2552
Langmuir 1992,8, 2552-2559
Chronoabsorptometric Determination of Adsorption Isotherms for Cytochrome c on Tin Oxide Electrodes Maryanne Collinson and Edmond F. Bowden* Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204 Received April 6,1992. In Final Form: June 29, 1992
The adsorption isotherms for ferri- and ferrocytochrome c on tin oxide electrodes were measured as a function of ionic strength and electrolyteusing the spectroelectrochemicaltechniquechronoabsorptometry. In low ionic strength pH 7.0 phosphate buffers, a high-affinityadsorption isotherm was obtained. As the ionic strength of the buffer was increased,the adsorption affinity of cytochrome c for tin oxide diminished, and a Langmuir-type isotherm was obtained. No apparent hysteresis was observed in the adsorption isotherms of ferricytochrome c upon dilution with buffer or higher ionic strength solutions. In contrast, the adsorptionisotherms for ferrocytochromec in low ionic strengthphosphate buffer exhibited considerable hysteresis,with some denaturation of ferrocytochrome c being evident. The saturation surface coverage (I'mh) and equilibrium adsorption constant ( K ) for ferricytochrome c on tin oxide were determined by fitting the Langmuir adsorptionmodel to the data. A ca. 2-fold reductionin rmh and a ca. 20-fold reduction in K was observed as the ionic strength of the phosphate buffer was changed from 40 to 150 mM due to decreased electrostatic attractions. Slightly larger rratn and K values were obtained for ferricytochrome c in solutions containingonly phosphate compared to solutions in which chloride or nitrate was a major component. The ionic strength dependence of K for the phosphate data was analyzed in terms of simple Debye-Huckel theory, and a value of 5 X 108 M-l at zero ionic strength was obtained. These results indicate that the major driving force for spontaneous adsorption is the strong electrostatic attraction between positively charged cytochrome c and the negatively charged tin oxide electrode.
Introduction that cytochrome c strongly adsorbs to tin oxide electrodes in low ionic strength buffers while maintaining near-native The strong adsorption of redox proteins on electrode properties. Consistent with the isoelectric points of 10 surfaces'-5 can result in attractive diffusionless systems and 5.5 for cytochrome c18 and tin oxide,l9 respectively, for investigating biologicalelectron tran~ferl-~ and protein the adsorption appears to be primarily electrostatic in adsorption at the solid-liquid interface5and for developing ~ r i g i n . ~Recently J~ the conformational state and total amperometric biosensors and biocatalytic device^.^^^ Sigsurface coverage were evaluated from visible absorption nificant progress has recently been made in characterspectra of adsorbed cytochrome cell Adsorbed ferricyizing the conformational electron transfer b e h a v i ~ r , ~ -redox ~ J ~ Jand ~ adsorption thermodynamics? tochrome c was found to adsorb in a conformationally surface coverage,ll and orientation14 of adsorbed redox stable, predominantly electroactive state with a surface proteins on electrode surfaces using resonance Raman coverage of ca. one monolayer (ca. 16 pmol/cm2).l1 Some spectroscopy,UV-visible absorptionspectroscopy,internal unexplained features, however, have been observed in the reflection fluorescence, ac impedance analysis, and cyclic cyclic voltammetry (CV) of this ~ystem.~J'Specifically, voltammetry. the Cv's are broader than those predicted by Laviron's Our laboratory has been primarily interested in monomode120based on a Langmuir isotherm, and a noticeable hemic proteins adsorbed on solid electrode s u r f a ~ e s , 2 * ~ , ~ Jscan ~ ~ rate ~ dependence of the electron transfer rate constant with specific emphasis being placed on the cytochrome has been consistently observed using this same model.20 c/tin oxide system.2J1J7 In previous work, we have shown One of the objectives of the present work was to obtain a more fundamental understanding of the binding of (1) Hildebrandt, P. J . Mol. Struct. 1991,242, 379-395. (2) Willit, J. L.; Bowden, E. F. J. Electroanal. Chem. 1987,221,265cytochrome c to the tin oxide electrodes by an in-depth 274. examination of the adsorption thermodynamics. Specif(3) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. SOC.1991,113,1847ically, the determination of the adsorption isotherm of 1849. (4) Butt, J. N.; Armstrong, F. A.; Breton, J.; George, S. J.; Thomson, cytochrome c on tin oxide may help explain the observed A. J.; Hatchikian, E. C. J. Am. Chem. SOC.1991,113,6663-6670. nonideal CV behavior since the shape of the voltammetric (5) Willit, J. L.; Bowden, E. F. J . Phys. Chem. 1990,94,8241-8246. (6) Turner, A. P. F., Karube, I., Wileon, G. S.,Eds. Biosensors: waves depends on the adsorption In addition, Fundamentals and Applications;Oxford University Press: Oxford,1987. a study of the adsorption isotherm can provide information (7) Tarasevich, M. R. In Comprehensiue Treatise OfElectrochemistry; concerning the amount adsorbed,type of binding involved, Srinivesan, S.,Chizmadzhev, Yu. A., Bockris, J. OM., Conway, B. E.,
Yeager, E., Eds.; Plenum: New York, 1985; Vol. 10, pp 231-295. (8) Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 67226728. (9) Roependowski, B. N.; Schlegel, V. L.; Holt, R. E.; Cotton, T. M. in Charge and Field Effects in Biosystem II; Allen, M. J., Cleary, S. F., Hawkridge, F. M.; Eds.; Plenum: New York, 1989; pp 43-58. (10) Cotton, T. M.; Schultz, S.G.;VanDuyne,R. P.J. Am. Chem. SOC. 1980,102, 7962-7965. (11) Collinson, M.; Bowden, E. F. Anal. Chem. 1992, 64, 1470-1476. (12) Sagara, T.; Niwa, K.; Sone, A.; Hinnen, C.; Niki, K. Langmuir 1990,6, 254-262. (13) Sagara, T.;Murakami, H.; Igarashi, S.;Sato, H.; Niki, K. Langmuir 1991, 7,3190-3196. (14) Fraaije, J. G. E. M.; Kleijn, J. M., van der Graaf, M.; Dijt, J. C. Biophys. J . 1990, 57, 965-975.
0743-7463/92/2408-2552$03.00/0
(15) Paddock, R. M.; Bowden, E. F. J. Electroanal. Chem. 1989,260,
487-494. - - . ._ .
(16) Senaratne, V.; Bowden, E. F. Biochem. Biophys. Res. Commun. 1988,157, 1021-1026. (17) Collinson, M.; Willit, J. L.; Bowden, E. F. In Charge and Field Effects in Biosystem II; Allen, M. J., Cleary, S.F.; Hawkridge, F. M., Eds.; Plenum: New York, 1989; pp 69-79. (18) Ferguson-Miller, S.;Brautigan, D. L.; Margoliash, E. In The Porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1979; Vol. VII-B, pp 149-240. (19) Ahmed, S. M. In Oxides and Oxide F i l m ; Diggle, J. W., Ed.; Marcel Dekker: New York, 1972; Vol. 1, pp 319-519. (20) Laviron, E. J . Electroanal. Chem. 1979, 101, 19-28.
0 1992 American Chemical Society
Cytochrome c on Tin Oxide Electrodes
Langmuir, Vol. 8, No. 10, 1992 2553
role of solvent, thermodynamic adsorption parameters, Heineman and K ~ w a n extended a~~ eq 2 by introducing two additional terms to account for changes in ionic excess and reversibility of adsorption under different solution at the electrode/solution interface: ~onditions.229~3 Little information regarding cytochrome c adsorption thermodynamics on electrode surfaces is available. Previously Hildebrandt and Stockberger,using resonance Raman spectroscopy,determinedthe adsorption The first term on the right-hand side of eq 4 reflects the isotherm for cytochrome c adsorbed on silver diffusion-controlledreduction of the electroactive species A Frumkin isotherm model was fit to these data, and the in solution, the second term reflects the immediate free energy of adsorption and the interaction parameter reduction of electroactive species in the adsorbed state were determined.24 In addition, Hill and co-worker8 have (Fox),and the last term represents potential-induced determined the adsorption isotherm for cytochrome c optical changes in the electrode/solution interface (A,,). weakly adsorbed on a 4,4'-bipyridine-modifiedgold electrode using an ac ring-disk electrochemical e ~ p e r i m e n t . ~ ~ A plot of absorbance vs t1l2will yield a straight line with a nonzero absorbance intercept.29 The slope of this line A Langmuir isotherm was used to fit their data, and a is directly proportional to the concentration of the surface coverageand an equilibriumbinding constant were electroactive species in solution whereas the intercept is obtained. a direct measure of both the concentration of the elecIn this paper, chronoabsorptometrywas used to detertroactive species adsorbed on the electrode surface and mine, in situ, the adsorption isotherms of ferri- and any potential-induced optical changes to the electrode/ ferrocytochrome c on tin oxide electrodes under several solution interface (A,,,). A,, can be determined from the different solution conditions. To our knowledge, this intercept of an absorbance vs plot obtained in the representsthe first in-depth investigationof the adsorption absence of an electroactive and/or adsorbed species.29 In thermodynamicsfor cytochrome c at an electrode surface. the presence of an electroactive, adsorbing species, the From these data, the saturation surface coverages and intercept should increase above A,, by an amount proequilibrium binding constants for ferri- and ferrocytoportional to the electroactive coverage. Assuming that chrome c absorbed on tin oxide were obtained using the adsorption does not significantly alter A,,, an in situ Langmuir adsorption model. The adsorption isotherms determination of surface coverage can thus be made in for ferri- and ferrocytochrome c were compared in order the presence of solution species.29 Heineman and coto discern any possible adsorptive differencesbetween the workers used this technique to successfully examine30131 oxidized and reduced forms. In addition, the equilibrium and quantify29the specific adsorption of anions on a Hgbinding constants obtained for ferricytochrome c in Pt optically transparent electrode. different ionic strength solutions were analyzed in terms of the Debye-Huckel theory in order to assess the charges Experimental Section involved in the interaction with tin oxide.
Theory Chronoabsorptometry. Chronoabsorptometry (CA) ia a spectroelectrochemical which involves optically monitoring the reduction (or oxidation) of a chromophoric species in solution following application of a potential step. For monitoring a simple reaction under diffusion-controlled conditions via normal transmission at a planar electrode ox(e=O) + ne-* red(c>O) (1) the following equation can be derived26to relate the subsequentincrease in adsorbance (A) to the concentration (CoJ and diffusion coefficient (D,) of the oxidized species, and to the molar absorptivity of the optically monitored reduced species (er&
A = (2Cox~,,dDox1/2t1/2)/a1/2(2) If the potential is then returned to its initial value for an identical time interval, Le., double potential step CA, the ratio of the adsorbances measured at the end of the forward and back steps should give
A d A , = (Ar&,27) - ArJX,7)j/Ar&,d = 0.586 (3) if the reduction reaction is diffusion controlled and chemically u n ~ o m p l i c a t e d . ~ ~ * ~ ~ (21) Laviron, E. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1982; Vol. 12, pp 53-157. (22) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267-340. (23) Parfitt, G. D., Rochester, C. H., Eds. Adsorption from Solution at the SolidlLiquid interfaces; Academic Prese: New York, 1983. (24) Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1986,90,60176024. (25) Albery,W. J.; Eddowe, M. J.; Hill, H. A. 0.;Hillman, A. R. J.Am. Chem. SOC. 1981,103,3904-3910. (26) Kuwana, T.; Winograd, N. Electroanal. Chem. 1974, 7, 1-78. (27) Heineman, W.; Hawkridge, F. M.; Blount, H. N. Electroanal. Chem. 1984,13, 1-113. (28) Li, C.-Y.; Wilson, G. S. Anal. Chem. 1973,45, 2370-2380.
Reagents and Materials. Horse heart cytochrome c (type VI) was obtained from Sigma Chemical Co. and chromatographically purified as previously described.32 Ferrocytochrome c was obtained by reducing ferricytochrome c with excess solid potassium dithionite. Prior to use, cytochromec was passed through a desalting gel (Biorad, P-6DG) previously equilibrated in the buffer used in the experiment. All buffers used in this work were pH 7.0,4.4-70 mM potassium phosphate (ionic strengths (h) of 10-150 mM). Reagent grade chemicals were used to prepare all solutions. Water was purifed using a Milli-Q system with an Organex-Q final stage (Millipore, Bedford, MA). The fluorinedoped tin oxide electrodes, which were provided by PPG, consisted of a 0.33-pm film on a 5-mm glass substrate with a sheet resistance of ca. 17 Q/square. Equipment. Spectroelectrochemistry was performed using 84526 diode array spectrophotometer a Hewlett-Packard (HP) and an EG&G PAR 273 potentiostat. A QuickBASIC program was written to simultaneously control both these instruments using a HP-IB interface card. Spectroelectrochemicaldata, cy+c voltammograms, and visible absorption spectra of both solution and adsorbed species were acquired with this program. The simultaneous control of both the spectrophotometer and the potentiostat proved especially crucial in the spectroelectrochemical subroutine. In order to obtain accurate chronoabsorptometry data, it was imperative that the acquisition measurements be synchronized with the potential step excitation pulse. This was achieved by triggering the H P 8452A with a pulse from the PAR 273 and experimentally determining the delays associated with this triggering procedure.33 The spectroelectrochemical cell, which has previously been described," consists of a lucite cell body and two tin oxide electrodes. The cell volume is ca. 1.8mL, the combined electrode area is 1.34 cm2, and the distance between (29) Heineman, W.; Kuwana, T. Anal. Chem. 1972,44, 1972-1978. (30) Heineman, W. R.; Goelz,J. F. J. Electroanal. Chem. 1978, 89, 437-441. (31) Goelz,J. F.; Heineman, W. R. J. Electroanal. Chem. 1979, 103, 155-163. (32) Brautigan, D. L.; Ferguson-Miller, S.; Margoliash, E. Methods Enzymol. 1978,530, 128-164. (33) Please consult authors for specific details concerning triggering and timing procedures.
Collinson and Bowden
2554 Langmuir, Vol. 8,No. 10,1992 the two electrodes, Le., path length, is 1.12cm. A HP air-driven magnetic stir module and in-house fabricated stir bars were used to stir the solution in the cell cavity. The stir module was secured on a base plate mounted in the sample compartment of the spectrophotometer. Transmission spectroscopic measurements were obtained by passing light through the cell normal to the electrode surfaces. 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 the Ag/AgCl electrode, which is 0.23 V vs NHE. The temperature of the cell was maintained at 18 f 1 OC using a thermostated cell holder. Procedures. The tin oxide electrodes were cleaned by successive 10-min sonications in Alconox, 95% ethanol, and Milli-Q water. This procedure was repeated, and the electrodes were then equilibratad overnight in the pH 7.0 phosphate buffer used in the experiment. After electrode equilibration, the spectroelectrochemicalcell was assembled and filled with buffer. The cell was then seated on the stirring module, aligned in the spectrophotometer, and secured in place. Once aligned and secured, the spectroelectrochemical cell remained undisturbed throughout the length of the experiment. A blank background spectrum of the tin oxide electrodesin buffer was initially acquired at 0.40 V. This spectrum waa internally subtracted from all subsequently obtained absorbance measurements. The CA potential step excitationwaveform for femcytochrome c consisted of holding the potential at 0.40 V for 10-30 8, stepping the potential to -0.20V for ca.2.2s,and then returning the potential to0.40V. Thia waveform was repeated 5-10 times, and the reaults were averaged to reduce some of the random noise associated with the low absorbance values. For ferrocytochrome c , a similar waveform was applied except that the initial potential was held at -0.20V and the potential was stepped to 0.40V. In both cases the absorbance at 418 nm was monitored. This wavelength was chosen%because it corresponds to the greatest molar absorptivity difference between the reduced and oxidized spectra of cytochrome c, Acrle = 57 OOO M-l cm-1.96-36Initial CA experiments showed that practical equilibrium is established within ca. 15 min of introducing cytochrome c to the cell cavity. Therefore, equilibration of cytochrome c was allowed to proceed for 30-45 min prior to the determination of surface coverage and solution concentration. Adsorption isotherms were obtained for ferri- and ferrocytochrome c using the followingCA procedure. In each case, results from 3-5 CA experiments were averaged together to obtain a more accurate intercept from the absorbance vs t1l2plots. A,. was fist determined by performing CA 3-5 times with only buffer present in the sample cavity. A 5-1O-hL aliquot of ca. 30 r M ferricytochrome c was then added to the ca. 1.8 mL of buffer in the sample cavity. The solution was continuously stirred for ca. 30-45 min, and the stirrer was then turned off 3-5 min prior to CA measurementa. A visible absorption spectrum of cytochrome c was acquired, and CA was then performed 3-5 times. Stirring was then resumed, another small aliquot of concentrated cytochrome c was added?’ and the above procedure was repeated. A total of 6-10 additions of cytochrome c were made over a 10-12-h time period. Reversibility with respect to dilution was evaluated by replacing an aliquot of cytochrome c in the cell with buffer and repeating the previously described procedure. In order to maintain cytochrome c in ita desired oxidation state, 0.40 V was applied during the adsorption of ferricytochrome c and -0.20 V was applied during ferrocytochrome c adsorption. The concen(34) In this work, the absorbance at 418 nm was monitored because it experimentally corresponded to the greatest difference between the reduced and oxidized spectra of adsorbed cytochrome c. Lass than a 5% difference was noted between the absorbance at 416 nm compared to 418 nm, and therefore, the molar absorptivity difference of 57 OOO M-1 cm-1 at 416 nmu was used in these calculations. (35) Van Buren, K. J. H.; Van Gelder, B. F.; Wilting, J.; Braams, R. Biochem. Biophys. Acta 1974,333, 421. (36) Koller, K. B.; Hawkridge, F. M. J. Electroanal. Chem. 1988,239, 291-306. (37) No significant differences, o;ther than electrode-to-electrode variability, wera noted in the adsorption isotherms when cytochrome c waa added in a stepwiee fashion to the same pair of tin oxide electrodes compared to using a fresh pair of electrodes for each concentration of cytochrome c.
0.007
0.001
0.000 -0.001 0 .o
1.o
0.5
1.5
2.0
Time, sec 0.008 0.007
T
B
/d
0.001
0.0
1 .o
0.5 (Time)”’,
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Figure 1. Absorbancetime (panel A)and absorbancet1/2 (panel
B)data for the diffusion-controlled reduction of ( 0 )0.680 mM
potassium ferricyanide in buffer and (A)buffer only at tin oxide electrodes. The solid lines represent the linear regression fit to the experimental data. Buffer conditions: 0.50 M glycine/HCl, pH 2.5,containing 0.50 M potassium nitrate. Chronoabsorptometry conditions: single potential step from +0.50 to -0.10V for ca. 2.2 s, absorbance monitored at 420 nm. tration of cytochrome c in solution was determined from the visible absorption spectrum using a molar absorptivity of 106 100 M-1 cm-l 38 at 410 nm for ferricytochrome c and 129 100M-1 cm-1 at 416 nmS for ferrocytochrome c. The electroactive surface coverage was determined, using eq 4, from the absorbance intercept of the absorbance-t1/2 plot, previously corrected for A,,&.The right-hand side of this equation was multiplied by a factor of 2 to account for the two tin oxide electrodes used in these experiments.
Results and Discussion Evaluation of Spectroelectrochemical Data Acquisition. In order to validate the experimental setup, CA of two Ymodelnsystems was examined, one diffusing and the other adsorbed. Potassium ferricyanide (e420 = 1020M-l cm-l 39 dissolved in pH 2.5,0.50M glycine buffer containing 0.5 M potassium nitrate was chosen as the diffusing model system. Equation 2 was modified to account for the electrochemical removal, rather than the formation, of the optically monitored species. Figure 1A shows the absorbance-time plots for 0.680 mM potassium ferricyanide and the buffer solutions after stepping the potential from +0.50 to 4.10 V for ca. 2.2 s. The upper curve in Figure 1A reflects the diffusion-controlled re(38) Margoliash, E.; Frohwirt, N. Biochem. J. 1969, 71,570-672. (39) Winograd, N.; Blount, H. N.; Kuwana, T. J. Phys. Chem. 1969, 73, 3456-3462.
Cytochrome c on Tin Oxide Electrodes
Langmuir, Vol. 8, No.10, 1992 2666
duction of ferricyanide in solution. Upon plotting these data as absorbancevs t1l2,good straight lines with slightly negative intercepts were obtained, Figure 1B. The absorbance intercept obtained when only buffer is present in the sample cavity reflects nonfaradaic optical changes 0.0°2 in the tin oxide electrodelbuffer interface due to the change in applied p 0 t e n t i a l , 2 ~i.e., ~ ~ *A,,. ~ ~ At the wavelength 0.001 monitored, i.e., 420 nm, A,, was found to be small, ca. -5 X AU. The y-intercept obtained from the absorbance-t1/2 plot for ferricyanide is indistinguishable from 0.000 this value. From the slope of the absorbance-N2 plot, a diffusion coefficient of 7.4 X lo+ cm2/swas obtained for ferricyanide,which is in agreement with literature values.42 Double potential step CA yielded an absorbance ratio -0.001 described by eq 3 of 0.582. This value is in excellent 0.0 agreement with the theoretical value of 0.586, indicating that ferricyanide reduction under these conditions is diffusion controlled and chemically u n c ~ m p l i c a t e d . ~ ~ ~ ~ These results verify that the experimental CA setup is operating correctly for diffusing ferricyanide. Further verification was provided by evaluating the adsorption of cytochrome c in 4.4 mM phosphate buffer m (ionic strength ( p ) of 10 mM). Under these conditions C cytochrome c adsorbs strongly on tin oxide electrodes at approximately monolayer c ~ v e r a g e . ~Equation J~ 4 was modified slightly to account for the chromophoricproperties of both the reduced and oxidized species at 418 nm. Figure 2A, plot a, shows the absorbance-time plots for ca. 0.000 1’ 3 pM cytochrome c equilibrated at the tin oxide electrodes. Followingthe potential step t0-0.20 V, an abrupt increase in the absorbance is observed due to the rapid reduction of ferricytochrome c molecules adsorbed on the electrode surface (see below). This is then followed by a gradual increase in absorbance due to the reduction of diffusing cytochrome c in solution. If the cytochrome c solution is then removed from the cell cavity and the cell thoroughly rinsed with the same buffer,5J1the absorbance-time data presented in Figure 2A, plot b, are obtained. In this plot, only the initial abrupt increase in absorbance is observed due to the immediate reduction of strongly adsorbed cytochrome c molecules. These data from both experiments are plotted as absorbance-t1/2 in Figure 2B. With the exception of the first point following application of the potential step,(3 good linearity is obtained. The absorbance42 plot obtained when only buffer is present in the cell cavity shows a slight negative intercept. As previously described, this negative intercept reflects nonfaradaic optical changes in the tin oxide electrodes due to the potential being stepped from +0.40 to -0.20 V, i.e., A,,,. After correcting the absorbance intercept obtained from plot b for A,,,, an electroactive surface coverage of 16.2 f 0.5 pmol/cm2was calculated. This value is in very good agreement with the value of electroactive coverage obtained from background-subtracted cyclic voltammograms, 15.5 f 1pmol/cm2,and from the visible absorption spectrum of adsorbed cytochrome c,ll 16 f 1pmol/cmZ.
f
1
(40)Winograd, N.; Kuwana, T. J . Electroanal. Chem. 1969,23,333342. (41)Srinivaaan, V. S.;Kuwana, T. J. Phys. Chem. 1968,72, 11441148. (42)Kawiak, J.;Kulesza, P. L.; Galus, Z. J. Electroanuf. Chem. 1987, 226,305-314. (43)In the absorbance-tl/*plots it is apparent that the first data point deviates considerably from the linear least-squaresregression line drawn through the data. This can be attributed to the quasi-reversibleelectron transfer kinetics of adsorbed cytochrome c (i.e., cytochrome c is not ’immediately” reduced upon stepping the potential to -0.20 V), v d to the high resistance of the 4.4 mM phosphate buffer. A rough estimate of the time delay for the reduction of adsorbed cytochrome c can be made wing the rate constant of 40 8-1 at this overpotential (Song, S.;Bowden, E. F. Unpublished result& and a solution resistance of ca. lo00 n. The resulting value of ca. 50 ms is in agreement with the time frame for the deviant point(s) in Figure 2B.
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2556 Langmuir, Vol. 8, No. 10, 1992
Collinson and Bowden
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tochrome c at tin oxide electrodes. Ferricytochrome c concen0.0, (A)0.09,(+) 0.34,( 0 )1.1,(*) 2.3,(0) 3.6, trations (pM): (0) and (X) 5.2. The solid lines represent the linear regression fits to the experimental data. Solution conditions: pH 7.0, p = 40 mM potassium phosphate. Same chronoabsorptometry conditions as described in Figure 2. 0
10
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20
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Figure 4. Adsorption isotherm of ferricytochrome c on tin oxide in p = 10 mM, pH 7.0 phosphate buffer. An approximate fit = 18.7 using a Langmuir isotherm with K = lo00 pM-l and rMtn pmol/cm2 is shown as the solid line.
due to background perturbations was estimated from results described in previous work." These two effects are, however, small, ca. ~of the Langmuir premises are not obeyed and the adsorption process is irreversible. In almost all situations involving protein adsorption on polymeric substrates, the protein is conformationally altered and adsorption is irreversible or, at best, only partially r e ~ e r s i b l e .The ~ ~ ~system ~ ~ described in this paper, however, consists of a conformationally stable adsorbed redox protein in practical equilibrium with solution. The adsorption is completely reversible with respect to dilution with buffer or higher ionic strength solutions, and no hysteresis is apparent in the desorption isotherm data. Although cytochrome c adsorption on tin oxide electrodes clearly does not satisfy criterion 4, eq 4 can be fit, at least empirically,to the adsorption isotherm data in order to extract useful parameters. Table I lists the saturation coverage (rmtn) and the equilibrium adsorption constant for ferricytochrome c adsorbed on tin oxide as a function of ionic strength and solution composition. As the ionic strength increases,both K and rsatn drop. This can be largely attributed to the decreased electrostatic attraction of cytochrome c to tin oxide as a result of increased ion hi el ding.^^ The ionic strength dependence on K can be very crudely analyzed in terms of simple Debye-Huckel theory. The following (54) Grainger, D. W.; Okano, T.; Kim, S. W. J. Colloid Interface Sci. 1989,132, 161-175. (55) Saavedra, S. S.; Reichert, W. M. Langmuir 1991, 7,995-999. (56) Mura-Galelli,M. J.; Voegel, J. C.; Behr, S.;Bres, E. F.; Schaaf, P. R o c . Natl. Acad. Sci. U.S.A. 1991,88,5557-5561. (57) The equilibrium adsorption constant K described in this paper is related to the equilibrium adsorption and desorption rate constants; Le., K = k.dJkdp = 0/(1- 0)C. The magnitude of K will therefore depend on the concentration units employed. In this paper, molar concentration units are used for C, and hence, K has units of M-l. These unita were chosen in order to facilitate the comparison of K to association constants determined for protein/protein electrostatic complexesmb5and other adsorption con~tants.*5~63-E5 (58) Chan, B. M. C.; Brash, J. L. J. Colloid Interface Sci. 1981, 82, 217-225. (59) Stuart, M. A. C.; Fleer, G . L.; Lyklema, J.; Norde, W.; Scheutjens, J. M. H. M. Adu. Colloid Interface Sci. 1991,34,477-535. (60) Koutsoukos, P. G.; Mumme-Young,C. A.; Norde, W.; Lyklema, J. Colloids Surf. 1982, 5 , 93-104.
Collinson and Bowden
2558 Langmuir, Vol. 8, No. 10, 1992 8 -
7.5 -7
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5.5 4 0.1
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0.3
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SQRT (14
Figure 8. Logarithm of equilibrium adsorptionconstant5' as a function of ionic strength for the cytochrome c/tin oxide electrostatic complex. Data points are from Table I. Solid line represents regressionfit of experimentaldata. Linear regression yields a slope of -7, a y-intercept of 8.7, and a regression coefficient of 0.997.6' Solution conditions: pH 7.0 phosphate buffer.
equation, based on the interaction between simple ions in the limit of zero ionic strength?' was used log K = log + Z,Zcp1'2 (6) where KO is the equilibrium adsorption constant at zero ionicstrength, 2,is the charge associated with cytochrome c, and 2,is the charge associated with an adsorption site on the tin oxide electrode (see below). A plot of log K vs p1/2should yield a straight line with a slope equal to Z& and a y-intercept equal to log KO. Figure 8 shows the phosphate data from Table I plotted in this form. Using eq 6, an equilibrium adsorption constant at zero ionic strength of 5 X 108M-l was obtained from the y-intercept. This large value for KO is consistent with the strong binding of cytochrome c to tin oxide that has been observed in low ionic strength solutions2~5J1J7 and is comparable within an order of magnitude to values determinedfor cytochrome c binding to soluble protein partners.62* The negative slope of the line in Figure 8 demonstrates the attractive electrostatic nature of cytochrome c binding to tin oxide electrodes. The absolute value of the slope, i.e., 7, reflecta in some manner the magnitudes of positive and negative interacting charge, but a straightforward interpretation of 2, and 2, is not available due to the complexity of the interaction. The linearity of the data in Figure 8 may indeed be somewhat fortuitous. A value for 2,(or 2,)calculated from the slope of this line cannot be expected to represent the total charge on cytochrome c (or an adsorption site). Instead, 23, must, at a minimum, reflect the complex interaction of localized charges on cytochrome c with localized and smeared out charge on the tin oxide surface. In addition, phosphate binding to cytochrome ~'3'3367 would further complicate this analysis. As can be observed in Table I, somewhat larger cytochrome c surface coverages are obtained for phosphate (61) The ionic strengtWequilibriumadmrptionconstant data were also fitted by the extended form of the Debye-Huckel equation with similar resulte; Le., Zse= -11.7; log KO = 9.3;.R* = 0.996. (62) Eman, J. E.; Vitello, L. B. J. Btol. Chem. 1980,255,6224-6227. (63) Kornblatt, J. A.; English, A. M. Eur. J. Biochem. 1986,155,505511. (64)Vitello, L. B.; Erman, J. E. Arch. Biochem. Biophys. 1987, 258, 621-629. (65) Shaw, D. C.; Hartzell, C. R. Biochemistry 1976, 15, 1909-1914. (66)Oaheroff, N.; Brautigan, D. L.; Margoliash, E. Proc. Natl. Acad. Sci. U.S.A. 1980. 77.4439-4443. (67) Gopal, D.'; Wileon, G. S.; Earl, R. A.; Cusanovich, M. A. J.Biol. Chem. 1988,263, 11652-11656.
buffer compared to cytochrome c in phosphatelnitrate or phosphatelchloride buffer solutions of identical ionic strength. Although subtle differences in ionic strength among the buffers cannot be ruled out, the most likely explanation for this difference is different degrees of anion bindings' to the cytochrome c molecules. Phosphate and chloride, in particular, bind to cytochrome c at different locations with different affinities.@*67Specifically, phosphate has been shown to bind to the front face of cytochrome c and stabilize the heme crevice whereas chloride does not.@ The slightly larger cytochrome c coverages obtained in the presence of phosphate alone may reflect this stabilization through the reduction in intramolecular repulsions and possibly intermolecular electrostatic repulsions. Another factor may be the role played by secondary binding sites for phosphate and chloride on cytochrome c.& In previous ~ o r kanion , ~ ~ ~ ~ binding effects have been observed for bovine serum albumin (BSA) adsorption on polystyrene lattices (PS). Specifically a substantially greater surface coverage of BSA at acidic p H s was obtained in the presence of SCNcompared to acetate or chloride.68@ This was attributed to the formation of a more compact structure of BSA due to decreased intramolecular repulsions resulting from the greater binding affinity of SCN- to BSA in comparison to chloride or a ~ e t a t e . The ~ ~ .relationship ~~ of these studies to the present one is, however, rather ambiguous because cytochrome c exhibits greater structural rigidity than BSA. Adsorption Isotherms for Ferrocytochromec. Figure 9Ashowsthe adsorption isotherms for ferrocytochrome c adsorbed on tin oxide electrodes in pH 7.0,p = 10,80, and 150 mM potassium phosphate. The adsorption isotherm obtained at the highest ionic strength is similar in nature to those previously described for ferricytochrome c, i.e., Figures 5 and 6. In general, however, we found the determination and analysis of the adsorption isotherms for ferrocytochrome c to be more problematical. In particular for the p = 10 or 80 mM buffers, when ferrocytochrome c was removed from the solution and replaced with buffer, the calculated electroactive surface coverages were smaller than the original values obtained prior t o d i l ~ t i o n This . ~ ~ result contrastswith dataobtained for ferricytochrome c which showed nearly identical adsorption and desorption isotherm points for all ionic strengths evaluated. For ferrocytochrome c at p = 10mM, a noticeable drop in surface coverage occurred over time as the solution concentration was incremented to higher values (Figure QA). The surface coverage continued to drop as the ferrocytochromec solution concentration was returned to lower values by dilution with the same buffer.7O Visible absorption spectra, obtained after the complete removal of solution cytochrome c, indicated that a conformational change had occurred in at least some of the adsorbed cytochrome c, Le., the Soret band was blueshifted ca. 1-2 nm. While adsorbed ferricytochrome c appears to be stable for at least 20 h at E = 0.4 V,adsorbed ferrocytochrome c in the p = 10 or 80 mM buffer is stable for only ca. 4-5 h at E = -0.2 V before noticeable changes in the electroactive surface coverage are apparent. This observation is, however, in direct contrast to solution studies which show that the reduced form of cytochrome c is more stable than the oxidized form.47 The more negative electrostatic field present at the electrode surface (68) Shirahama, H.; Takeda, K.; Suzawa, T. J. Colloid Interface Sci.
1986. ~ .,.109.552-556. -.- - -,- - - - - -
(69) Suzawa, T.; Shirahama, H. Adu. Colloid Interface Sci. 1991,35, 139-172. (70) As stated in the Experimental Section, ferrocytochrome c was prepared by reducing ferricytochrome c with excess dithionite. Similar results were obtained by electrochemically reducing ferricytochrome c at the electrode surface by maintaining an applied potential of 4 - 2 0 V.
Cytochrome c on Tin Oxide Electrodes
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Langmuir, Vol. 8, No.10, 1992 2569
A
16 14 N
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1400 1200
6
8
MM
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B
800
i 600 400
I/"
200 A
0
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Figure 9. Adsorption isotherms (panel A) for ferrocytochrome
c on tin oxide in pH 7.0 phosphate buffer. Ionic strength (mM):
(0)10, ( 0 ) 80,and (A) 146. Desorption points obtained by dilution are shown as darkened symbols. Solid lines in panel A ( p = 10,80 mM) were added to guide the eye. The solid line for panel A ( p = 146 mM) represents the Langmuir fit to the experimental data. Panel B: Langmuir plot for ferrocytochrome c adsorption in p = 146 mM pH 7.0 phosphate buffer. The solid line in panel B represents the linear regression fit of the experimental data.
when -0.20 V is applied may be adversely affecting the long-term conformational stability of adsorbed ferrocytochrome c. Other workers have observed that, when cytochrome c denatures upon strong adsorption to bare silverlP8 or g0ld'~J3electrodes, its redox potential shifts negative0.4-0.5 V. A similar type of denaturation behavior could account for the gradual loss of electroactive ferrocytochrome c in our experiments. That is, at an initial applied potential of -0.2 V vs Ag/AgCl, any denatured cytochrome c would be present in the ferric form and would then no longer be detectable in the chronoabsorptometric transient to E = 0.40 V. As can be seen in Figure 9B, good linearity is obtained when the adsorption data for ferrocytochrome c at p = 146 mM are plotted in the linear form of the Langmuir equation, i.e., eq 4. The value of K determined from the regression line, 1.0 f 0.2 pM-l, is indistinguishable from that obtained at an identical ionic strength for ferricytochrome c (Table I). In a previous study? we found that the reduced form binds somewhat less strongly than the oxidized form at the formal potential (ca. 0 V). The
adsorptions of ferri- and ferrocytochrome c were examined in the present study, however, at +0.4 and -0.2 V, respectively, which could account for this different result. The value of rmtnat p = 150 mM obtained from the regression line in Figure 9B is 6.8 i 0.1 pmol/cm2,which is significantly less than the 11.1pmol/cm2measured for ferricytochromec (Table I). One possible explanation for the lower apparent coverage of ferrocytochrome c is an anion binding effect. Phosphate has been shown to bind less strongly to the ferrocytochrome c than to ferricytochrome c.67 As a result, smaller surface coverages could be obtained for ferrocytochromec (vide supra). However, given the questionable stability of ferrocytochrome c at thissurface,which is clearlyevident at lower ionicstrengths (Figure 9A),we can speculate that cytochrome c spreading and/or denaturation on the tin oxide surface may also be a factor. Further investigationswould be required to fully understand these results.
Conclusions Chronoabsorptometryis a useful in situ technique for determining adsorption isotherms of an electroactive species adsorbedon planar solid electrodes. Theoretically, this technique can be used to quantify both the amount of electroactive species adsorbed on an electrode surface as well as the amount of species in solution. However, a highly absorbing sample in contact with an optically transparent electrode is necessary for transmission chronoabsorptometry. Signal averaging can be used to eliminate much of the random noise inherently present in the typically small absorbance values. In this paper, the absorption isotherms for electroactive ferri- and ferrocytochrome c electrostatically adsorbed on tin oxide electrodes were determined using chronoabsorptometry. For the conditions used in voltammetric experiments, i.e., low ionic strength and neutral pH phosphatebuffers, high-affinity adsorption isotherms were obtained. In higher ionic strength solutions, Langmuirtype adsorption isotherms were obtained for both ferriand ferrocytochromec. The adsorption isotherms for ferriand ferrocytochrome c appeared similar, although some differences were noted in the plateau surface coverage values. Although these results do not suggest an obvious explanation to account for the nonideal voltammetric features that have been previously observed,7l they do provide a clearer picture of the adsorption behavior of cytochrome c on tin oxide electrodes. Future work will involve pH- and temperature-dependent investigations of the adsorption isotherm for ferricytochrome c. Acknowledgment. We thank Jimmy Reeves, John Evans, Glenn Hennessee, and Richard Beck for helpful discussions regarding interfacing procedures, William Patrick for the design and fabrication of the thermostated cell holder used in these measurements, and John Sopko at PPG for providing the tin oxide. We gratefully acknowledge support of this work by the National Science Foundation (Grant CHE-8820832). Registry No. KC1,7447-40-7;KN03,7757-79-1; cytochrome c, 9007-43-6; tin oxide, 1332-29-2;potassium phosphate, 16068-
46-5.
(71) Recent chronoamperometric measurements have revealed significant chargetransfer limitations associatedwith the tin oxideelectrodes (Song,S.;Willit, J. L.; Bowden,E. F. Unpublished results). This appears to be the primary cause of nonideal voltammetry.
