electrochemical cells used in this study are described elsewhere.29 A Flex-Column gravity liquid chromatography column was purchased from Kontes Glass Co. (Vineland, NJ) and used for the size exclusion chromatographic purification of ferritin and apoferritin. A Perkin-Elmer (Norwalk, CT) Lambda 552 UVvisible spectrophotometer was used for the determination of ferritin concentration. XPS analysis was performed using a Physical Electronics model PHI 5800 spectrometer. This system was equipped with a monochromatic Al KR X-ray source (1486.6 eV), a hemispherical analyzer, and a resistive multichannel detector. A low-energy (5 eV) electron gun was used for charge neutralization. The composition of the samples was determined from 0 to 1000 eV using survey scans acquired at an analyzer pass energy of 187.5 eV. The high-resolution spectra were collected at a pass energy of 23.5 eV, with 0.1 eV steps, at a 45° takeoff angle. XPS scans were collected following different stages of electrochemical transformation of adsorbed ferritin. The ITO/ferritin electrode was removed from the electrochemical cell and rinsed with purified water prior to placing the electrode into the XPS chamber. The XPS chamber was evacuated to a pressure of 10-9 Torr or lower before collecting XPS spectra. The N 1s, Fe 3p, and (21) Endo, K.; Sachdera, R.; Araki, Y.; Ohno, H. Proceedings of the First International Conference on Shape Memory and Inelastic Technologies; SMST International Committee: 1995; pp 197-201. (22) Schakenraad, J. M.; Van Der Mei, H. C.; Rouxhet, P. G.; Busscher, H. J. Cell Biophys. 1992, 20, 57-67. (23) Lee, J. H.; Kopeckova, P; Kopecek, J; Andrade, J. D. Biomaterials 1990, 11, 455-464. (24) Klomp, A. J. A.; Engbers, G. H. M.; Mol, J.; Terlingen, J. G. A.; Feijen, J. Biomaterials 1999, 20, 1203-1211. (25) Lhoest, J.-B.; Detrait, E.; Van Den Bosch De Aguilar, P.; Bertand, P. J. Biomed. Mater. Res. 1998, 41, 95-103. (26) Caruso, F.; Furlong, D. N.; Kingshott, P. J. Colloid Interface Sci. 1997, 186, 129-140. (27) Yan, C.; Zharnikov, M.; Golzhauser, A.; Grunze, M. Langmuir 2000, 16, 6208-6215. (28) Nelson, A. J.; Aharoni, H. J. Vac. Sci. Technol., A 1987, 5, 231233. (29) Martin, T. D.; Monheit, S. A.; Niichel, R. J.; Peterson, S. C.; Campbell, C. H.; Zapien, D. C. J. Electroanal. Chem. 1997, 420, 279290.

Langmuir, Vol. 19, No. 14, 2003 5809 In 3d5/2 peaks were used to monitor relative amounts of nitrogen, iron, and indium, respectively. Typically 15 spectra per element of interest were signal averaged to give the resultant highresolution spectrum. The Shirley background correction was applied to all spectra, and binding energies were corrected against a C 1s energy of 285.0 eV. Elemental percentages were calculated from peak areas and relative sensitivities. Phenylmethylsulfonyl fluoride (PMSF) (99.0%) and sodium azide (99.9%) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Bovine serum albumin (fraction 5 powder), HEPES (99.5%), horse spleen ferritin (>85%), horse spleen apoferritin (>85%), ferrous sulfate heptahydrate (99% minimum), and phenanthroline monohydrate (99% minimum) were obtained from Sigma Chemical Co. (St. Louis, MO). EDTA (disodium salt) was purchased from Thiokol (Davis, MA), and Bio-Rad Protein Assay G-250 Dye from Bio-Rad Laboratories (Hercules, CA). Sephadex G-200 (protein fraction range, 5000-600 000 Da) and NAP-5 columns (containing medium DNA grade G-25 Sephadex) were obtained from Pharmacia Biotech (Piscataway, NJ). All chemicals with the exception of ferritin and apoferritin were used without further purification. Water was purified by distillation though a heated platinum catalyst in the presence of oxygen.30 ITO on glass (Applied Films Corp., Longmont, CO) was cut into 6 × 10 mm pieces and then cleaned by sonication in a saturated solution of Alconox in 95% ethanol for 10 min.31 The electrodes were rinsed and sonicated twice, for 10 min each, in pyrolytically distilled water, and soaked for at least 24 h in purified water prior to use. In all experiments, the lower 6 × 6 mm portion of the ITO electrode was immersed into the sample solution. Each electrode was clipped on the top 4 mm portion and suspended by an electrical lead wire. The bare ITO electrodes were soaked in pH 7.1 phosphate buffer (µ ) 1 M) for 60 h prior to XPS analysis. Ferritin and apoferritin were purified using size exclusion chromatography. A Flex-Column with dimensions 2.5 cm by 50 cm was packed to approximately 15 cm high with a gel filtration medium of Sephadex G-200 and equilibrated with a G-200 buffer (20 mM pH 7.0 phosphate buffer, 0.9% NaCl, 0.2 mM PMSF, and 0.05% NaN3). Collected fractions of ferritin were combined and passed though a NAP-5 column to remove non-ferritin iron. Ferritin and apoferritin concentrations were determined by the Bradford method.32 ITO electrodes were soaked in a solution of 0.1 mg/mL ferritin or apoferritin in a pH 7.2 phosphate buffer (µ ) 1 M) for a period of 60 h at room temperature. Current-potential curves of ITO/ferritin electrodes were obtained as follows. The ITO/ferritin electrodes were rinsed free of dissolved ferritin with purified water, clipped to the lead wire, and immersed in a deaerated pH 7.2 phosphate buffer (µ ) 1 M). The potential was then cycled nominally between 0.20 and -0.90 V (versus Ag/AgCl) at 100 mV/s. Prior to XPS analysis, the potential was cycled once between 0.20 and -0.80 V. For the pH dependence study, current-potential curves were scanned in solutions of deaerated phosphate buffers of varying pH in the range of 5.0-8.0. In the iron release experiments, the potential of an ITO/ferritin electrode was immersed into 10 mM EDTA in phosphate buffer (µ ) 1 M) at 0.20 V. The potential was cycled once between 0.20 and -0.80 V, and the electrode was removed from solution and rinsed with purified water. The reactivity of adsorbed ferritin toward an iron-chelating agent, under nonreducing conditions, was accomplished by soaking the ITO/ferritin electrodes in 10 mM EDTA in pH 7.0 phosphate buffer at open circuit potential for 22 h, rinsing with purified water, and scanning the current-potential curve as described above. The electrochemically induced iron uptake trial was performed as follows: The ITO/ferritin electrode was first immersed in 10 mM 1,10-phenanthroline in pH 7 phosphate buffer (µ ) 1 M) at 0.20 V. The potential was scanned to -0.80 V and held at that value for 1 min, and then the electrode was removed from the (30) Conway, B. E.; Angerstein-Kozlowska, H.; Sharp, W. B. A.; Criddle, E. E. Anal. Chem. 1973, 45, 1331. (31) Bowden, E. F.; Hawkridge, F. M.; Blount, H. N. J. Electroanal. Chem. Interfacial Electrochem. 1984, 161, 355-376. (32) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254.

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Table 1. Relative Surface Elemental Percentages of In, N, and Fe for Various Electrochemical Transformationsa sample

% In

%N

% Fe

ITO clean ITO/ferritin ITO/ferritin (cycled once) ITO/apoferritin ITO/ferritin (emptied) ITO/ferritin (reconstituted)

37 5.4 3.5 34 15 0.4

0 6.5 5.9 1.1 5.0 5.1

0 5.9 5.3 0 2.6 8.2

Fe/N 0.91 0.90 0.52 1.6

a The % N in the bare ITO samples served as the background for nitrogen and was subtracted from the % N in each experiment.

Figure 2. Peak shape analysis of the Fe 3p signal from adsorbed ferritin. The high-resolution spectrum is the average of 15 scans.

Figure 1. High-resolution XPS spectra of bare ITO and ITO/ ferritin electrodes. (A) Bare ITO electrodes were immersed in pH 7 phosphate buffer (µ ) 1 M) for 60 h and rinsed prior to XPS analysis. (B) Bare ITO electrodes were immersed in 0.1 mg/mL ferritin in pH 7.2 phosphate buffer (µ ) 1 M) for 60 h. Each spectrum is the average of 15 scans. cell. The ITO/ferritin (“empty”) electrode was rinsed with purified water and immersed in 0.10 mM ferrous sulfate in pH 7.0 HEPES buffer (0.5 M HEPES and 1.0 M NaCl), at 0.20 V for 20 min. The electrode was removed from solution, rinsed with purified water, and placed into the XPS spectrometer. To examine the voltammetry of an ITO electrode exposed to iron, a clean ITO electrode was immersed at 0.20 V, in deaerated 0.10 mM ferrous sulfate in pH 7.0 HEPES buffer (0.5 M HEPES and 1.0 M NaCl), for 20 min. The electrode was rinsed with purified water and reimmersed in deaerated pH 7.0 phosphate buffer, and the current-potential curve was scanned between 0.20 and -0.90 V at 100 mV/s. In a pH dependence study, the current-potential curves were scanned in solutions of deaerated phosphate buffers of varying pH in the range of 5.0-8.0. The reactivity of an ITO/iron electrode toward an iron-chelating agent was examined in the same manner described above for ITO/ ferritin.

Results and Discussion The percentages of In, N, and Fe and the Fe/N ratios for the different electrochemical transformations of adsorbed ferritin are summarized in Table 1. ITO/ferritin electrodes were rinsed free of dissolved ferritin and evacuated, and the XPS spectra were collected. The highresolution spectra, highlighting the In 3d5/2, N 1s, and Fe 3p peaks, for bare ITO and ITO/ferritin are shown in parts A and B of Figure 1, respectively. The spectra show a dramatic increase in the N 1s and Fe 3p compared to that of bare ITO. This change, in addition to the attenuation of the substrate (In 3d5/2 peak), confirms that ferritin is adsorbed on ITO. Although the cross sections for N 1s and Fe 3p are relatively small,33 the peak intensities of these signals are easily measured because of the large amounts of N and Fe present in the ferritin layer. The N 1s signal does not show a chemical shift due to the nitrogen in amide linkages versus the nitrogen in the amine side chains of (33) Czanderna, A. W. Methods of Surface Analysis; Elsevier Scientific: New York, 1975.

Figure 3. Peak shape analysis of the C 1s and O 1s signals from adsorbed ferritin. The high-resolution spectra are the average of 15 scans. (a) O 1s spectrum of bare ITO. (b) C 1s spectrum of bare ITO. (c) O 1s spectrum of ITO/ferritin. (d) C 1s spectrum of ITO/ferritin.

the lysine and arginine residues. The peak shape analysis for the Fe 3p signal, shown in Figure 2, reveals the contributions of the Fe 3p1/2 and Fe 3p3/2 transitions at 57.76 and 55.73 eV, respectively, but there is no evidence of an oxidation state other than Fe(III). The Fe 2p3/2 (not shown) at 711.86 eV has a binding energy characteristic of an iron oxide of the type (FeOOH).34 Parts a and c of Figure 3 give the O 1s high-resolution spectra for bare ITO and ITO/ferritin electrodes, respectively. The O 1s signal at 530.5 eV is assigned to In2O3, while the signals at 531.4 and 532.0 are probably due to the oxygen in carbonyl and carbohydroxy-bearing contaminants on the ITO surface. Following adsorption of ferritin, the O 1s signal at 530.5 is attenuated by the ferritin layer, as expected. In the O 1s band of the ITO/ ferritin electrode, there is signal intensity at lower binding energy (530.0 eV) that is not present in the bare ITO spectrum, which can be attributed to the oxygen from the iron oxide core. Parts b and d of Figure 3 show the C 1s spectra for ITO and ITO/ferritin, respectively. The C 1s signal on bare ITO is due to adventitious carbon. The peak at 285.0 eV on bare ITO is assigned to hydrocarbons; the peak at 286.1 eV is probably due to carbohydroxy groups, while the signal at 288.3 eV is likely from carbonyl groups. The C 1s binding energies from the ITO/ferritin electrodes are similar, with the exception that the peak (34) Moulder, J. F.; Stickle, W. F.; Sobol, P. E.; Bomben, K. D. Handbook of X-ray Photoelectron Spectroscopy; Physical Electronics, Inc.: Eden Prairie, MN, 1995.

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Figure 4. Current-potential curve of an ITO/ferritin electrode. The ferritin layer was prepared as in Figure 1. Electrode area, 0.36 cm2; electrolyte, pH 7.0 phosphate buffer (µ ) 1 M); scan rate, 100 mV/s.

Figure 5. Current-potential curve of ITO/ferritin scanned in 10 mM EDTA in pH 7.0 phosphate buffer (µ ) 1 M). The ferritin layer was prepared as in Figure 1. Electrode area, 0.36 cm2; scan rate, 100 mV/s.

at 286.4 eV is probably due to a contribution by the R-carbons in the protein backbone. The peak area ratio of the amide type carbon to R-type carbon approaches unity. The i-E curve of an ITO/ferritin electrode in pH 7.0 phosphate buffer is shown in Figure 4. The peak at -0.62 V represents the reduction of the adsorbed protein layer. It is unlikely that this peak is due to the reduction of free iron in the ferritin sample because the free iron was removed when the sample was passed through a G-25 Sephadex column. An anodic peak appears at -0.20 V, and a new cathodic peak occurs at -0.36 V. The more positive midpoint potential and faster kinetics of the new electrochemical couple are indications that significant changes had occurred in the adsorbed protein layer as a result of electrochemical reduction. A more detailed discussion of what these changes might entail is discussed elsewhere.5 In addition to the kinetic and the thermodynamic changes, the electrolytic charge represented by the anodic peak or new cathodic peak is substantially smaller than that of the initial cathodic process. The disparity in the charges is not clear and is the subject of another study. XPS spectra of ITO/ferritin electrodes were collected after cycling the potential once between 0.20 and -0.80 V. The Fe 3p and N 1s signals are both approximately 10% smaller than those before potential cycling, but the Fe/N signal ratio remains the same. These results may suggest that some ferritin desorbs with electrochemical cycling. On the other hand, the protein may have assumed a conformation that is more compact as a result of the electrochemical change in the iron core. In either case, the results suggest that iron remains in the protein core, after its initial reduction, since the Fe/N ratio does not change. The overall inference that can be made is that the substantial decrease in electrolytic charge following the initial reduction is probably due to a loss of electroactivity of adsorbed ferritin rather than an electrochemically induced release of iron. Figure 5 is the i-E curve of an ITO/ferritin electrode in the presence of EDTA. It has been suggested that the absence of the anodic and new cathodic peaks is due to the release of iron from the protein and the subsequent complexation of the iron by EDTA.6 The N 1s signal decreases by 23%, and the Fe 3p signal decreases by 56%, from their values prior to the reduction of the ferritin layer in the presence of EDTA. In addition, the substrate signals increased relative to those in the ITO/ferritin spectrum. The decrease in the % N may be due to the desorption of the protein during or following iron release

since considerable space on the ITO surface had been surrendered. In a control experiment, ITO electrodes were exposed to apoferritin under the same conditions as those used for dosing ITO with ferritin. The XPS spectrum shows that the N 1s contribution by apoferritin is 17% of that of ferritin and that the In 3d5/2 signal is only slightly attenuated compared with that of bare ITO. These results indicate that apoferritin does not adsorb on ITO as readily as ferritin and suggest that the presence of iron within the protein influences the adsorption properties of the protein. The presence of the N and Fe following the scan of ITO/ferritin in EDTA may be due to the possibility that not all of the adsorbed ferritin releases iron following reduction. In earlier work, it was suggested that adsorbed ferritin exists in more than one state.5 Perhaps only some adsorbed states readily release iron when reduced, while other states do not. The possibility of different adsorption states possessing different iron release kinetics is the subject of another study. The fact that protein still remains on the surface following iron release does not preclude the possibility of a desorption process. Desorption may be occurring but is kinetically slow. It has been previously reported that ferritin can be electrochemically reconstituted by exposing ITO/ferritin (“emptied”) to ferrous ion at oxidizing potentials.6 An ITO/ ferritin electrode was emptied of its iron and then placed into 0.10 mM ferrous sulfate in pH 7 HEPES buffer, at 0.20 V for 20 min. The electrode was removed from solution and rinsed, and the XPS spectrum was recorded. The Fe 3p signal is higher than that observed with ITO/ferritin, as expected, if ferritin had been reconstituted. However, the In 3d5/2 signal in the spectrum of the ITO/ferritin (“reconstituted”) electrode is greatly attenuated compared with that of the ITO/ferritin (emptied) electrode, suggesting that iron is depositing directly on the ITO surface. In a control experiment, a bare ITO electrode was exposed to ferrous ion in the same manner as the ITO/ ferritin (emptied) electrode. The i-E curve of the ITO/ ferritin (reconstituted) electrode is shown in Figure 6a, while that of the ITO/Fe electrode is shown in Figure 6b. The initial cathodic peak potentials are identical at -0.57 V, suggesting similar reactivities of the initial layer, and the initial cathodic peak area for each electrode is large, reflecting large amounts of deposited iron. While it is not clear from the data in the uptake experiment that ferritin is being reconstituted, it appears that iron is depositing directly on the ITO surface, probably in the spaces between the ferritin molecules. The general similarity of the i-E curves of ITO/ferritin (Figure 4) and ITO/Fe (Figure 6b) may be an indication

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Figure 6. (A) Current-potential curve of ITO/ferritin (reconstituted). An ITO/ferritin electrode was scanned in the presence of EDTA, as in Figure 4. The electrode was then rinsed and immersed in a solution of 10-4 M ferrous sulfate in 0.50 M HEPES/1.0 M NaCl for 20 min at 0.20 V. The electrode was then rinsed and scanned under the same conditions as in Figure 3. (B) Current-potential curve of ITO/Fe. The electrode was treated with 10-4 M ferrous sulfate and scanned under the same conditions as in Figure 5.

that one is observing the electrochemical reactions of the same species. That is, the voltammetric signatures are similar enough to raise the question as to whether iron remains in the protein sheath when ferritin adsorbs onto the electrode or if iron is released as a result of ferritin undergoing extensive unfolding. If the latter were the case, the i-E signature of ITO/ferritin would be similar to that of the ITO/Fe electrode. It has been shown that upon adsorption proteins can undergo various degrees of unfolding.35,36 However, ferritin is known to stay intact at temperatures as high as 80 °C and does not fully degrade until the pH is as low as 3 or as high as 10.37,38 Given its robust nature, ferritin would not be expected to undergo extensive unfolding. It is important, however, to ascertain that the voltammetric signature of adsorbed ferritin is due to iron inside the ferritin core rather than iron which has merely been expelled onto the ITO surface. A pH dependence in the reactivity can sometimes be used to distinguish between two species. One of the notable electrochemical properties of ferritin is that two protons are transferred for every electron transferred.39,40 Adsorbed ferritin exhibits the same pH dependence as

Martin et al.

dissolved ferritin.23 A voltammetric experiment, in which the pH was varied, showed that both ITO/ferritin and ITO/Fe exhibited about a 120 mV change in initial cathodic peak potential per pH unit, though the dependence of ITO/Fe is offset in potential by approximately 100 mV. This result alone does not distinguish the two systems. The iron contained in ferritin is an iron oxide approximating a ferrihydrite structure.2,41 It is possible that iron adsorbed directly on ITO is also an iron oxide given the similarities in its voltammetry with that of ITO/ ferritin. XPS spectroscopic results support this hypothesis. The Fe 2p3/2 peak (not shown) of the ITO/ferritin (reconstituted) electrode spectrum has a binding energy (711.9 eV) characteristic of an FeOOH type iron oxide. In the case of ITO/Fe, the iron is certainly located directly on the surface. If the iron in ITO/ferritin resides inside a protective protein sheath, then the reactivities of iron in ITO/ferritin versus ITO/Fe toward an iron-chelating agent should be different. Exposure of an ITO/Fe electrode to 10 mM EDTA in pH 7.0 phosphate buffer, at open circuit potential for 22 h, results in a dramatic reduction in the area of the voltammetric peaks indicating that iron had reacted with EDTA. However, the voltammetric peaks of ITO/ferritin electrodes under similar conditions were not reduced in size, indicating that the iron on the ITO/ferritin electrode was protected by the protein coat and that ferritin in the adsorbed state remained intact. Although the i-E curves of ITO/Fe and ITO/ferritin suggest similar electrochemical behavior, the differences in reactivity toward EDTA clearly indicate that they are different species. Conclusions Horse spleen ferritin adsorbs from solution, at controlled potential, on ITO surfaces. However, apoferritin does not adsorb as readily as ferritin, a behavior that may hold information about the folding free energy properties of ferritin. Following the electrochemical reduction of ferritin in the presence of an iron-chelating agent, iron is released from the protein core, accompanied by desorption of ferritin. Ferritin adsorbs onto ITO as the intact proteiniron complex; that is, iron is not released upon adsorption. Exposure of emptied ferritin to ferrous ion at oxidizing potentials results in the deposition of an iron species, probably an iron oxide, on regions of the ITO surface between ferritin molecules. This casts doubt on the conclusion from previous studies that ferritin adsorbed on ITO can be electrochemically reconstituted. Acknowledgment. The authors gratefully acknowledge the National Science Foundation (Grant Number CHE-0070875) for support of this work. The authors thank Applied Films Incorporated (Longmont, CO) for donating the tin-doped indium oxide. The authors also acknowledge Professor Edmond F. Bowden at North Carolina State University for his helpful suggestions. LA020098Q

(35) Sagara, T.; Niwa, K.; Sone, A.; Hinnen, C.; Niki, K. Langmuir 1990, 6, 254-262. (36) Collinson, M.; Bowden, E. F.; Tarlov, M. J. Langmuir 1992, 8, 1247-1250. (37) Theil, E. C. In Advances in Inorganic Biochemistry; Theil, E. C., Eichhorn, G. L., Marzilla, L. G., Eds.; Elsevier: New York, 1983; Vol. 5, pp 1-38. (38) Feder, J.; Giaever, I. J. Colloid Interface Sci. 1980, 78, 144.

(39) Yang, X.; Chen-Barret, Y.; Arosia, P.; Chasteen, N. D. Biochemistry 1998, 37, 9743-9750. (40) Jacobs, D. L.; Watt, G. D.; Frankel, R. B.; Papeathymiou, G. C. Biochemistry 1989, 28, 1650-1655. (41) Mann, S.; Williams, J. M.; Treffrry, A.; Harrison, P. M. J. Mol. Biol. 1987, 198, 405-416.