Anal. Chem. 1992, 64, 1470-1476
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UV-Visible Spectroscopy of Adsorbed Cytochrome c on Tin Oxide Electrodes Maryanne Collinson and Edmond F. Bowden* Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204
The conformational state and surface coverage of eiectrostatically adsorbed cytochrome con tin oxide electrodes were examined by udng visible absorption spectroscopy. Spectral analysis revealed that no malor structural changes occur in ferri- or ferrocytochrome c as a result of adsorption from pH 7, low ionic strength, phosphate buffers. Adsorbed ferricytochrome cwas found to be conformationallystable at applied potentials below 0.70 V vs Ag/AgCi (1 M KCI). At applied potentials above 0.70 V, adsorbed cytochrome c was irreverdbly oxidized beyond the ferric state with the resulting oxidation product remaining tenaciously adsorbed on the electrode surface. Fromthe close similarity betweenthe total surface coverage, determined from the Soret absorbance, and the eiectroactive coverage, determined from voltammetry, lt was concluded that adsorbed cytochrome c resides in a predominantly eiectroactive form. Due to the small signals involved and the presence of adsorption-induced optical perturbationsin the tin oxide/buffer background absorbance, the developmentof backgroundsubtractionproceduresproved necessary. These procedures are discussed in detail.
INTRODUCTION During the past decade, considerable attention has been focused on redox proteins irreversibly adsorbed on electrode surfaces at (sub)monolayer c~verages.l-~ These studies have been stimulated by the potential for obtaining fundamental information concerningbiological electron transfer,2-4protein adsorption at the solid-liquid interface? and the development of biosensors and biocatalytic devices.7~8Significant developments have been made in the characterization of adsorbed redox proteins using such techniques as resonance Raman spectroscopy,182,9-11electroreflectance,12 total internal reflection fluorescence,13 ac impedance,12 and cyclic voltammetry.5r6 These investigations have contributed greatly toward (1)Hildebrandt, P. J. Mol. Struct. 1991,242,379-395. (2)Hildebrandt, P.; Stockburger, M. Biochemistry 1989,28,6722-6728. (3)Willit, J. L.;Bowden, E. F. J . Electroanal. Chem. 1987,221,265274. (4)Tarlov, M. J.; Bowden, E. F. J. Am. Chem. SOC. 1991,113,18471849. (5)Armstrong, F. A.; Butt, J. N.; George, S. J.; Hatchikian, E. C.; Thomson, A. J. FEBS Lett. 1989,259, 15-18. (6)Willit, J. L.;Bowden, E. F. J . Phys. Chem. 1990,94,8241-8246. (7)Turner, A. P. F., Karube, I., Wilson, G. S., Eds.; Biosensors: Fundamentals and Applications; Oxford University Press: Oxford, 1987. (8) Tarasevich, M. R. In Comprehensiue Treatise OfElectrochemistry;
Srinivasan, S., Chizmadzhev, Yu. A., Bockris, J. O’M., Conway, B. E., Yeager, E., Eds.; Plenum: New York, 1985;Vol. 10,pp 231-295. (9)Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 67106721. (10)Rospendowski, B. N.; Schlegel, V. L.; Holt, R. E.; Cotton, T. M. In Charge and Field Effects in Biosystems II; Allen, M. J.; Cleary, S. F., Hawkridge, F. M., Eds.; Plenum: New York, 1989;pp 43-58. (11)Cotton, T. M.; Schultz, S. G.; Van Duyne, R. P. J. Am. Chem. SOC. 1980.102.7962-7965. (12)Sagara, T.; Niwa, K.; Sone, A.; Hinnen, C.; Niki, K. Langmuir 1990,6,254-262. (13)Fraaije, J. G.E. M.; Kleijn, J. M.; van der Graaf, M.; Dijt, J. C. Biophys. J . 1990,57,965-975. 0003-2700/92/0364-1470$03.00/0
understanding the structure, function, orientation, and electron-transfer behavior of redox proteins in the adsorbed state. However, several fundamental issues remain to be addressed or examined in sufficient detail. These include protein orientation,adsorption thermodynamics, total vs electroactive surface coverage, influence of applied potential, and retention of activity and native properties after adsorption. Our laboratory and others have been examiningthese issues in greater detail using cytochrome c adsorbed to charged interfaces.’-4,634-15 Cytochrome c is a very useful protein to study owing to its availability, ease of purification, documented crystallographic structure,16and high degree of physicochemical characteri~ation.~~ In previous voltammetric work, we have shown that cytochrome c adsorbs strongly on tin oxide electrodes in low ionic strength, neutral pH, phosphate buffers. Cytochrome c is believed to be adsorbed electrostaticallyvia the positively charged lysine groups near the exposed heme edge,presumably the left to left-frontface.14 The electroactive surface coverage,estimated from cyclic voltammograms, is approximately a monolayer, with reported values in the range of 13-18 pmol/cm2. Previous work has shown this value to be comparable, within a factor of 2, to the total surface coverage determined from measurements using long optical pathlength thin-layer spectroelectrochemistry.18 The close similarity between the surface formal potential, which has been determined from the cyclic volta”etry,l6J4J5 and the solution formal potential argues that adsorbed cytochrome c is in a native or near-native state.3 Some recent results, however, have shown that cytochrome c, when electrostatically adsorbed under certain conditions to silver9 and other charged interfaces,ZJg does not exist in a nativelike state but is structurally altered. In light of these results, additional characterization of the surface coverage and the degree to which cytochrome c remains native when adsorbed to tin oxide electrodes are desirable. Visible absorption spectroscopy can be used to provide insight into these two critical issues. The visible absorption spectrum of cytochromec is sensitive to the spin and coordination states of the heme iron20 and, as a result, has been used to examine the conformational state of cytochromec after adsorption or immobilization.19~21~22 Schafer and Wilson, for example, compared the visible spectrum of cytochrome c covalently attached to Sepharose (14)Collinson, M.; Willit, J. L.; Bowden, E. F. In Charge and Field Effects in Biosystems II; Allen, M. J., Cleary, S. F., Hawkridge, F. M., Eds.; Plenum: New York, 1989;pp 63-76. (15)Willit, J. L.;Bowden, E. F. In Redor Chemistry and Interfacial Behavior of Biological Molecules; Dryhurst, G., Niki, K., Eds.; Plenum: New York, 1989;pp 69-79. (16)Takano, T.; Kalla, 0. B.; Swansen, R.; Dickerson, R. E. J . Biol. Chem. 1973,248,5234-5255. (17)Dickerson, R. E.; Timkovich, R. In The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1975;Vol. XIA, pp 397-547. (18)Collinson, M.; Bowden, E. F. Unpublished results. (19)Chottard, G.; Michelon, M.; Herve, M.; Herve, G. Biochim. Biophys. Acta 1987,916,402-410. (20) Smith, D. W.; Williams, R. J. P. Struct. Bond. 1970, 7, 1-45. (21)Greenwood, C.;Moore, T. A. Biochem. J . 1976,153, 159-163. (22) Schafer, M. A.; Wilson, G. S. J. Biol. Chem. 1983,258,1283512841. 0 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64,
6MB to that of native, solution cytochrome ceZ2They found little difference between the two spectra and concluded that no major structural changes occurred in cytochrome c as a result of this particular immobilization.22 On the other hand, Chottard and co-workers did detect structural modifications in cytochrome c electrostatically bound to heteropolytungstates by using spectroscopic te~hniques.1~ The extent of these structural modifications was apparent in visible absorption spectra as significant wavelength and intensity variations.19 The objective of the present work was to examine and characterize the conformational state and surface coverage of electrostatically adsorbed cytochrome c on tin oxide through the application of visible absorption spectroscopy. The wavelengths and intensities of the absorption bands can provide information on possible conformational changes induced by adsorption or applied potential. Furthermore, from the Soret absorbance, a value for the total surface coverage of adsorbed cytochrome c can be obtained and compared to the electroactive coverage determined from cyclic voltammetry. Due to the small signals involved and the large tin oxide background absorbance, considerable attention to background subtraction and signal optimization proved necessary in this work. EXPERIMENTAL SECTION Reagents and Materials. Horse heart cytochrome c (type 100000) were VI) and poly-L-lysine hydrobromide (MW purchased from Sigma Chemical Co. Cytochrome c was chromatographically purified as previously described.6~23Reagentgrade chemicals were used to prepare all solutions. Water was purified using a Milli-Q system with an Organex-Q final stage (Millipore, Bedford, MA). With the exception of Figure 6 (see legend), the fluorine-doped tin oxide electrodes were in the form of a 0.33-pmfilm on a 5-mm glass substrate witha sheet resistance of ca. 17 Wsquare. All tin oxide electrodes were donated by PPG Industries. Approximate sheet resistances were determined from a two-point conductivity measurement. Film thicknesses were determined by using a step profiler after chemically etching a step using a zinc dust/hydrochloric acid slurry. Equipment. Visible absorption spectra were acquired with a Hewlett Packard 8452A diode array spectrophotometer. This computer-controlled instrument has a single-beam configuration and employs a deuterium lamp for spectral acquisition from 190 to 820 nm. The spectrograph consists of 328 photodiodes spaced 2 nm apart. A QuickBASIC program was written to acquire, signal average, and subtract absorbance data as desired. This program also controlled an EG&G Princeton Applied Research potentiostat, Model 273, used for electrochemical measurements. The spectroelectrochemicalcell, shown in Figure 1, consists of a lucite cell body and two tin oxide electrodes. Brass foils,used for electrical contact, were isolated from solution through the use of Viton O-rings. Visible absorption spectra were obtained by passing light through the cell normal to the electrode surfaces. All spectra, simulated (vide infra) or experimental (Figures 2-10), are for transmission through two electrodes. The reference electrode was Ag/AgCl in 1M potassium chloride, and the auxiliary electrode was a platinum wire. These electrodes were positioned in the spectroelectrochemical cell so as not to interfere with light passing through the cell. All potentials refer to a Ag/AgC1(1M KC1) reference electrode,which is 0.23 V vs NHE. Procedures. The tin oxide electrodes were cleaned by successive 10-minsonications in alconox,95% ethanol, and MilliQ water. This procedure was repeated, and the electrodes were then equilibrated overnight in the pH 7.0 phosphate buffer used for the experiment. The phosphate buffers used were either 2.2 or 4.4 mM potassium phosphate with ionic strengths ( p ) of 5 and 10 mM, respectively. The p = 5 mM, pH 7.0, phosphate buffer was used in some experiments to ensure the minimization of any
-
(23) Brautigan, D. L.; Ferguson-Miller, S.; Margoliash, E. Methods Enzymol. 1978,530, 128-164.
NO. 13, JULY 1, 1992
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\E Figure 1. Simplified cross-sectional view of spectroelectrochemical cell composed of a luclte cell body (C), two tin oxide electrodes (D) with brass contacts (F) isolated from solution by Vtton O-rings (E), a Ag/AgCI reference electrode in 1 M KCI (A), and a platinum auxiliary electrode (8). The cell volume is ca. 1 mL, the comblned electrode area is 1.34 cm2,and the distance between the two electrodes is 0.77 cm. desorption of cytochrome c from the tin oxide electrodes. No
differences were observed in the spectra of adsorbed cytochrome c in p = 5 vs p = 10 mM phosphate buffer. After electrode equilibration,the spectroelectrochemicalcell was assembled,fiied with buffer, aligned in the sample compartment of the spectrophotometer, and secured in place. Once aligned and secured, the spectroelectrochemical cell remained in place throughout the entire length of the experiment. A blank background spectrum of the tin oxide electrodes in buffer was initiallyacquired at 0.40 V. This tin oxide/buffer blank was internally subtracted from all subsequently obtained spectra to give what we will refer to as “apparent”spectra. Ferricytochrome c was adsorbed to the tin oxide electrodes at open circuit by removing the buffer from the sample cavity and adding ca. 1mL of a 5-10 pM solution of cytochrome c in pH 7.0, p = 5-10 mM, phosphate buffer. After 10-20 min, the cytochrome c solution was removed, and the spectroelectrochemical cell was carefully rinsed at least five times with phosphate buffer and then refilled. Spectra of adsorbed ferricytochrome c, unless otherwise noted, were acquired at an applied potential of 0.40 V while spectra of ferrocytochrome c were acquired at an applied potential of -0.20 V. Each spectrum was obtained by signal averaging 100-500 times. The temperature of the cell was maintained at 18 f 1 “C using a thermostated cell holder. The electroactive surface coverage of cytochrome c was determined with an estimated precision of 6-10 % by integrating the cathodic peak of a background-subtracted cyclic voltammogram. The geometric electrode area of 1.34 cm2was used in this calculation. This level of precision arises primarily from nonreproducibility associated with the CV background subtraction. The spectroscopic surface coverage (I?) of adsorbed cytochrome c, taken to be the total surface coverage,was determined from the following equation,24using a molar absorptivity (e) of 106 100 M-l cm-l at 410 nm for solution cytochrome A = 2(1000m Simulated absorption spectra were calculated by using this (24) Heineman, W. R.; Kuwana, T. Anal. Chem. 1972,44,1972-1978.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992 0.50
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Flgure 2. Simulated absorption spectra of adsorbed cytochrome c o n tin oxide at a coverage of 15 pmol/cm2. Ferricytochrome c(iight line); ferrocytochrome c (heavy line). Spectrum Is shown for transmission through two electrodes.
formula and a tabulated list of molar absorptivitie~.~~ For these calculations,molar absorptivities for adsorbed cytochrome c were assumed to be identical to the solution values. However, it is recognized that small differences are almost certainly present (vide infra).
O . O O ! ' , : " , " " ' : " : " 1 350 410 470 530 590 650 710 Wavelength, nm Figure 3. Background absorbance of tin oxide in pH 7.0, ~c = 10 mM, phosphate buffer vs air. Spectrum acquired using HP 89531A UVV I S operating software. The small "spikes" appearing at 487 and 658 nm are lamp lines originating from the deuterium lamp used In these experiments. 0.006
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RESULTS AND DISCUSSION Visible Absorption Spectroscopy. Figure 2 shows the simulated spectra of adsorbed ferri- and ferrocytochrome c a t a coverage of 15 pmol/cm2. Ferricytochrome c exhibits two major bands, the Soret band at 410 nm and the 0 band located at 530 nm, while ferrocytochrome c exhibits three bands, the Soret at 416 nm, the a band a t 550 nm, and the fl band at 520 nmaZ5The Soret bands, with molar absorptivities exceeding 100 000 M-1 cm-1, are predicted to yield absorbance values of 3-4 mAU for transmission through two electrodes while the a and fl bands are predicted to have absorbance values below 1 mAU.25 The weak 695-nm band (e -800 M-1 cm-1)20is predicted, in the case of adsorbed ferricytochrome c, to fall below the detection limit of this technique and indeed was not observed. In order to obtain the experimental spectrum of adsorbed cytochrome c, the background absorbance due to the two tin oxide electrodes must be considered. A typical absorption spectrum of tin oxide in buffer is shown in Figure 3. This spectrum, which arises from reflection and scattering losses, free carrier absorption within the film, and interference effects,26,27 is ca. 100 times larger than the expected absorbance signal due to adsorbed cytochrome c. As described in the Experimental Section, the tin oxide/buffer blank was subtracted from each spectrum to give what is referred to as an "apparent" spectrum. Pictured in Figure 4 are the apparent spectra thus obtained for adsorbed ferri- and ferrocytochrome c. As can be seen, these spectra exhibit some similarities to the simulated spectra in Figure 2, but there are significant differences. Notably, the Soret band for adsorbed ferricytochrome c does appear near 410 nm but is considerably larger than expected, a new feature near 540 nm appears which is much broader and more intense than the now obscured 530-nm band, and a negative baseline is obvious. We attribute these new features to a ~
~
(25) Margoliash, E.; Frohwirt, N. Biochem. J. 1959, 71, 570-572. (26) Vossen, J. L. In Physics of Thin Films; Hass, G., Francombe, M. H., Hoffman, R. W., Eds.; Academic Press: New York, 1977; Vol. 9, pp 1-71. (27) Chopra, K. L.; Major, S.; Pandya, D. K. Thin Solid Films 1983, 102, 1-46. ~
350
410
410
530
590
650
710
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Flgure 4. Appareni absorption spectra of adsorbed ferrlcytochrome c (light line) and adsorbed ferrocytochrome c (heavy line) on tin oxide. Solution conditions: pH 7.0, p = 5 mM phosphate buffer.
difference in the interference fringe pattern between the sample and reference spectra induced by protein adsorption. Upon electrochemical reduction, the sharp a and /3 bands of adsorbed ferrocytochrome c appear superimposed on the new feature near 540 nm. The slight differencesobservedbetween these two spectra,notably in the baseline and the broad feature near 540 nm, are due to potential dependent variations in the tin oxidelbuffer background absorbanceaZ8A blank difference spectrum of the tin oxide electrodes at 0.4 and -0.2 V supports this claim (figure not shown). These slight potential dependent baseline shifts have been eliminated in all remaining cytochrome c spectra through a double-subtraction procedure associated with eq 1 (vide infra). The discrepancies observed between the simulated spectra of adsorbed cytochrome c (Figure 2) and the apparent spectra (Figure 4) can be attributed primarily to optical perturbations induced in the tin oxide/buffer blank as a result of protein adsorption. It is well known that the modification of metal and semiconductorelectrode interfaces by variation of applied potential2"30 or ion adsorption24Jlwill result in perturbation of optical properties, e.g., refractive index. In the specific (28) Winograd, N.; Kuwana, T . J . Electroanal. Chem. 1969,23,333342. (29) Hansen, W. N.; Prostak, A. Phys. Rev. 1968, 174, 500-503. (30) Srinivasan, V. S.; Kuwana, T. J. Phys. Chem. 1968, 72, 11441148. (31) Takamura, T.; Takamura, K.; Yeager, E. J . Electroanal. Chem. 1971, 29,279-291.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992 0.006 /
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Flgure 5. Apparent absorption spectrum of poly+-lysine adsorbed on tin oxide acquiredat an applied potentialof 0.4 V. Solution conditions: pH 7.0, 1.1 = 10 mM phosphate buffer.
case of tin oxide, Winograd and Kuwana2*attributed perturbations observed in the visible spectrum primarily to changes in the refractive index resulting from alterations in free electron concentration as well as ion concentrations in the double-layer region. Changes in the refractive index of the tin oxide electrode/buffer, either through variations in applied potential or ion adsorption, will shift the interference spectrum of the tin oxide electrode and cause the absorbance to change as a function of wavelength.28 In order to test the hypothesis that the specific adsorption of cytochrome c similarly perturbs the optical constants of the tin oxide interface, a polycation, poly-L-lysine, was investigated. Previous work has shown polylysine to adsorb strongly on tin oxide in low ionic strength phosphate buffer.14 However, in contrast to cytochrome c, polylysine does not absorb light in the visible region. If our hypothesis is valid, an apparent spectrum of adsorbed polylysine should exhibit spectral anomalies similar to those observed in the apparent spectrum of adsorbed cytochrome c (Figure 4) since both molecules adsorb to tin oxide via positively charged lysine groups. The apparent spectrum of adsorbed polylysine is shown in Figure 5. This spectrum exhibits two apparent bands, one near 405 nm and the other, a broader band, near 540 nm. In addition, a negative baseline is also evident. Since polylysine does not absorb light in the visible region, this spectrum undoubtedly reflects changes in the tin oxidelbuffer blank induced by adsorption of this polycation. The appearance of background perturbations of this type would indeed account for the increased size of the Soret band, the new broad feature near 540 nm, and the negative baseline that are present in the apparent spectrum of adsorbed cytochrome c (Figure 4). To further substantiate the presence of adsorption-induced optical perturbations, tin oxide electrodes of different thicknesses and sheet resistances were examined. The magnitude and wavelength of these optical perturbations, being a function of the tin oxide film, would be expected to differ among tin oxide electrodes having different film properties. As can be seen in Figures 6 (panels A and Bj and 4,dissimilar apparent spectra are obtained for cytochrome c adsorbed on different tin oxide electrodes. In Figure 6A, the Soret absorbance has a larger value than that theoretically expected while the 530-nmband is obscured by the slopingbackground. In Figure 6B, the Soret absorbance is close to the value theoretically expected but is superimposed on a broad feature extending between 350 and 600 nm. The corresponding apparent spectra of adsorbed polylysine, shown in Figure 6, panels A and B, confirm that these anomalies result from optical perturbations induced by polycation adsorption. As
410
470 530 590 Wavelength, nm
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...............4 ............... 4 .............. 0 004 ............... i................I _.__.__.__.___.~
350
410
470 530 590 Wavelength, nm
650
710
Flgure 6. Apparent absorption spectra of ferricytochrome c (heavy line) and polylysine (light line) adsorbed on tln oxMe electrodes having different thicknesses and sheet resistances. (Panel A): 0.25 pm, 30 Wsquare. (Panel B): 0.17 pm, 50 ahquare. All spectra wereacquired at an applied potential of 0.4 V. The spike appearing at 658 nm Is a lamp line originating from the deuterlum lamp used in these experiments. Solution conditions: pH 7.0, p = 10 mM phosphate buffer.
a final point, we find that tin oxide film thicknesses estimated from the positions of the features in the apparent polylysine spectra are in agreement with the known values. Thus, using the formula t = 1/[2n& - &)I, where t is film thickness, n, is refractive index (taken to be 226,27),and is the wavenumber frequency of the maxima or minima, the appropriate values o f t (in pm) determined from Figures 5,6A, and 6B are 0.38, 0.27, and0.18, respectively. These compare satisfactorily with the known values (0.33, 0.25, 0.17 pm), which confirms our assignment of these adsorption-induced features to an alteration in thin-film interference patterns. From these results, we conclude that the apparent spectra of adsorbed cytochrome c , shown in Figures 4 and 6, are composed primarily of two components: absorbance due to the chromophore of the adsorbed protein and induced perturbations in the tin oxidelbuffer blank. This can be represented by the following equation where Aappis the apparent absorbance of adsorbed cytochrome c, Acfl is the true absorbance of adsorbed cytochromec, and Awfirepresents the background perturbations due to variation in applied potential andlor polyion adsorption:
This equation can be used to obtain A, by subtracting the adsorption-induced and potential-induced background perturbations, Le., Ape,, from Aapp. Potential dependent baseline variations, see for example, Figure 4,can be eliminated
ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992
1474
0.006 ............... ................ > ............... > ............... 4 ..........................
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470 530 590 Wavelength, nm
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Figure 7. Corrected absorption spectra of adsorbed ferricytochrome
c (light line) and ferrocytochrome c (heavy line) on tin oxide. The spectrum of adsorbed ferricytochrome c was corrected by subtracting the apparent spectrum of adsorbed polylysine acquired at 0.40 V (see text for details). Likewise, the spectrum of adsorbed ferrocytochrome c was corrected by subtractlng the apparent spectrum of adsorbed polylysine acquired at -0.20 V. Solution conditions: pH 7.0, p = 5 mM phosphate buffer. 0.006 0 , 005
