Langmuir 1997, 13, 4119-4125
4119
Electrochemistry and Catalysis with Myoglobin in Hydrated Poly(ester sulfonic acid) Ionomer Films Naifei Hu† and James F. Rusling* Department of Chemistry, Box U-60, University of Connecticut, Storrs, Connecticut 06269-4060 Received February 7, 1997X Thin films made from the ionomer poly(ester sulfonic acid) Eastman AQ38 and the protein myoglobin (Mb) on pyrolytic graphite (PG) electrodes formed stable hydrated gels in water. While interactions between Mb and the ionomer undoubtedly control retention of the protein, Soret absorbance band positions suggest that Mb retains a conformation similar to the native state in the medium pH range. Cyclic voltammograms were reproducible for several months when Mb-AQ38 films were stored dry or immersed in aqueous buffer. The formal potential of Mb heme Fe(III)/Fe(II) in AQ films shifted -50 mV/pH between pH 4 and 10, suggesting that one electron and one proton are involved in the electrochemical reaction. Square wave voltammograms of Mb in the AQ films were fit by nonlinear regression analysis using a model featuring dispersion of formal potentials. The effective electron transfer rate between PG electrodes and the iron heme of Mb in AQ38 films was comparable to those in surfactant films, but much faster than on bare PG in Mb solutions. Oxygen and trichloroacetic acid were catalytically reduced by Mb in AQ films with significant decreases in the electrode potential required.
Introduction Films containing proteins in water-containing microenvironments are useful for applications to biosensors and biocatalysis. One of our particular interests is to develop stable films for studies of chemical pollutant activation catalyzed by the family of iron heme cytochrome P450 (cyt P450) enzymes.1 Since cyt P450s are not commercially available and require considerable effort to obtain, our approach has been to develop films using the heme protein myoglobin (Mb)2-7 as a model. While Mb is less completely stereo- and regiospecific as a catalyst, it has reactivity similar to that of cyt P450s. The long-term goal is to make stable films with good enzyme activity which are amenable to a variety of electrochemical and spectroscopic experiments. Once useful films are developed using Mb, they can then be applied to cyt P4508 and other enzymes. We recently reported lamellar liquid crystal surfactant films in which electron transfer rates between electrodes and the heme proteins myoglobin (Mb) and hemoglobin (Hb) are greatly enhanced compared to those for bare electrodes with the protein in solution.2-5 When coated onto carbon, gold, or platinum electrodes, these films were stable for a month when stored in buffers but were less stable on indium tin oxide (ITO) electrodes.3 Good catalytic activity for organohalide reduction was achieved with films of Mb and didodecyldimethylammonium bromide (DDAB).6 However, the Mb-DDAB films were subject to mechanical damage in stirred electrolytic reactors. † Permanent address: Chemistry Department, Beijing Normal University, Beijing 100875, China. X Abstract published in Advance ACS Abstracts, July 15, 1997.
(1) (a) Ortiz de Montellano, P. R., Ed. Cytochrome P450, 2nd ed.; Plenum: New York, 1995. (b) Schenkman, J. B.; Greim, H., Eds. Cytochrome P450; Springer-Verlag: Berlin, 1993. (2) Rusling, J. F.; Nassar, A.-E. F. J. Am. Chem. Soc. 1993, 115, 11891. (3) Nassar, A-E. F.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1995, 67, 2386. (4) Nassar, A-E. F.; Narikiyo, Y.; Sagara, T.; Nakashima, N.; Rusling, J. F. J. Chem. Soc., Faraday Trans. 1995, 91, 1775. (5) Lu, Z.; Huang, Q.; Rusling, J. F. J. Electroanal. Chem. 1997, 423, 59-66. (6) Nassar, A.-E. F.; Bobbitt, J. M.; Stuart, J. D.; Rusling, J. F. J. Am. Chem. Soc. 1995, 117, 10986. (7) Huang, Q.; Lu, Z.; Rusling, J. F. Langmuir 1996, 12, 5472. (8) Zhang, Z.; Nassar, A.-E. F.; Lu, Z.; Schenkman, J. B.; Rusling, J. F. J. Chem. Soc., Faraday Trans. 1997, 93, 1769.
S0743-7463(97)00129-7 CCC: $14.00
An ionomer is an ionic polymer that contains only a fraction of ionizable groups per monomer unit. Mb and Hb incorporated into composite films made from DDAB and the fluorocarbon sulfonate ionomer Nafion showed good electron transfer properties and improved stability compared to simple protein-surfactant films.7 However, films of Nafion alone did not incorporate these proteins. Poly(ester sulfonic acid) ionomers, with the trade name Eastman AQ, have also generated interest as film-forming materials. Eastman AQ38 and AQ29 ionomers have 11 mol % aromatic sulfonate groups (see structure below).9 Unlike Nafion, these thermoplastic, amorphous polymers give translucent, low viscosity dispersions in water without adding organic solvent. AQ films cast onto electrodes and dried do not dissolve in water. Similar to Nafion, AQ films bind hydrophobic cations preferentially and exclude negatively charged species.10-12
Enzymes can be added directly to aqueous dispersions of AQ ionomers and deposited onto solid surfaces to form films without denaturation or significant loss of activity.13,14 AQ films can also incorporate protein from solution. Bianco et al.15a successfully incorporated the redox enzymes cytochrome c and cytochrome c3 into AQ films coated onto pyrolytic graphite (PG) electrodes. These AQ films facilitated faster electron transfer than for dissolved c type cytochromes on bare PG electrodes. In (9) Raynolds, P. W. Surface Phenomena and Fine Particles in Waterbased Coatings and Printing Technology, Proceedings of the Fine Particles Society Symposium; Sharma, M. K., Micale, F. I., Eds.; Plenum: New York, 1991; pp 275-281. (10) Wang, J.; Golden, T. Anal. Chem. 1989, 61, 1397. (11) Wang, J.; Lu, Z. J. Electroanal. Chem. 1989, 266, 287. (12) Gennett, T.; Purdy, W. C. Anal. Chem. 1990, 62, 2155. (13) Wang, J.; Leech, D.; Ozsoz, M.; Martinez, S. Anal. Chim. Acta. 1991, 245, 139. (14) Fortier, G.; Beliveau, R.; Leblond, E.; Belanger, D. Anal. Lett. 1990, 23, 1607. (15) (a) Bianco, P.; Taye, A.; Haladjian, J. J. Electroanal. Chem. 1994, 377, 299. (b) Hahn, C. E. W.; Hill, H. A. O.; Ritchie, M. D.; Sear, J. W. J. Chem. Soc., Chem. Commun. 1990, 125.
© 1997 American Chemical Society
4120 Langmuir, Vol. 13, No. 15, 1997
Hu and Rusling
contrast, these proteins were not incorporated from solution by Nafion films.15 In this paper, we show that myoglobin incorporated into AQ films facilitates direct electron transfer with PG electrodes. Mb-AQ films take up large amounts of water and form stable gels. Electrochemical catalysis with MbAQ films was demonstrated for reduction of oxygen and trichloroacetic acid. Experimental Section Chemicals. Myoglobin from horse heart (Sigma) was dissolved in water or buffer and passed through Amicon filters (30 000 MW cutoff) to remove macromolecular impurities.3 Concentrations after ultrafiltration were estimated by absorption spectroscopy.2 The poly(ester sulfonic acid) ionomers used,9 AQ38 (MW ) 14 000, Tg ) 38 °C), AQ55 (MW ) 14 000, Tg ) 55 °C), and AQ29 (MW ) 16 000, Tg ) 29 °C), were gifts from Eastman Chemical Co. and used as received. AQ dispersions in water were obtained by sonication. Nafion, DDAB, and dimyristoylphosphatidylcholine (DMPC) were obtained from previously named sources.7,8 Buffers were 0.1 M acetate, 0.05 M tris(hydroxymethyl)aminomethane hydrochloride (TRIS‚HCl), or 0.025 M sodium dihydrogen phosphate + 0.025 M boric acid + 0.025 M citric acid. Buffers contained 0.1 M or 0.05 M NaBr. Water was purified with a Barnstead Nanopure System to specific resistance >15 MΩ cm. All other chemicals were reagent grade. Preparation of Mb-AQ films. Prior to coating, the PG electrodes were abraded with 600-grit SiC paper, polished successively with aqueous slurries of 0.3 µm and 0.05 µm alumina using a metallographic polishing wheel on billiard cloth, and then polished on clean billiard cloth with pure water. Electrodes were sonicated in water for 20 s after each polishing step. Mb-AQ films were prepared on PG electrodes by two methods. In method I, 10 µL of a1% AQ dispersion was spread evenly with a microsyringe onto a PG electrode and dried in air overnight. Afterward, this coated electrode was placed into a pH 5.5 buffer containing 0.16 mM Mb for incorporating Mb into the films. In method II, 15 µL of a dispersion of 1 part 0.13 mM Mb and 2 parts 1% AQ was deposited onto a PG electrode. A small bottle was tightly fit over the electrode so that the water evaporated slowly. Overnight drying in the small, closed bottle was needed to optimize the voltammetric response. Films that were dried without covering gave smaller CV peaks. Mb-AQ films made by either method gave similar voltammograms. Method II was more convenient and was used for most of the work reported. Apparatus and Procedures. A Bioanalytical Systems BAS100B/W electrochemical analyzer was used for cyclic16 and square wave voltammetry.17 The three-electrode cell featured a saturated calomel electrode (SCE) as reference, a Pt wire as counter electrode, and a basal plane pyrolytic graphite (PG) disk (HPG99, Union Carbide; geometric area 0.16 cm2) as working electrode. The cell was thermostated at 25 ( 0.2 °C. Voltammetry on electrodes coated with Mb-AQ films was done in buffers containing no Mb. Buffers were purged with purified nitrogen for g20 min prior to a series of experiments. A nitrogen environment was kept over solutions in the cell for exclusion of oxygen. In aerobic experiments, measured volumes of air were added via a syringe to solutions in a sealed cell which had been previously degassed with purified nitrogen. UV-vis absorption spectroscopy was done with a Perkin-Elmer Lambda-6 spectrophotometer. Films were prepared by depositing a 1:2 (v/v) mixture of 0.13 mM Mb and 1% AQ38 dispersion onto indium tin oxide coated slides (ITO, from Delta Technologies) and drying them in air. Scanning electron microscopy (SEM) was done with a DSM 982 Gemini microscope (LEO-ZEISS). Sample electrodes were fixed on the SEM mounting stage with low resistance carbon transfer adhesive (Ernest F. Fullam). For cross-sectional views, electrodes were fractured after immersion in liquid nitrogen. Prior to SEM analyses, about 3 nm of Au-Pd was coated onto samples with a Polaron E5100 sputter coater. (16) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New York, 1980. (17) Osteryoung, J.; O’Dea, J. J. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1986; Vol. 14, p 209.
Figure 1. Cyclic voltammograms at 0.1 V s-1 for AQ38 films prepared by method I and immersed in pH 5.5 buffer containing 0.16 mM Mb for (a) 1; (b) 5; (c) 9; (d) 20; (e) 45; (f) 70; (g) 120, and (h) 400 min.
Results Cyclic Voltammetry (CV) of Mb-AQ Films. We first illustrate voltammetric results for films prepared by method I described in the Experimental Section. Basal plane pyrolytic graphite (PG) electrodes coated with AQ38 were placed into a pH 5.5 Mb solution. At this pH, Mb has a surface charge of +6.18 Coulombic attraction of Mb and the AQ sulfonate groups is expected. CV scans at different immersion times revealed the growth of a cathodic-anodic peak pair near -0.3 V vs SCE, characteristic of the Mb heme Fe(III)/Fe(II) redox couple2 (Figure 1). These peaks began to appear 4. All changes in CV peak potentials and currents with pH were reversible. For example, CVs for Mb-AQ films at pH 7 were reproduced after immersion in pH 4 buffer and then returning the film to the pH 7 buffer. CV data were used to investigate the pH dependence of the formal potentials (Eo′) of the Mb Fe(III)/Fe(II) redox couple, estimated as the midpoint of cathodic and anodic peak potentials. Plots of Eo′ vs pH were linear between pH 4 and 10 with slope of -50 mV per pH unit (Figure 5). This suggests that a single proton transfer is coupled to the electron transfer.22,23 An inflection point appears in the plot at pH 4.0. At pH < 4, Eo′ was nearly independent of pH. The surface concentration (Γ*) of Mb in AQ films estimated by integration of CV reduction peaks, was essentially constant between pH 5 and 10, with an average value of (2.38 ( 0.03) × 10-10 mol cm-2. However, Γ* decreased by about 20% when the pH was lowered from 5.0 to 3.2. UV-vis Spectroscopy. Positions of the Soret absorption band of iron(III) heme provide information about (22) Meites, L. Polarographic Techniques, 2nd ed.; Wiley: New York, 1965; p 278. (23) Bond, A. M. Modern Polarographic Methods in Analytical Chemistry; Marcel Dekker: New York, 1980; p 27.
Electrochemistry and Catalysis with Myoglobin
Langmuir, Vol. 13, No. 15, 1997 4123
Figure 6. Visible spectra of Mb-AQ38 films on indium tin oxide (ITO) slide in different pH buffer. Vertical line at 411 nm represents wavelength of peak at pH 7.
possible denaturation of Mb.24 Mb dissolved in pH 5.5 buffer has a Soret band at 408 nm. Films cast from Mb alone in neutral buffers gave a Soret band at about 410 nm.7 Dry Mb-AQ films on transparent ITO glass showed a Soret band at 408 nm, suggesting that Mb in AQ films has a secondary structure similar to the native state in solution. The position of the Soret band depended on pH when Mb-AQ films were placed into buffer solutions (Figure 6). At pH 7.0, the Soret band appeared at 411 nm. This band shifted blue to 405 nm when the pH was changed to 4.0 or 3.2. At pH 10.0, the Soret band was at 408 nm. While Mb-AQ films did not adhere as well to ITO-coated glass as to PG electrodes, Soret bands remained constant for 4-5 days when the ITO was treated with dilute acetic acid prior to film deposition. Scanning Electron Microscopy (SEM). Top views of an AQ film (Figure 7a) on a PG electrode revealed an uneven, porous structure with small cubic particles on it. Top SEM views of polished PG electrodes appeared relatively flat and featureless at the same magnification. AQ films containing Mb, however, showed much different top SEM views (Figure 7b), which revealed a leaflike pattern. SEM views of Mb-AQ film cross-sections after freeze-fracturing were employed to estimate an average dry film thickness of 1-3 µm. Catalytic Reactivity. The possibility of electrochemical catalysis of the reduction of oxygen by Mb-AQ films was examined by CV with oxygen present in the external solution. After air was passed through a pH 5.5 buffer solution, a substantial increase in the MbFeIII reduction peak at about -0.3 V was observed (Figure 8d) for MbAQ films compared to the peak for MbFeIII with no oxygen present (Figure 8b). This increase in reduction current was accompanied by the disappearance of the oxidation peak for MbFeII. For AQ films without Mb, the peak for direct reduction of oxygen was observed at about -1.0 V (Figure 8c). The oxidation peak for MbFeII does not appear because it has reacted with oxygen (Figure 8d,e). An increase in the amount of oxygen in solution increases the height of the reduction peak (Figure 8e). Catalytic efficiency, expressed as the ratio of the MbFeIII peak reduction current in the presence (ic) and absence of oxygen (id),16 decreased, as the scan rate was increased (Figure 9). All of these results are characteristic of reduction of oxygen by electrochemical catalysis.16 Electrochemical catalytic reduction of trichloroacetic acid (TCA) using Mb-AQ films was also demonstrated. (24) (a)Theorell, H.; Ehrenberg, A. Acta Chem. Scand. 1951, 5, 823. (b) George, P.; Hanania, G. Biochem. J. 1952, 52, 517. (c) Herskovits, T. T.; Jaillet, H. Science 1969, 163, 282.
Figure 7. Top SEM views of coated PG electrodes: (a) AQ38 film; (b) Mb-AQ38 film.
Figure 8. Cyclic voltammograms at 0.1 V s-1 in 6 mL of pH 5.5 buffer solution containing 50 mM NaBr: (a) AQ38 film with no Mb and no oxygen present; (b) Mb-AQ38 film with no oxygen present; (c) AQ38 film with no Mb after 40 mL of air was injected into a sealed cell; (d) Mb-AQ38 film after 40 mL of air was injected; (e) Mb-AQ38 film after 90 mL of air was injected.
When TCA was added to the buffer, an increase in the MbFeIII reduction peak in Mb-AQ films was observed (Figure 10). This was accompanied by a decrease and disappearance of the MbFeII oxidation peak as the TCA concentration was increased. These results indicate reaction of MbFeII with TCA in a catalytic cycle, presumably resulting in the reductive dechlorination of the acid.6 MbFeII is known to react with TCA, giving less chlorinated acetic acids and acetic acid itself.2,6 Compared to AQ films
4124 Langmuir, Vol. 13, No. 15, 1997
Figure 9. Influence of scan rate on catalytic efficiency, ic/id, for Mb-AQ38 electrode in pH 7.5 buffer, where id is the CV reduction peak current in buffer without oxygen and ic is the CV cathodic peak current in 6 mL buffer after 10 mL of air was injected into a sealed cell.
Figure 10. Cyclic voltammograms at 0.01 V s-1 in pH 5.5 buffers containing 50 mM NaBr: (a) AQ38 film without Mb in buffer containing 70 mM trichloroacetic acid; (b) Mb-AQ38 film in buffer containing no trichloroacetic acid; (c) Mb-AQ38 film in buffer containing 70 mM trichloroacetic acid.
without Mb (Figure 10a), Mb-AQ films lowered the potential required for reduction of TCA by about 0.6 V (Figure 10c). The catalytic efficiency decreased with increasing scan rate, and the reduction peak current for Mb increased with increasing trichloroacetic acid concentration in solution, characteristic of electrochemical catalysis.16 Discussion Stability and Structural Factors. AQ ionomers were shown to form dispersions in water which feature spherical colloids having outer negative charges facing the water. A microgel-like model for the colloidal particle has been proposed.9 While cast dispersions of these polyester ionomers dry to give clear films, they rehydrate in water to form opaque gels containing nearly 90% water. We expect that these gels retain the charge segregation of the colloidal particles from which they were made. This is consistent with reported cation binding and exclusion of anions by AQ films.10-12 The water content of AQ films is the same in the presence or absence of the protein Mb. Thus, the environment of the protein in these films contains considerable water. Reversible changes in spectra (Figure 6) and voltammograms with changes in pH (Figures 4 and 5) show that the properties of the protein are controlled by the pH of the external solution in which the films reside. Soret band spectra are quite similar to those in water (Figure 6) and suggest that Mb has a secondary structure in the films similar to that of the native protein in the medium pH range.
Hu and Rusling
A significant amount of Mb remains electroactive in the films and is stable for long periods. SEM pictures of AQ films with and without Mb (Figure 7) show a very different, patterned structure when Mb is present. This suggests interactions between Mb and AQ38 which govern morphology of the dry films. Such interactions may also be responsible for retention of Mb in the films for long periods (Table 1). The large water content (87%) of AQ films compared to films of the more widely used Nafion (28% water) may be correlated to the ability of AQ films to take up significant amounts of Mb, while Nafion films do not take up Mb.7 While both types of films have porous structures, AQ films are expected to have a larger water-filled volume than Nafion. This may enable easier access of the protein to enter the film. Once in the film, Mb’s interactions with the polyester backbone and arenesulfonate groups of the AQ ionomers are apparently stronger than those with Nafion, which has a fluorocarbon backbone and aliphatic sulfonate groups. AQ films preferentially bind large hydrophobic cations.10-12 At pH 5.5 Mb (Figure 1) has a +6 charge18 and seems to fall into this category. While the stability and retention of Mb in AQ films are likely to depend on hydrophobic, Coulombic, and hydrogen-bonding interactions between the two macromolecules, the exact nature of these interactions are as yet unknown. Hydrophobic interactions may contribute significantly to Mb-AQ interactions, consistent with the stability of the films at pH 7.5 (Table 1). At this pH, Mb, with an isoelectric point25,26 (pI) at pH 6.8, is nearly neutral and Coulombic interactions would not be strong. Despite interactions with AQ in the films, Soret band positions (Figure 6) suggest that Mb retains a near-native conformation when solutions are in the medium pH range. Mb-AQ films seem to be somewhat more stable than films of Mb and insoluble surfactants or Nafion-surfactant composites (Table 1). Mb-AQ films retained larger CV signals upon storage than films of Mb-DDAB or MbDDAB-Nafion. Results suggests that AQ films have better adhesion to PG electrodes and strong interactions with Mb. Mb-AQ films on ITO-glass slides were less stable than on PG, but adhered to acid-treated ITO for 4-5 days without significant losses of Soret band absorbance. Electrochemical Properties. Nearly reversible cyclic voltammograms for Mb were obtained in AQ films prepared in two ways (Figures 1 and 2a), suggesting direct electron transfer between Mb in the AQ gel films and the electrode. Rapid appearance of peaks after immersion of AQ films in Mb solutions (method I, Figure 1) suggested physical diffusion of Mb within these films, as also found for Mb-DDAB films.2 The issue of physical diffusion vs electron self-exchange as mechanisms for charge transport has not been specifically addressed in this paper. However, the above evidence for Mb diffusion, and the Mb film concentrations of roughly 1 mM estimated from electroactive Mb surface concentrations and film thickness, would argue against a predominant role for electron self-exchange. Tentatively, the AQ films can be viewed as gel phases with a high water content and affinity for Mb, which may remain relatively mobile within them. Electron transfer was much faster in the AQ films than for Mb in solution on bare PG,2,3 on which electron transfer (25) Bellelli, A.; Antonini, G.; Brunori, M.; Springer, B. A.; Sligar, S. G. J. Biol. Chem. 1990, 265, 18898. (26) Rossi-Fenelli, A.; Antonini, E.; Povoledo, D. In Symposium on Protein Structure; Neuberger, A., Ed.; Methuen & Co. Ltd.: London, 1958; p 144.
Electrochemistry and Catalysis with Myoglobin
Langmuir, Vol. 13, No. 15, 1997 4125
is so slow and irreversible as to be difficult to observe. Subject to the interpretation that ks is an apparent electron transfer rate constant only, its value for Mb-AQ films was similar to those in films of dimyristoyl- or dilauroylphosphatidylcholine (DMPC and DLPC, respectively), and slightly larger than that in DDAB and DDAB-Nafion films (Table 2). The formal potentials of the heme Fe(III)/Fe(II) couples of Mb in AQ films were similar to those in DMPC and DLPC films, but they were considerably more negative than those in DDAB and DDAB-Nafion films (Table 2). This confirms a specific influence of film environment on Eo′ of heme proteins which had been reported previously.8,20 Film components may shift potentials via interactions with the protein or by their influence on the electrode double-layer.20 The dependence of Eo′ and Soret band spectra on pH (Figures 4-6) is similar in Mb-AQ films to that of MbDDAB and Mb-phosphatidylcholine films.20b The slope of -50 mV/pH for plots of Eo′ vs pH at pH > 4 is close to the theoretical value at 25 oC of -59 mV/pH for a reversible, proton-coupled single electron transfer.22,23 This suggests that a single protonation accompanies electron transfer between the electrode and MbFeIII, represented by
MbFeIII + H+ + e- h MbFeII
(7)
where the charges on Mb species have been omitted. Soret bands of Mb in AQ films shifted blue from 411 to 405 nm when the pH changed from 7.0 to 4.0 or 3.2 (Figure 6). This suggests the partial denaturation of Mb in AQ films at pH
