Langmuir 1998, 14, 1971-1973
1971
Direct Electron Transfer of Ferritin Adsorbed at Tin-Doped Indium Oxide Electrodes Ryan J. Cherry, A. Jason Bjornsen, and Donald C. Zapien* Department of Chemistry, University of Colorado at Denver, Denver, Colorado 80217 Received June 26, 1997. In Final Form: January 28, 1998 In this work, the electron transfer reactions of horse spleen ferritin at tin-doped indium oxide electrodes were investigated for the first time. Cyclic voltammetry reveals that ferritin adsorbs from phosphate solution into a layer composed of two adsorbed states, the relative packing density of the states depending strongly on ionic strength. Upon reduction, the initial layer reconstructs into a new one with faster electron-transfer kinetics. Reduction of the adsorbed layer in the presence of ethylenediaminetetraacetic acid results in the disappearance of any further voltammetric response indicating that transport of iron from the protein shell had been induced electrochemically.
Introduction Cellular ferritin is a protein that is responsible for the storage of iron in organisms. It has been suggested that an electron-transfer step is involved in the loading and unloading of iron into the apoferritin shell.1,2 Describing the role of electron transfer is key to understanding the mechanism by which iron is sequestered by cellular ferritins. Electrochemical methods have shown to be useful in the study of the redox properties and reactions of both dissolved and surface-bound proteins. Many proteins exhibit direct electron transfer at tindoped indium oxide electrodes.3-6 Cytochrome c has been shown to irreversibly adsorb at In2O3 electrodes as evidenced by a fairly well-defined current-potential curve obtained after the exposed electrode had been rinsed free of dissolved protein.7 In another study, it was suggested that the positively charged lysine residues hydrogen bond to the oxide groups of the electrode surface based on the adsorption strength dependence on pH.8 Koller and Hawkridge have pointed out that upon reduction, the adsorbed cytochrome c intermediate undergoes a conformation change that may facilitate its desorption.9 The immobilization of cytochrome c onto a similar material, tin oxide, allowed the determination of unimolecular electron-transfer rate constants.10 Daido and Akaike showed that there was a strong dependence of packing density of cytochrome c with ionic strength. In addition, the anodic peak was smaller than the cathodic * To whom correspondence should be addressed. (1) Thiel, E. C. Annu. Rev. Biochem. 1987, 56, 289-315. (2) (a) Xu, B.; Chasteen, N. D. J. Biol. Chem. 1991, 266, 1996-1970. (b) 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. (c) Watt, G. D.; Frankel, R. B.; Jacobs, D.; Huang, H.; Papaefthymiou, G. C. Biochemistry 1992, 31, 5672-5679. (3) Bowden, E. F.; Hawkridge, F. M.; Chlebowski, J. F.; Bancroft, E. E.; Thorpe, C.; Blount, H. N. J. Am. Chem. Soc. 1982, 104, 7641-4. (4) Taniguchi, I.; Watanabe, K.; Tominaga, M.; Hawkridge, F. M. J. Electroanal. Chem. 1992, 333, 331-8. (5) Nassar, A.-E. F.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1995, 67, 2386-92. (6) Sagara, T.; Niwa, K.; Sone, A.; Hinnen, C.; Niki, K. Langmuir 1990, 6, 254-262. (7) Bowden, E. F.; Hawkridge, F. M.; Blount, H. N. J. Electroanal. Chem. Interfacial Electrochem. 1984, 161, 355-76. (8) Koller, K. B.; Hawkridge, F. M. J. Am. Chem. Soc. 1985, 107, 7412-17. (9) Koller, Kent B.; Hawkridge, Fred M. J. Electroanal. Chem. Interfacial Electrochem. 1988, 239, 291-306. (10) Willit, J. L.; Bowden, E. F. J. Electroanal. Chem. 1987, 265274.
peak, and upon subsequent scans, the peak areas remained unchanged, indicating a reversible reorientation of the adsorbed protein.11 We have been exploring the direct electron transfer of ferritin at electrodes and have previously found an electrochemical response at a gold electrode modified with 3-mercaptopropionic acid (MPA).12 Evaluation of ferritin’s electrochemistry at other electrodes has revealed unusual behavior at tin-doped indium oxide (TIO) electrodes. We have discovered that ferritin can be immobilized at submonolayer coverages, that its electroactive center can be redox cycled, and that a hydrophobic interaction may be responsible for its adsorption. In addition, when ferritin is adsorbed at a TIO electrode, iron can be induced to exit the protein shell by applying a sufficiently reducing potential in the presence of an iron chelating agent. In this Letter, the surface-electrochemical behavior of horse spleen ferritin at tin-doped indium oxide electrodes is reported for the first time. Experimental Section The electrochemical cell used in this study has been described elsewhere.12 Tin-doped indium oxide (100 µm on glass) was donated by Applied Films Corp. (Boulder, CO). Electrodes were cut to 10 mm × 6 mm pieces and cleaned according to published methods.7 In the voltammetric scans described below, only a 6 mm × 6 mm area was immersed in solution. Horse spleen ferritin (Sigma, St. Louis, MO) was purified by size exclusion chromatography using a 25 cm × 2.5 cm column packed with Sephadex G-200 (protein fractionation range, 5000600000 Da). A standard curve was prepared with bovine serum albumin standards complexed with a coomassie Blue G-250 dye (Bio-Rad, Hercules, CA). The concentration of ferritin was determined by measuring the absorbance of the ferritin-dye complex at 595 nm against the albumin-dye standard curve.13 Purified ferritin was diluted with pH 7 phosphate buffer (0.18 M unless otherwise stated) and then deaerated with purified nitrogen for 5 min prior to any scan. Electrodes were immersed into respective ferritin solutions at open circuit potential for 12 h. The treated electrode was rinsed in pure electrolyte, followed by placement in pure electrolyte solution (pH 7 phosphate buffer) at 0.00 V (vs Ag/AgCl). The potential was nominally scanned at 100 mV/s to -0.70 V and returned to 0.00 V, followed by a sweep back to -0.70 V. Ionic strengths of the solutions used were modified by varying the concentrations of the phosphate buffer, (11) Daido, T.; Akaike, J. J. Electroanal. Chem. 1993, 344, 91-106. (12) Martin, T. D.; Monheit, R. J.; Niichel, S. A.; Peterson, S. C.; Campbell, C. H.; Zapien, D. C. J. Electroanal. Chem. 1997, 420, 279190. (13) Bradford, M. M. Anal. Biochem. 1976, 72, 248-54.
S0743-7463(97)00685-9 CCC: $15.00 © 1998 American Chemical Society Published on Web 03/20/1998
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Figure 1. Current-potential curves of ferritin adsorbed at a tin-doped indium oxide electrode: (s) solid curve, first cycle; (- - -) dashed curve, second cycle; (‚‚‚) dotted curve, third cycle. Electrode was immersed at open circuit potential into 1.15 mg/mL purified ferritin in a pH 7.0 phosphate buffer for 12 h, followed by rinsing, and immersion in pure electrolyte; electrode area, 0.36 cm2; ionic strength, 0.40 M; temperature, 25 °C; scan rate, 100 mV/s. while maintaining neutral pH. All potentials mentioned throughout this report have been measured against the Ag/AgCl reference electrode. Experimental packing densities, Γ, were determined by integrating the charge from the cathodic peaks of adsorbed ferritin’s current-potential curve. A baseline was drawn from the leading edge to the tailing edge of a peak, followed by measuring the peak area by cutting and weighing. The packing density was calculated using the Faraday law; the determination of n (1910 ( 190) by controlled potential electrolysis is described elsewhere.12 The theoretical packing density of ferritin was estimated from the geometric electrode area and the “footprint” area of ferritin. To induce iron to exit the adsorbed ferritin following reduction, voltammetric scans of the modified electrode were run in the presence of ethylenediaminetetraacetic acid (EDTA). The electrode was treated as described above, rinsed in electrolyte, and immersed into a solution of 10 mM EDTA in electrolyte at 0.00 V. The current-potential curves were then scanned to -0.80 V and back to 0.00 V.
Results and Discussion Following immersion in the ferritin sample for 12 h, the treated tin-doped indium oxide electrode was transferred into a cell containing pure electrolyte and rinsed free of dissolved ferritin. The electrode was then transferred into another cell containing pure electrolyte, and the potential was scanned; the result is shown in Figure 1. Cathodic peaks appear at -0.44 and -0.60 V, and an anodic peak appears at -0.16 V. On the return scan of the first cycle, the anodic peak is smaller than either of the two initial cathodic peaks. In the second cycle the initial cathodic peaks are no longer present, but a new
Letters
Figure 2. Current-potential curves of ferritin adsorbed at a tin-doped indium oxide electrode: (s) solid curve, first cycle; (- - -) dashed curve, second cycle. Scan conditions were same as in Figure 1. Scan direction was switched before the second cathodic peak.
cathodic peak appears at -0.32 V. On the return scan of the second cycle, the anodic peak potential has not changed. On the third cycle, the new cathodic peak persists with some decrease in the electrolytic charge. These results suggest that upon reduction of the initial layer adsorbed ferritin undergoes a reconstruction/partial desorption leading to a new layer, which is more stable to redox cycling than the initial states. Since the anodic and cathodic peak potentials do not change with repeated cycles, it is inferred that these peaks correspond to the redox couple of the new layer. The peaks appear asymmetric, with the anodic branch being smaller than the cathodic branch. However, since the negative scan is performed after the positive scan, the disparity in peak area is not merely due to desorption of the oxidized form. The presence of a return wave and the narrowness of the peak potential difference indicate that the electrontransfer kinetics of the new layer are faster than those of the initial layer. Though it is not clear what might be responsible for the reconstruction of the initial layer upon its reduction, conformational changes and possible reorientation in the global protein occurring with changes to the metallic centers are known for other proteins.8,9 Little is known about how the quaternary structure or reactivity of ferritin changes when reduction of the iron core commences. In the initial negative scan, the two cathodic peaks signify two different adsorption states. To investigate whether the two states behave independently of one another, the potential scan was stopped just short of the second peak and the scan continued in the positive direction, as shown in Figure 2. During the second cycle, the scan is continued to -0.70 V, and the second of the two initial peaks appears. This experiment shows that not only each state interacts directly with the surface but
Letters
Figure 3. Current-potential curves of ferritin adsorbed at a tin-doped indium oxide electrode in the presence of dissolved EDTA: (s) solid curve, first cycle; (- - -) dashed curve, second cycle. Electrode was immersed at open circuit potential into 0.12 mg/mL purified ferritin in phosphate buffer for 12 h, followed by rinsing, and immersion in pure electrolyte containing 10 mM EDTA: electrode area, 0.36 cm2; ionic strength, 0.40 M; temperature, 25 °C; scan rate, 100 mV/s.
also each state reacts independently of the other. However, the increase to the anodic current following the reduction of the second state is slight, indicating that the formation of the new layer following reduction occurs principally from the reduction of the first state. Using the reported diameter of tissue ferritin of 12.0 nm1 and given that the quaternary structure of ferritin is nearly spherical, the theoretical area occupied per ferritin molecule is calculated to be 1.5 × 10 -12 cm2. With this estimate, a theoretical packing density for ferritin is calculated to be 1.1 pmol/cm2. An experimental packing density can be determined for the two initial states collectively and another for the reconstructed layer following reduction. At an ionic strength of 0.4 M, and a pH of 7.0, the experimentally determined packing density is 0.16 pmol/cm2 for the composite initial layer and 0.10 pmol/cm2 for the reconstructed layer. Clearly, some desorption or loss in electroactivity has occurred following reduction of the initial layer as evidenced by the difference (14) Clegg, G. A.; Fitton, P. M.; Harrison, P. M.; Treffery, A. Prog. Biophys. Mol. Biol. 1980, 36, 56-86. (15) Jones, T.; Spencer, R.; Walsh, C. Biochemistry 1978, 17, 40114017.
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in packing density. At lower ionic strength (0.1 M), the peak at -0.44 V is much larger than the peak at -0.60 V. As the ionic strength is increased to 1 M, the packing density for the composite layer increases to 1.2 pmol/cm2. In addition, the area of the second peak increases relative to that of the first. The distribution of negative charge on metal oxide surfaces is known to be nonuniform.7 It is possible that the patchy surface of indium oxide is responsible for the two different adsorption states observed. That is, the peak at -0.44 V may represent the reduction of ferritin, a negatively charged protein (pI ) 4.5), adsorbed in regions containing a higher surface concentration of anionic groups, and the peak at -0.60 representing the reduction of ferritin residing in regions with fewer anionic groups. The ionic strength dependence suggests that the interaction responsible for ferritin’s adsorption is primarily hydrophobic in both cases. However, in the regions of greater charge density, electrostatic repulsion between ferritin and negative surface groups is possible, leading to the destabilization of the hydrophobic interaction. Iron Transport from Adsorbed Ferritin. It is known that iron can be induced to exit the mineral core following the reduction of ferritin iron.14,15 Since adsorbed ferritin is electroactive at the indium oxide electrode, it is of interest to see whether reduction can induce iron transport from ferritin in the adsorbed state. Figure 3 shows the i-E curve of a similar layer of ferritin immersed in electrolyte containing 10 mM EDTA. The cathodic peaks of the initial layer are present, though attenuated, possibly due to competition with EDTA for adsorption sites on the surface. If displacement of ferritin by EDTA is occurring, then the second adsorption state is less resistant to displacement than the first state. What is most interesting, however, is the absence of the anodic branch of the reconstructed layer, indicating that following reduction, the iron has apparently exited the protein shell, and has been complexed by EDTA surrounding adsorbed ferritin. This result also suggests that the cathodic peak is absent because the iron is no longer present in the protein core. One of the central goals of ferritin research is to explain how iron is transported to and from the protein shell. Reported here for the first time, ferritin in the adsorbed state is shown to give a current-potential response. As a result of accomplishing the direct electron transfer of ferritin, we can observe first-hand via voltammetry, the mobilization of iron through the electrochemical reduction of adsorbed ferritin in the presence of a complexing agent. Clearly, these results present an electrochemical system with promise for examining the loading and unloading of iron in ferritin by controlling the potential of the electrode. This system thus provides the opportunity to study in greater detail iron mobilization kinetics and mechanisms of iron transport. Acknowledgment. The authors acknowledge the Research Corporation (Cottrell Research Grant Number C-3735) for support of this research and Applied Films Incorp. (Boulder, CO) for donating the tin-doped indium oxide. The authors also wish to thank Professor Edmond F. Bowden (North Carolina State University) for his many helpful suggestions. LA970685P
