The Effect of Charge on the Volume Change of DNA Binding with Intercalator DAPP

Xuesong Shi and Robert B. Macgregor, Jr.*Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, UniVersity of Toronto,144 College Street, Toronto, Ontario M5S 3M2, Canada

ReceiVed: December 7, 2006; In Final Form: February 19, 2007

To study the effect of ligand charge on DNA-ligand binding, we measured the difference in volume changeof the noncovalent complex formed between calf thymus DNA and the charged and uncharged form of 3,8-diamino-6-phenylphenanthridine (DAPP). We found that the volume change for binding with the chargedDAPPH+ is about 7.8( 1.5 cm3 mol-1 more positive than with the neutral DAPP. We hypothesize that thislarge difference in interaction volume originates from partial desolvation of the charge of DAPPH+ when itis bound to DNA.

1. Introduction

Most DNA-binding ligands are positively charged; thisenhances their solubility in water and their affinity to DNA.The contribution of the electrostatic interaction between thepositively charged ligand and the negatively charged DNA tothe binding affinity is generally considered to be responsiblefor the favorable entropy change arising from counter ionrelease.1,2 The change of hydration environment of the chargeof the ligand upon binding with DNA (i.e., the dehydration)should also have an impact on the thermodynamics of thecomplex; however, this change has been largely ignored inthermodynamic studies of DNA-ligand interactions. In sometreatments of this issue, the contribution arising from thedehydration of the ligand has been considered to be canceledout by the hydration of the released counter ions.3

We have measured the volume change difference for DNAbinding with neutral ligand 3,8-diamino-6-phenylphenanthridine(DAPP) and its protonated form DAPPH+ and analyzed theresults from the point of view of changes in hydration. In asolution of neutral pH, free DAPP is primarily in its neutralform and bound DAPP is mainly in its charged protonated statedue to an increase in pKa upon intercalation into DNA.4 Fourequilibria among DAPP, DAPPH+, DAPP‚DNA, and DAPPH+‚DNA coexist in such a solution4 (Figure 1a), providing a rareopportunity to compare binding of DNA with charged anduncharged ligands that are structurally very similar.

2. Materials and Methods

DAPP and calf thymus DNA were obtained from Sigma-Aldrich Co. DAPP was used without further purification. Calfthymus DNA was dialyzed against pH 7.2 buffer solutionconsisting of 20 mM Tris-HCl, 0.1 mM EDTA with 200 mMadded NaCl. The same buffer solution was used in theexperiments except when it is specifically stated otherwise. TheDNA concentration was determined spectroscopically usingε260

) 13 200 cm-1 M-1 (bp) as the molar extinction coefficient.5

The concentration of DAPP was determined by weight. Theabsorption maxima of neutral DAPP and charged DAPPH+ arewell separated spectroscopically at about 400 and 472 nm,respectively, for the free ligands (determined in this work) andabout 412 and 515 nm, respectively, for the bound ligand.4 Thewell-separated absorption peaks of the neutral and chargedspecies enabled us to monitor the concentration changes ofmultiple components using fluorescence spectroscopy.

Our experiments were carried out on solutions containing 819µM calf thymus DNA and 17µM DAPP. According to Jonesand Wilson,4 the pKa values of free and bound DAPP are about5.8 and 7.9, respectively, at the salt concentration used in theseexperiments. At pH 7.2, we would expect the equilibrium of

* Corresponding author. E-mail: rob.macgregor@utoronto.ca. Tele-phone: 416 978 7332. Fax: 416 978 8511.

Figure 1. (a) Coupled equilibria of DAPP and its binding to calfthymus DNA in the protonated and unprotonated forms. (b) Weberfree energy level scheme21 for the system in aqueous solution containing200 mM salt. From this analysis, one finds thatδG ) ∆G3 - ∆G1 )∆G4 - ∆G2 ) -3.3 kcal mol-1 < 0, which implies binding of DNAand H+ with DAPP facilitate each other.21

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the free DAPP to favor strongly the neutral form and that neutralDAPP‚DNA would be a small but non-negligible fraction ofthe total bound DAPP, i.e., charged and neutral. The fourequilibria, shown in Figure 1a, are related as according toK1K2

) K3K4 and ∆V1 + ∆V2 ) ∆V3 + ∆V4. We calculated thevolume changes from the pressure dependence of associationconstant,Ka ) [complex]/[DNA][ligand], using the standardequation (∂ ln Ka/∂P)T ) -∆V/RT, whereR is the gas constant.The experimental temperature was maintained at 25°C. Thefluorescence was monitored by a Spex FluoroMax3 fluorometer(Jobin Yvon, Inc., Edison, NJ). Greater detail concerning theexperimental setup can be found in our previous publications.6,7

We used excitation/emission wavelength pairs of 410/535,410/630, and 480/630 nm to monitor concentration changes ofDAPP, DAPP‚DNA, and DAPPH+‚DNA, respectively. Thesewavelengths were chosen in order to minimize the spectraloverlap for the signals of DAPP (410 and 535 nm) andDAPPH+‚DNA (480 and 630 nm). However, the fluorescencesignals arising from DAPP and DAPPH+‚DNA do overlap withthe signal of DAPP‚DNA at the excitation/emission wavelengthpair of 410 and 630 nm. The contributions of the signals ofDAPP and DAPPH+‚DNA were determined as percentages oftheir strong signals at 410 and 535 nm and 480 and 630 nm,respectively. The fractional contribution of DAPP to the totalsignal was determined from separate pressure measurements onsolutions containing only DAPP and buffer with pH adjustedto 11.3 to eliminate DAPPH+. On the basis of these measure-ments, the contribution of DAPP,fDAPP, was set equal to (I410,630/I410,535)DAPP. Similarly, for DAPPH+‚DNA, measurements werecarried out on solutions of a mixture of 819µM calf DNA and17 µM DAPP at low salt concentration (∼36 mM) and low pH(5.7). Under this condition, DAPPH+‚DNA is the dominant formof DAPP in the solution andfDAPPH·DNA ) (I410,630/I480,630)DAPPH·

DNA. With the two percentages known, we calculated the signalof DAPP‚DNA as IDAPP·DNA ) I410,630- fDAPPI410,535- fDAPPH·

DNAI480,630. Representative spectra are shown in Figure 2.Furthermore, under our experimental conditions, pH) 7.2 andhigh DNA concentration, equilibria 1 and 2, favor depletion ofDAPPH+. This, combined with the fact that the quantumefficiency of free DAPPH+ is several times lower than its boundform, the signal arising from free DAPPH+ is estimated to beless than 1/300 of that arising from DAPPH+‚DNA. Althoughwe are unable to monitor directly the concentration of DAPPH+,we assume its contribution to the observed fluorescence signalof DAPPH+‚DNA to be negligible.

3. Results and Discussion

With knowledge of the concentrations of DAPP, DAPP‚DNA,and DAPPH+‚DNA, we were able to obtain the pressuredependence ofK3 andK4, shown in Figure 3. The data in Figure3 were fitted with second-order polynomial using Origin(OriginLab Corp., Northampton, MA), and the first derivativewas used to calculate volume change,∆V3 ) 3.2 ( 1.0 cm3

mol-1 and∆V4 ) -11.7 ( 1.1 cm3 mol-1.To obtain∆V1, we carried out separate measurements at ele-

vated pressure on aqueous solutions containing of only DAPPand buffer (Table 1). Excitation/emission wavelength pairs of410/535 nm and 480/630 nm were used to monitor concentrationchanges of DAPP and DAPPH+, respectively. The protonationprocess (Reaction 1) was favored by pressure with∆V1 equal toabout-4.6( 1.1 cm3 mol-1. Lepori and Gianni8 proposed thatthe relative contribution to partial molar volume is 7.19(0.2 cm3 mol-1 for an uncharged aromatic nitrogen and-1.73(0.6 cm3 mol-1 for a charged aromatic nitrogen. Coupled with par-

tialmolecularvolumeof theproton,V°(H+))-5.4cm3mol-1,9,10

the molar volume change of a protonation process similar toReaction 1 can be predicted to be about-3.5( 0.6 cm3 mol-1,which is in good agreement with our experimental result.

With ∆V1, ∆V3, and∆V4 measured,∆V2 ) ∆V3 + ∆V4 -∆V1 ) -3.9 ( 1.8 cm3 mol-1. Reaction 2 is the intercalationof positively charged DAPPH+ with DNA and resembles thewell-studied intercalation of ethidium bromide (EB) with DNA.The volume change of-3.9 ( 1.8 cm3 mol-1 compares wellwith literature data on intercalation of EB with natural DNAobtained using high-pressure methods.11,12

Reactions 2 and 4 correspond to the intercalation of chargedDAPPH+ and neutral DAPP with DNA, respectively. Assumingthat the structure of the DNA complexes formed by DAPPH+

and DAPP are similar, then the difference between∆V2 and∆V4 represents the effect of the positive charge of DAPPH+ onthe volume change of DNA-ligand binding. The effect of thepositive charge is given by∆∆V(+) ) ∆V2 - ∆V4 ) ∆V3 -∆V1 ) 7.8 ( 1.5 cm3 mol-1, which is a large positive value.There are several possible molecular origins of the∆∆V(+).We can write∆∆V(+) ) (f1 - 1)∆∆V(+)ligand + ∆∆V(+)counterion

+ ∆∆V(+)other; in this expression, the first term on the right-hand side,∆∆V(+)ligand, is the difference between partial molarvolumes of charged and neutral forms of free ligands, i.e.,DAPPH+ and DAPP. This term will always have the largestmagnitude of the three and will be negative due to electros-triction of the water molecules in the vicinity of the charge.Relocating the positively charged ligand from bulk solution tothe minor groove of DNA will cause its charge to be partiallyneutralized by DNA phosphate and the charge will presumablyhave reduced accessibility to water. Consequently, the volumeeffect of the charge,∆∆V(+)ligand, decreases to a fraction,f1,relative to its value in free solution. The volume change thatarises from the protonation of an aromatic amine dissolved in

Figure 2. Representative spectra of the ionized and un-ionized formsof DAPP, free and bound to calf thymus DNA. The black curve is theemission spectrum of DAPP plus DNA, pH 7.2, excited at 410 nm.The red curve is the spectrum of DAPP alone, also excited at 410 nm.The intensity at 535 nm arises mostly from DAPP andI410, 535is usedto monitor the concentration change of DAPP. The green curve is thedifference between the black curve and the red curve. The blue curveis the spectrum of DAPPH+‚DNA alone. The intensity at 630 nm isthe sum of DAPP‚DNA and overlapping signals from DAPP andDAPPH+‚DNA. The signal from DAPP equalsfDAPPI410,535and the signalfrom DAPPH+‚DNA equals fDAPPH‚DNAI480,630. The small differencebetween I410,535 and I410,535 for the DAPP signal arises from thecontribution of DAPP‚DNA and is not as sensitive to pressure as DAPP.Therefore, the effect of this small discrepancy on the pressuredependency and volume result is ignored for simplicity.

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water is reported to be-8.9( 0.6 cm3 mol-1.8 Comparing thisvalue with our result of∆∆V(+) ) 7.8 ( 1.5 cm3 mol-1, it isevident that a significant portion, 1- f1, of the volume effectof a ligand’s charge is lost upon intercalation with DNA.

The second term,∆∆V(+)counter ion, is the volume changearising as a consequence of the counter ion release thataccompanies ligand binding. However, because univalent counterions, such as Na+, are considered to be mostly fully hydratedand delocalized around DNA,13 we presume that the ion-waterinteractions of the condensed counter ions do not differsignificantly from those of the free ions. Thus, the volume effectof counter ion release,∆∆V(+)counter ion, is likely to be negligiblefor univalent counter ions. It is interesting to note that localizedunivalent counter ions have been found in the minor groove ofsome narrow AT-rich sequences at low temperature.14,15How-ever, such binding events became negligibly rare at roomtemperature.16 Furthermore, one study suggests that counter ions

might be partially dehydrated around poly[d(A-T)]‚poly[d(A-T)] and poly[d(G-C)]‚poly[d(G-C)].17 However the reportedvolume effect of the dehydration of ions is smaller than theerror in our experiments.

Finally, all other potential contributions to∆∆V(+) arecollected in the term,∆∆V(+)other. For example, if significantconformational differences existed between complexes of DNAwith charged and neutral ligands, the difference might result ina sizable∆∆V(+)other.

The binding of a charged ligand with DNA is associated withan unfavorable free energy due to dehydration and a favorablefree energy arising from the formation of electrostatic interac-tions between the ligand and the DNA. We call the sum of thesetwo free energies∆∆Gdehy. This term and the free energy ofcounter ion release,∆∆Gcir, are the main sources of thedifference in the free energy of the binding of charged andneutral ligands with DNA in the absence of conformationaldifferences. According to counter ion release theory,∆∆Gcir

disappears at 1 M salt, thus, under this condition,∆∆Gdehy canbe obtained as the free energy difference between DNA bindingwith charged and neutral ligand at 1 M salt, ∆∆G1M. In fact,for DAPP,∆∆Gdehy ) (∆G2 - ∆G4)1M ) (∆G3 - ∆G1)1M )-2.3RT[pKa(DAPPH+‚DNA) - pKa(DAPPH+)]1M. Accordingto Jones and Wilson,4 the difference between the pKa ofionization of bound and free DAPP, i.e., (pKa(DAPP‚DNA) -pKa(DAPP))1M, is about 1.6. Thus,∆∆Gdehy for DAPP is about-2.2 kcal mol-1, which is a surprisingly large favorable freeenergy. However, it is important to note that if the binding ofDAPP and DAPPH+ also differ in some aspect not related totheir charge difference such as orientation of the ligand informed complex, then these differences will contribute to theobserved value of∆Gdehy.

There are few other cases in the literature that allow us tocalculate∆∆Gdehy.In one study, exchanging the charged aminegroup of doxorubicin with a neutral hydroxyl group had beenfound to make the DNA-binding free energy,∆G1M, ap-proximately 1 kcal mol-1 more unfavorable,18 and∆∆G1M isreported to be-1 kcal mol-1. This difference was interpretedas the result of stronger interactions between DNA and theamine group (without the charge) than the hydroxyl group.18

Evidently, ∆∆Gdehy likely also contributed to the observeddifferences. A study of neutral-red (NR) showed that this dye,in a manner similar to DAPP, also undergoes pKa shift uponintercalation with DNA.19 From these results, we estimated the∆∆Gdehy to be about-1.3 kcal mol-1 for NR. Clearly, theobserved values of∆∆Gdehydepend on the ligand. We proposethat the differences may originate from the position of the chargein the minor groove, which in turn would result in differencesin charge DNA interactions and accessibility to water. We arecurrently using computational methods to test this hypothesis.

The thermodynamic effect of charge dehydration has alsobeen discussed in Misra and Honig’s computational work onthe same DAPP‚DNA system.20 They studied the electrostaticfree energy of the system using a model based on the nonlinearPoisson-Boltzmann equation. These authors found that desol-vation of DAPPH+ is 0.8 kcal mol-1 more unfavorable thandesolvation of neutral DAPP. They also proposed that thisdesolvation free energy could vary depending on the locationof the charge. However, the desolvation free energy of Misraand Honig is simply the electrostatic free energy of placingcharge into an environment with a smaller dielectric constantthan water. The water reorganization process, which is the maincause of volume change, probably is not fully counted by theirdesolvation free energy. Therefore, volumetric studies and

Figure 3. (a) Pressure dependence of lnK3. (b) Pressure dependenceof ln K4.

TABLE 1

reaction stepa ∆V (cm3mol-1)

1 L + H+ T LH+ -4.6( 1.12 LH+ + DNA T DNA‚LH+ -3.9( 1.8b

3 D‚L + H+ T DNA‚LH+ 3.2( 1.04 L + DNA T DNA‚L -11.7( 1.1

a L is the ligand, DAPP, LH+ is DAPPH+, and DNA is calf thymusDNA. b ∆V2 ) ∆V3 + ∆V4 - ∆V1

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computational methods could complement each other in under-standing the charge dissolvation process and DNA-ligandbinding in general.

Finally, from the point of view of the volume change, adifferent ligand and DNA may have different values off1 and∆∆V(+)ligand and consequently lead to a different value of∆∆V(+). Data of ∆∆V(+) complement∆∆Gdehy very wellbecause∆∆V(+) mainly comes from the dehydration part of∆∆Gdehy and helps to further dissect∆∆Gdehy. Unfortunately,∆∆V(+) data are even rarer than∆∆Gdehy data. Furthervolumetric and thermodynamic studies of ligand pairs similarto DAPP and DAPPH+ will help us better understand the effectof charge on DNA-ligand binding.

References and Notes

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20, 1887.

(6) Wu, J. Q.; Macgregor, R. B.Anal. Biochem.1993, 211, 66.(7) Shi, X. S.; Macgregor, R. B.Biophys. J.2006, 90, 1729.(8) Lepori, L.; Gianni, P.J. Solution Chem.2000, 29, 405.(9) Zana, R.; Yeager, E.J. Phys. Chem.1966, 70, 954.

(10) Zana, R.; Yeager, E.J. Phys. Chem.1967, 71, 521.(11) Heremans, K.; Nuland, Y. V.High Temp.sHigh Pressures1977,

9, 539.(12) Sugimoto, N.; Sasaki, M.Chem. Express1988, 3, 487.(13) Anderson, C. F.; Record, M. T.Annu. ReV. Phys. Chem.1995, 46,

657.(14) Shui, X. Q.; McFail-Isom, L.; Hu, G. G.; Williams, L. D.

Biochemistry1998, 37, 8341.(15) Shui, X. Q.; Sines, C. C.; McFail-Isom, L.; VanDerveer, D.;

Williams, L. D. Biochemistry1998, 37, 16877.(16) Denisov, V. P.; Halle, B.Proc. Natl. Acad. Sci. U.S.A.2000, 97,

629.(17) Tikhomirova, A.; Chalikian, T. V.J. Mol. Biol. 2004, 341, 551.(18) Chaires, J. B.; Satyanarayana, S.; Suh, D.; Fokt, I.; Przewloka, T.;

Priebe, W.Biochemistry1996, 35, 2047.(19) Walz, F. G.; Terenna, B.; Rolince, D.Biopolymers1975, 14, 825.(20) Misra, V. K.; Honig, B.Proc. Natl. Acad. Sci. U.S.A.1995, 92,

4691.(21) Weber, G.Protein Interactions; Chapman & Hall: New York, 1992;

p 33.

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