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Myoglobin Coadsorbed on Electrodes from Microemulsions Provides Reversible Electrochemistry and Tunable Electrochemical Catalysis Geoffrey N. Kamau,† Momanyi P. Guto,†,‡ Bernard Munge,‡ Venkateswarlu Panchagnula,‡ and James F. Rusling*,‡,§ Department of Chemistry, University of Connecticut, U-60, 55 North Eagleville Road, Storrs, Connecticut 06269-3060, Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06032, and Department of Chemistry, University of Nairobi, P.O. Box 30197, Nairobi, Kenya Received March 21, 2003. In Final Form: May 27, 2003 The iron heme protein myoglobin (Mb) coadsorbs with surfactant onto glassy carbon, pyrolytic graphite, and platinum electrodes from microemulsions of sodium dodecyl sulfate or cetyltrimethylammonium bromide, water, oil, and pentanol. Adsorbates on all electrodes gave reversible heme FeIII/FeII voltammetry for Mb, which spectroscopy showed to be in near-native conformation. The nature of the adsorbed films was highly dependent on the electrode material. The carbon electrodes gave nonideal thin-film voltammetry, but Pt electrode voltammetry was more consistent with thick films featuring in-film diffusion of protein. Mb films on all electrodes supported electrochemical catalytic reduction of 1,2-dibromocyclohexane (DBCH) and trichloroacetic acid (TCA). Results on carbon electrodes were consistent with Michaelis-Menten enzyme kinetics. The catalytic rate and binding of reactant to Mb were more efficient for DBCH in microemulsions than in aqueous buffer but were more efficient for TCA in aqueous buffer. Microemulsions exerted a tuning effect on the reactivity of these reactants according to their hydrophobicity. The more hydrophobic DBCH had better access to Mb in the surfactant films and reacted at a faster rate, while the hydrophilic TCA had poor access to the catalyst in the film.
Introduction Public health concerns are driving the development of environmentally friendly chemical technologies. We have been exploring mediated electrochemical syntheses in microemulsions,1 which provide less toxic and less expensive reaction media2 than conventional organic solvents often used for electro-organic synthesis. Microemulsions are clear, thermodynamically stable isotropic mixtures of oil and water with nanoheterogeneous structures. Surfactants reside at oil-water (o/w) interfaces and stabilize the fluid by decreasing interfacial free energy to very small values.3 Our recent work showed how microemulsions can be used to enhance rates and control pathways of electrochemical syntheses with dissolved and surface-bound mediators.1,4 Microemulsions are excellent solvents for polar and nonpolar molecules and polymers. While this property serves well for bringing together dissolved ionic mediators with nonpolar reactants, it also dictates special stability requirements for catalytic electrode coatings. We found that covalent linkage of a cobalt corrin-poly(Llysine) polyion to oxidized carbon cathodes was necessary to provide films stable enough for catalytic electrochemical synthesis in microemulsions.4-7 However, another useful * To whom correspondence should be addressed. E-mail:
[email protected]. † University of Nairobi. ‡ University of Connecticut. § University of Connecticut Health Center. (1) Rusling, J. F. In Reactions and Synthesis in Surfactant Systems; Texter, J., Ed.; Marcel Dekker: New York, 2001; pp 323-335. (2) Friberg, S. Adv. Colloid Interface Sci. 1990, 32, 167-182. (3) Bourrel, M.; Schechter, R. S. Microemulsions and Related Systems; Marcel Dekker: New York, 1988. (4) Rusling, J. F.; Campbell, C. J. In Encyclopedia of Surface and Colloid Science; Hubbard, A., Ed.; Marcel Dekker: New York, 2002; pp 1754-1770.
approach in special situations is coadsorption of mediator and surfactants to electrodes.8 The heme protein myoglobin (Mb) is responsible for oxygen transport in mammalian muscle but can also catalyze organic oxidations9 and reductions.10,11 We used Mb in thin polyion or lipid films on electrodes to catalyze a number of such reactions in aqueous emulsions.12,13 In this paper, we show that the characteristics of Mb films coadsorbed onto electrodes from microemulsions depend strongly on the electrode material. These adsorbed Mb films were used to catalyze the reduction of trans-1,2dibromocyclohexane and trichloroacetic acid in bicontinuous microemulsions made with anionic or cationic surfactants by a Michaelis-Menten enzyme catalysis pathway. Experimental Section Chemicals and Microemulsions. Cetyltrimethylammonium bromide (CTAB, 99%) and sodium dodecyl sulfate (SDS, 99%) tetradecane were from Acros. Trichloroacetic acid (TCA) (5) Zhou, D.-L.; Njue, C. K.; Rusling, J. F. J. Am. Chem. Soc. 1999, 121, 2909-2914. (6) Njue, C. K.; Rusling, J. F. J. Am. Chem. Soc. 2000, 122, 64596463. (7) Njue, C. K.; Rusling, J. F. Electrochem. Commun. 2002, 4, 340343. (8) Rusling, J. F. Acc Chem. Res. 1991, 24, 75-81. (9) Ortiz de Montellano, P. R.; Catalano, C. E. J. Biol. Chem. 1985, 260, 9265-9271. (10) Wade, R. S.; Castro, C. E. J. Am. Chem. Soc. 1973, 95, 231-234. (11) Bartnicki, E. W.; Belser, N. O.; Castro, C. E. Biochemistry 1978, 17, 5582-5586. (12) (a) Rusling, J. F.; Zhang, Z. In Handbook of Surfaces and Interfaces of Materials. Vol. 5. Biomolecules, Biointerfaces, and Applications; Nalwa, R. W., Ed.; Academic Press: San Diego, 2001; pp 33-71. (b) Rusling, J. F.; Zhang, Z. In Biomolecular Films; Rusling, J. F., Ed.; Marcel Dekker: New York, 2003; pp 1-64. (13) Rusling, J. F.; Zhang, Z. In Electroanalytical Methods for Biological Materials; Chambers, J. Q., Bratjer-Toth, A., Eds.; Marcel Dekker: New York, 2002; pp 195-231.
10.1021/la0344940 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/28/2003
Myoglobin Coadsorbed on Electrodes was from Janssen Chimica, and n-pentanol and trans-1,2dibromocyclohexane (DBCH) were from Aldrich. Horse skeletal muscle myoglobin was from Sigma and was dissolved in a pH 4.6 buffer (0.01 M acetate/acetic acid + 0.05 M NaCl) or in microemulsions of the following compositions: SDS/0.1 M NaCl/ n-pentanol/tetradecane (13/52/27/8, by wt) and CTAB/water/npentanol/tetradecane (5/88.6/5.4/1, by wt). These fluids were characterized previously6 with the following results: SDS microemulsion: bicontinuous, η ) 12.5 cp, κ ) 6 MΩ-1 cm-1; CTAB microemulsion: oil/water, η ) 5.7 cp, κ ) 4 MΩ-1 cm-1. Water was purified to a specific resistance of >18 MΩ cm. All other chemicals were reagent grade. Apparatus and Procedures. CHI 660 and 430 Workstations (CHI Instruments) were used for cyclic and rotating disk voltammetry (RDV, Pine Instruments) and electrolysis. A threeelectrode cell was used with a saturated calomel electrode (SCE) as reference. A platinum wire was used as the counter electrode, and pyrolytic graphite (PG, 0.17 cm2), glassy carbon (GC, 0.10 cm2), or platinum (0.018 cm2) disks were used as working electrodes. All experiments were at 22 ( 2 °C, except bulk electrolysis was at 4 °C. Solutions were purged with pure nitrogen for ∼25 min prior to experiments. Initially all electrodes were polished while cooling with water using 400 grit, then 600 grit SiC paper (Carbimet), then 0.3 µm alumina on Mark V Laboratory green billiard cloth on a polishing wheel for 1 min, followed by ultrasonication in water for 1 min. Before each experiment, electrodes were polished on green billiard cloth (1 min), followed by rinsing in water and drying in nitrogen. RDV was done at 500 rpm and 50 mV s-1. A JASCO 710 spectropolarimeter was used to obtain circular dichroism (CD) spectra. Absorption spectroscopy was done using a Hewlett-Packard 8453 UV-vis diode array spectrophotometer. Films for spectroscopy were spread onto fused silica slides from 0.07 mM Mb in the microemulsion, dried, and then dipped in the respective microemulsion before recording the spectra.
Results Spectroscopic Characterization. The iron heme Soret absorption band characteristic of native Mb14 occurs at λmax ) 409 nm at neutral pH. In pH 4.6 buffer, we obtained a Soret band at 398 nm (see Supporting Information). However, in an SDS microemulsion made with pH 4.6 buffer, a blue shift in the absorption band occurred, giving a peak at λmax ) 392 nm. This is in the same range as λmax values observed in microemulsions made with CTAB or didodecyldimethylammonium bromide.15 These decreases in λmax from the value for the native protein suggest a partial unfolding of the protein to a stable molten globule conformation of Mb that still retains the iron heme cofactor and about half of its native R-helix content.16 Mb in n-pentanol had a Soret band at λmax ) 401 nm. CD spectra confirmed partial denaturation of the Mb in microemulsions. Films of Mb prepared on fused silica from the above microemulsions gave UV-vis spectra with the Soret band at 411 nm characteristic of native protein in surfactant and polyion films17,20 (Figure 1a). CD spectra in the UV region (Figure 1b) gave double minima at 210 and 224 nm in CTAB films and 214 and 227 nm in SDS films characteristic of R-helices in the Mb backbone18 that (14) (a) Brunori, M.; Giacometti, G. M.; Antonini, E.; Wyman, J. J. Mol. Biol. 1972, 63, 139-152. (b) Takahashi-Ushijima, E.; Kihara, H. Biochem. Biophys. Res. Commun. 1982, 105, 965-968. (c) Puett, D. J. Biol. Chem. 1973, 248, 4623-4634. (15) (a) Onuoha, A. C.; Rusling, J. F. Langmuir 1995, 11, 32963301. (b) Onuoha, A. C.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1997, 119, 3979-3986. (16) (a) Goto, Y.; Fink, A. L. J. Mol. Biol. 1990, 214, 803-805. (b) Stigter, D.; Alonso, D. O. V.; Dill, K. A. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4176-4180. (c) Yang, A.-S.; Honig, B. J. Mol. Biol. 1994, 237, 602-614. (17) Panchagnula, V.; Kumar, C. V.; Rusling, J. F. J. Am. Chem. Soc. 2002, 124, 12515-12525. (18) Holzwarth, G.; Doty, P. J. Am. Chem. Soc. 1965, 87, 218-228.
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Figure 1. Spectra of wet films of Mb cast onto fused silica slides from CTAB and SDS microemulsions: (a) visible absorption spectra and (b) UV circular dichroism spectra (spectra are offset for clarity).
represent 76% of the native conformation in solution (see Supporting Information).19 These results suggest that although Mb is partly denatured in the microemulsions, in the adsorbed surfactant films it refolds into near-native conformations. Cyclic Voltammetry. Voltammograms of Mb in an anaerobic SDS microemulsion at a polished GC electrode gave a pair of reduction-oxidation peaks centered at -0.2 V versus SCE (Figure 2a). The peaks are in the correct potential range for the MbFeIII/FeII redox couple in films on carbon electrodes.13,20 Cyclic voltammograms (CVs) of Mb at PG electrodes in an SDS microemulsion gave similar peaks. In CTAB microemulsions, both types of carbon electrodes gave reversible voltammograms for Mb centered at -0.2 V versus SCE, as illustrated for pyrolytic graphite (Figure 2b). For both types of carbon electrodes in both microemulsions, the nearly symmetric oxidation and reduction peaks of Mb with peak widths of >90 mV were separated by about 20-40 mV at scan rates (ν) of less than 100 mV s-1. Plots of peak current versus ν were all linear. Scan rate studies (ν ) 25-800 mV s-1) at GC electrodes in the SDS system did not show any significant peak shift, while slight shifts in peaks were found in CTAB microemulsions. Such behavior is characteristic of nonideal, quasireversible thinlayer voltammetry of redox proteins,12,13 suggesting adsorption of Mb onto the electrodes. The peak separations were larger than the ideal theoretical value of 0 mV for a monomolecular electroactive film on an electrode. However, this is typical for Mb in thin surfactant films20 and may reflect known small differences in conformation21 between oxidized and reduced forms of the protein.22 Voltammograms indicated that adsorbed Mb films on GC and PG electrodes were stable in SDS and CTAB (19) Rusling, J. F.; Kumosinski, T. F. Nonlinear Computer Modeling of Chemical and Biochemical Data; Academic Press: New York, 1996; pp 117-134. (20) Rusling, J. F.; Nassar, A.-E. F. J. Am. Chem. Soc. 1993, 115, 11891-11897.
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Figure 3. Cyclic voltammogram of 0.07 mM Mb in the SDS microemulsion on a Pt electrode at 0.3 V s-1. Table 1. Parameters for Mb on Various Electrodes from Cyclic Voltammetry
Figure 2. Cyclic voltammogram of 0.07 mM Mb (a) on polished glassy carbon electrodes in the SDS microemulsion at scan rates of 25, 50, 100, 300, 500, and 800 mV s-1 for successively larger peaks and (b) on polished pyrolytic graphite electrodes in the CTAB microemulsion at 40, 50, 60, 70, 80, and 90 mV s-1 for successively larger peaks.
microemulsion
electrode
Eo′, V vs SCE
SDS SDS SDS CTAB CTAB CTAB pH 4.6 buffer
PG GC Pt PG GC Pt PG
-0.21 -0.18 -0.52 -0.20 -0.19 -0.57 -0.21
a
D′,a cm2 s-1
2.5 × 10-3 1.4 × 10-3
Γo,b nmol cm-2 0.12 0.006 0.06 0.0046 0.0007
Apparent values from the slopes of peak current vs ν1/2 and the
Randles-Sevcik equation. b From integration of slow-scan CVs, rds (20%.
microemulsions and in acetate buffer solution containing 70 µM Mb for several days. When electrodes were transferred to microemulsions with no dissolved Mb, peak currents decrease 20-70% after a day. Thus, all further experiments were done with 70 µM Mb present in the fluid. Complete removal of adsorbed myoglobin from GC and PG electrodes previously exposed to Mb in microemulsions required extensive polishing, starting from 400 grit silicon carbide paper (see Experimental Section). The voltammetry of Mb at platinum electrodes in microemulsions was very different from that on carbon electrodes, with the FeIII/FeII peak appearing 300 mV more negative on Pt than on carbon. CVs in SDS microemulsions gave a pair of unsymmetric oxidation-reduction peaks with nearly equal height centered at about -0.52 V versus SCE (Figure 3). The oxidation-reduction peak separation was ∼60 mV at scan rates up to 100 mV s-1 and increased to larger values as the scan rate increased. Reduction peak currents on Pt electrodes were proportional to ν1/2 in the range 25-300 mV s-1. All these results are consistent with a quasireversible, diffusion-controlled electrode process.23 Similar voltammetry was observed on Pt electrodes for Mb in CTAB microemulsions, but the peaks were centered at -0.57 V versus SCE. The unsymmetric, diffusion-controlled shapes of the peaks in both microemulsions were maintained at very low scan rates, even at 1 mV s-1. Formal potentials, apparent diffusion coefficients (D′) for Pt electrodes, and surface concentrations on the carbon
electrodes were obtained from CV data by standard procedures12,23 (Table 1). D′ values could be obtained only from the data on Pt electrodes. In microemulsions, there are two reasons why apparent diffusion coefficients may be significantly different from values in water, that is, 5 × 10-7 cm2 s-1 for Mb.24 Smaller diffusion coefficients are found if the electroactive solute is bound to diffusing microemulsion nanostructures such as droplets.25 A larger apparent diffusion coefficient can result from preconcentration of the electroactive solute into a surface film on the electrode.8 The latter situation seems pertinent for Mb in microemulsions on Pt electrodes, as will be discussed later. Catalytic Reductions. Electrochemical reductions of the organic halides DBCH and trichloroacetic acid can be catalyzed by Mb films to yield dehalogenated products.26 These reactions were examined by voltammetry to assess the catalytic ability of adsorbed Mb in the microemulsions. Figure 4 shows cyclic voltammograms of Mb in SDS microemulsions with and without DBCH on GC and Pt electrodes. For both electrodes, the chemically reversible CVs reported earlier are seen in the absence of DBCH. In the presence of DBCH, reduction peaks increased and oxidation peaks disappeared. No reverse peak was observed except at scan rates exceeding 2 V s-1, above which reversibility began to be reestablished. Similar results were found in CTAB microemulsions. These results are consistent with electrochemical catalytic reduction of DBCH by reaction with electrochemically generated MbFeII,20 and this is why the oxidation peak is not seen.
(21) Nassar, A.-E. F.; Zhang, Z.; Hu, N.; Rusling, J. F.; Kumosinski, T. F. J. Phys. Chem. B 1997, 101, 2224-2231 and references therein. (22) El Kasmi, A.; Leopold, M. C.; Galligan, R.; Robertson, R. T.; Saavedra, S. S.; El Kacemi, K.; Bowden, E. F. Electrochem. Commun. 2002, 4, 177-181. (23) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, 2nd ed.; Wiley: New York, 2001.
(24) (a) King, B. C.; Hawkridge, F. M.; Hoffman, B. M. J. Am. Chem. Soc. 1992, 114, 10603-10608. (b) Taniguchi, I.; Watanabe, K.; Tominaga, M.; Hawkridge, F. M. J. Electroanal. Chem. 1992, 333, 331-338. (25) Rusling, J. F. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1994; Vol. 19, pp 1-88. (26) Nassar, A.-E. F.; Bobbitt, J. M.; Stuart, J. D.; Rusling, J. F. J. Am. Chem. Soc. 1995, 117, 10986-10993.
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Figure 5. Rotating disk voltammetry at 500 rpm on a pyrolytic graphite electrode in 0.07 mM Mb in the SDS microemulsion with various concentrations of trichloroacetic acid as labeled on the curves.
Figure 4. Cyclic voltammogram at 0.05 V s-1 of 0.07 mM Mb in the SDS microemulsion (a) on a GC electrode and (b) on a Pt electrode with (1) no DBCH added and (2) 60 mM DBCH added. Direct reduction of DBCH in the SDS microemulsion occurs at -1.86 V.
In SDS and CTAB microemulsions, direct reduction of DBCH occurs irreversibly at -1.86 V versus SCE at 0.2 V s-1. Also, CVs of the Mb FeIII/FeII couple were reproduced within ∼5% in microemulsions containing 70 mM Mb after the electrode had been used for the catalytic reactions. The current efficiency of the catalytic reductions (Icat/ Id), defined as the ratio of the peak current for Mb in the presence (Icat) to that in the absence of the reactant (Id), decreased with the scan rate between 0.05 and 2 V s-1 at GC electrodes as expected for an electrochemical catalytic reduction.23 However, it remained relatively constant at platinum electrodes, with an average value of 2.2 ( 0.4 for DBCH/SDS/Pt and 3.3 ( 0.8 for the DBCH/CTAB/Pt system. Similar results were also obtained with trichloroacetic acid in either microemulsion (direct reduction -1.7 V vs SCE). Rotating Disk Voltammetry. This method was used to control reactant mass transport to carbon electrodes having thin films of adsorbed Mb to achieve steady-state limiting currents proportional to the protein turnover rate.27 Figure 5 illustrates steady-state currents from RDV in the SDS microemulsions and shows that these limiting currents increase with increasing concentration of trichloroacetic acid. The RDV of the surface-bound Mb did not give a steady-state current. This voltammogram remains in a peak shape similar to Figure 2b because rotation of the electrode does not influence the voltammetry of the adsorbed film.23 RDV was also used in buffer solutions (Figure 6), for which a very small Mb peak could be obtained after multiple scanning (see Supporting Information). Despite the small FeIII/FeII peaks, steady-state currents were readily measured in the presence of TCA and DBCH. RDV can also be used to extract kinetic parameters. The Michaelis-Menten model of enzyme kinetics assumes (27) Heering, A. H.; Hirst, J.; Armstrong, F. A. J. Phys. Chem. B 1998, 102, 6889-6902.
Figure 6. Rotating disk voltammetry at 500 rpm on a pyrolytic graphite electrode for 0.07 mM Mb in pH 4.6 buffer with various concentrations of trichloroacetic acid as labeled on the curves.
that the first step is reversible formation of a complex between the enzyme and the reactant; then this complex breaks up to give product and regenerate the enzyme. For RDV with a thin film of immobilized enzyme on an electrode surface, the electrochemical version of the Michaelis-Menten equation becomes27,28
Icat ) [nFAΓkcatCs]/[Cs + Km]
(1)
where Icat is the steady-state RDV current, Cs is the substrate concentration in solution, Γ is the total surface concentration of enzyme, A is the electrode surface area, F is Faraday’s constant, Km is the Michaelis dissociation constant of the enzyme-substrate complex, and kcat is the turnover rate constant. Equation 1 may be linearized to eq 2 for the estimation of kcat and Km,
1/Icat ) [Km/(nFAΓkcatCs)] + [1/(nFAΓkcat)]
(2)
An electrochemical Lineweaver-Burke plot of 1/Icat versus 1/Cs provides Km/(nFAΓkcat) as the slope and 1/(nFAΓkcat) as the intercept on the 1/Icat axis. Thus, a series of RDV scans on PG and GC electrodes were made in the two microemulsions at different concentrations of TCA and DBCH. Figure 7 shows the Icat data plotted according to eq 2. Excellent linearity was obtained in all cases. The kinetic parameters obtained from this analysis are shown in Tables 2 and 3. The results for TCA in microemulsions were essentially independent of microemulsion or electrode type. Thus, the DBCH reaction was analyzed for only one microemulsion and a (28) Armstrong, F. A. In Bioelectrochemistry of Biomacromolecules; Lenz, G., Milazzo, G., Eds.; Birkhauser Verlag: Basel, Switzerland, 1997; pp 205-255.
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Table 2. Apparent Michaelis Parameters kcat, Km, and kcat/Km from RDV for Reduction of TCA by Adsorbed Mb parameter (s-1)
kcat Km (M) × 10-4 kcat/Km (M-1 s-1)
pH 4.6 buffer on PG
SDS on PG
CTAB on PG
SDS on GC
CTAB on GC
(8.2 ( 0.7) × (4.1 ( 0.6) × 10-3 (2.0 ( 0.2) × 102
6.8 ( 1.2 3.2 ( 0.2 2.2 ( 0.2
9.9 ( 0.8 2.8 ( 0.1 3.5 ( 0.2
8.9 ( 2.5 2.9 ( 0.4 3.1 ( 0.4
9.2 ( 0.2 2.9 ( 0.1 3.2 ( 0.2
103
Figure 7. RDV Lineweaver-Burke plots for catalytic reduction of trichloroacetic acid on (a) a PG electrode in the SDS microemulsion and (b) a GC electrode in the CTAB microemulsion. Table 3. Apparent Michaelis Parameters kcat, Km, and kcat/Km from RDV for Reduction of DBCH by Adsorbed Mb parameter (s-1)
kcat Km (M) kcat/Km (M-1 s-1)
pH 4.6 buffer on PG
SDS on PG
0.41 ( 0.01 (6.0 ( 0.1) × 10-3 (6.7 ( 0.1) × 101
2.0 ( 0.8 (1.7 ( 0.3) × 10-5 (1.2 ( 0.1) × 105
buffer emulsion on a PG electrode. Similar studies were not applicable to Pt electrodes because thick layers of Mb form on these electrodes in microemulsions. Results show that for TCA kcat was much larger and Km was much smaller for the reaction in the buffer solution compared to the microemulsions. For DBCH, the opposite was true. Discussion Voltammetry of Adsorbed Mb. We previously reported the voltammetry and catalysis of Mb in microemulsions of didodecyldimethylammonium bromide (DDAB), water, and dodecane (13/28/59 by wt). Very small CV peaks were found on PG electrodes for Mb adsorbed onto PG from this microemulsion.15b Larger CV peaks for Mb were obtained by casting Mb-DDAB films on PG, drying, and then doing cyclic voltammetry in the DDAB
microemulsions, but these films slowly degraded under hydrodynamic conditions.15a Results of the present work show that Mb can be adsorbed onto electrodes from microemulsions of the common surfactants CTAB and SDS, pentanol, oil, and water (52-87%), which are more than 10-fold less expensive than DDAB microemulsions and contain much more water. The resulting films gave stable, well-defined, chemically reversible voltammetry characteristic of adsorbed films of Mb (Figures 2 and 3) that were relatively stable under hydrodynamic conditions. Furthermore, Mb appears to assume a near-native conformation in these surfactant films (Figure 1). The nature of the adsorbed films depended strongly on the electrode material. The two carbon electrodes gave similar formal potentials (Table 1) and rather typical nonideal surface voltammograms characteristic of thin redox protein films12 (Figure 2). The PG electrode gave the largest surface concentration of Mb of 0.12 nmol cm-2, slightly larger than the final surface concentration reported for the steady-state value of 0.1 nmol cm-2 for cast Mb-DDAB films in DDAB microemulsions. PG electrodes adsorbed more protein than the GC electrodes, consistent with the better adhesive properties of PG for surfactant films.29 Finally, more protein was adsorbed onto the carbon electrodes from SDS microemulsions than from CTAB microemulsions. Surfactant is typically adsorbed onto carbon and metal electrodes in microemulsions, and coadsorption of other molecules into a surface layer of surfactant is common.4,8,25 Our results suggest that Mb is coadsorbed along with surfactant onto the PG, GC, and Pt electrodes in SDS and CTAB microemulsions. Apparently, surfactant facilitates Mb adsorption from these microemulsions. This view is supported by amounts of Mb adsorbed onto PG from the microemulsions that were 80-170-fold larger than that adsorbed from pH 4.6 buffer (Table 1). The larger amount of Mb adsorbed from SDS than from CTAB is also consistent with coadsorbed Mb-surfactant layers on the electrodes. At the effective microemulsion water phase pH ∼ 5, Mb has a +7 charge.16 Thus, it is likely that Coulombic attraction between the negative DSions and the positive protein contribute to the stability of the films adsorbed from SDS. With CTAB, the charges on the protein and the surfactant are the same. However, hydrophobic interactions were identified as major factors in stabilizing films of Mb and insoluble cationic surfactants such as DDAB30 and may contribute to Mb-CTAB surface film stability in the CTAB microemulsions as well. CVs on Pt electrodes had diffusion-controlled shapes (Figure 3), rather than the symmetric voltammetric signatures of adsorbed thin films on carbon electrodes. Furthermore, Mb reduction occurred 300-350 mV more negative on Pt than on carbon. Calculation of the apparent diffusion coefficient D′ from the slope of peak current (Ip) versus ν1/2 using the Randles-Sevcik equation and the concentration of Mb in the microemulsion (CMb) gave impossibly large values (Table 1). According to the Randles-Sevcik equation, D′1/2 is proportional to Ip/ (29) Nassar, A. E. F.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1995, 67, 2386-2392. (30) Rusling, J. F. Acc. Chem. Res. 1998, 31, 363-369.
Myoglobin Coadsorbed on Electrodes
[ν1/2CMb]. If diffusion is not from the solution to the electrode but occurs within a film on the electrode with Mb concentration . CMb, then the D′ value will be large because the Mb concentration in the films is grossly underestimated. This was documented for 9-phenylanthracene adsorbed in thick films of micellar CTAB on mercury electrodes for which D′ values were found8,31 in the same range as those in Table 1 for Mb on Pt. Using this analogy, we can interpret the results in terms of a thick film of Mb and surfactant on Pt electrodes. Thinfilm voltammetry cannot be achieved with the available scan rates. Why such thick films form on Pt electrodes but not on carbon electrodes is unclear at this time. Electrochemical Catalysis. Mb films on all three electrodes catalyzed the electrochemical reduction of a hydrophilic substrate, TCA, and a hydrophobic substrate, DBCH. Much higher current densities were found on Pt electrodes than on the carbon electrodes. For example, from Figure 4 obtained in SDS microemulsions, current density in the presence of 60 mM DBCH was 830 µA cm-2 on Pt and 20 µA cm-2 on GC. These results suggest larger amounts of Mb in the thicker films on Pt than in the thin films on carbon electrodes. As current densities are proportional to reaction rates,23 Pt electrodes might be a better choice for practical electrolyses in these systems. However, only the carbon electrodes gave the typical thin-film protein voltammetry amenable to MichaelisMenten analyses of steady-state catalytic currents (Figures 5 and 6). Fits of the data to eq 2 were excellent (Figure 7). Thus, reductive electrochemical catalysis with Mb on electrode surfaces is consistent with the MichaelisMenten model. TCA is reduced catalytically by Mb in DDAB films on electrodes in stepwise dechlorinations.26 In the present work, we find that the apparent parameters kcat, Km, and kcat/Km do not depend significantly on the type of carbon electrode or microemulsion (Table 2). However, when results in buffer are compared to those in microemulsions, kcat is 3 orders of magnitude larger. Km is 3 orders of magnitude smaller in the buffer, suggesting much better TCA binding to Mb in the buffer than in the microemulsion. kcat/Km having the units of a second-order rate constant is 100-fold larger in the buffer than in microemulsions for TCA. The situation is the reverse for DBCH (Table 3), which is reduced to cyclohexene by electrochemical catalysis with Mb.26 kcat is 5-fold larger in the SDS microemulsion than in buffer. Km is several orders of magnitude larger in the microemulsion. kcat/Km is 2000-fold larger in the microemulsion than in buffer. Thus, the apparent reaction rate is faster and the binding of DBCH to Mb is stronger in the microemulsions. (31) Rusling, J. F.; Shi, C.-N.; Gosser, D. K.; Shukla, S. S. J. Electroanal. Chem. 1988, 240, 201-216.
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We can rationalize these kinetic results by considering the nature of the coadsorbed films of Mb and surfactant on the electrode. TCA is a strong acid present mainly as the trichloroacetate anion in the microemulsions and the buffer. Its access to Mb in the microemulsions may be inhibited by the internal hydrophobic nature of the film. Thus, from the perspective of eq 2, the apparent binding to Mb becomes weaker and the reaction rate decreases (Table 2), presumably through limiting the local concentration of TCA in the vicinity of the active catalyst Mb. DBCH is water insoluble and resides exclusively in the oil phase of microemulsions.1,4 It is very likely to be preconcentrated into the surfactant films and reside in close proximity to Mb in high concentrations. Thus, binding to Mb appears stronger, and the apparent rate of the catalytic reduction increases (Table 3). Similar observations on the reactivity of TCA and DBCH were made in DDAB microemulsions with metallophthallocyanine catalysts adsorbed on carbon electrodes.32 As with adsorbed Mb, the rate of reduction of TCA was larger in homogeneous acetonitrile/water than in the microemulsion, and the reaction rate for DBCH was faster in the microemulsion than in acetonitrile/water. Rationalization was based on access of the reactant to the catalyst in a surfactant-catalyst film on the surface, similar to that suggested for the present work. Conclusions Mb coadsorbed with surfactant onto GC, PG, and platinum electrodes from microemulsions of SDS and CTAB gave reversible heme FeIII/FeII voltammetry for Mb, which is probably in a partly unfolded, stable, “molten globule” conformation. The voltammetry of the adsorbed films depended strongly on the electrode material. Mb films on all of the electrodes catalyzed electrochemical reduction of TCA and DBCH. The apparent rates of reduction and binding affinities to Mb were larger in microemulsions for the hydrophobic DBCH but were larger in aqueous buffer for hydrophilic TCA. The microemulsions exerted a tuning effect on the reactivity of the substrates according to their hydrophobicity. That is, the more hydrophobic reactant had better access to Mb in the surfactant films and reacted at a faster rate. Acknowledgment. The authors are grateful for financial support of this work by Grants INT-0096456 and CTS-9982854 from the National Science Foundation. Supporting Information Available: Three additional figures showing absorption and CD spectra of Mb in microemulsions and reversible Mb voltammetry in pH 4.6 buffer. This material is available free of charge via the Internet at http://pubs.acs.org. LA0344940 (32) Kamau, G. N.; Hu, N.; Rusling, J. F. Langmuir 1992, 8, 10421044.
