J. Phys. Chem. B 2000, 104, 6851-6859
6851
Electrocatalysis of Guanine Electron Transfer: New Insights from Submillimeter Carbon Electrodes Veronika A. Szalai and H. Holden Thorp* Department of Chemistry, UniVersity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290 ReceiVed: March 8, 2000; In Final Form: May 10, 2000
Electrocatalytic oxidation of guanines in DNA by Ru(bpy)33+/2+ (bpy ) 2,2′-bipyridine) has been detected at 86 µm-radius carbon electrodes. The electrocatalysis, which is apparent as a current enhancement of the Ru(bpy)33+/2+ couple in the presence of DNA, is dependent on the ionic strength. The ionic strength effect is due not to a change in the mediator-electrode surface interaction, but rather to a change in the mediatorDNA interaction. The small size of the electrode allows examination of the contribution of the time-independent current, which is a function of the diffusion coefficients of the electroactive species, to the total current generated by guanine oxidation by Ru(bpy)33+. The second-order rate constants from digital simulation of the experimental cyclic voltammograms collected at low ionic strength are (in terms of moles of guanine) (5.6 ( 1.4) × 104 M-1 s-1 at 5 mV/s and (8.8 ( 2.2) × 104 M-1 s-1 at 250 mV/s. At high ionic strength, the second-order rate constants for voltammograms collected at 5 mV/s or 250 mV/s are both 2 × 104 M-1 s-1. Digital simulation of the time-independent current reveals that weak electrostatic association of Ru(bpy)32+ or Ru(bpy)33+ with DNA produces maximal currents; extensive association of either the 2+ or the 3+ form inhibits the electrocatalysis and depresses the catalytic currents. Therefore, Ru(bpy)32+, which binds to DNA with a weaker affinity than does Ru(bpy)33+, is a near-ideal catalyst for electrocatalytic guanine oxidation in DNA.
Introduction The chemistry of nucleic acid oxidation leading to DNA damage plays a recognized role in disease.1-4 Recent emphasis has been placed on determining the rate of initial oxidation and subsequent charge migration in DNA, partly as a means to understand mechanisms of mutagenesis.5-21 Because guanine is the nucleobase with the lowest one-electron redox potential (1.34 V vs normal hydrogen electrode22), study of the redox chemistry of guanine, particularly when stacked in guanine doublets and guanine triplets, has been a major focus.6,10,11,13,23-30 In most cases, relative reactivities of these guanine species are obtained from gel electrophoresis experiments; however, use of this approach to assign relative rates of vertical, one-electron guanine oxidation is subject to concerns about the follow-up chemistry that produces strand scission.5-7,23-25 Nevertheless, gel electrophoresis experiments offer a significant advantage in comparing multiple reactions that produce strand scission under a variety of conditions.9-11,13,31 Our laboratory has developed a sensitive electrochemical technique, using tris(bipyridine)ruthenium(III/II), Ru(bpy)33+/2+, that incorporates the well-documented oxidation chemistry of guanine into an electrocatalytic scheme.15,32-34 Rate constants for guanine oxidation can be obtained by digital simulation of the catalytic cyclic voltammograms, provided that adequate mechanistic insight is available. The efficiency of the reaction of Ru(bpy)33+/2+ with DNA depends on the salt concentration of the solution, and as a result, the reaction must be modeled using one of two different mechanisms.15,20,21 Optical spectroscopy has demonstrated that Ru(bpy)33+/2+ does not intercalate * Corresponding author. E-mail:
[email protected]; FAX: (919) 9622476.
between base pairs in DNA, which means that purely electrostatic binding constants best describe the interaction between Ru(bpy)33+/2+ and DNA.35 Under high-ionic-strength conditions where Ru(bpy)32+ does not associate significantly with DNA, the reaction between guanines in DNA and Ru(bpy)33+/2+ can be described by the two-step mechanism15
Ru(bpy)32+ f Ru(bpy)33+ + ek2
Ru(bpy)33+ + DNA 98 Ru(bpy)32+ + DNAox
(1) (2)
where DNAox is a DNA strand in which guanine has been oxidized by one electron. The DNA oxidation step (eq 2) is catalytic in ruthenium, which means that very small concentrations of Ru(bpy)32+ effect oxidation of multiple guanines. When the ionic strength of the medium is lowered, binding of Ru(bpy)33+/2+ to DNA must be included in the mechanism, as shown in Scheme 1.15 In this case, binding is diffusion-controlled (ka and ka′), the association constants (K2+ and K3+) of Ru(bpy)33+/2+ are known,15 and second-order rate constants for the oxidation reaction are derived from the relationship15
k2 )
[
]
K3+ k1 K [DNA] + 1 3+
(3)
Understanding the importance of electrostatic association between a catalyst and its substrate has broad implications for biology, especially in the realms of enzyme catalysis,36-38 biomolecular recognition,39,40 and directed targeting of therapeutic drugs.41,42 Our system offers the opportunity to study nonspecific electrostatic association between the small Ru-
10.1021/jp000912n CCC: $19.00 © 2000 American Chemical Society Published on Web 06/29/2000
6852 J. Phys. Chem. B, Vol. 104, No. 29, 2000 SCHEME 1
Szalai and Thorp is controlled by the salt concentration in solution. Simulations of cyclic voltammograms collected under nonlinear diffusion conditions enabled by the small electrodes show that maximum catalysis occurs when Ru(bpy)33+/2+ is not associated extensively with DNA. Under these conditions, unbound Ru(bpy)32+ is available to migrate to the electrode surface and serve as an electrochemical mediator; extensive association of Ru(bpy)33+/2+ and DNA produces fewer catalytic turnovers and less current. A larger consequence of these results is that the reaction trajectory for guanine oxidation is controlled partly by electrostatic attraction between unbound, electrogenerated Ru(bpy)33+ and DNA. Thus, lack of association between Ru(bpy)33+/2+ and DNA results in collision-controlled guanine oxidation; extensive association of Ru(bpy)33+/2+ with DNA results in decreased electrocatalysis; and modest association of Ru(bpy)33+/2+ with DNA results in near-maximal detected currents from the catalytic reaction. These features are explained readily by the model used for digital simulation. Experimental Section
(bpy)33+ catalyst and the large DNA template in which substrate guanines are distributed randomly. Unlike the irregularly shaped surfaces of DNA-binding proteins, the positive charge on Ru(bpy)33+/2+ is spherically symmetric, so the electrostatic association is determined predominantly by the charge topology of the DNA surface. This simplicity stands in contrast to the many subtle geometries and interactions that exist between proteins and DNA during macromolecular recognition. Although Ru(bpy)33+/2+ and DNA-binding proteins are different in size and shape, both experience multiple electrostatic encounters with a length of DNA many times larger than the target reaction site during catalysis.20,40,42 Thus, a detailed understanding of the events through which Ru(bpy)33+/2+ catalyzes guanine electron transfer may illuminate our understanding of site-specificity within a uniform charge topology. In this paper, we use carbon electrodes to reduce the electroactive area of the ITO electrodes that we have used previously14,19-21,32,33,43 by a factor of 600, from 0.32 cm2 to 0.0005 cm2. Digital simulation of the resulting cyclic voltammograms shows that the efficiency of electrocatalytic oxidation of guanine mediated by Ru(bpy)32+ is determined in large part by the extent of association of Ru(bpy)33+/2+ with DNA, which
Materials. Type XIV DNA from herring testes, 3-hydroxytyramine, and L-ascorbic acid were purchased from Sigma. Potassium chloride, sodium chloride, citric acid, and sodium phosphate (mono- and dibasic) were purchased from Mallinckrodt. Racemic [Ru(bpy)3]Cl2 was purchased from Aldrich and purified by ion-exchange chromatography (Sephadex SP C25, Aldrich). Potassium ferricyanide was obtained from Aldrich and used as received. Deionized water was obtained from a Milli-Q Plus water-purification system from Millipore. Stock solutions of sheared herring-testes DNA in 50 mM NaPi at pH 7 were prepared by room-temperature sonication for 90 min and then passed through a 22-gauge syringe needle four times. The average length of the sheared DNA was 700 ( 200 base pairs as confirmed by agarose-gel electrophoresis.21 The concentration of DNA (per nucleotide phosphate) was determined spectrophotometrically by monitoring the absorbance at 260 nm, ) 6600 M-1 cm-1 on a Hewlett-Packard 8452A diode-array spectrometer. Buffer solutions for DNA experiments contained 50 mM sodium phosphate at pH 7 and 0, 0.1, 0.4, or 0.8 M NaCl as indicated in the figure legends. Solutions of 0, 0.25, 0.5, 1.0, 1.5, and 2.0 mM DNA correspond to 0, 62.5, 125, 250, 375, and 500 µM guanine because herring-testes DNA contains 25% guanine.44 DNA solutions with 100 µM Ru(bpy)32+ were stored in the dark at 4 °C overnight before use. The buffer solution for the dopamine/ascorbic acid experiments contained 9.2 mM sodium citrate and 181.6 mM sodium phosphate at pH 7.4. The solution was degassed for 15 min with water-saturated N2 before use.45 Stock solutions (10 mM) of dopamine and ascorbic acid were prepared fresh for each experiment by placing the solid in a septum-capped vial and introducing degassed citrate-phosphate buffer with a gastight syringe. Solutions of 0, 0.25, 0.5, 1, and 1.5 mM ascorbic acid and 100 µM dopamine were prepared by dilution of the stock solutions immediately prior to data collection. Electrochemistry. Cyclic voltammetry, chronocoulometry, and chronoamperometry were performed on a Bioanalytical Systems 100B potentiostat interfaced to a microcomputer. The glass-encapsulated carbon minielectrodes were purchased from Quanteon, Inc., Denver, CO. Removal of the tip of the electrode followed by six cyclic voltammetry scans (25 mV/s, 400-1200 mV) in buffer generated a fresh electrode surface. The Ag/AgCl reference electrode was purchased from Cypress Systems, Inc. A Pt-wire auxiliary electrode was purchased from Aldrich. To determine electrode area, chronoamperometry experiments with
G Oxidation at Carbon Electrodes
J. Phys. Chem. B, Vol. 104, No. 29, 2000 6853
a step time of 250 ms (Estep ) 500 to 0 mV) were performed with 1 mM K3Fe(CN)6 (D ) 7.6 × 10-6 cm2 s-1)46 in 0.1 M KCl in water. With a step time of 250 ms, the diffusioncontrolled current is 80% of the total current (eq 4). The radii of the electrodes were calculated from the slopes of the Anson plots (current vs t-1/2) and gave an average value of 86 ( 4 µm. Hemispherical diffusion was assumed because simulation45 of the steady-state current response for 1 mM Ru(bpy)32+ gave a good fit to the time-independent cyclic voltammogram. Double-step chronocoulometry experiments with step times of 250 ms or 5 s verified that adsorption of mediator or nucleic acid was insignificant. In all cases, solutions of mediator or mediator with nucleic acid gave the same Anson plot y-intercept as buffer alone. For DNA electrochemistry, 30 µL of solution was placed in an air-jacketed glass electrochemical cell. Anaerobic dopamine/ascorbic acid experiments were carried out on 1 mL of solution in an N2-purged septum-capped vial. Cyclic voltammograms at 1 or 2 mV/s are the average of two scans; all other traces are the average of four scans. In all cases, the solution was mixed between each scan. Independent experiments using fresh electrodes were conducted to generate average values and assess errors. Unless otherwise noted, linear fits are leastsquares regressions using Kaleidagraph. Cyclic voltammograms were simulated using the Digisim version 2.1 software package from Bioanalytical Systems. Simulations were carried out as described in the work of Johnston and Thorp,15 except that the electrode geometry was hemispherical and diffusion coefficients of Ru(bpy)32+ and Ru(bpy)33+ in 50 mM NaPi buffer were set to 6 × 10-6 cm2/s.15 The binding constants for Ru(bpy)32+ and Ru(bpy)33+ in 50-mM NaPi buffer were 700 M-1 and 3500 M-1, respectively.15 The numerical values of K2+ and K3+ were varied for simulations, but the K3+/K2+ ratio was kept constant at 5.35 All second-order rate constants are in terms of guanine concentration. Results Previously, electrocatalytic guanine oxidation mediated by Ru(bpy)32+ has been studied only at ITO electrodes with large surface areas (0.32 cm2).15,32,33,43 Under these conditions, the current associated with the catalytic reaction is described by a linear-diffusion model where planar electrode geometry is assumed. Wightman45,47-49 and others50-52 have shown that when the radius of the electrode is small compared to the rate of diffusion of the electroactive species, the current response of Ru(bpy)32+ alone can be described by:
i ) -nFCmedbDmed
[
2π1/2r2 + 2πr (Dmedt)1/2
]
(4)
where n is the number of electrons per mole of mediator, F is Faraday’s constant, Cmedb is the concentration of mediator in the bulk solution in mol cm-3, Dmed is the diffusion coefficient of the mediator in cm2 s-1, r is the radius of the electrode in cm, and t is time in s. An experimentally verifiable consequence of eq 4 is that catalytic currents arising from a solution reaction such as that in eq 2 are decreased at electrodes with small radii.45 To observe catalytic guanine oxidation in solution using commercially available potentiostats that detect 1 nA of current as a lower limit, the appropriate electrode radius is in the range of 50100 µm.49 At this electrode size, the volume of the assay can be decreased significantly, and the response should indicate nonplanar diffusion at slow scan rates. Figure 1 shows that catalytic guanine oxidation by Ru(bpy)32+ is observed at carbon
Figure 1. Cyclic voltammograms at (A) 5 or (B) 250 mV/s of 100 µM Ru(bpy)32+ in 50 mM NaPi (s), pH 7 with 2 mM sheared herringtestes DNA and 0 (‚‚‚‚‚) or 0.8 (- - -) M NaCl.
electrodes with radii of 86 ( 4 µm. Because the conventional definition of a microelectrode is an electrode with a radius of 10 µm or less, we refer to these 50-100 µm radius electrodes as minielectrodes. At slow scan rates, the minielectrode gives a sigmoidal, time-independent voltammogram (Figure 1A), whereas at higher scan rates, linear-diffusion controlled cyclic voltammograms are obtained (Figure 1B). In both cases, the peak current in the presence of DNA is enhanced relative to that of Ru(bpy)32+ alone. Voltammograms collected with 0 or 0.8 M of added NaCl show that the current enhancement from guanine oxidation by Ru(bpy)33+ is more pronounced when no sodium chloride is present, as we have observed at larger electrodes.15,19,28,43 The scan rates at which the time-dependent current contributes to the observed current can be predicted using the parameters F50 or σ51 (eq 5):
σ)
( ) Dmed
2
nf νr
1/2
[ ( )]
F ) nf
r2ν Dmed
1/2
(5)
where ν is the scan rate in V/s and f is F/RT where F is Faraday’s constant, R is the gas constant, and T is temperature. Almost all of the current is expected to be time-dependent when σ is less than 0.151 or F is greater than 17.50 This analysis predicts that the time-dependent currents should predominate at scan rates above 100 or 285 mV/s, respectively. Accordingly, the voltammograms in Figure 1B collected at a scan rate of 250 mV/s reflect the low σ, high F regime of the current
6854 J. Phys. Chem. B, Vol. 104, No. 29, 2000
Figure 2. Oxidative peak current of 100 µM Ru(bpy)32+ in 50 mM NaPi, with 0 (‚) or 0.8 (o) M NaCl, pH 7. (A) Plot of the average oxidative peak current intensity versus the square root of the scan rate. (B) Plot of the ratio of the oxidative peak current to the square root of the scan rate versus the scan rate. Error bars are for one standard deviation and are derived from at least three experiments.
response, whereas those in Figure 1A are in the timeindependent regime, as predicted by eq 5. As shown in Figure 1, increasing the salt concentration lowers the extent of catalytic enhancement, which we have observed at large electrodes and ascribed to a decrease in the catalytic rate constant due to suppression of electrostatic binding. To confirm that the salt effects are not due to differences in the interaction of Ru(bpy)32+ with the electrode surface, the Ru(bpy)33+/2+ couple was investigated in the absence of DNA as a function of salt concentration at the carbon minielectrodes. Figure 2 shows that the oxidative peak current for Ru(bpy)32+ alone in 50 mM sodium phosphate and 0 or 0.8 M NaCl gives results that are the same within error.53 From the peak currents of the voltammograms, we determined the relative contributions of the time-dependent and time-independent currents to the overall current. When the time-dependent current is significant, the quantity i*ν-1/2 should decrease as a function of scan rate.51 Figure 2B shows that in both high- and low-salt buffer, i*ν-1/2 becomes less negative as the scan rate increases from 2 to 20 mV/s and then becomes constant. At the same scan rates where the i*ν-1/2 plot levels off (20 mV/s), the data in Figure 2A deviate from linearity. This deviation from linearity occurs when the time-independent current becomes significant and is characteristic of nonlinear diffusion contributions to the net current,54,55 which is consistent with the sigmoidal voltammograms obtained in Figure 1A at lower scan rates.
Szalai and Thorp
Figure 3. Guanine-concentration dependence of the oxidative peak current from cyclic voltammograms at 5 (×), 50 (+), and 250 (]) mV/s of 100 µM Ru(bpy)32+ in 50 mM NaPi, pH 7 with 0 (A) or 0.8 (B) M NaCl and DNA.
Because we obtain ample electrochemical oxidation of guanine by Ru(bpy)33+ at micromolar concentrations of guanine, a large excess of substrate or reactant relative to the catalyst or mediator is unnecessary. Although this feature is convenient for conserving DNA substrate, our experimental voltammograms cannot be evaluated quantitatively using equations derived for pseudo-first-order catalytic current responses. However, the equations for the pseudo-first-order case can be used to differentiate second-order from pseudo-first-order behavior and to indicate under what conditions electrocatalytic guanine oxidation approaches the classic catalytic case. Under pseudofirst-order conditions, the current from steady-state catalysis is described by54,56
i ) nFACmedb(DmedkcatCsubb)1/2
(6)
where A is the area of the electrode in cm2, Csubb is the solution concentration of substrate in mol/cm3, and kcat is the rate constant of the catalytic reaction (eq 2) in cm3 (mol-s)-1. Thus, the current should vary linearly with the square root of the substrate concentration. Plots of the oxidative peak current for Ru(bpy)33+ oxidation of DNA versus the square root of the guanine concentration are linear at all scan rates (Figure 3). The linear relationship between peak current and substrate concentration is expected because digital simulations of cyclic voltammograms collected at large ITO electrodes give apparent rate constants for guanine oxidation that are independent of guanine concentration.20
G Oxidation at Carbon Electrodes
J. Phys. Chem. B, Vol. 104, No. 29, 2000 6855
Figure 4. Plot of the apparent catalytic-rate constant (eq 6) versus scan rate for 100 µM Ru(bpy)32+ and 2 mM DNA in 50 mM NaPi, pH 7 with 0 (‚), 0.1 ([), 0.4 (2), and 0.8 (1) M NaCl. The lines connecting the points are guides to the eye. Data for the reaction of 100 µM dopamine with 1 mM ascorbic acid (0) in citrate-phosphate buffer are also shown with a linear fit. Note that y-axis is on a logarithmic scale.
Added salt decreases the slopes of the lines in the current versus [guanine]1/2 plots shown in Figure 3. Further, contrary to eq 6, the slopes of the best-fit lines change more dramatically from one scan rate to the next with no added salt (Figure 3A) than with 0.8 M salt (Figure 3B). This finding is consistent with the digital simulations of Sistare et al.,20 which have revealed that the rate constants are scan-rate-dependent. The scan-rate dependence arises from biphasic kinetics of the reaction of Ru(bpy)32+ with DNA due to multiple, non-interconvertible binding geometries of the cation to the DNA polyanion.12,20,31,57-61 Because binding of Ru(bpy)32+ to DNA is more pronounced at low salt concentrations, the oxidative peak current becomes less dependent on scan rate in Figure 3 as the NaCl concentration is increased. To test whether the voltammograms collected with 0.8 M NaCl mimic a pseudo-first-order catalytic reaction, we extracted an apparent catalytic rate constant from the slopes of the current versus substrate1/2 plots for the reaction of Ru(bpy)33+ with DNA. The apparent catalytic rate constant for the experiments with DNA becomes independent of scan rate only when the buffer contains 0.8 M NaCl. The largest apparent catalytic rate constant is 4.5 × 104 M-1 s-1 at 500 µM guanine concentration and 0 M added NaCl. As discussed previously,20 this effect is due to sampling of the fast phase of the catalytic reaction at high scan rates in low salt buffers. To confirm that the scan-rate dependence is due to factors related to the Ru(bpy)33+/DNA reaction, we also studied the reaction of dopamine with ascorbic acid. Dopamine oxidizes ascorbic acid through a two-electron catalytic reaction that has been studied in detail by Dayton et al.45 As expected for a pseudo-first-order catalytic reaction, the catalysis of ascorbic acid oxidation by dopamine yields a scanrate-independent catalytic rate constant (Figure 4, squares). Based on the results shown in Figures 3 and 4 and those presented in the work of Sistare et al.,20 we expect the current for guanine oxidation by Ru(bpy)33+ at minielectrodes to be scan-rate-dependent (Figure 5). In fact, the slopes of the bestfit lines for the low-salt case (Figure 5A) change more rapidly with scan rate than do those for the high-salt case (Figure 5B). Figure 5 highlights the sampling of fast-reaction kinetics at higher scan rates and distinct binding modes of Ru(bpy)33+/2+ to DNA at low salt. The insight we have gained from digital simulation of cyclic voltammograms collected at ITO electrodes led us to attempt
Figure 5. Scan-rate dependence of the oxidative peak current from cyclic voltammograms of 100 µM Ru(bpy)32+ with 0.25 (o), 0.5 ([), 1.0 (4), 1.5 (1), and 2.0 (0) mM DNA in 50 mM NaPi, pH 7 with 0 (A) or 0.8 (B) M NaCl.
to fit the voltammograms collected at minielectrodes under nonlinear (5 mV/s) and linear (250 mV/s) diffusion conditions. Figure 6A shows fits and cyclic voltammograms collected at 5 mV/s in low-salt buffer. The voltammograms for the reaction of Ru(bpy)33+ with DNA were simulated using a mechanism in which Ru(bpy)33+/2+ bound to DNA exchanges with Ru(bpy)33+/2+ free in solution (Scheme 1).15 The binding constants are 700 M-1 and 3500 M-1 for the 2+ and 3+ forms, respectively. The first-order rate constants obtained from fitting the voltammograms are converted to second-order rate constants using the expression in Johnston and Thorp (eq 3).15 Our laboratory has demonstrated previously that voltammograms collected at slow scan rates are best fit with a mechanism containing multiple one-electron oxidation steps, whereas voltammograms collected at fast scan rates are often fit adequately using a single one-electron oxidation step.15,19-21 At the minielectrodes, two oxidation steps are required to fit the data collected at 5 mV/s, and the second-order rate constants are (5.6 ( 1.4) × 104 M-1 s-1 for the first oxidation step and (2.3 ( 0.4) M-1 s-1 for the second oxidation step. Voltammograms collected at 250 mV/s at minielectrodes are similar to those collected at ITO electrodes15,19-21,43 because both are collected under linear-diffusion conditions. Accordingly, rate constants from fits to the voltammograms collected at 250 mV/s are weighted toward the fast component of the reaction of Ru(bpy)32+ with DNA, as discussed in Sistare et al.20 The voltammograms collected at 250 mV/s at minielectrodes can be
6856 J. Phys. Chem. B, Vol. 104, No. 29, 2000
Figure 6. Simulations of cyclic voltammograms collected at carbon minielectrodes. (A) Experimental cyclic voltammograms (solid lines) and simulations (dotted lines) at 5 mV/s with 100 µM Ru(bpy)32+ and 2 mM DNA in 50 mM NaPi buffer, pH 7. An 86 µm radius hemispherical electrode geometry and the binding mechanism in Scheme 115 were used to fit the data. (B) Measured peak currents of simulations for the oxidation of 2 mM DNA by 100 µM Ru(bpy)32+. The scan rate was 5 mV/s and the electrode geometry was hemispherical with a radius of 86 µm. The solid symbols are for the binding case in which the values of K3+ and K2+ were varied; the K3+/K2+ ratio was 5 in all simulations.35 The triangle is the simulated peak current for the second-order reaction between Ru(bpy)33+ and DNA in which binding is excluded from the mechanism. The square is the simulated current of Ru(bpy)32+ alone. Note that the simulated peak currents are in good agreement with the peak currents for the data from Figures 1A and 6A.
fit using a single oxidation step with a rate constant of (8.8 ( 2.2) × 104 M-1 s-1. The rate constants for the first oxidation step at 5 mV/s or 250 mV/s are similar to the maximum rate constant from Figure 4, revealing that the peak currents of voltammograms collected at carbon minielectrodes are less scanrate-dependent than those collected at ITO electrodes for the
Szalai and Thorp reaction of Ru(bpy)33+ with DNA.20 The conditions for fitting and the rate constants obtained are summarized in Table 1. One of the potential advantages of simulating the voltammograms collected at minielectrodes is that two scenarios for the oxidation of guanines in DNA at low salt can be differentiated. The first possibility is that the majority of the current arises from oxidation of free Ru(bpy)32+ at the electrode that then collides with DNA to effect the catalytic reaction. In this scenario, lowering the salt concentration increases the rate constant for the reaction of free Ru(bpy)33+ with DNA by increasing the binding affinity. The second case is that bound Ru(bpy)32+ is oxidized directly at the electrode surface, and electron transfer from guanine to pre-bound Ru(bpy)33+ results in efficient turnover. In this case, lowering the salt concentration increases the active Ru(bpy)32+‚DNA fraction. These situations are differentiated by collecting and simulating voltammograms at slow scan rates at minielectrodes because, when timedependent contributions to the observed current are minimal, the current depends linearly on the diffusion coefficient of the electroactive species.35,62 The change in diffusion coefficient for the two cases is a factor of 30 and can be simulated by changing the association constants of Ru(bpy)33+/2+ from the nonbinding to the binding regime. Using the binding mechanism for guanine oxidation and the results of Figure 6A, the peak currents can be obtained from digital simulations in which K3+, the association constant of Ru(bpy)33+, is varied from 0.35 M-1 to 1 × 109 M-1 (Figure 6B). The value of K2+ is set to be smaller by a factor of 5 in all cases, as shown previously from polyelectrolyte theory.35 The peak current reaches a maximum when K3+ is 500 M-1 and decreases markedly at higher or lower values of K3+, confirming that if direct electrode oxidation of bound Ru(bpy)32+ were a primary determinant of catalytic efficiency, the current would be smaller than that which we observe in the large K3+ regime of Figure 6B. Although the peak currents plotted in Figure 6B were generated from the fits to the voltammograms in Figure 6A, the peak current (12 nA) obtained from simulation of the nonbinding mechanism is in good agreement with the peak current of the high-salt voltammogram shown in Figure 1A. Discussion Properties of the Minielectrodes. Although guanine in adsorbed DNA has been detected using anodic-stripping voltammetry at mercury electrodes,63 catalytic oxidation of guanines in native DNA in solution has been limited to ITO electrodes with large electroactive areas.15,32,33,43 One of the benefits of carbon electrodes is that the accessible potential range for electrochemical measurements is comparable to that for ITO electrodes.53,62 The active surface areas of the carbon minielectrodes used here are more than 600 times smaller than the ITO electrodes previously used to detect DNA in solution.15,19,21,32,33,43 The 86 µm-radius electrode was critical in detecting mediated guanine oxidation because smaller electrode radii would preclude observation of catalytic currents.45 The carbon minielectrodes behave like true microelectrodes only at scan rates equal
TABLE 1: Rate Constants for Guanine Oxidation by Ru(bpy)33+ at Carbon Minielectrodes
a
[NaCl] mM
ν (mV/s)
k2 (M-1 s-1)
k2′ (M-1 s-1)
K3+ (M-1)
K2+ (M-1)
kappa (M-1 s-1)
0 0 800 800
5 250 5 250
(5.6 ( 1.4) × 104 (8.8 ( 2.2) × 104 2.0 × 104 2.1 × 104
(2.3 ( 0.4) × 103 c
3500b 3500
700b 700
2.9 × 103 4.5 × 104 1.1 × 103 4.1 × 103
Calculated from data in Figure 4. b From ref 15. c Not required for fitting.
G Oxidation at Carbon Electrodes to or below 2 mV/s. It is instructive to calculate the fraction of current that arises from the time-independent term in eq 4. For Ru(bpy)32+ alone, if the total experiment time is 100 s, the timeindependent portion of the current contributes 83% of the total current, whereas at a total experiment time of 1 s, the timeindependent contribution decreases to 30%. At the scan rate used to collect the electrochemical traces in Figure 1B, each trace is complete in about 30 s and contains significant contributions from both the time-independent and the timedependent terms in eq 4. Note that under conditions used for typical electrochemical measurements, eq 4 cannot be simplified to neglect the time-dependent term unless the electrode size is reduced significantly. Scan-Rate Dependence. Ideally, when the catalysis is fast or the substrate concentration is high, the total current, including the catalytic reaction at a microelectrode, is a function of the time-dependent and time-independent currents for Ru(bpy)32+ itself and the current generated by the steady-state catalytic reaction. At slow scan rates at small electrodes, the timedependent current can be ignored, and the total current is proportional to the radius of the electrode and the steady-state catalytic current. Straightforward evaluation of these contributions gives the catalytic rate constant for a reaction carried out under pseudo-first-order conditions. The apparent rate constant kapp should be scan-rate-independent, but for mediated guanine oxidation at minielectrodes it is not (Figure 4), demonstrating that pseudo-first-order kinetics do not apply. The most noticeable trend is that the reaction at low ionic strength is highly scanrate-dependent and, therefore, not described accurately by conventional electrochemical equations for an ECE mechanism. Recent studies in our laboratory have shown that the scan-rate dependence of kapp arises from biphasic reaction kinetics for the Ru(bpy)33+/guanine reaction, which is more pronounced at low ionic strength.20,28 Second-order rate constants obtained from digital simulation of cyclic voltammograms and direct analysis of chronoamperomograms show that two kinetic regimes exist.20 At slow scan rates in cyclic voltammetry experiments, the apparent catalytic rate constant approaches that of the slow phase of the reaction and vice versa for voltammograms collected at fast scan rates at ITO electrodes.20 When cyclic voltammograms are collected at carbon minielectrodes at 5 mV/s and at low salt concentration, the rate constant for the slower second oxidation step extracted from the digital simulations is within error of the 3 × 103 M-1 s-1 kapp obtained from Figure 4 at the same scan rate, representing the slow phase of the kinetics for the reaction of Ru(bpy)33+ with guanines in DNA. The maximum kapp we obtain in this work occurs at the highest scan rates, in accord with our previous results.15,20,21 The rate constants derived from digital simulation of cyclic voltammograms collected at ITO or carbon minielectrodes differ most in their dependence on salt concentration and scan rate. At high salt, the rate constants are similar to one another for both electrodes, and are all in the range of 103 (5 and 25 mV/s) to 104 (250 mV/s) M-1 s-1. However, at low ionic strength, the rate constant for guanine oxidation at ITO is 3 × 105 M-1 s-1 at 25 mV/s and 1 × 106 M-1 s-1 at 250 mV/s,20 whereas at carbon minielectrodes the rate constants are 5.6 × 104 M-1 s-1 (5 mV/s) and 8.8 × 104 M-1 s-1 (250 mV/s). The biphasic nature of guanine oxidation by Ru(bpy)33+ is more evident in cyclic voltammograms collected at ITO electrodes than at the carbon minelectrodes. At carbon electrodes, the switch from low to high salt has a much smaller effect on the rate constant for the initial guanine oxidation step, as evidenced by the small
J. Phys. Chem. B, Vol. 104, No. 29, 2000 6857 decrease in rate constant from 8.8 × 104 M-1 s-1 to 2.1 × 104 M-1 s-1 (250 mV/s). Role of the Electrode Surface. Some of the disparity between the rate constants for high- and low-salt conditions derives from the differences between the surfaces of the ITO and carbon electrodes. The heterogeneous electron-transfer rate constant is typically lower for carbon than for ITO, which limits the rate at which electrons are transferred to and from the Ru(bpy)32+ mediator. Like ITO, oxidized carbon electrode surfaces contain numerous functional groups (i.e., hydroxyl or carboxylate64) that cause the current from the electroactive species to depend on the ionic strength of the solution. The current for Ru(bpy)33+/2+ at the glass-encapsulated carbon electrodes used for this work did not change noticeably when the ionic strength was increased dramatically. Significant salt effects are observed at ITO for Ru(bpy)33+/2+ alone, because an increase in the ionic strength of the buffer solution screens the Ru(bpy)32+ cation from the negatively charged ITO surface.28,65 Further, the rate constants for guanine oxidation at ITO differ dramatically from low-salt conditions (105-106 M-1 s-1) to high-salt conditions (103-104 M-1 s-1), suggesting that the ITO surface at low-salt concentrations accelerates the oxidation of Ru(bpy)32+. In contrast, the salt effects observed in the presence of DNA at the minielectrodes must be due solely to a change in the mediator-DNA interaction and do not include a change in the mediator-electrode surface interaction because the rate constants for DNA oxidation by Ru(bpy)33+/2+ at low and high ionic strength do not change significantly. Contributions of Bound and Free Ru(bpy)32+. Because the carbon surface is insensitive to changes in the bulk salt concentration, and because both time-dependent and steady-state regimes are accessible, minielectrodes offer the opportunity to investigate the steady-state dynamics of guanine oxidation in DNA by Ru(bpy)33+. At slow scan rates, only the time-independent part of eq 4 contributes to the observed current, so experiments at minielectrodes and slow scan rates distinguish whether the observed current enhancement obtained in buffers containing low concentrations of NaCl arises from Ru(bpy)33+ exchanging with Ru(bpy)32+ near the DNA, or from bound Ru(bpy)32+ being oxidized at the electrode surface. In the expression for the total current (eq 4), Dmedb should be replaced by Deff, an effective diffusion coefficient containing contributions from the diffusion coefficients of both the free and bound forms of the mediator. When the metal complex is in fast exchange between bound and free forms, Deff is the sum of the diffusion coefficients of the free and bound forms scaled by the appropriate mole fractions.35,62 The diffusion coefficient of free Ru(bpy)32+ is 6 × 10-6 cm2 s-1;15 the diffusion coefficient of the bound Ru(bpy)32+ can be approximated as the diffusion coefficient of DNA.62,66,67 The diffusion coefficient of sheared herring-testes DNA cannot be calculated using a theoretical treatment because the size of the DNA exceeds 200 bp;68-70 however, normal-pulse voltammetry of sheared herring-testes DNA with an osmium intercalator at an ITO electrode35,62 gave an apparent diffusion coefficient for DNA of 2 × 10-7 cm2 s-1 (results not shown). If oxidation at the electrode surface of bound Ru(bpy)32+ constitutes the major contribution to the current, the effective diffusion coefficient should decrease and the total current also should decrease. In fact, for solutions containing DNA and Ru(bpy)32+, the total current observed when cyclic voltammograms are collected at slow scan rates is higher than that for Ru(bpy)32+ alone (Figure 1A). When we model the decrease in diffusion coefficient as an increase in the binding constant of Ru(bpy)33+/2+, we find that the experimentally observed
6858 J. Phys. Chem. B, Vol. 104, No. 29, 2000 current enhancement cannot arise exclusively from oxidation of bound Ru(bpy)32+ at the electrode surface. Therefore, the current enhancement at low ionic strength derives largely from oxidation of free Ru(bpy)32+ at the electrode and association and reaction of Ru(bpy)33+ with DNA, followed by dissociation and migration of Ru(bpy)32+ to the electrode surface to repeat the catalytic cycle as outlined in Scheme 1. Thus, the primary effect of the electrostatic association of the catalyst with the DNA is acceleration of the reaction of free, electrogenerated Ru(bpy)33+ with the guanine reaction site and not preassociation of Ru(bpy)32+ with DNA. In fact, the latter reaction actually depresses the catalytic effect, as shown in Figure 6B. Control of reaction rates by electrostatic binding equilibria has been invoked to explain the rates of reaction between Pt(II) complexes and oligonucleotides.41,42,71 Elmroth and Lippard found that the rates of reactivity between a cationic platinum complex and DNA decrease as the salt concentration is increased, which is similar to what we report here. They ascribe the decreased reaction yield at high ionic strength to disruption of a preassociation pathway that normally allows directed diffusion of the reactant cationic platinum complex along the DNA strand. One conclusion drawn from including directed diffusion in a mechanism describing cation/DNA reactions is that as the length of the DNA strand increases, reactivity should also increase. The simple case where an increase in the length of the DNA substrate correlates with increased yield does not hold in our case because tunneling has been implicated in guanine oxidation in double-stranded DNA, which increases the size of the critical reaction site.5-7,9,11,14,15,17,19-21,25,27,29,30,34,43 Further, because our system involves a redox change of the catalyst, the catalytic efficiency is determined by the differential affinities of Ru(bpy)33+ and Ru(bpy)32+ and not the simple association of a single redox state. Suitability of Ru(bpy)32+. The combined data point to an optimum catalytic scenario where significant concentrations of the free form of Ru(bpy)32+ are available for oxidation by the electrode but where there is sufficient affinity of Ru(bpy)33+ to drive the homogeneous reaction. Our group has spent considerable effort showing that Ru(bpy)32+ is one of the few complexes whose affinities in both the 3+ and 2+ forms are driven entirely by electrostatics.35 Thus, the binding constants for the 2+ and 3+ forms of 700 M-1 and 3500 M-1 and the ratio of binding affinities of K3+/K2+ are close to the ideal values predicted by polyelectrolyte theory,72,73 with no additional contributions from nonelectrostatic modes of interaction such as hydrophobic effects or intercalation. As shown here, this scenario is nearly optimized for the DNA oxidation catalysis. The higher affinity of the 3+ form compared to the 2+ form allows for significant concentrations of the free 2+ form under conditions where the 3+ form is attracted strongly to the DNA. The reduced affinity of the 2+ form subsequently leads to efficient product release, which is vital for efficient catalysis.74 The plot in Figure 6B shows that the highest current would be obtained for the case where the affinity of the 3+ form was 500 M-1; however, this affinity is below that for the purely electrostatic case for a 3+ binder. Thus, there is no real room for improvement in Ru(bpy)32+ in the absolute affinity. More hydrophobic complexes such as Ru(phen)32+ actually show higher affinities for the 2+ form than for the 3+ form,35,66,67 because the hydrophobic interactions are more pronounced for the lower-valent forms. Thus, these complexes should be poorer electrocatalysts, because the 2+ forms will bind more strongly than the 3+ forms, leading to product inhibition and poor availability of the free catalyst to the electrode.75
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