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Direct electron transfer of ferritin adsorbed at bare gold electrodes
www.elsevier.nl/locate/jelechem Journal of Electroanalytical Chemistry 494 (2000) 114– 120
Direct electron transfer of ferritin adsorbed at bare gold electrodes Donald C. Zapien *, Michael A. Johnson Department of Chemistry, Uni6ersity of Colorado at Den6er, Den6er, CO 80217, USA Received 27 January 1998; received in revised form 19 July 1999; accepted 21 July 2000
Abstract In this work, the direct electron transfer of ferritin adsorbed on bare gold is investigated. Clean gold electrodes were exposed to purified horse spleen ferritin in phosphate buffer at controlled potential. Cyclic voltammetry was the principal method used to investigate the reactivity of the adsorbed layer. Ferritin adsorbs at fairly negative potentials and exhibits reasonably well-defined current–potential curves. The packing density increases with ionic strength, indicating a hydrophobic interaction between adsorbed ferritin and the gold electrode surface. Potential dependent studies suggest that an electroactive layer is formed only with reduced ferritin. An adsorption time dependence study shows that a limiting packing density is reached within 90 s exposure time. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Ferritin; Direct electron transfer; Adsorption
1. Introduction Cellular ferritin is a protein which is responsible for the storage of iron in organisms. Ferritin is composed of 24 subunits, and has a molar mass of 450 000 g mol − 1. The subunits are associated in a 4:3:2 symmetry, resulting in eight hydrophilic and six hydrophobic channels which link the exterior with the interior of the shell. It is through the hydrophilic channels that iron passes into the protein shell [1]. In vitro experiments have revealed that under certain conditions, iron can also exit the protein [2 – 5]. When iron, as Fe(II), enters the protein it is converted to Fe(III). Moreover, core iron can be mobilized from the protein shell provided the iron is first reduced to Fe(II). Though electron transfer steps are involved in the loading and unloading of iron, the nature of the electron transfer is not clear [6 – 8]. Microcoulometric techniques have been used effectively for determining n-values and estimating formal potentials of horse spleen ferritin [9 – 13]. However, direct electrochemical measurements are needed to examine the nature of ferritin-electrode interactions. The direct electron transfer of horse spleen ferritin at modified gold electrodes has recently been reported * Corresponding author. Fax: +1-303-5564776. E-mail address: [email protected] (D.C. Zapien).
[14]. Gold electrodes were modified by the formation of self-assembled monolayers of 3-mercaptopropionic acid (MPA) on the surface yielding reasonably well-defined current –potential curves for ferritin. Controlled potential electrolysis experiments yielded a measurement of approximately 1900 electrons transferred per molecule of ferritin. When a Au/MPA electrode is immersed in ferritin solution at −0.45 V, rinsed and re-immersed in pure electrolyte, the cyclic voltammogram reveals the presence of an electroactive adsorbed layer [15]. Evaluation of ferritin’s electrochemistry at other electrodes has revealed unusual behavior at tin-doped indium oxide (ITO) electrodes [16]. After immersion into ferritin solution, followed by removal of the residual dissolved ferritin, the current –potential scan in pure electrolyte shows the presence of two cathodic peaks indicating two adsorption states of ferritin. The reduction of the two states of the initial layer results in the reconstruction of a single, new layer which can be redox cycled. Ferritin adsorbed (open circuit) on gold has recently been examined using surface-sensitive methods including X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) [17]. It was found that ferritin does not adsorb at monolayer coverages; height measurements indicated flattening of the molecules compared with their natural state, and that aggregates
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of up to 20 ferritin molecules were formed on the surface. Given what is known about its adsorption on gold and its electroactivity at other surfaces, it is of interest to investigate ferritin’s electrochemical behavior while adsorbed at gold. An electrochemical study enables the probing of the ferritin – surface interactions; comparing these interactions with those of other types of surfaces will lead to a better understanding of the relationship between ferritin’s iron core electron transfer reactivity and the chemistry of the protein sheath. Information on the direct electron transfer of other proteins at bare electrode surfaces is useful in the interpretation of ferritin’s electrochemistry at bare gold. Differential pulse methods have been used to enhance the typically small voltammetric signals of cytochrome c adsorbed at gold electrodes [18 – 21]. Most redox proteins unfold as a consequence of their strong adsorption at bare electrode surfaces and lose their inherent electron transfer characteristics. However, there are various stages of unfolding, which occur depending on the nature of the surface [22,23]. Studies using surfaceenhanced Raman spectroscopy have shown that the redox properties of cytochrome c3 in the bulk are well-preserved for the adsorbed states at the silver electrode [24,25]. This behavior contrasted with that observed with cytochrome c on gold electrodes, in which potentials (bulk versus adsorbed) differed by 500 mV [25]. Sagara et al. showed that cytochrome c3 on mercury is similar to the native protein, both in structure and reactivity [26]. Cytochrome c has also been shown to be electroactive while adsorbed at metal oxide electrodes [27 –32]. In this work, ferritin is adsorbed at clean polycrystalline gold electrodes at controlled potentials. Cyclic voltammetry is used to evaluate the electroactivity of the adsorbed layer and to determine the type and degree of surface interaction under a variety of conditions.
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The cell in which the adsorbed layer of ferritin on gold was formed is shown in Fig. 1. The cell was constructed of Teflon, machined to contain a solution volume of 3.5 ml. Both reference (Ag AgCl) and auxiliary (gold wire) electrodes were encased in separate Teflon tubes, each employing porous Vycor rods [22]. The electrode sheath was suspended in the cell by a Teflon thermometer adapter; the adapter in turn, fitted into the cell via a 24/40 ground glass joint. In this cell configuration, the ferritin solution was gently stirred under a blanket of nitrogen, gradually purging the oxygen from the sample. The cells used in the voltammetric experiments are described elsewhere [14]. Voltammetric scans were performed with a Cypress model Omni 90 potentiostat (Lawrence, KS) and a BioAnalytical Systems model RXY recorder (West Lafayette, IN). A Perkin –Elmer (Norwalk, CT) model 552 UV –vis spectrophotometer was used to measure the concentration of ferritin following size-exclusion chromatography. G-200 Sephadex was purchased from Pharmacia (Alameda, CA). A gravity liquid chromatography column was purchased from Kontes Glass Co. (Vineland, NJ). Sodium hydroxide (Analytical Reagent), sulfuric acid (98%), and sodium phosphate, monobasic (Analytical Reagent) were purchased from Mallinkrodt Specialty Chemicals Co. (Paris, KY). Bio-Rad Protein Assay Dye was purchased from Bio-Rad Laboratories (Hercules, CA) and PMSF (phenylmethyl-sulfonyl fluoride) (\ 99%) from Aldrich Chemical Company (Milwaukee, WI). Sigma Chemical Company (St. Louis, MO) was the source of bovine albumin (fraction 5 powder) and horse spleen ferritin (\85%). All of the chemicals except for ferritin were used as obtained without fur-
2. Experimental The working electrode was prepared by fusing polycrystalline gold foil (99.99%, Alfa-Johnson Matthey, Danvers, MA), to 0.254 mm diameter gold wire, and shaping the foil portion into a cylinder. The gold electrode assembly was inserted into a 5 mm i.d. glass sheath, through which nitrogen is fed by way of a Teflon stopcock. The electrode was annealed in a natural gas+ air flame, soaked in chromic acid cleaning solution, followed by electrochemical cycling between − 0.35 and 1.50 V (versus Ag AgCl KCl (sat)) in 1 M sulfuric acid. The electrode area was determined by integrating the charge of the electrocatalytic oxidation of adsorbed iodine [33].
Fig. 1. Low volume adsorption cell. (A) Nitrogen valve. (B) Luer T. (C) Gold wire lead. (D) 6 mm i.d. glass sheath. (E) 10 mm i.d. thermometer adapter. (F) 24/40 ground glass joint. (G) Teflon cell body. (H) Gold auxiliary electrode. (I) Ag AgCl reference electrode. (J) Gold working electrode. (K) Teflon-coated stirring bar.
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ther treatment. Water was purified by distilling water vapor through a heated platinum catalyst in the presence of oxygen [34]. The pyrolytically distilled water was used for cleaning purposes as well as for the preparation of all solutions. The liquid chromatography gel bed of dimensions 2.5 diameter ×20 cm consisted of Sephadex G-200 (protein fractionation range, 5000 – 600 000 g mol − 1). The column was equilibrated with 500 ml of G-200 buffer (20 mM sodium dihydrogen phosphate+ 0.9% NaCl + 0.2 mM PMSF +0.02% NaN3). A standard curve was prepared with bovine serum albumin standards complexed with a Coomassie blue G-250 dye. The concentration of ferritin was determined by measuring the absorbance of the ferritin – dye complex at 595 nm against the albumin – dye standard curve [35]. Ferritin samples were prepared in pH 7.2 phosphate buffer (nominally 0.46 M). The ionic strength of the buffer was nominally 1.0 M, however, when different ionic strengths were required, the concentrations of sodium dihydrogen phosphate and sodium hydroxide were varied while a constant ratio between the two components was maintained. The ionic strength of the buffer from which ferritin was adsorbed, and the buffer in which the current – potential curve was scanned was the same. For the current – potential curves of adsorbed ferritin, a clean gold electrode was immersed into ferritin solution at − 0.00 V, the potential scanned to −0.50 V at 100 mV s − 1, then the electrode was transferred into a cell containing deaerated buffer at −0.50 V, where the electrode was rinsed free of dissolved ferritin. Finally, the electrode was immersed at −0.50 V into another cell containing deaerated buffer, and the potential cycled between − 0.50 and 0.20 V. The charge represented by the anodic peak area was used to estimate an experimental packing density at the optimum adsorption time and ionic strength. A layer of ferritin was formed at −0.50 V as described above and the current –potential curve scanned from − 0.50 V at 100 mV s − 1. The anodic peak area was cut and weighed, and compared to that of 1 cm2 of area (representing 5 mC), yielding the electrolytic charge from the ferritin layer. Estimates for packing densities, Y, were determined substituting the electrolytic charge into the Faraday law. The concentration of iron in a ferritin sample was determined by atomic absorption spectroscopy using a Perkin –Elmer 5000 (Norwalk, CT) atomic absorption spectrometer at 248.3 nm. The ratio of iron concentration to ferritin concentration (using UV – vis spectroscopy) yielded the average number of iron atoms per ferritin molecule, NFe.
3. Results and discussion
3.1. Voltammetry of adsorbed ferritin When a clean gold electrode is immersed at −0.50 V, rinsed, re-immersed at −0.50 V, and the potential cycled between − 0.50 and 0.20 V, a fairly well-defined current –potential curve results indicating that adsorbed ferritin undergoes direct electron transfer. As is shown in Fig. 2, the anodic peak appears at − 0.05 V, and the cathodic peak at − 0.13 V. Whereas the faradaic currents observed with cytochrome c (on bare gold) are very small [21], the large number of electrons transferred per ferritin molecule generates easily measured currents (\ 25 mA cm − 2) permitting the use of normal cyclic voltammetry to study current –potential behavior. As is shown in Fig. 3, the anodic peak current of the second cycle is smaller than that of the first, suggesting that either some ferritin desorbs, or ferritin has become less electroactive with each cycle. It is not clear why the cathodic peak is smaller than the anodic peak. An anodic peak is larger than its preceding cathodic peak, indicating that the smaller size of the cathodic peak is not merely due to desorption of ferritin. This result may indicate that the orientation of adsorbed ferritin in the reduced form is different from that of the oxidized form. It is not known how many electrons are transferred for adsorbed ferritin. Assuming that adsorbed ferritin is completely electroactive, an estimate for the packing density of 0.409 0.05 pmol cm − 2 is calculated from the integrated current and the Faraday law. Consecutive cycling of the I–E curve, shown in
Fig. 2. Current– potential curve of ferritin at a bare polycrystalline gold electrode. The clean electrode was immersed at −0.50 V into 2.6 mg ml − 1 purified ferritin in a pH 7.0 phosphate buffer for 3 min, rinsed in buffer at − 0.50 V, and the potential cycled between − 0.50 and 0.2 V. NFe, 1500 Fe/ferritin; ionic strength, 1.0 M; electrode area, 1.40 cm2; scan rate, 100 mV s − 1.
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Fig. 3, causes the peak area to diminish. The reasons for this behavior are not clear. However, this result may indicate that with multiple cycles, the protein gradually assumes an orientation which is electroinactive. This behavior has been observed for cytochrome c3 at a bare mercury electrode [26]. The current –potential curves of adsorbed ferritin have been recorded at scan rates of 50, 100, 200, and 400 mV s − 1. The plot of anodic peak current versus scan rate is shown in Fig. 4 (NFe was not determined, but is estimated to be 1700\NFe \1500). The linear regression slope of the data is 380 mF, with a correlation coefficient of 0.998 indicating that the peak current has a linear dependence on scan rate; a correlation which, according to theory, indicates that the electroactive species is adsorbed ferritin. The relationship be-
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tween peak current and scan rate for a reversible reaction is given by the following equation [36]: Ip = (n 2F 2Y/4RT)6 Assuming that n=1500, the calculated slope should be 0.85F, roughly 2000 times the experimental slope. The reason for the discrepancy may be that the equation applies strictly to those systems for which n is known and whose voltammetry behaves according to the known n-value. Ferritin is a complex system in that potentially 1500 electrons are transferred. However, no model has been developed which describes the degrees or the types of interactions between the electroactive centers in the protein. Clearly, if the system behaved as though n=1500, the anodic and cathodic branches would be extremely narrow, and the peak currents very large. The large currents would then give a large slope, as predicted by theory. Judging from Fig. 2, this is not the case. Rather, the voltammetry resembles that of a one-electron transfer system as though 1500 individual one-electron transfer reactions are occurring. There are several examples in the literature in which electrons are transferred to and from molecules containing multiple redox centers (up to 1200 per molecule in some electroactive polymers) while the voltammetry has the appearance of a one-electron transfer system [37 –40]. In addition, the equation is derived on the assumption that the reacting species are non-interacting. Perhaps the discrepancy is due to interaction between ferritin molecules.
3.2. Potential dependent adsorption of ferritin
Fig. 3. Multiple scan cycles for ferritin adsorbed at a bare polycrystalline gold electrode. Scan conditions as in Fig. 2.
Fig. 4. Scan rate dependence of ferritin at a bare polycrystalline gold electrode. The clean electrode was immersed at − 0.50 V into 2.6 mg ml − 1 purified ferritin in a pH 7.2 phosphate buffer for 2 min, rinsed in buffer at − 0.50 V, and the potential cycled between − 0.50 and 0.20 V. NFe, estimated to be 1700 \ NFe \ 1500; ionic strength, 1.0 M; electrode area, 1.62 cm2; scan rate (a) 50; (b) 100; (c) 200 and (d) 400 mV s − 1.
When the clean gold electrode is immersed in ferritin solution at 0.20 V, rinsed under nitrogen, re-immersed in pure phosphate buffer, and the potential cycled between − 0.50 and 0.20 V, no faradaic current is observed as is shown in Fig. 5(a). Adsorption was also carried out at −0.10 and − 0.30 V while the scan of the adsorbed layer was cycled between − 0.50 and 0.20 V. It can be seen that the amount of faradaic charge from adsorbed ferritin is potential dependent. The observed increase in the faradaic current indicates that more ferritin is adsorbed as the adsorption potential becomes more negative. Clearly, adsorbed ferritin is in the oxidized form at 0.20 V, and is in the reduced form at − 0.50 V. The data suggest that ferritin adsorbs in an electroactive form only when it is first reduced. It cannot be determined from these data alone whether electroinactive ferritin is adsorbed at 0.20 V, or if ferritin is not adsorbed at all. Blocking experiments have been used to assess whether an adsorbed protein layer is present on the surface [32]. In a control experiment, a clean gold electrode was exposed to ferritin at 0.20 V, rinsed under nitrogen, immersed in 10 − 4 M ferricyanide in 1 M KCl, and the potential cycled
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dized or reduced forms of ferritin are not known. However, considering the electrochemical data presented here, the reduced form of ferritin may be less stable to unfolding than the oxidized form.
3.3. Packing density dependence on ionic strength, ferritin concentration and adsorption time
Fig. 5. Current– potential curves of ferritin adsorbed at (a) 0.20; (b) −0.10; (c) − 0.30 and (d) − 0.50 V Other scan conditions as in Fig. 4.
Fig. 6. Dependence of ferritin packing density on ionic strength. Ferritin concentration 1.6 mg ml − 1. Other conditions in Fig. 4.
between 0.60 and − 0.10 V. The current – potential curve showed essentially the same currents for ferricyanide as that obtained when a clean electrode was used, suggesting that ferritin does not adsorb at 0.20 V. It is conceivable that the reduction of the ferritin core may induce conformational changes in the protein quaternary structure facilitating its adsorption on gold. The folding Gibbs energy of a protein can be different depending on the oxidation state of the electroactive center. For proteins such as cytochrome c [41], and myoglobin [42], the reduced forms have a larger folding Gibbs energy than the oxidized forms, thus the reduced forms are more stable to unfolding. For other proteins, such as azurin, the reduced form is more stable to unfolding [43]. The folding Gibbs energies for the oxi-
Fig. 6 shows relative ferritin packing density plotted against ionic strength. As the ionic strength is increased from 0.3 to 1.5 M, the packing density reaches a maximum value beginning at about 1.0 M. The increase in packing density with ionic strength indicates that the interaction with the surface is more favorable when the ionic concentration is high, a behavior which suggests that the protein –gold interaction is hydrophobic in nature. The surfaces of globular proteins are at least partially hydrophobic [44]. Hydrophobic regions of ferritin may be interacting with the bare gold surface allowing ferritin to be immobilized while its electroactivity is preserved. The type of electrode-protein behavior observed with ferritin on bare gold is also found with ferritin at tin-doped indium oxide (ITO) electrodes, in which a limiting packing density is measured at about 1 M ionic strength [16]. Fig. 7 displays the adsorption isotherm for ferritin at a bare gold electrode. The lowest protein concentration represents the detection limit of the method, while the maximum limit is near the highest ferritin concentration obtainable following purification by size-exclusion chromatography. Well-defined plateau regions are found in the isotherms of globular proteins adsorbed from dilute to semi-dilute solution [44]. Fig. 7 shows that packing density is proportional to concentration in the ferritin concentration range studied, typical behavior for compact molecules at low concentration. However, if higher concentrations were available, a plateau is expected.
Fig. 7. Dependence of ferritin packing density on dissolved ferritin concentration. Other scan conditions as in Fig. 4.
D.C. Zapien, M.A. Johnson / Journal of Electroanalytical Chemistry 494 (2000) 114–120
The bare gold electrode was exposed to ferritin solution at − 0.50 V for various periods of time. Fig. 8 shows that a limiting packing density is reached after 90 s adsorption time. Adsorption processes usually involve transport to the electrode surface, anchoring to the surface, structural rearrangement, disengagement, and transport from the electrode. With regard to structural rearrangement, crowding of the protein on the surface may induce changes in the structure/orientation of the adsorbed molecules [44]. The time dependent adsorption data appear to be consistent with the adsorption rate being limited more by the diffusion of ferritin to the gold surface and/or surface attachment rather than structural or orientational changes occurring as more ferritin molecules adsorb. This result is in sharp contrast to the long periods of time (h) required for ferritin to reach a limiting surface concentration at tin-doped indium oxide electrodes [16]. The reasons for the large differences are not clear, however, it appears that the negative charge on the surface oxides is not solely responsible. Since potentials negative of the pzc are used, some electrostatic repulsion is also expected between the gold surface and the negatively charged ferritin molecules.
3.4. Possible oligomerization of ferritin at bare gold The peaks of the Au/ferritin electrode are broader than those of the Au/MPA/ferritin electrode though the process of forming the ferritin layer and the scanning conditions are essentially the same [15]. In addition, a small, reproducible cathodic shoulder appears at 0.11 V in Figs. 2 and 3. The broadness of the peaks may indicate that at the bare gold electrode more than one species is reacting. Images obtained by atomic force microscopy (AFM) showed that ferritin aggregates at a bare gold surface to form structures of various sizes, most having diameters which were 10 – 15 times larger than that of a single ferritin molecule. Szu¨cs and Nova´k have reported that in the current – potential curve of
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cytochrome c adsorbed at a bare gold electrode, another peak appeared at a potential more positive than that of the principal adsorbed species, [19,20] and was attributed to the presence of adsorbed, oligomerized cytochrome c. Oligomerization causes changes in the globular network, causing changes in the energy level of the redox center, which is manifested as a different redox potential. However, the presence of surface groups, such as those inherent to the electrode or from adsorbed molecules, anchors the proteins to the electrode surface, preventing lateral movements, which in turn, prevents the aggregation of adsorbed protein. Considering these reports together, the broadness of the peaks and the presence of peak shoulders may be due to the oxidation/reduction of ferritin monomers and oligomers. In contrast, when ferritin is adsorbed on the Au MPA surface, the anionic mercaptopropionate groups may be anchoring ferritin in such a way that aggregation is prevented.
4. Conclusions Ferritin adsorbs from phosphate solution onto clean polycrystalline gold electrodes at controlled potential. The large number of electrons transferred, perhaps as many as 1500, facilitates study of the electrochemistry of adsorbed ferritin using normal cyclic voltammetry. Ferritin does not appear to adsorb at oxidizing potentials. In contrast, at potentials that sustain ferritin in the reduced state, the protein adsorbs in an electroactive form. The packing density increases with ionic strength, indicating a hydrophobic interaction between adsorbed ferritin and the bare gold electrode surface. Time dependence data suggest that the rate-limiting step in the adsorption process is probably diffusion to the electrode surface and/or surface binding.
Acknowledgements The authors acknowledge the Research Corporation (Cottrell Research Grant Number C-3735) for support of this research. The authors also wish to thank Professor Edmond F. Bowden at North Carolina State University for his helpful suggestions.
References
Fig. 8. Dependence of ferritin packing density on adsorption time. Other scan conditions the same as in Fig. 4.
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Direct electron transfer of ferritin adsorbed at bare gold electrodes Donald C. Zapien *, Michael A. Johnson Department of Chemistry, Uni6ersity of Colorado at Den6er, Den6er, CO 80217, USA Received 27 January 1998; received in revised form 19 July 1999; accepted 21 July 2000
Abstract In this work, the direct electron transfer of ferritin adsorbed on bare gold is investigated. Clean gold electrodes were exposed to purified horse spleen ferritin in phosphate buffer at controlled potential. Cyclic voltammetry was the principal method used to investigate the reactivity of the adsorbed layer. Ferritin adsorbs at fairly negative potentials and exhibits reasonably well-defined current–potential curves. The packing density increases with ionic strength, indicating a hydrophobic interaction between adsorbed ferritin and the gold electrode surface. Potential dependent studies suggest that an electroactive layer is formed only with reduced ferritin. An adsorption time dependence study shows that a limiting packing density is reached within 90 s exposure time. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Ferritin; Direct electron transfer; Adsorption
1. Introduction Cellular ferritin is a protein which is responsible for the storage of iron in organisms. Ferritin is composed of 24 subunits, and has a molar mass of 450 000 g mol − 1. The subunits are associated in a 4:3:2 symmetry, resulting in eight hydrophilic and six hydrophobic channels which link the exterior with the interior of the shell. It is through the hydrophilic channels that iron passes into the protein shell [1]. In vitro experiments have revealed that under certain conditions, iron can also exit the protein [2 – 5]. When iron, as Fe(II), enters the protein it is converted to Fe(III). Moreover, core iron can be mobilized from the protein shell provided the iron is first reduced to Fe(II). Though electron transfer steps are involved in the loading and unloading of iron, the nature of the electron transfer is not clear [6 – 8]. Microcoulometric techniques have been used effectively for determining n-values and estimating formal potentials of horse spleen ferritin [9 – 13]. However, direct electrochemical measurements are needed to examine the nature of ferritin-electrode interactions. The direct electron transfer of horse spleen ferritin at modified gold electrodes has recently been reported * Corresponding author. Fax: +1-303-5564776. E-mail address: [email protected] (D.C. Zapien).
[14]. Gold electrodes were modified by the formation of self-assembled monolayers of 3-mercaptopropionic acid (MPA) on the surface yielding reasonably well-defined current –potential curves for ferritin. Controlled potential electrolysis experiments yielded a measurement of approximately 1900 electrons transferred per molecule of ferritin. When a Au/MPA electrode is immersed in ferritin solution at −0.45 V, rinsed and re-immersed in pure electrolyte, the cyclic voltammogram reveals the presence of an electroactive adsorbed layer [15]. Evaluation of ferritin’s electrochemistry at other electrodes has revealed unusual behavior at tin-doped indium oxide (ITO) electrodes [16]. After immersion into ferritin solution, followed by removal of the residual dissolved ferritin, the current –potential scan in pure electrolyte shows the presence of two cathodic peaks indicating two adsorption states of ferritin. The reduction of the two states of the initial layer results in the reconstruction of a single, new layer which can be redox cycled. Ferritin adsorbed (open circuit) on gold has recently been examined using surface-sensitive methods including X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) [17]. It was found that ferritin does not adsorb at monolayer coverages; height measurements indicated flattening of the molecules compared with their natural state, and that aggregates
0022-0728/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 7 2 8 ( 0 0 ) 0 0 3 6 4 - 8
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of up to 20 ferritin molecules were formed on the surface. Given what is known about its adsorption on gold and its electroactivity at other surfaces, it is of interest to investigate ferritin’s electrochemical behavior while adsorbed at gold. An electrochemical study enables the probing of the ferritin – surface interactions; comparing these interactions with those of other types of surfaces will lead to a better understanding of the relationship between ferritin’s iron core electron transfer reactivity and the chemistry of the protein sheath. Information on the direct electron transfer of other proteins at bare electrode surfaces is useful in the interpretation of ferritin’s electrochemistry at bare gold. Differential pulse methods have been used to enhance the typically small voltammetric signals of cytochrome c adsorbed at gold electrodes [18 – 21]. Most redox proteins unfold as a consequence of their strong adsorption at bare electrode surfaces and lose their inherent electron transfer characteristics. However, there are various stages of unfolding, which occur depending on the nature of the surface [22,23]. Studies using surfaceenhanced Raman spectroscopy have shown that the redox properties of cytochrome c3 in the bulk are well-preserved for the adsorbed states at the silver electrode [24,25]. This behavior contrasted with that observed with cytochrome c on gold electrodes, in which potentials (bulk versus adsorbed) differed by 500 mV [25]. Sagara et al. showed that cytochrome c3 on mercury is similar to the native protein, both in structure and reactivity [26]. Cytochrome c has also been shown to be electroactive while adsorbed at metal oxide electrodes [27 –32]. In this work, ferritin is adsorbed at clean polycrystalline gold electrodes at controlled potentials. Cyclic voltammetry is used to evaluate the electroactivity of the adsorbed layer and to determine the type and degree of surface interaction under a variety of conditions.
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The cell in which the adsorbed layer of ferritin on gold was formed is shown in Fig. 1. The cell was constructed of Teflon, machined to contain a solution volume of 3.5 ml. Both reference (Ag AgCl) and auxiliary (gold wire) electrodes were encased in separate Teflon tubes, each employing porous Vycor rods [22]. The electrode sheath was suspended in the cell by a Teflon thermometer adapter; the adapter in turn, fitted into the cell via a 24/40 ground glass joint. In this cell configuration, the ferritin solution was gently stirred under a blanket of nitrogen, gradually purging the oxygen from the sample. The cells used in the voltammetric experiments are described elsewhere [14]. Voltammetric scans were performed with a Cypress model Omni 90 potentiostat (Lawrence, KS) and a BioAnalytical Systems model RXY recorder (West Lafayette, IN). A Perkin –Elmer (Norwalk, CT) model 552 UV –vis spectrophotometer was used to measure the concentration of ferritin following size-exclusion chromatography. G-200 Sephadex was purchased from Pharmacia (Alameda, CA). A gravity liquid chromatography column was purchased from Kontes Glass Co. (Vineland, NJ). Sodium hydroxide (Analytical Reagent), sulfuric acid (98%), and sodium phosphate, monobasic (Analytical Reagent) were purchased from Mallinkrodt Specialty Chemicals Co. (Paris, KY). Bio-Rad Protein Assay Dye was purchased from Bio-Rad Laboratories (Hercules, CA) and PMSF (phenylmethyl-sulfonyl fluoride) (\ 99%) from Aldrich Chemical Company (Milwaukee, WI). Sigma Chemical Company (St. Louis, MO) was the source of bovine albumin (fraction 5 powder) and horse spleen ferritin (\85%). All of the chemicals except for ferritin were used as obtained without fur-
2. Experimental The working electrode was prepared by fusing polycrystalline gold foil (99.99%, Alfa-Johnson Matthey, Danvers, MA), to 0.254 mm diameter gold wire, and shaping the foil portion into a cylinder. The gold electrode assembly was inserted into a 5 mm i.d. glass sheath, through which nitrogen is fed by way of a Teflon stopcock. The electrode was annealed in a natural gas+ air flame, soaked in chromic acid cleaning solution, followed by electrochemical cycling between − 0.35 and 1.50 V (versus Ag AgCl KCl (sat)) in 1 M sulfuric acid. The electrode area was determined by integrating the charge of the electrocatalytic oxidation of adsorbed iodine [33].
Fig. 1. Low volume adsorption cell. (A) Nitrogen valve. (B) Luer T. (C) Gold wire lead. (D) 6 mm i.d. glass sheath. (E) 10 mm i.d. thermometer adapter. (F) 24/40 ground glass joint. (G) Teflon cell body. (H) Gold auxiliary electrode. (I) Ag AgCl reference electrode. (J) Gold working electrode. (K) Teflon-coated stirring bar.
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ther treatment. Water was purified by distilling water vapor through a heated platinum catalyst in the presence of oxygen [34]. The pyrolytically distilled water was used for cleaning purposes as well as for the preparation of all solutions. The liquid chromatography gel bed of dimensions 2.5 diameter ×20 cm consisted of Sephadex G-200 (protein fractionation range, 5000 – 600 000 g mol − 1). The column was equilibrated with 500 ml of G-200 buffer (20 mM sodium dihydrogen phosphate+ 0.9% NaCl + 0.2 mM PMSF +0.02% NaN3). A standard curve was prepared with bovine serum albumin standards complexed with a Coomassie blue G-250 dye. The concentration of ferritin was determined by measuring the absorbance of the ferritin – dye complex at 595 nm against the albumin – dye standard curve [35]. Ferritin samples were prepared in pH 7.2 phosphate buffer (nominally 0.46 M). The ionic strength of the buffer was nominally 1.0 M, however, when different ionic strengths were required, the concentrations of sodium dihydrogen phosphate and sodium hydroxide were varied while a constant ratio between the two components was maintained. The ionic strength of the buffer from which ferritin was adsorbed, and the buffer in which the current – potential curve was scanned was the same. For the current – potential curves of adsorbed ferritin, a clean gold electrode was immersed into ferritin solution at − 0.00 V, the potential scanned to −0.50 V at 100 mV s − 1, then the electrode was transferred into a cell containing deaerated buffer at −0.50 V, where the electrode was rinsed free of dissolved ferritin. Finally, the electrode was immersed at −0.50 V into another cell containing deaerated buffer, and the potential cycled between − 0.50 and 0.20 V. The charge represented by the anodic peak area was used to estimate an experimental packing density at the optimum adsorption time and ionic strength. A layer of ferritin was formed at −0.50 V as described above and the current –potential curve scanned from − 0.50 V at 100 mV s − 1. The anodic peak area was cut and weighed, and compared to that of 1 cm2 of area (representing 5 mC), yielding the electrolytic charge from the ferritin layer. Estimates for packing densities, Y, were determined substituting the electrolytic charge into the Faraday law. The concentration of iron in a ferritin sample was determined by atomic absorption spectroscopy using a Perkin –Elmer 5000 (Norwalk, CT) atomic absorption spectrometer at 248.3 nm. The ratio of iron concentration to ferritin concentration (using UV – vis spectroscopy) yielded the average number of iron atoms per ferritin molecule, NFe.
3. Results and discussion
3.1. Voltammetry of adsorbed ferritin When a clean gold electrode is immersed at −0.50 V, rinsed, re-immersed at −0.50 V, and the potential cycled between − 0.50 and 0.20 V, a fairly well-defined current –potential curve results indicating that adsorbed ferritin undergoes direct electron transfer. As is shown in Fig. 2, the anodic peak appears at − 0.05 V, and the cathodic peak at − 0.13 V. Whereas the faradaic currents observed with cytochrome c (on bare gold) are very small [21], the large number of electrons transferred per ferritin molecule generates easily measured currents (\ 25 mA cm − 2) permitting the use of normal cyclic voltammetry to study current –potential behavior. As is shown in Fig. 3, the anodic peak current of the second cycle is smaller than that of the first, suggesting that either some ferritin desorbs, or ferritin has become less electroactive with each cycle. It is not clear why the cathodic peak is smaller than the anodic peak. An anodic peak is larger than its preceding cathodic peak, indicating that the smaller size of the cathodic peak is not merely due to desorption of ferritin. This result may indicate that the orientation of adsorbed ferritin in the reduced form is different from that of the oxidized form. It is not known how many electrons are transferred for adsorbed ferritin. Assuming that adsorbed ferritin is completely electroactive, an estimate for the packing density of 0.409 0.05 pmol cm − 2 is calculated from the integrated current and the Faraday law. Consecutive cycling of the I–E curve, shown in
Fig. 2. Current– potential curve of ferritin at a bare polycrystalline gold electrode. The clean electrode was immersed at −0.50 V into 2.6 mg ml − 1 purified ferritin in a pH 7.0 phosphate buffer for 3 min, rinsed in buffer at − 0.50 V, and the potential cycled between − 0.50 and 0.2 V. NFe, 1500 Fe/ferritin; ionic strength, 1.0 M; electrode area, 1.40 cm2; scan rate, 100 mV s − 1.
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Fig. 3, causes the peak area to diminish. The reasons for this behavior are not clear. However, this result may indicate that with multiple cycles, the protein gradually assumes an orientation which is electroinactive. This behavior has been observed for cytochrome c3 at a bare mercury electrode [26]. The current –potential curves of adsorbed ferritin have been recorded at scan rates of 50, 100, 200, and 400 mV s − 1. The plot of anodic peak current versus scan rate is shown in Fig. 4 (NFe was not determined, but is estimated to be 1700\NFe \1500). The linear regression slope of the data is 380 mF, with a correlation coefficient of 0.998 indicating that the peak current has a linear dependence on scan rate; a correlation which, according to theory, indicates that the electroactive species is adsorbed ferritin. The relationship be-
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tween peak current and scan rate for a reversible reaction is given by the following equation [36]: Ip = (n 2F 2Y/4RT)6 Assuming that n=1500, the calculated slope should be 0.85F, roughly 2000 times the experimental slope. The reason for the discrepancy may be that the equation applies strictly to those systems for which n is known and whose voltammetry behaves according to the known n-value. Ferritin is a complex system in that potentially 1500 electrons are transferred. However, no model has been developed which describes the degrees or the types of interactions between the electroactive centers in the protein. Clearly, if the system behaved as though n=1500, the anodic and cathodic branches would be extremely narrow, and the peak currents very large. The large currents would then give a large slope, as predicted by theory. Judging from Fig. 2, this is not the case. Rather, the voltammetry resembles that of a one-electron transfer system as though 1500 individual one-electron transfer reactions are occurring. There are several examples in the literature in which electrons are transferred to and from molecules containing multiple redox centers (up to 1200 per molecule in some electroactive polymers) while the voltammetry has the appearance of a one-electron transfer system [37 –40]. In addition, the equation is derived on the assumption that the reacting species are non-interacting. Perhaps the discrepancy is due to interaction between ferritin molecules.
3.2. Potential dependent adsorption of ferritin
Fig. 3. Multiple scan cycles for ferritin adsorbed at a bare polycrystalline gold electrode. Scan conditions as in Fig. 2.
Fig. 4. Scan rate dependence of ferritin at a bare polycrystalline gold electrode. The clean electrode was immersed at − 0.50 V into 2.6 mg ml − 1 purified ferritin in a pH 7.2 phosphate buffer for 2 min, rinsed in buffer at − 0.50 V, and the potential cycled between − 0.50 and 0.20 V. NFe, estimated to be 1700 \ NFe \ 1500; ionic strength, 1.0 M; electrode area, 1.62 cm2; scan rate (a) 50; (b) 100; (c) 200 and (d) 400 mV s − 1.
When the clean gold electrode is immersed in ferritin solution at 0.20 V, rinsed under nitrogen, re-immersed in pure phosphate buffer, and the potential cycled between − 0.50 and 0.20 V, no faradaic current is observed as is shown in Fig. 5(a). Adsorption was also carried out at −0.10 and − 0.30 V while the scan of the adsorbed layer was cycled between − 0.50 and 0.20 V. It can be seen that the amount of faradaic charge from adsorbed ferritin is potential dependent. The observed increase in the faradaic current indicates that more ferritin is adsorbed as the adsorption potential becomes more negative. Clearly, adsorbed ferritin is in the oxidized form at 0.20 V, and is in the reduced form at − 0.50 V. The data suggest that ferritin adsorbs in an electroactive form only when it is first reduced. It cannot be determined from these data alone whether electroinactive ferritin is adsorbed at 0.20 V, or if ferritin is not adsorbed at all. Blocking experiments have been used to assess whether an adsorbed protein layer is present on the surface [32]. In a control experiment, a clean gold electrode was exposed to ferritin at 0.20 V, rinsed under nitrogen, immersed in 10 − 4 M ferricyanide in 1 M KCl, and the potential cycled
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dized or reduced forms of ferritin are not known. However, considering the electrochemical data presented here, the reduced form of ferritin may be less stable to unfolding than the oxidized form.
3.3. Packing density dependence on ionic strength, ferritin concentration and adsorption time
Fig. 5. Current– potential curves of ferritin adsorbed at (a) 0.20; (b) −0.10; (c) − 0.30 and (d) − 0.50 V Other scan conditions as in Fig. 4.
Fig. 6. Dependence of ferritin packing density on ionic strength. Ferritin concentration 1.6 mg ml − 1. Other conditions in Fig. 4.
between 0.60 and − 0.10 V. The current – potential curve showed essentially the same currents for ferricyanide as that obtained when a clean electrode was used, suggesting that ferritin does not adsorb at 0.20 V. It is conceivable that the reduction of the ferritin core may induce conformational changes in the protein quaternary structure facilitating its adsorption on gold. The folding Gibbs energy of a protein can be different depending on the oxidation state of the electroactive center. For proteins such as cytochrome c [41], and myoglobin [42], the reduced forms have a larger folding Gibbs energy than the oxidized forms, thus the reduced forms are more stable to unfolding. For other proteins, such as azurin, the reduced form is more stable to unfolding [43]. The folding Gibbs energies for the oxi-
Fig. 6 shows relative ferritin packing density plotted against ionic strength. As the ionic strength is increased from 0.3 to 1.5 M, the packing density reaches a maximum value beginning at about 1.0 M. The increase in packing density with ionic strength indicates that the interaction with the surface is more favorable when the ionic concentration is high, a behavior which suggests that the protein –gold interaction is hydrophobic in nature. The surfaces of globular proteins are at least partially hydrophobic [44]. Hydrophobic regions of ferritin may be interacting with the bare gold surface allowing ferritin to be immobilized while its electroactivity is preserved. The type of electrode-protein behavior observed with ferritin on bare gold is also found with ferritin at tin-doped indium oxide (ITO) electrodes, in which a limiting packing density is measured at about 1 M ionic strength [16]. Fig. 7 displays the adsorption isotherm for ferritin at a bare gold electrode. The lowest protein concentration represents the detection limit of the method, while the maximum limit is near the highest ferritin concentration obtainable following purification by size-exclusion chromatography. Well-defined plateau regions are found in the isotherms of globular proteins adsorbed from dilute to semi-dilute solution [44]. Fig. 7 shows that packing density is proportional to concentration in the ferritin concentration range studied, typical behavior for compact molecules at low concentration. However, if higher concentrations were available, a plateau is expected.
Fig. 7. Dependence of ferritin packing density on dissolved ferritin concentration. Other scan conditions as in Fig. 4.
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The bare gold electrode was exposed to ferritin solution at − 0.50 V for various periods of time. Fig. 8 shows that a limiting packing density is reached after 90 s adsorption time. Adsorption processes usually involve transport to the electrode surface, anchoring to the surface, structural rearrangement, disengagement, and transport from the electrode. With regard to structural rearrangement, crowding of the protein on the surface may induce changes in the structure/orientation of the adsorbed molecules [44]. The time dependent adsorption data appear to be consistent with the adsorption rate being limited more by the diffusion of ferritin to the gold surface and/or surface attachment rather than structural or orientational changes occurring as more ferritin molecules adsorb. This result is in sharp contrast to the long periods of time (h) required for ferritin to reach a limiting surface concentration at tin-doped indium oxide electrodes [16]. The reasons for the large differences are not clear, however, it appears that the negative charge on the surface oxides is not solely responsible. Since potentials negative of the pzc are used, some electrostatic repulsion is also expected between the gold surface and the negatively charged ferritin molecules.
3.4. Possible oligomerization of ferritin at bare gold The peaks of the Au/ferritin electrode are broader than those of the Au/MPA/ferritin electrode though the process of forming the ferritin layer and the scanning conditions are essentially the same [15]. In addition, a small, reproducible cathodic shoulder appears at 0.11 V in Figs. 2 and 3. The broadness of the peaks may indicate that at the bare gold electrode more than one species is reacting. Images obtained by atomic force microscopy (AFM) showed that ferritin aggregates at a bare gold surface to form structures of various sizes, most having diameters which were 10 – 15 times larger than that of a single ferritin molecule. Szu¨cs and Nova´k have reported that in the current – potential curve of
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cytochrome c adsorbed at a bare gold electrode, another peak appeared at a potential more positive than that of the principal adsorbed species, [19,20] and was attributed to the presence of adsorbed, oligomerized cytochrome c. Oligomerization causes changes in the globular network, causing changes in the energy level of the redox center, which is manifested as a different redox potential. However, the presence of surface groups, such as those inherent to the electrode or from adsorbed molecules, anchors the proteins to the electrode surface, preventing lateral movements, which in turn, prevents the aggregation of adsorbed protein. Considering these reports together, the broadness of the peaks and the presence of peak shoulders may be due to the oxidation/reduction of ferritin monomers and oligomers. In contrast, when ferritin is adsorbed on the Au MPA surface, the anionic mercaptopropionate groups may be anchoring ferritin in such a way that aggregation is prevented.
4. Conclusions Ferritin adsorbs from phosphate solution onto clean polycrystalline gold electrodes at controlled potential. The large number of electrons transferred, perhaps as many as 1500, facilitates study of the electrochemistry of adsorbed ferritin using normal cyclic voltammetry. Ferritin does not appear to adsorb at oxidizing potentials. In contrast, at potentials that sustain ferritin in the reduced state, the protein adsorbs in an electroactive form. The packing density increases with ionic strength, indicating a hydrophobic interaction between adsorbed ferritin and the bare gold electrode surface. Time dependence data suggest that the rate-limiting step in the adsorption process is probably diffusion to the electrode surface and/or surface binding.
Acknowledgements The authors acknowledge the Research Corporation (Cottrell Research Grant Number C-3735) for support of this research. The authors also wish to thank Professor Edmond F. Bowden at North Carolina State University for his helpful suggestions.
References
Fig. 8. Dependence of ferritin packing density on adsorption time. Other scan conditions the same as in Fig. 4.
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