Langmuir 1996,11, 3296-3301
3296
Articles Electroactive Myoglobin-Surfactant Films in a Bicontinuous Microemulsion Anthony C. Onuoha and James F. Rusling" Department of Chemistry, University of Connecticut, Box U-60, Storrs, Connecticut 06269-3060 Received January 12, 1995. In Final Form: May 11, 1995@ Films cast from DDAB microemulsions containing the protein myoglobin (Mb) onto pyrolytic graphite electrodes gave good electron-transfer properties when used in these same microemulsions. The films became thinner during the first 10 min after initial insertion into the microemulsion. A film < 1pm thick remained on the surface of the electrode in DDAB microemulsions and exhibited direct electron transfer between the electrode and the Fe(III)/Fe(II)redox couple of myoglobin. Mb-DDAB films could be used for nearly a week in an unstirred DDAB/water/dodecane (13/28/59)microemulsion. The position of the Soret electronic absorbance band suggests that Mb in these films is partly denatured. Mb-DDAB films were used to facilitate redox reactions of polar and nonpolar solutes in DDAB microemulsions, as illustrated by reductions of the polar trichloroacetic acid and the nonpolar oxygen.
Introduction We recently reported the preparation of stable films of the protein myoglobin and surfactants on carbon, noble metal, and indium tin oxide electrodes.1,2 When these films were used in buffer solutions, electron-transfer rates between electrodes and myoglobin's heme Fe(III)/Fe(II) couple were enhanced by as much as 1000-foldcompared to bare electrodes with Mb in solution. This rate enhancement is caused at least in part by strong adsorption of surfactant at the film-electrode interface, which inhibits adsorption of proteins and provides a pathway for electron exchange.2 These films feature surfactants ordered in stacked bi1ayers.l Thus, protein is contained in a biomembranelike environment, and these films provide models for the study of membrane-bound protein chemistry. Cast Mbsurfactant films were stable in aqueous buffers for up to a month and can catalytically dehalogenate organohalide pollutants dissolved in water.l Microemulsions are clear, microheterogeneous mixtures of oil, water, and surfactant with intriguing possibilities as media for chemical reactions, including kinetic control of reactions and rate We are currently exploring microemulsions as media for electrochemical Abstract published inAdvance ACSAbstracts, August 1,1995. (1)(a)Rusling, J. F.; Nassar, A.-E. F. J . A m . Chem. SOC.1993,115, 11891-11897. (b) Rusling, J. F.; Nassar, A.-E. F.; Kumosinski, T. F. In Molecular Modeling; Kumosinski, T. F.; Leibman, M. N., Ed.; ACS Symposium Series 576;American Chemical Society: Washington, DC, 1994;pp 250-269. (2)Nassar, A.-E. F.; Willis, W. S.; Rusling, J. F. Anal. Chem., in press. (3) Fendler, J. H. J . Phys. Chem. 1980,84, 1485-1491. (4)O'Connor, C. J.;Lomax, T. D.; Ramage, R. E.Adv. Colloid Interface Sci. 1984,20,21-97. (5)Fendler, J.H.Membrane Mimetic Chemistry; Wiley: New York, 1982. (6) Luisi, P. J.;Magid, L. J. CRC Crit.Rev. Biochem. 1987,20,409472. (7)Rusling, J. F. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1994,Vol. 18,pp 1-88. (8)Rusling, J.F. In Modern Aspects ofElectrochemistry; Conway, B. E., Bockris, J. O.'M., Eds.; Plenum: New York, 1994;No. 26,pp 49104. (9)Friberg, S.E. Adv. Colloid Interface Sci. 1990,32,167-182. (10)Owlia, A.;Wang, Z.; Rusling, J. F. J . Am. Chem. SOC.1989,111, 5091-5098. @
catalysis and ~ y n t h e s e s . ~Microemulsions t~ are being considered as less toxic and less expensive replacements for organic solvents in many application^.^*^ They are excellent solvents for both polar and nonpolar comp o u n d ~ . ~Development -~ of catalytic films on electrodes usable in microemulsions would enable applications to reactants that are not soluble in water. Thus far, nearly all studies of electrochemical catalysis in microemulsions have involved catalysts which are soluble in the medium or which form relatively unstable adsorbed films on e1ectrodes.l0-l2 It would be highly desirable to have reusable catalytic films on electrodes which are stable in microemulsions. This would be especially important if the catalyst were an expensive protein. In this paper, we describe films cast from myoglobin solutions in microemulsions of didodecyldimethylammonium bromide (DDAB)onto pyrolytic graphite electrodes. These films were usable for up to a week in a microemulsion made from water, oil, and 13% DDAB. Mediation of the electrochemical reduction of polar and nonpolar compounds by these films is demonstrated.
Experimental Section Chemicals. Lyophilized horse heart myoglobin was from Sigma. Didodecyldimethylammoniumbromide (DDAB,99%) and dodecane (99%) were from Eastman Kodak. The 0.05 M acetate buffer (pH4.8)contained 0.05 M NaBr. Trichloroacetic acid was from Janssen Chimica. Microemulsions were prepared as described previously.13 Their compositions by weight were DDAB/water/dodecane (13/ 28/59)(13% DDAB microemulsion, specific conductivity 0.12 x W 1cm-l) and DDAB/water/dodecane(21/39/40) (21% DDAB, specific conductivity 1 x Q-l cm-l). Apparatus and Procedures. A Princeton Applied Research Corp. (PARC)Model 273 electrochemical analyzer was used for cyclic voltammetry. The three-electrode cell employed a Vycor tipped saturated calomel reference electrode, a Pt wire counter electrode, and an ordinary (not highly ordered) basal plane (11)JSamau, G. N.; Hu, N.; Rusling, J. F. Langmuir 1992,8,10421044. (12)Zhang, S.;Rusling, J. F. Enuiron. Sci. Technol. 1993,27,13751380. (13)Iwunze, M.0.;Sucheta, A,; Rusling, J. F.Ana1. Chem. 1990,62, 644-649.
0743-746319512411-3296$09.00/00 1995 American Chemical Society
Langmuir, Vol. 11, No. 9, 1995 3297
Electroactive Myoglobin-Surfactant Films 20 I
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pyrolytic graphite (HPG-99from Union Carbide,A = 0.28 cm2) or a glassy carbon (A = 0.071 cm2) working electrode. Carbon electrodes were polished with a 0.3-pm a-alumina dispersionon a billiard cloth on a metallographic polishingwheel for 2 min. The electrode was subsequently sonicated for 2 min in distilled water and then wiped dry with a Kimwipe. Myoglobin(Mb)-DDAB films were deposited from a solution of Mb in microemulsion. Unless otherwise mentioned, 10 pL of 0.5mM Mb dissolved in the 13% DDAB microemulsion solution was deposited onto the polished mirrorlike surface of a bare electrode and dried overnight in air. To obtain reproduciblevoltammograms,freshly prepared MbDDAB electrodes were placed in an electrochemical cell containing microemulsion for an initial period of 5-10 min. Following this, CV measurements were made using a delay of 3 s at the initial potential before each scan. Resistance of the cells was 2000-2500 Q in the 13% microemulsion and 200-300 C2 in acetate buffer. Ohmic drop was fully compensated by the PARC 273 system in all experiments. Voltammetry was done at 25 "C. In experiments on the reductionof oxygen, a gas-tightsyringe was used to inject a known volume of 0 2 into an air-tight electrochemical cell, containing5 mL ofmicroemulsionor acetate buffer. CV measurements were made a few seconds after the oxygen was injected into the medium.
Results Voltammetry of Myoglobin in Microemulsions. Cyclic voltammetry of myoglobin dissolved in DDAB microemulsions was observable on bare pyrolytic graphite (PG) electrodes. However, the curve shapes and large anodic-cathodic peak separations suggest rather slow kinetics and complicated electrode processes (Figure 1). On the other hand, films deposited from Mb-DDAB microemulsions and used in DDAB microemulsions without dissolved Mb gave well-defined symmetric cathodic and anodic peaks with similar peak potentials at low scan rates (Figure 2a). These CVs are characteristic of thinlayer e l e c t r ~ c h e m i s t r yin , ~which ~ all of the electroactive Mb in the film is reduced or oxidized during a given halfcycle. Peaks appeared at potentials consistent with those of the MbFe(III)/MbFe(II) redox c0up1e.l~ At scan rates above 1V s-l, the peak shapes for the films became characteristic of diffusion-kinetic-controlled electrochemistry (Figure 2b) in which only part of the electroactive material in the film is electrolyzed during a single half-cycle. This type of behavior was found previously above 50 mV s-' for 20-pm-thick Mb-DDAB films in aqueous (14) (a) Bard, A. J.;Faulkner, L. R. Electrochemical Methods; Wiley: New York, 1980. (b) Murray, R. W. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1984;Vol. 13,pp 191-368. (15)King, B. C.;Hawkridge, F. M.; Hoffman, B. M. J. Am. Chem. SOC.1992,114, 10603-10608.
I 0.60
-0.20
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Figure 1. Cyclic voltammograms on bare PG electrodes for 0.5 mM Mb dissolved in 13% DDAB microemulsion at (a) 100 and (b) 50 mV s-'.
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Figure 2. Cyclic voltammograms of Mb-DDAB films on PG electrodes in 13% DDAB microemulsion: (a) at scan rates 5100 mV s-l; (b) at scan rates between 2 and 5 V s-'.
The cathodic peak current for the films was directly proportional to scan rate below 200 mV s-I (Figure 3a), consistent with thin-layer electr~chemistry.'~CV peak current separations a t scan rates between 20 and 100 mV s-l were used to estimate a n average electron-transfer rate constant of about 2 s-l by using the method of Laviron.'6 The formal potential estimated as the midpoint between anodic and cathodic peak potentials was -0.19 V vs SCE or 0.05 V vs NHE. This value is in the same range as those of the Mb Fe(III)/Fe(II) couple in MbDDAB films in aqueous buffers of pH 5.5(0.09 V vs NHE) and pH 7.5 (0.050Vvs NHEY as well as for Mb in solution at pH 7 on indium-tin oxide electrodes (0.055Vvs NHE).15 It is also similar to the value of 0.03 V vs NHE obtained for films deposited from Mb in a DDAB microemulsion in pH 4.8 acetate buffer. At scan rates > 0.5 V s-', the peak current of the films in 13% DDAB microemulsion is proportional to the square root of the scan rate, characteristic of diffusion control (Figure 3b). In previous work in aqueous buffers, Mb-DDAB films about 20-pm thick gave thin-layer CVs only between 1 and 6 mV In contrast, the Mb-DDABfilms deposited from microemulsions in the present work were initially about 30-pm thick as estimated from the molecular volumes and amounts of the film molecules deposited.'" Nevertheless, thin-layer behavior extended to 200 mV s-l. This suggests that the films in the DDAB microemulsion are considerably thinner than 30 pm. Immediately after film deposition and drying, visual inspection revealed rather thick, opaque white coatings on electrodes. After use in a microemulsion, films are visibly thinner and appear as a translucent thin coating on the underlying carbon electrode. Consistent with this idea, we find a n average of 1x mol of Mb in the films (16)Laviron, T.in Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1982;Vol. 12, pp 53-157.
Onuoha and Rusling
3298 Langmuir, Vol. 11, No. 9, 1995 /
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’.OO
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60
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2
8
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Figure 4. Stability of a Mb-DDAB film cast from the microemulsion onto a PG electrode and stored continuouslyin a DDAB microemulsion. CVs at 1V s-l with numbers on curves denoting days elapsed after preparation of the film.
Scan Rate (mVl8)
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Figure 3. Influence of scan rate (v) on the peak current of Mb-DDAB-coated PG electrode: (a)linear dependenceon Y at low scan rates; (b) linear dependence on vu2 at higher scan rates.
by integration of CV peaks at scan rates < 40 mV s-l after films have been soaked in a microemulsion, although 5 x mol of Mb was initially deposited in the film. While it is possible that some Mb leaches out of the films, this cannot explain the characteristic thin-layer CVs (Figure 2a). The above results suggest that the films become thinned by dissolution of their outer layers in the microemulsion. An electroactive layer, roughly estimated to be < 1-pm thick from the amount of Mb present, remains adhered onto the electrode and is responsible for the observed peaks (Figure 2). Most of the thinning takes place within about the first 5-10 min after the electrode is placed into the microemulsion. Such a thin film is consistent with the symmetric thin-layer CVs observed up to 200 mV s-l. Stability of the Films. Mb-DDAB films were more stable in the 13%DDAB microemulsion than in the 21% DDAB microemulsion. Films prepared on pyrolytic graphite (PG) electrodes were more stable than on glassy carbon. Thus, most of the work reported in this paper was done with films on PG electrodes in the 13%DDAB microemulsion. In a 13%DDAB microemulsion stirred only by occasional bubbling with nitrogen, CV scans at 5-min intervals on films prepared on PG gave identical cathodic peak currents a t 1V s-l for 2.5 h. To illustrate longer term stability, CVs for 4 successive days of a n electrode in 13% DDAB microemulsion are compared (Figure 4). These CVs are very similar. Such electrodes can be used for nearly a weekin unstirred 13% DDAB microemulsionwithout large decreases in peak current. With continuous stirring and bubbling with nitrogen in a 13%DDAB microemulsion,
Wavelength (nm)
Figure 5. UV-vis spectra of Mb-DDAB film cast from the microemulsion onto indium-tin oxide coated glass. Labels indicate the number of minutes the film was soaked in a 13% DDAB microemulsion before removal and recording of the spectrum.
the cathodic peak decreased by about 10% in the first 30 min and then remained relatively constant for several hours. Absorption Spectra. Films deposited onto conductive glass from a Mb-DDAB microemulsion had a Soret band from absorption of the heme group a t 408 nm (Figure 5). Mb dissolved in the 13%microemulsion had a Soret band a t 393 nm. Native Mb dissolved in buffers between pH 5.5 and 7.5 has a Soret band at 409 nm. Films prepared previously from Mb and DDAB only and placed in aqueous buffers had Soret bands at 413 nm a t pH 5.5 and 415 nm at pH 7.5. Infrared spectroscopy showed that Mb-DDAB films in aqueous pH 5-8 buffers retain the native Mb conformation. New films prepared on quartz and then soaked in the 13%microemulsion for various times had Soret bands at about 407 nm. The shorter wavelengths for Soret bands of Mb dissolved in the DDAB microemulsion and in the Mb-DDAB microemulsion films are consistent with partial denaturation of the protein.17 Absorption spectra were also used to confirm thinning of the films. Soret bands decreased in intensity with time of soaking in the microemulsion (Figure 5). After 10 min, the peak was less than 10% of its original intensity in the dry film. Reduction of Oxygen. CVs of Mb-DDAB microemulsion films on PG electrodes were examined in the presence of dissolved oxygen in both 13%DDAB micro(17) (a) Goto, Y.;Fink, A. L.J.Mol. B i d . 1990,214,803-805. (b) Stigter, D.;Alonso, D.0.V.;Dill, K. A. Proc. Natl. Acad. Sci. U S A . 1991,88,4176-4180.
Langmuir, Vol. 11, No. 9, 1995 3299
Electroactive Myoglobin-Surfactant Films
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Figure 7. Influence of a limiting amount of oxygen on CVs at 100 mV s-l of Mb-DDAB film on PG in 13% DDAB microemulsion: (a)in the absence of oxygen; (b) after passing 1mL of 0 2 through the fluid.
'
-1.50
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Figure 6. Influence of oxygen on CVs on PG electrodes at 200 mV s-l in 13%DDAB microemulsion: (a) Mb-DDAB film in the absence of oxygen; (b)Mb-DDAB film after passing 5 mL of 0 2 through the fluid; (c) on bare PG electrode after passing 5 mL of 0 2 through the fluid; (d)DDAB film (no Mb) in oxygenfree fluid; (e)DDAB film after passing 5 mL of 0 2 through the fluid.
emulsion and pH 4.8 acetate buffer. This buffer pH was chosen because it is close to the estimated pH of the water phase of the microemulsion.l8 Experiments were done after passing a fixed volume of oxygen through the solution, since saturation with oxygen produced currents which were too large for meaningful analysis. A typical cyclic voltammogram obtained with a MbDDAB film electrode after passing 5 mL of oxygen through a 13% DDAB microemulsion is shown in Figure 6b, while Figure 6a is the CV obtained in the absence of oxygen. The addition of oxygen caused a n increase in cathodic current at the potential of the reduction of MbFe(II1).This is accompanied by a shift of the peak by about 40 mV to more positive potentials and the disappearance of the anodic peak for the oxidation of MbFe(I1). The second reduction peak in Figure 6b at a potential of -0.6 V vs SCE is due to the direct reduction of oxygen in the film at the PG electrode. This occurs a t a more positive potential than the direct reduction of oxygen on bare PG, which is responsible for a broad peak a t about -1.0 V (Figure 6c). This view was confirmed by voltammograms for reduction of oxygen on an electrode coated with a DDAB microemulsion film not containing Mb. Such electrodes showed overlapped peaks for the oxygen reduction at -0.6 V vs SCE, as well as the second peak near -1 V. The first peak corresponds to the direct reduction of oxygen in the film, while the second probably reflects reduction at uncoated portions of the PG electrode. As reported earlier,l* DDAB films on PG are much less stable than DDAB-Mb films. It is probable the DDAB microemulsion film develops some bare spots during expose to the microemulsion, which give rise to the peak at -1 V.
-1.50
-1.00
-0.50
0.00
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1.00
log (scan rate, V l s )
Figure 8. Influence of scan rate for MbFe(II1) reduction in Mb-DDAB films in 13%DDAB microemulsion on the ratio of cathodic peak current for MbFe(II1)reduction in the presence of a reactant to that in the absence of any reactant: (0)0.1 M trichloroacetic acid; (+) 5 mL of oxygen passed into 5 mL of microemulsion; (0) 5 mL of oxygen passed into 5 mL of pH 4.8 acetate buffer.
The above results suggest that Mb facilitates the reduction of oxygen by lowering its reduction overpotential in some way. The excess 0 2 in the film not reduced in the peak at -0.18 V can be reduced directly a t the electrode surface. Under the conditions in Figure 6b, the reaction is limited by the amount ofMb in the films, and the second peak for the direct reduction of oxygen is observed at -1 V. A limiting amount of oxygen in the microemulsion should not give this direct reduction peak a t -0.6 V a t a Mb-DDAB electrode, and only the enhanced reduction peak a t -0.18 V was observed when 1mL of oxygen was used (Figure 7). The second direct reduction peak was observed only when the dissolved 0 2 concentration was increased to higher levels (cf. Figure 6b). The ratio of the peak current of the Fe(II1) reduction of Mb-DDAB microemulsion films in the presence and absence of oxygen vs the log of the scan rate at constant oxygen concentration showed values tending toward 3 a t low scan rates. This peak ratio decreased and tended toward 1 with increasing scan rate (Figure 8). This behavior is consistent with a n overall three-electron reduction at the low scan rates. DDAB microemulsions are excellent solvents for oxygen.lg However, oxygen is about 10-fold less soluble in aqueous pH 4.8 acetate buffer. Thus, when the same volume of oxygen is passed through the buffer, much less oxygen dissolves compared to the microemulsion. A CV obtained after 5 mL of oxygen was passed through the
~~
(18)Gounili, G.; Miaw, C. L.; Bobbitt, J. M.; Rusling, J. F. J.Colloid Interjface Sci. 1992, 153, 446-456.
(19) Schweizer, S.;Huang, Q.; Rusling, J. F. Chemosphere 1994,28, 961-970.
Onuoha and Rusling
3300 Langmuir, Vol. 11, No. 9, 1995 15
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Figure 9. Influence of oxygen on CVs at 200 mV s-l for MbDDAB film on PG (a and b) in pH 4.8 acetate buffer: (a) in the absence of oxygen; (b) after passing 5 mL of 02 through the fluid; (c) on bare PG electrode after passing 5 mL of 0 2 through the solution.
buffer (Figure 9) shows a peak for the facilitated reduction of oxygen positive of the MbFe(II1)reduction peak. Since a smaller fraction ofthe oxygen injected into the acetatebuffer medium gets dissolved, the Mb in the film becomes depleted of oxygen. Such double-peak behavior is characteristic20 of the reduction of two species a t slightly different potentials, possibly hIbFe(II1) and oxygen, when limited amounts of oxygen are present. Also, a small anodic peak is seen in Figure 9b for the MbFe(I1) which has not participated in a reaction with oxygen. When a larger volume of 0 2 (40 mL) was injected into the fluid, a large single cathodic wave with no anodic peak resulted. Direct CV reduction of oxygen a t bare pyrolytic graphite in this buffer medium gave an irreversible cathodic peak at -0.6 V vs SCE (Figure 9c). As in the microemulsion, a plot of the ratio of the peak current of the Fe(II1)reduction in the presence and absence of oxygen [i,(O&(Mb)l vs the log of the scan rate a t constant [ 0 2 l approached 3 a t smaller scan rates. The ratio decreased and tended toward 1 a t higher scan rates (Figure 8). Mediated Reduction of TrichloroaceticAcid. CVs of Mb-DDAB electrodes aRer the addition of trichloroacetic acid (TCA) to microemulsions showed increases in cathodic current for the reduction of MbFe(II1) and decreases of the anodic peak for the oxidation of MbFe(I1) (Figure lob-e) when compared with the nearly reversible CV of Mb-DDAB in the absence of TCA (Figure loa). This cathodic current increased linearly with increasing concentration of TCA. Direct reduction of trichloroacetic acid a t bare PGE in 13%DDAB microemulsion gave a n irreversible peak a t a potential of about -1.5 V vs SCE (Figure 100. As the scan rate was increased, the ratio i,(Mb TCA)/ i,(Mb) decreased in microemulsions containing TCA from about 6 at scan rate of 0.05 V s-l to about 1 a t scan rates between 1 and 5 V s-l (Figure 8). This behavior is consistent with the catalytic dechlorination of trichloroacetic acid.21,22
+
Discussion Films used in this work were cast from a Mb-containing microemulsion and were prepared a bit differently from cast Mb-DDAB films reported previously,1+2which were unstable in microemulsions. Cyclicvoltammetry, vidual, (20) Andrieux, C. P.; Blocman, C.;Dumas-Bouchiat,J.-M.;M'Halla,
F.;Saveant, J.-M. J.Electroanal. Chem. 1980,113, 19-40.
(21)Rusling, J. F.; Miaw, C. L.; Couture, E. C. h o g . Chem. 1990,
29, 2025-2027.
"
(22) (a)Nassar, A.-E. F.; Bobbitt, J. M.; Stuart, J. D.; Rusling, J. F. In preparation. (b) Onuoha, A. C.; Rusling, J. F. In preparation.
2ooo
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Figure 10. Influence of trichloroacetic acid on CVs at 100 mV s-l ofMb-DDAB film (a-e) on PGin 13%DDAB microemulsion with different concentrationsof trichloroacetic acid: (a)0 mM, (b) 2.8 mM, (c) 4.7mM, (d) 7.3 mM, (e) 11.7 mM, and (0100 mM trichloroacetic acid on a bare PG electrode. and spectroscopic observations indicated that the films prepared in the present work become thinned when placed into DDAB microemulsions. After about 5-10 min, enough of the film remains on the surface of the pyrolytic graphite electrode to obtain well-defined voltammograms which demonstrate direct electron transfer between the electrode and the Mb Fe(III)/Fe(II) redox couple. This thinned Mb-DDAB film is stable under operating conditions in 13% DDAB microemulsions for nearly a week, presumably because of adequate adhesion between hydrophilic groups in the SurfactantJprotein layer and the hydrophilic surface' of the PG electrode. The film is slightly less stable when the solution is stirred continuously. The position of the Soret band a t 407 nm in UV-vis spectra of films which had been soaked in microemulsion suggests that Mb is not in its native state.17 Native Mb in films gives a Soret band at 410-415 nm.la Soret bands in buffer solutions for the partly denatured stable "molten globule" form of Mb existing below pH 5 are found a t wavelengths smaller than 408 nm. Thus, the position of the Soret band suggests that Mb in the films is partly denatured. The reasonable stability of the Mb-DDAB films and their relatively good electron-transfer properties allowed them to be used to facilitate the reduction of polar and nonpolar molecules in the 13%DDAB microemulsion. The reduction of oxygen in the Mb-DDAB films takes place with a 0.8-V decrease in overpotential, which is proportional to activation free energy, compared to bare PG. Reduction of trichloroacetic acid occurs with a 1.3-V decrease in overpotential. CV data for the reduction of both oxygen and trichloroacetic acid are consistent with reductions facilitated by Mb. Although hemin, the iron heme cofactor of myoglobin,can catalyze these reductions, it is retained in DDAB films for only a few hours.22a Reduction of trichloroacetic acid by Mb-DDAB films on electrodes in aqueous buffers involves catalytic, step-
Electroactive Myoglobin-Surfactant Films
Langmuir, Vol. 11, No. 9, 1995 3301
wise loss of chloride to yield acetic acid.zz The reaction is probably similar in the microemulsion. The pathway of reduction of oxygen in the Mb-DDAB films is undoubtedly complex. While a detailed mechanistic discussion is beyond the scope of this paper, the results presented are consistent with the reduction of MbFe(III1, followed by fast reaction of MbFe(I1) with oxygen. The latter reaction is well-known and occurs with a rate constant of 2 x lo7 M-l s-l at neutral pH.23 This explains the disappearance of the anodic peak for MbFe(11)in the presence of oxygen. Results also suggest a further two-electron reduction of oxygen, supported by the current ratios for the MbFe(II1) reduction peak in the presence and absence of oxygen (Figure 8) that tend toward 3 at low scan rates. Preliminary results of spectroelectrochemical studieszzbsuggested a Mb-facilitated two-electron reduction of oxygen to hydrogen peroxide, a process which can be catalyzed by simple transition-metal porphyrins.24 The increase in cathodic peak current at -0.18 V, reflecting a decreased overpotential for the reduction of
oxygen (Figures 6 and 7), and the value of 3 approached by the ratio ofthe MbFe(II1)peakin the presence ofoxygen to that in its absence at low scan rates (Figure 8) are all consistent with some type of myoglobin-facilitated reduction of oxygen. It is uncertain a t present in what form oxygen is reduced. It was previously reported that oxygen can be transported by native MbFe(II1) for the reduction at electrodes in buffer solutions.z5 A similar process might occur in the films. A second possibility is that the MbFe(II)-Oz complex is reduced. Furthermore, oxidation of deoxy-MbFe(I1)by hydrogen peroxide is well-known and occurs with a second-order rate constant of 3.6 x lo3M-l s-l a t pH 7 and 25 oC.z6 It is unclear whether this latter reaction contributes to the CV signals. The present work establishes that the Mb-DDAB microemulsion films prepared as described are useful to facilitate reductions ofboth polar and nonpolar solutes in microemulsions. However, further studies are needed to establish detailed mechanisms for the small molecule reductions discussed in this paper. A detailed investigation of the pathway for reduction of oxygen in the MbDDAB films will be presented in a separate paper.2zb
(23)(a)Armstrong, G. D.; Sykes, A. G.Znorg. Chem. 1986,25,31353139. (b) Wazawa, T.; Matsuoka, A,; Tajimi, G.; Sugawara, Y.; Nakamura, K.; Shikama, K. Biophys. J. 1987,26,6684-6688. (24)(a)Collman, J.P.; Marrocco,M.; Denisevich, P.; Koval, C.;Anson, F. J. Electroanal. Chem. 1979, 101, 117-122. (b) Collman, J. P.; Denisevich, P.; Konai, Y.; Marrocco, M.; Koval, C.; Anson, F. J. Am. Chem. SOC.1980.102.6027-6036 and references therein. (c) Liu. H. Y.; Weaver, M. J.; Wang, C.-B.; Chang, C. K. J. Electroanal. Chem. 1983,145,439-447.
Acknowledgment. We are grateful for financial support of this work by the National Science Foundation (NSF) through Grant CTS9306961. LA9500235 (25)King, B. C.;Hawkridge, F. M. Tulanta 1989,36,331-334. (26)Yusa, K.;Shikama, K. Biochemistly 1987,26,6684-6688and references therein.
