Distribution of Metal Complexes Bound to DNA Determined by Normal

J. Phys. Chem. 1996, 100, 13829-13836

13829

Distribution of Metal Complexes Bound to DNA Determined by Normal Pulse Voltammetry Thomas W. Welch† and H. Holden Thorp* Department of Chemistry, UniVersity of North Carolina, Chapel Hill, North Carolina 27599-3290 ReceiVed: January 25, 1996X

The effects of DNA binding on the normal pulse voltammetry of metal complexes have been investigated. Studies were performed both for oxidation of OsL32+/3+ and for reduction of CoL33+/2+ (L is bpy ) 2,2′bipyridine or phen ) 1,10-phenanthroline). The diffusive current obtained from voltammograms at potentials well past E1/2 gives an accurate measure of the extent to which the complexes codiffuse with DNA or are free in solution, and this response is not affected by kinetic factors resulting from slow heterogeneous electron transfer. Analysis of the diffusion-limited current using the appropriate binding isotherm provides binding constants in good agreement with those measured by other methods. For the bpy complexes, the ionic strength dependence, the relative binding constants for the 2+ and 3+ forms, and the associated change in E1/2 upon DNA binding are in good agreement with the predictions of polyelectrolyte theory where the 3+ ion binds more strongly. For the phen complexes, the reverse trend is observed and is consistent among the absolute binding constants, ionic strength dependence, and E1/2 shift; this behavior is ascribed to a hydrophobic interaction. The technique is also applied to two-electron couples based on [(tpy)(L)RuOH2]2+/[(tpy)(L)RuO]2+ that exhibit slow heterogeneous electron transfer; however, these kinetic complications do not prohibit accurate determination of the binding energetics using normal pulse voltammetry. Taken together, the data provide a comprehensive picture of the effects of partial DNA binding on voltammetry, which provides a basis for determining homogeneous kinetic rate constants for electrocatalytic DNA oxidation from voltammograms.

The binding of proteins, enzymes, and metal ions to nucleic acids is a field of intense study because these binding reactions regulate gene expression, initiate strand cleavage and linkage reactions, and determine nucleic acid secondary and tertiary structure.1-5 A ligand may bind via one or several modes ranging in strength from covalent attachment6-10 to weaker electrostatic attraction.11-14 Strong binding affinity may also result through intercalation of aromatic moieties, hydrogen bonding, and mutual association of hydrophobic regions of the ligand and nucleic acid strand.15-21 Complete understanding of such binding reactions requires an accurate method of determining binding equilibrium constants and the decomposition of this constant into electrostatic and nonelectrostatic contributions. The sugar-phosphate backbone of a nucleic acid strand is completely ionized at physiological pH and thereby comprises a polyanion whose linear charge density varies with secondary structure. Linear duplexes can be considered rigid over at least 200 base pairs,22 and binding of small cations can therefore be treated according to polyelectrolyte theory.4,11,13,23,24 Polyelectrolyte theory describes the energetics of attraction between linear polyions and small counterions.4,11 The requirement of counterion condensation on a linear polyion as a function of linear charge density has been addressed by Manning.11 The theory can be derived from the equilibrium expression for the general case of a charged ligand binding to a polyanion:

B + DNA h B‚DNA + ∆rM+

(1)

where M+ is the buffer cation and ∆r is the number of counterions released upon binding of the cationic B. The † Present address: Beckman Institute, California Institute of Technology 139-74, Pasadena, CA 91125. X Abstract published in AdVance ACS Abstracts, June 1, 1996.

S0022-3654(96)00251-1 CCC: $12.00

contributions to the equilibrium constant for the reaction in eq 1 have been summarized by Record et al. in the following expression:4

ln Kobs ) ln K°t + Zξ-1(ln(γ(δ)) - ZΨ(ln([M+])) (2) where Kobs is the observed binding constant, Z is the charge on the binding ligand K°t is the thermodynamic contribution due to nonelectrostatic forces, γ( is the mean activity coefficient at cation concentration, ξ and δ equal 4.2 and 0.56, respectively, for B-form DNA, and Ψ ) 0.88 for B-form DNA.4 The second term is negligible with respect to the contributions from thermodynamic and electrostatic terms for DNA, so the expression can be shortened to:

ln Kobs ∼ ln K°t - ZΨ(ln([M+]))

(3)

Linear regression of ln Kobs as a function of natural logarithm of the excess counterion concentration gives a slope equal to -ZΨ. The contribution to Kobs from effects such as intercalation are calculated from the intercept, ln K°t. The intercept for a ligand binding solely through electrostatics will be zero, i.e., K°t ) 1. Classical biochemical techniques for determining binding constants of transition metal complexes are severely limited in the case of very low (K < 1000 M-1) and very high (K > 105 M-1) affinites. High binding constants are difficult to measure via equilibrium dialysis because of the difficulty in maintaining accurate concentrations of nucleic acid in the presaturation concentration regime coupled with the low absorbance of free complexes once saturation obtains.12,25,26 Equilibrium dialysis gives reliable results for weak binders given the collection of enough data points.27 Absorption titration requires small amounts of nucleic acid but relies on large differences in the near-UV visible spectra of free and bound species;25,28,29 such © 1996 American Chemical Society

13830 J. Phys. Chem., Vol. 100, No. 32, 1996 differences are typically only seen with organic chromophores that exhibit high molar absorptivities. An alternative method devised by Kalsbeck and Thorp uses the different rate constants exhibited by free and bound metal complexes in quenching the excited state of Pt2(pop)44- (pop ) P2O5H22-).13 Although this technique provides accurate values of K > ∼600 M-1, it is the most labor intensive technique and requires the largest amounts of nucleic acid. Research on transition metal complexes that are electrocatalytic DNA cleavage agents indicates that selectivity occurs through a combination of the strength and specificity of the binding reaction described in eq 1 influenced by the subsequent oxidation kinetics.30-32 The electropotentiated forms of these complexes can be inner-sphere oxidants, such as [(tpy)(L)RuO]2+ where L is bpy ) 2,2′-bipyridine, phen ) 1,10phenanthroline, or dppz ) dipyridophenazine.30-32 Outer-sphere oxidants, such as RuL33+, damage DNA by one-electron oxidation of guanine.33 While both free and bound forms of these complexes are electrochemically active, the current from bound complexes is limited by the diffusion of the biopolymer.14,32 Valid kinetic analysis of the electrocatalytic current enhancement seen in voltammetry of these systems requires accurate knowledge of the diffusion of bound species as well as the fraction of complexes bound at specific DNA and buffer concentrations.29,34 We have recently shown that catalytic rate constants determined from cyclic voltammograms can be used to detect single base mismatches in DNA oligomers.33 We report here a strategy for the electrochemical determination of the DNA binding constants of electroactive complexes of osmium, cobalt, and ruthenium. The technique quantitates the concentrations free and bound during DNA titrations based on the change in mass-transfer-limited current and the current expected upon saturation of binding. The order of magnitude difference in free and bound diffusion coefficients allows determination of binding constants from 100 to 105 M-1. The validity of the technique is demonstrated by a study of the electrostatic binders Os(bpy)32+ and Co(bpy)33+. Nonelectrostatic affinities for complexes containing phenanthroline ligands are consistent with those determined by other techniques13,23,27,35,36 and implicate a hydrophobic component of the binding affinity. Finally the binding of Co(bpy)33+ to the synthetic homoduplexes poly[dA]‚poly[dT] and poly[rA]‚poly[dT] shows that the electrostatic properties of both A- and B-form DNA are identical for relatively large cations. Experimental Section Materials. Calf thymus DNA was purchased from Sigma and used according to published procedures.37 The synthetic homopolymers poly[dA]‚poly[dT] and poly[rA]‚poly[dT] were purchased from Pharmacia. Water was purified with a MilliQ purification system. Ligands and metal salts were purchased from Aldrich. Published procedures were used to prepare the metal complexes [Os(bpy)3](PF6)2, [Os(phen)3](PF6)2,38 [Co(bpy)3](ClO4)3, [Co(phen)3](ClO4)3,39 [(tpy)(bpy)RuOH2](ClO4)2, and [(tpy)(phen)RuOH2](ClO4)2.40 Methods and Instrumentation. All solution concentrations were determined spectrophotometrically using a Hewlett-Packard HP 8452 diode array spectrophotometer. The extinction coefficients used were 490 ) 12 900 M-1 cm-1 for [Os(bpy)3]2+, 476 ) 19 000 M-1 cm-1 for [Os(phen)3]2+, 318 ) 31 000 M-1 cm-1 for [Co(bpy)3]3+, 303 ) 19 200 M-1 cm-1 for [Co(phen)3]3+, 476 ) 9600 M-1 cm-1 for [(tpy)(bpy)RuOH2]2+, 474 ) 9600 M-1 cm-1 for [(tpy)(phen)RuOH2]2+, 257 ) 8600 M-1 cm-1 for poly[dA]‚poly[dT] and poly[rA]‚poly[dT], and 260 ) 6600 M-1 cm-1 for calf thymus DNA where concentration of nucleic acids is measured in nucleotide phosphate.

Welch and Thorp

Figure 1. Normal pulse voltammograms of 100 µM Os(bpy)32+ obtained during titration with calf thymus DNA in 500 µM increments. Inset: plot of slope vs tp-1/2 from COOL algorithm fits of voltammograms in the presence of 0 (9) and 3.5 mM (2) DNA.

Electrochemical measurements were performed in a cell that has been described previously.41 The working electrode was tin-doped indium oxide obtained from the Donnelly Corp. The electrode was cleaned by sequential 10 min sonications in Alkonox, 95% ethanol, water twice, and finally the desired buffer. The potential was applied using a platinum wire auxiliary electrode and a Cypress Systems Ag/AgCl electrode as a reference. Voltammograms were collected using an EG&G Princeton Applied Research 273A potentiostat and analyzed using the COOL Algorithm software package. Electrochemical titrations of metal complexes with DNA were performed by first preparing two solutions containing 100 µM metal complex with one solution also containing 6.0 mM nucleic acid. Voltammograms were collected on pure buffer and the metal complex in the absence of nucleic acid. The solution containing nucleic acid was added in aliquots to raise the concentration of nucleic acid in 0.5 mM increments with a voltammogram collected after each addition. Results Os(bpy)32+ and Co(bpy)33+. The normal pulse voltammograms obtained during the titration of 100 µM Os(bpy)32+ with calf thymus DNA are shown in Figure 1. The potential was applied in pulses of 100 ms duration at a frequency of 1 pulse s-1 and raised sequentially by 5 mV. As the potential reaches the E1/2 of the Os(III/II) couple, the voltammetric current exhibits a sigmoidal transition to a potential-independent plateau. The current response prior to the inflection point is identical with that observed in cyclic staircase voltammetry and illustrates the potential-dependent rate of the oxidation under kinetic control. The current in the plateau region reflects the mass-transfer limited rate of the oxidation and can be calculated from theory via the Cottrell equation:

i ) nFACt(D/πtp)1/2 where i is current in amperes, n is the number of electrons in the redox couple, A is electrode area in cm2, Ct is concentration in mol cm-3, D is the diffusion coefficient in cm2 s-1, and tp is the pulse duration in seconds. Adherence to the Cottrell equation is demonstrated by the linearity of the plot of limiting current vs tp-1/2. The decrease in magnitude of the limiting current observed during the DNA titration reflects a decrease in the effective diffusion coefficient as the complex binds to DNA.

Effects of DNA Binding on NPV of Metal Complexes

J. Phys. Chem., Vol. 100, No. 32, 1996 13831

Figure 2. Plots of i2 - io2 during titration of Os(bpy)32+ with calf thymus DNA in the presence of 15 mM (A) and 75 mM (B) excess Na+ and nonlinear least-squares fits to eq 6 using the territorial binding model (eq 7).

The effective diffusion coefficient of a complex rapidly exchanging between free and bound states is the sum of the diffusion coefficients of the free and bound complexes weighted by their respective mole fractions:

Deff ) DbXb + DfXf

(4)

Electron transfer between free and bound complexes can influence the apparent diffusion coefficient;42 however, we have shown previously that these effects are not important for metal complexes in fast exchange with DNA.29 Substitution of 1 Xb for Xf and rearrangement yields an expression for the mole fraction of complex bound:

(Deff - Df)/(Db - Df) ) Xb

(5)

Because of the direct proportionality of current to D1/2, eq 5 can be rewritten in terms of voltammetric current:

i2 - io2 ) (isat2 - io2)Xb

(6)

where io2 is the current observed prior to the addition of nucleic acid and isat2 is the current expected upon complete saturation of binding. The current function i2 - io2 will therefore approach isat2 - io2 as Xb approaches unity. The value for isat can be calculated based on diffusion coefficients obtained for nucleic acid fragments via direct measurements29,43,44 or theoretical calculation.45,46 The appropriate expression of Xb for Os(bpy)33+/2+ is that for the weak, territorial binding model:

Xb ) K[DNA]/(K[DNA] + 1)

(7)

This model is valid only when the binding equilibrium in eq 5 lies far enough to the left that binding of individual complexes does not significantly decrease the free nucleotide concentration. Substitution of this expression for Xb in eq 6 yields a function that can be fit to a plot of i2 - io2 versus DNA concentration via optimization of K. Figure 2 shows values of i2 - io2 collected during DNA titrations of Os(bpy)32+ in the presence of 15 and 75 mM excess sodium ion. The notable difference in the binding curves is the extent to which the values of i2 - io2 approach that value

Figure 3. Plots of ln(K) for Os(bpy)32+ (2) and Co(bpy)33+ (9) vs ln([Na+]).

expected for isat2 - io2 (-1.62 × 10-10 A2). At 4.0 mM DNA, Os(bpy)32+ is ∼69% bound in 15 mM excess sodium and ∼12% bound in 75 mM excess sodium as determined from eq 6. Shown superimposed are nonlinear least-squares fits to eq 6 using the weak binding isotherm of eq 7 and the value of isat2 - io2 calculated from previous studies. The binding constants obtained are 950 and 98 M-1 in the presence of 15 and 75 mM excess sodium, respectively. Alternatively, a two-parameter fit can be used to optimize both K and isat2. This approach gives binding constants of 650 and 72 M-1 and isat2 values that give Db ) 4.5 × 10-7 and 9.0 × 10-7 cm2 s-1. Although the diffusion coefficients are somewhat higher than the known value of 2.1 × 10-7 cm2 s-1, the binding constants are in reasonable agreement with those from the fit where isat2 is determined by the known diffusion coefficient. The technique is therefore robust in the ability to determine consistent binding constants without data in the regime of high levels of binding, which cannot be obtained for these weak binders. Similar experiments for Co(bpy)33+ under identical conditions gave binding constants of 5800 and 260 M-1 in 15 and 75 mM excess sodium, respectively, using a stipulated value for isat2. These values reflect the higher binding affinity of the tricationic cobalt complex compared to that of Os(bpy)32+. Binding constants of Os(bpy)32+ and Co(bpy)33+ were also determined in the presence of 7.5 and 30 mM excess sodium to determine whether the relationship between ln(Kobs) and ln([Na+]) is linear as predicted by eq 3. Plots of ln(Kobs) vs ln([Na+]) are presented in Figure 3 along with linear least-squares regression. The regression parameters for Os(bpy)32+ correspond to ZΨ ) 1.78 and ln(K°t) ) -0.20. The parameters for Co(bpy)33+ are ZΨ ) 2.46 and ln(K°t) ) -0.47. The corresponding values of K°t are close to unity, as expected for purely electrostatic binding, and are similar, as expected for isostructural complexes. The values for ZΨ are in agreement with the accepted values of 1.76 and 2.64 based on Ψ ) 0.88.4 Given the agreement of these parameters with those predicted solely by the linear distribution of charge on the DNA strand, normal pulse voltammetric titrations provide an accurate means of measuring the extent of binding of an electroactive metal complex. Simple inspection of the plots of ln(Kobs) vs ln([Na+]) suggests that ln(K3+), where K3+ represents the binding constant of the trication, is approximately 1.5 ln(K2+) over the entire range of excess sodium concentration. The ratio KOs(II)/KOs(III) can be

13832 J. Phys. Chem., Vol. 100, No. 32, 1996

Welch and Thorp

A

Figure 5. Plot of Xb obtained during titration of Os(phen)32+ with calf thymus DNA in the presence of 15 mM excess Na+ and nonlinear leastsquares fits to eq 6 using the site-binding model (eq 9).

B

Figure 4. (A) Plots of E1/2 determined by cyclic voltammetry vs R for Os(bpy)32+ in the presence of 15 mM ([) and 75 mM (2) excess Na+. (B) Plots of E1/2 vs R for Os(phen)32+ in the presence of 75 mM excess Na+.

predicted by the shift in the E1/2 of the Os(II/III) couple in the binding equilibrium outlined in Scheme 1.

The Nernst equation predicts the following shifts in the formal potentials of free and bound complexes:

Eb° - Ef° ) 0.059 log(KOs(II)/KOs(III))

the trication versus the dication that is as expected based on polyelectrolyte theory and the observed binding constants of Os(bpy)32+ and Co(bpy)33+. Os(phen)32+ and Co(phen)33+. Normal pulse voltammograms of the tris-phen complexes closely resemble those of trisbpy analogues but exhibit more marked decreases in limiting currents as similar amounts of DNA are added. Fitting of plots of i2 - io2 for Os(phen)32+ and Co(phen)33+ in 75 mM excess Na+ yields binding constants of 2400 and 1200 M-1 for Os(phen)32+ and Co(phen)33+, respectively. Immediately noteworthy is that the trication binds less strongly than the dication, indicating a greater K°t for the dication. If eq 3 is solved using the known values of ZΨ from the bpy complexes, the values of K°t are 26 and 2 M-1 for Os(phen)32+ and Co(phen)33+, respectively. The stronger binding of the dication is supported by the shift in the E1/2 of Os(phen)32+ from 652 to 661 mV upon addition of DNA, as shown in Figure 4B. The 9 mV difference predicts a ratio K2+/K3+ of 0.7 from eq 8, in reasonable agreement with the observed ratio of 0.52. In the presence of 15 mM excess Na+, the magnitude of limiting current during DNA titrations of Os(phen)32+ falls more drastically to values that are consistent with saturation of binding. The measured diffusion coefficient of the bound species is 2.2 × 10-7 cm2 s-1, which is within experimental error of the accepted value for DNA.43 The plot of Xb in Figure 5 calculated via rearrangement of eq 6 indicates that saturation is achieved upon addition of ∼10 stoichiometric equivalents of nucleotide phosphate, and the territorial binding model is therefore invalid in these circumstances. The values of Xb vs [DNA] can be fit to the equation describing binding of highaffinity complexes to DNA assuming noncooperative binding to a discrete site:

(8)

The E1/2 of the Os(III/II) couple determined by cyclic voltammetry is plotted versus DNA concentration in Figure 4A. The ratios in 4.0 mM DNA calculated from the shifts of 13 and 35 mV in 75 and 15 mM Na+ are 1.7 and 3.9, respectively. The values extrapolated to saturation of binding are 4.2 and 10.9, respectively. Thus the shift in E1/2, like the effective diffusion coefficient, exhibits a smooth transition between limiting values for free and bound complexes and a difference in binding for

(

b - b2 Xb )

)

2K2Ct[DNA] s 2KCt

b ) 1 + KCt + K[DNA]/2s

1/2

(9a) (9b)

where K is the binding constant, Ct is total metal complex concentration, [DNA] is concentration of nucleotide phosphate and s is the site size in base pairs. The optimized values of K

Effects of DNA Binding on NPV of Metal Complexes

J. Phys. Chem., Vol. 100, No. 32, 1996 13833

A

Figure 6. Normal pulse voltammograms of 100 µM [(tpy)(bpy)RuOH2]2+ obtained during titration with calf thymus DNA in 500 µM increments.

and s for Os(phen)32+ at 15 mM are 200 000 M-1 and 4 base pairs, respectively (Figure 5). Although binding saturation is less distinct in the titration of Co(phen)33+, the highest quality fit to eq 9 gives K ) 74 000 M-1 and s ) 4.6 base pairs. Site sizes of ∼4 bp are typical for intercalators containing phen ligands.12,13,28 Division of Kobs by exp(-ZΨ ln([Na+])) yields values of K°t equal to 206 and 3 M-1 for Os(phen)32+ and Co(phen)33+, respectively. [(tpy)(L)RuOH2]2+. Diffusion-limited currents can be measured for the two-electron, two-proton oxidation of aquaruthenium(II) complexes to the corresponding oxoruthenium(IV) complexes. These complexes offer an excellent opportunity to evaluate the sensitivity of our approach to heterogeneous kinetic complications because oxidation to the oxoruthenium(IV) form is slow.47 Normal pulse voltammograms obtained during the titration of [(tpy)(bpy)RuOH2]2+ with calf thymus DNA are presented in Figure 6. The faradaic current response as a function of potential falls into three distinct regimes. The response between the onset of faradaic current and ∼0.55 V corresponds to the first redox couple (eq 10) under kinetic control:

[(tpy)(bpy)RuOH2]2+ f [(tpy)(bpy)RuOH]2+ + H+ + e(10) The response then shallows, consistent with contributions from both the first couple under diffusion control and the second couple (eq 11) under kinetic control:

[(tpy)(bpy)RuOH]2+ f [(tpy)(bpy)RuO]2+ + H+ + e-

(11)

At ∼0.8 V, the current achieves a potential-independent response consistent with a two-electron oxidation of a species with a diffusion coefficient of 2.4 × 10-6 cm2 s-1. Catalytic current is observed in cyclic voltammetry of aquaruthenium complexes in the presence of DNA because the half-life of the DNA oxidation reaction is ∼10 s.31 The experimental time scale of normal pulse voltammetry (∼10-1 s) makes this contribution negligible. Plots of i2 - io2 for the titrations of [(tpy)(bpy)RuOH2]2+ and [(tpy)(phen)RuOH2]2+ in 15 and 75 mM excess Na+ were obtained. Nonlinear least-squares fits to eq 6 with isat2 stipulated

B

Figure 7. (A) Plots of i2 - io2 obtained during titration of Co(bpy)33+ with poly(dA)‚poly(dT) in the presence of 15 mM excess Na+ and nonlinear least-squares fits to eq 6 using the territorial binding model (eq 7). (B) Plots of i2 - io2 obtained during titration of Co(bpy)33+ with poly(rA)‚poly(dT) in the presence of 15 mM excess Na+ and nonlinear least-squares fits to eq 6 using the territorial binding model (eq 7).

for a two-electron couple give binding constants for [(tpy)(bpy)RuOH2]2+ of 700 and 100 M-1 in the presence of 15 and 75 M excess Na+, respectively. The corresponding values for [(tpy)(phen)RuOH2]2+ are 1500 and 150 M-1. Thus, the substitution of a single phen for bpy enhances the binding affinity of aquaruthenium complexes only slightly. Effect of Nucleic Acid Structure. The binding of Co(bpy)33+ to the synthetic polymers poly[dA]‚poly[dT] and poly[rA]‚poly[dT] was examined to assess any differences in these sequences’ affinities for proven electrostatic binders. The plots of i2 - io2 for poly[dA]‚poly[dT] and poly[rA]‚poly[dT] are presented in Figure 7 with nonlinear least-squares fits to the territorial binding model (eq 7). The binding constants in the presence of 15 mM excess Na+ are 2100 and 3000 M-1 for poly[dA]‚poly[dT] and poly[rA]‚poly[dT], respectively. Although these values are lower than that measured for calf thymus DNA, they are consistent with purely electrostatic binding, as discussed below.

13834 J. Phys. Chem., Vol. 100, No. 32, 1996

Welch and Thorp

TABLE 1: Diffusion Coefficients of Metal Complexes (cm2 s-1) complex

106 Dfa

107 D35a

106 Df

107 Db

Co(bpy)33+ Co(phen)33+ Os(bpy)32+ Os(phen)32+ Fe(bpy)32+ Fe(phen)32+ [(tpy)(bpy)RuOH2]2+ [(tpy)(phen)RuOH2]2+

4.1 3.3 7.3 5.5

5.4 1.5 5.2 2.2

4.5b 4.2b

12b 2.6b

2.8b 4.9b 15c 70c

1.8b 0.9b 9.6c 45c

a

2.5 4.7

3.5 5.3

This work. b Reference 14. c Reference 32.

TABLE 2: Binding Constants of Metal Complexes complex

15 mM Na+

5800 Co(bpy)33+ Co(phen)33+ 74 000d Os(bpy)32+ 945 Os(phen)32+ 202 000d Fe(bpy)32+ Fe(phen)32+ [(tpy)(bpy)RuOH2]2+ 700 [(tpy)(phen)RuOH2]2+ 1500

75 mM 50 mM 50 mM Na+ Na+ a Na+ s (bp) 260 1200 98 2360 100 150

920 14 000b 4300 26 000b 580 13 600 1400b 14 700b 590 15000c 930 78 000c

3b 5b 3b 4b 3c 3c

Interpolated using eq 3 for purposes of comparison. b Reference 14. Reference 32. d Fit using eq 9 and s ) 4; all others fit using eq 7. a

c

Discussion Voltammetric Measurement of Binding. The electrochemical response of a metal complex in DNA solution is a rich source of information about binding and reactivity. The diffusion rates of the free and bound forms of a complex differ by over 1 order of magnitude and, like any observable property that changes upon binding, define a scale on which the binding affinity can be quantified. If the metal complexes are assumed to diffuse as small spheres, the appropriate theoretical description of the diffusion coefficient is the Stokes-Einstein equation:

D ) kT/6πηor

(12)

where k is the Boltzmann constant, T is temperature in Kelvin, ηo is the native solvent viscosity, and r is the sphere’s radius. Diffusion of DNA fragments has been shown via light scattering43,44 and electrochemical labeling29 to conform to the theoretical treatment of Tirado and Garcia de la Torre for a rigidrod model:45,46,48

D ) (kT/3πηoL)(ln(p) + 0.312 + 0.565p-1 - 0.100p-2) (13) where p is the ratio of length to diameter. Thus, combination of the expressions for diffusion coefficients (eqs 12 and 13), binding constants (eqs 7-9), and salt dependence (eq 3) yields a rigorous mathematical model for the effects of binding on the electrochemical response. Determination of binding constants from electrochemically measured diffusion coefficients was first pursued by Carter and Bard14,49 in the cases of Co(bpy)33+, Co(phen)33+, Fe(bpy)32+, and Fe(phen)32+ and later by Grover et al. in the cases of [(tpy)(bpy)RuOH2]2+ and [(tpy)(phen)RuOH2]2+.32 Both studies relied on diffusion coefficients calculated via the expression for peak current in the cyclic voltammogram of a kinetically reversible couple. We have discussed elsewhere that cyclic voltammetry does not account for kinetic limitations on electron transfer when determining diffusion coefficients for metal complexes bound to macromolecules.29 In the RuOH22+ systems, potential kinetic limitations can occur due to slow electron transfer to the DNA-bound metal complex,29 the use of semiconducting ITO electrodes,29 and, in the case of the proton-coupled RuOH22+ systems, slow electron transfer to the metal complex itself.47 Diffusive currents measured by normal pulse voltammetry provide a reflection of the binding distribution that is not obfuscated by these kinetic complications.34 The diffusion coefficients of the complexes free in 15 mM excess Na+ and in the presence of calf thymus DNA at R ) 35 (R ) [DNA]/[complex]) are given in Table 1. Included for comparison are values from the previous studies mentioned above. The values for free tris chelates are consistent with those measured by Carter and Bard allowing for slightly faster diffusion of iron complexes in comparison to osmium ana-

logues.14 The errors in the diffusion coefficients reported by Grover et al. probably arise from the proximity of the Ru(IV/ III) and Ru(III/II) couples and the kinetic limitations imposed by the coupling of deprotonation to the electrochemistry. Fitting Xb vs [DNA] to obtain a binding constant requires distinction between the asymptotic binding curve of the territorial model and the biphasic saturation curve of the site-binding model. The two previous electrochemical studies considered only the site-binding model,14,32,49 the results of which are presented in Table 2 along with those from this study at 15 and 75 mM Na+ and interpolated at 50 mM Na+. This model may have seemed valid for the weaker-binding complexes because the peak current errors in cyclic voltammetry are larger for bound complexes, and therefore a large positive error is introduced in Xb as the titration proceeds. Calculation of theoretical binding curves from the reported parameters shows that Xb is overestimated in these studies. As mentioned in the Results section, only tris-phen complexes at low ionic strength appear to achieve codiffusion with DNA, as evidenced by the biphasic binding curves obtained during DNA titrations. The overestimated binding constants determined in the previous studies therefore result from an inappropriate use of the sitebinding equation (eq 9), which was exacerbated in the case of the two-electron Ru(IV/II) couples by the kinetic limitations discussed above. Binding Energetics. Polyelectrolyte theory requires that the nonelectrostatic binding energies remain constant as the ionic strength changes. Previous studies have demonstrated this criterion for complexes of dppz,13,50 bpy,24 and, over a relatively narrow ionic strength range, phen.23,35 The data in Figure 3 show clearly that electrochemical methods also allow for determination of the polyelectrolyte parameters. The values of ZΨ obtained from the plots are in excellent agreement with the theoretical values for 2+ and 3+ cations, and the values of the nonelectrostatic energies ∆G°t are consistent with nearly pure electrostatic binding, as suggested by numerous studies on other bpy complexes.12,13 Similarly, the binding affinities for the [(tpy)(L)RuOH2]2+ complexes adhere to the theoretical value of ZΨ and provide a very low ∆G°t, consistent with nearly pure electrostatic binding. The binding of the tris-phen complexes is interesting because the value of K°t for Os(phen)32+ is sufficiently larger than that of Co(phen)33+ to reverse the relative magnitudes compared to the tris-bpy analogues. This effect was first indicated by the limiting shift in E1/2 of Co(phen)33+ observed by Carter et al.14 We observe here a similar shift if the E1/2 of Os(phen)32+ by the normal pulse technique. Since Co(phen)33+ and Os(phen)32+ are isosteric, the higher affinity measured for Co(phen)33+ compared to Os(phen)32+ indicates the same effect as the shift in E1/2. A van der Waals or intercalation binding contact would be synergistic with electrostatic binding, and we have shown that

Effects of DNA Binding on NPV of Metal Complexes TABLE 3: Nonelectrostatic Binding Energies for phen Complexes ∆G°t (kcal/mol) complex

15 mM Na+

75 mM Na+

Co(phen)33+ Os(phen)32+ [(tpy)(phen)RuOH2]2+

1.5 3.2 0.5

1.0 1.9 0.3

the intercalating complex Os(bpy)2(dppz)2+ exhibits a negative shift in E1/2, implying tighter binding of the 3+ form compared to the 2+ form and a similar K°t for both redox forms.29 In addition, the dependence of the affinities of dppz complexes on added salt is consistent with intercalation as the sole component of the nonelectrostatic affinity.13,50 Table 3 lists the values of ∆G°t calculated as -RT ln K°t for complexes with phen ligands studied here. Values in 15 mM excess Na+ are higher than those in 75 mM excess Na+ by ∼50% for both Co(phen)33+ and Os(phen)32+. However, the ratio of binding energies is approximately 2:1 at both ionic strengths, as expected from the shifts of E1/2. As originally suggested by Carter et al.,14 the binding scenario most consistent with the data is a hydrophobic interaction wherein water is released upon binding of the tris-phen complex. The higher solvation energy of the trication therefore disfavors the elimination of water from the binding site; a similar assignment has been made to explain the relative binding affinities of the 2+ and 1+ forms of methylviologen to anionic micelles.51 The discrepancy in binding energies for the tris-phen complexes determined at 15 and 75 mM Na+ probably results from the need for a different binding isotherm for the two cases. Nonetheless, the relative binding energies of the 3+ and 2+ ions are independent of both the ionic strength and the binding isotherm, supporting the participation of a hydrophobic interaction in the binding mode. The low values of ∆G°t for binding of [(tpy)(phen)RuOH2]2+ probably reflect at most one or two van der Waals contacts, which are typically on the order of 0.25 kcal mol-1.52 Presumably these contacts are to the 5 and 6 positions of the phen ligand since tpy and aqua ligands are the same as in the bpy analogue. The relative binding constants of [(tpy)(bpy)RuOH2]2+ and [(tpy)(phen)RuOH2]2+ reported here correlate well with the trend in the “burst fraction” observed by Neyhart et al. in the reaction of the oxo complexes with DNA.53 This fraction represents the immediate reduction of bound complexes upon addition of DNA and is therefore proportional to Xb. Since [(tpy)(phen)RuOH2]2+ possesses the sterically most accessible phen ligand, the relative binding affinities of the three phen complexes imply a participation of at least two phen ligands in the binding of the tris complexes. Finally, the study of binding of Co(bpy)33+ to the synthetic polymers poly[dA]‚poly[dT] and poly[rA]‚poly[dT] confirms their similarity to calf thymus DNA in the binding of relatively large cations. The higher reactivity of AT-rich sequences has been ascribed to higher negative charge density in the minor groove,54,55 just as differences between DNA-DNA and RNADNA duplexes have been ascribed to a deeper minor groove in the A-form RNA-DNA structure.56 This study confirms that electrostatic territorial binding is a function of linear charge density as opposed to the local charge density that would affect site binding. The binding constants so obtained are actually slightly lower than those for calf thymus DNA. Black and Cowan have noted a lower electrostatic affinity of A-DNA for ammine complexes,57 but these complexes are much smaller and therefore probe more minute changes in the charge density. Implications. The large differences in the accurately measured diffusion coefficients of an electrochemically active metal complex and a macromolecule provide a basis for quantitation

J. Phys. Chem., Vol. 100, No. 32, 1996 13835 of binding. This study demonstrates the utility of modern pulse voltammetry to observe mass transport via the normal pulse mode and redox thermodynamics via the cyclic staircase mode. This approach to measuring binding energetics of an electrochemically active species with a nonactive macromolecule is widely applicable provided the electrode material is chosen to preclude adsorption of the macromolecule. Within this limitation, the only requirement is that the interfacial charge transfer occur with ∼100% kinetic efficiency at some potential, which is evident if the normal pulse voltammogram achieves a potential-independent current response. Because of the small amounts of macromolecule needed, many binding studies, such as those on A-form DNA shown here, are much more feasible. Thorough understanding of the binding equilibria of the less oxidizing tris chelates is a necessary complement to any voltammetric studies addressing metal-DNA reactivity, including recent studies by Johnston et al. of electrocatalytic oxidation of DNA by tris chelates of iron and ruthenium.33,58 In addition to the catalytic reaction itself, analysis of catalytic current for DNA oxidation mediated by metal complexes requires an understanding of the binding of both redox forms, the diffusion coefficients of the bound and free forms of both redox states, and the heterogeneous charge transfer rates for the bound and free forms. These studies of isosteric complexes of cobalt and osmium form a basis for determining electron transfer rates between DNA and more oxidizing metal mediators. Acknowledgment. This work was supported by the David and Lucile Packard Foundation. T.W.W. thanks the Department of Education for a graduate fellowship. H.H.T. is a Camille Dreyfus Teacher-Scholar. References and Notes (1) Theil, E. C. Biofactors 1993, 4, 87-93. Haile, D. J.; Rouault, T. A.; Harford, J. B.; Kennedy, M. C.; Blondin, G. A.; Beinert, H.; Klausner, R. D. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11735-11739. (2) O’Halloran, T. V. Science 1993, 261, 715-725. (3) Honig, B.; Nicholls, A. Science 1995, 268, 1144-1149. (4) Record, M. T., Jr.; Anderson, C. F.; Lohman, T. M. Q. ReV. Biophys. 1978, 11, 103. (5) Theil, E. C. New J. Chem. 1993, 18, 435-441. (6) Lippard, S. J. Acc. Chem. Res. 1978, 11, 211-217. (7) Barton, J. K.; Lolis, E. J. Am. Chem. Soc. 1985, 107, 708. (8) Clarke, M. J. Met. Ions Biol. Syst. 1980, 11, 231. (9) Grover, N.; Welch, T. W.; Fairley, T. A.; Cory, M.; Thorp, H. H. Inorg. Chem. 1994, 33, 3544-3548. (10) Grover, N.; Gupta, N.; Thorp, H. H. J. Am. Chem. Soc. 1992, 114, 3390. (11) Manning, G. S. Acc. Chem. Res. 1979, 12, 443. (12) Pyle, A. M.; Rehmann, J. P.; Meshoyrer, R.; Kumar, C. V.; Turro, N. J.; Barton, J. K. J. Am. Chem. Soc. 1989, 111, 3051. (13) Kalsbeck, W. A.; Thorp, H. H. J. Am. Chem. Soc. 1993, 115, 71467151. (14) Carter, M. T.; Rodriguez, M.; Bard, A. J. J. Am. Chem. Soc. 1989, 111, 8901. (15) Holmlin, R. E.; Barton, J. K. Inorg. Chem. 1995, 34, 7. (16) Dupureur, C. M.; Barton, J. K. J. Am. Chem. Soc. 1994, 116, 10286-10287. (17) Krotz, A. H.; Hudson, B. P.; Barton, J. K. J. Am. Chem. Soc. 1993, 115, 12577-12578. (18) David, S. S.; Barton, J. K. J. Am. Chem. Soc. 1993, 115, 2984. (19) Jeanette, K. W.; Lippard, S. J.; Vassiliades, G. A.; Bauer, W. R. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 3839. (20) Eriksson, M.; Leijon, M.; Hiort, C.; Norde´n, B.; Gra¨slund, A. Biochemistry 1994, 33, 5031-5040. (21) Hiort, C.; Lincoln, P.; Norde´n, B. J. Am. Chem. Soc. 1993, 115, 3448. (22) Tracy, M. A.; Pecora, R. Annu. ReV. Phys. Chem. 1992, 43, 525. (23) Satyanarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Biochemistry 1992, 31, 9319. (24) Kalsbeck, W. A.; Thorp, H. H. Inorg. Chem. 1994, 33, 34273429. (25) Sitlani, A.; Long, E. C.; Pyle, A. M.; Barton, J. K. J. Am. Chem. Soc. 1992, 114, 2303.

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