research article exploration of electrochemical intermediates ...research article exploration of...

Research ArticleExploration of Electrochemical Intermediates ofthe Anticancer Drug Doxorubicin Hydrochloride UsingCyclic Voltammetry and Simulation Studies withan Evaluation for Its Interaction with DNA

Partha Sarathi Guin1 and Saurabh Das2

1 Department of Chemistry Shibpur Dinobundhoo Institution (College) 4121 G T Road (South) Howrah 711102 India2Department of Chemistry Jadavpur University Raja SC Mullick Road Kolkata 700032 India

Correspondence should be addressed to Partha Sarathi Guin parthasggmailcom

Received 16 June 2014 Accepted 5 July 2014 Published 3 August 2014

Academic Editor Shengshui Hu

Copyright copy 2014 P S Guin and S DasThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Electrochemical behavior of the anticancer drug doxorubicin hydrochloride was studied using cyclic voltammetry in aqueousmedium using Hepes buffer (pHsim74) At this pH doxorubicin hydrochloride undergoes a reversible two-electron reduction with119864

12value minus665 plusmn 5mV (versus AgAgCl saturated KCl) Depending on scan rates processes were either quasireversible (at low

scan rates) or near perfect reversible (at high scan rates) This difference in behavior of doxorubicin hydrochloride with scan ratestudied over the same potential range speaks of differences in electron transfer processes in doxorubicin hydrochloride Attemptwasmade to identify and understand the species involved using simulationThe information obtained was used to study the interactionof doxorubicin hydrochloride with calf thymus DNA Cathodic peak current gradually decreased as more calf thymus DNA wasadded The decrease in cathodic peak current was used to estimate the interaction of the drug with calf thymus DNA Nonlinearcurve fit analysis was applied to evaluate the intrinsic binding constant and site size of interaction that was compared with previousresults on doxorubicin hydrochloride-DNA interaction monitored by cyclic voltammetry or spectroscopic techniques

1 Introduction

Anthracycline anticancer drugs are amongst the most activeagents in oncology [1 2] first recognized for their antibac-terial properties in 1939 Their chemical characterizationled to an increase in use after therapeutic value of theirantineoplastic activity was described in the early 1960sTwo anthracyclines daunomycin (daunorubicin) and dox-orubicin hydrochloride (hydroxyl daunomycin or doxoru-bicin) were initially isolated from Streptomyces peucetiusvar caesitue [3 4] and exhibit the widest spectrum ofantitumor activity [5 6]The presence of a side chain primaryalcohol group in doxorubicin hydrochloride has importantconsequences in antitumor activity that makes it more valu-able than other anthracyclines Doxorubicin hydrochlorideis used in the treatment of breast cancer childhood solidtumors soft tissue sarcomas and aggressive lymphomas

However its use is often hampered by cumulative dose-limiting cardiotoxicity resulting in cardiomyopathy and con-gestive heart failure that may be irreversible [7]

Though there is still some controversy with regard totheir mode of action anticancer activity or cytotoxic effectsinvolve interactionwith nuclear components especiallyDNAand type-II topoisomerase [8 9] The anthracyclines containa planar aglycone ring coupled with an aminosugar Theplanar anthraquinone intercalates between base pairs of DNAby 120587-120587 stacking and hydrophobic interaction Its long axisis nearly perpendicular to the axis of the double helixThe aminosugar interacts with negatively charged phosphategroups in the DNA major groove [10 11] In addition tostacking and hydrophobic interaction intercalation is furtherstabilized by hydrogen bonding between the bases of DNAand the drug molecule [12 13] In case of daunomycin thesingle positive charge contributes electrostatically to binding

Hindawi Publishing CorporationInternational Journal of ElectrochemistryVolume 2014 Article ID 517371 8 pageshttpdxdoiorg1011552014517371

2 International Journal of Electrochemistry

[12] Intercalation causes a change in the shape of the DNAdouble helix thereby hindering DNA replication and RNAtranscription [12 14 15] Doxorubicin hydrochloride anddaunomycin were observed to accumulate rapidly in livingcells in the nuclei and bind to chromosomal DNA [16 17] Inthe cell nuclear DNA is associated with a variety of proteinsforming a nucleoprotein complex called chromatin [18]Accumulating evidence suggests these drugs interact withchromatin leading to chromatin unfolding and aggregationthat causes a change in structure and function of chromatin[19 20] The structural disruption is likely to interfere withmetabolic processes of DNA (replication and transcription)contributing to apoptosis induced by these drugs in cancercells [19 20]

In addition inside the cell anthracyclines react withcytochrome reductase in the presence of reduced nicoti-namide adenine dinucleotide phosphate (NADPH) to formsemiquinone radical intermediates which participate inoxidation-reduction reactions generating superoxide radi-cal anion (O

2

∙minus) that are converted to hydrogen peroxide

(H2O2) and hydroxyl radical (OH∙) [21 22]The free radicals

induce membrane-lipid peroxidation DNA strand scissionand direct oxidation of purine-pyrimidine bases thiols andamines and also contribute to cardiotoxicity The heart tissuebeing devoid of glutathione peroxidase is unable to convertsuperoxide radicals to oxygen and is therefore handicappedin this regard [23 24] Therefore the study of the redoxbehavior of anthracyclines is an important aspect as it helpsin the understanding of toxicities and therapeutic efficienciesThere are many reports on the redox behavior of these drugscorrelated with biological findings [25 26]The present studyis an attempt to revisit the redox behavior of doxorubicinhydrochloride in aqueous media at physiological pH (74)using cyclic voltammetry trying to understand the mecha-nism by which the molecule copes with reduction

2 Materials and Methods

Doxorubicin hydrochloride [980ndash1000 (HPLC)] was pur-chased from Sigma-Aldrich USA and it was used withoutany purification Since the quinonemoiety is sensitive to lightsolutions were prepared just before experiment and kept inthe dark CT DNA was purchased from Sisco Research Lab-oratory India After dissolution of CT DNA in buffer puritywas checked from absorbance ratio A

260A280

For all solu-tions this ratio was in the range 18 lt A

260A280gt 19 and

provided a good estimate of purity [27] DNA obtained frombiological sources has proteins as contaminants that absorbmore heavily around 280 nm owing to tryptophanwith somecontribution from tyrosine residues that decrease A

260A280

ratio An A260A280

ratio of 18-19 is considered ldquocleanrdquo withregard toDNApurity indicating that no further deproteiniza-tion is required [27] Concentration of DNA (in bases) wasdetermined taking 120598

260= 6600Mminus1 cmminus1 for CT DNA

Analytical grade Hepes buffer (N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid 10mM) (Spectrochem Pvt LtdIndia) was used to maintain a pH of sim74 in all experimentsSodium chloride (AR grade) obtained fromMerck Germanywas used to maintain ionic strength It was also used as

the supporting electrolyte for electrochemical experimentsAll solutions were prepared in triple distilled water Cyclicvoltammetry experiments were done using the conventionalthree-electrode system at 25∘C A glassy carbon electrodeof surface area 01256 cm2 was the working electrode whileplatinum wire and AgAgCl saturated KCl were used asthe counter and reference electrode respectively Experi-ments were carried out using EG amp G Potentiostat Model263A Voltammetric experiments on doxorubicin hydrochlo-ride were done in aqueous buffer (pHsim74) in the pres-ence and absence of CT DNA All experimental solutionswere degassed for 30min with high-purity argon beforecyclic voltammetrywas doneMicroprocessor pHIONmeter(pMX 3000) was used for recording pH of solutions UV-Visspectroscopic studies were carried out using a spectropho-tometer (modelUNICAMUV500)Apair of 10times10mmpathlength quartz cuvettes was used Simulation of electrochem-ical data was done to explain the probable mechanism forthe reactions using esp24b software developed by Dr CarloNervi [28] Simulation was carried out in planar geometryand cathodic currents were taken positive

3 Results and Discussion

31 Electrochemical Reduction of Doxorubicin HydrochlorideIn 15mM Hepes buffer (pHsim74) containing 160mM NaCldoxorubicin hydrochloride (33 120583M) undergoes two-electronreversible reduction generating a cathodic peak at minus690 plusmn5mV (versus AgAgCl saturated KCl) with the correspond-ing anodic peak at minus640 plusmn 5mV (versus AgAgCl saturatedKCl) (Figure 1) at a scan rate of 100mVsminus1 The formalreduction potential (119864

12) was minus665plusmn5mV (versus AgAgCl

saturated KCl) The variation of peak potentials with scanrate is shown in Figure 2 and the corresponding electro-chemical parameters are summarized in Table 1 In Figure 2the cyclic voltammograms were taken in the potential range0 to minus085V since beyond minus085V there is no reductionor oxidation under the experimental condition which isclear from Figure 1 Previous studies [29 30] showed that atacidic pH and neutral pH there were two reduction peaksone reversible two-electron reduction of the quinone unitof doxorubicin hydrochloride to quinone dianion speciesat minus600mV (versus calomel electrode) that is minus644mV(versus AgAgCl saturated KCl) and another irreversiblepeak at much more negative potential owing to hydrogenevolution [31] However the second irreversible reductionpeak vanished at alkaline pH [31 32] Since the presentstudy was carried out at pH 74 that is slightly alkaline thesecond cathodic peak was not observed at more negativepotential which was in accordance with previous results[31 32] A plot of the ratio of the anodic to cathodic peakcurrent (119868pa119868pc) versus the logarithm of scan rate (log v)(Figure 3) shows that the 119868pa119868pc ratio is less than unityin case of lower scan rate while at higher scan rate itis almost unity [32] This suggests that the reduction ofdoxorubicin hydrochloride at physiological pH at lower scanrates was quasireversible while at higher scan rate it wasalmost reversible Using these observations for low and highscan rates a simulation study was performed to understand

International Journal of Electrochemistry 3

Table 1 Variation of peak potentials and formal reduction potential with scan rate for the reduction of doxorubicin hydrochloride in aqueousbuffer at pH 74 on glassy carbon electrode [Doxorubicin hydrochloride] = 33120583M [Hepes buffer] = 15mM and [NaCl] = 160mM andtemperature = 25∘C

Scan rate (mVsminus1) Cathodic peak potential 119864119901(mV) Anodic peak potential 119864

119886(mV) Formal reduction potential 119864

12(mV)

50 minus690 plusmn 5 minus640 plusmn 5 minus665 plusmn 5100 minus690 plusmn 5 minus640 plusmn 5 minus665 plusmn 5200 minus690 plusmn 5 minus640 plusmn 5 minus665 plusmn 5300 minus696 plusmn 5 minus636 plusmn 5 minus666 plusmn 5400 minus704 plusmn 5 minus634 plusmn 5 minus669 plusmn 5500 minus706 plusmn 5 minus630 plusmn 5 minus668 plusmn 5600 minus708 plusmn 5 minus614 plusmn 5 minus661 plusmn 51000 minus710 plusmn 5 minus610 plusmn 5 minus660 plusmn 5

whether there is any possibility of a chemical reaction alongwith the electrochemical reduction of the molecule leadingto the disappearance of the reduced species the quinonedianion For this reason the cyclic voltammograms at lowscan rates failed to show reversible nature In simulationstudy (Figure 2) the irreversibility of the electrochemicalreaction at lower scan rates was explained by assuminga comproportionation reaction between the quinone dian-ion (reduced species) and the quinone itself (doxorubicinhydrochloride) forming a semiquinone (Scheme 1) As aresult if more time was provided through the applica-tion of lower scan rate the dianion got sufficient time toreact and the concentration of the quinone dianion fallsleading to an irreversible reaction This is in accordancewith our previous results where we showed that at alkalinepH sodium 14-dihydroxy-910-anthraquinone-2-sulphonatea simple analogue of doxorubicin hydrochloride undergoes acomproportionation reaction with quinone dianion formingsemiquinone [32] It was observed that in the present studythe cathodic peak current (119868pc) has a linear relationship withthe square root of scan rate and passes through the origin(inset of Figure 1) following (1) [33] This clearly indicatesthat the reduction was diffusion controlled and there was noadsorption of doxorubicin hydrochloride onto the electrodesurface which was also in accordance with previous studies[31 32] Thus it was concluded that the reduction peakcurrent (119868pc) has a linear relationship with concentration ofdoxorubicin hydrochloride Consider

119868pc = 04463(119865

3

RT)

12

119899

32119860

0119863

0

12119862

0V12 (1)

where 119868pc 119899 1198600 1198630 1198620 and V refer to the cathodic peakcurrent (A) number of electrons involved in reduction areaof the electrode (in cm2) diffusion coefficient (cm2 sminus1)concentration (mol cmminus3) and scan rate (Vsminus1) respectivelyUsing this relation (see (1)) the diffusion coefficient for thereduction of doxorubicin hydrochloride was determined andit was found to be 307times10minus5 cm2 sminus1The reduced chi squarefor the plot (inset of Figure 1) was found to be 01135

32 Interaction of Doxorubicin Hydrochloride with Calf Thy-mus DNA Interaction of doxorubicin hydrochloride with

minus12 minus10 minus08 minus06 minus04 minus02 00minus2

0

2

4

6

8

10

Potential (V)

080604020

16

12

8

4

0Cu

rren

t (120583

A)

12 (Vsminus1)12

I pctimes106

(A)

Figure 1 Cyclic voltammogram of doxorubicin hydrochloride inaqueous buffer at pH 74 on glassy carbon electrode [Doxorubicinhydrochloride] = 33 120583M [NaCl] = 0160M scan rate = 01 Vsminus1and temperature = 25∘C Inset plot of cathodic peak currentversus square root of scan rate for the two-electron reduction ofdoxorubicin hydrochloride in aqueous buffer at pH 74

CT DNA was studied using cyclic voltammetry in aqueousbuffer at physiological pH (74) containing 160mM NaClDifferent solutions were prepared having a fixed concen-tration (33 120583M) of doxorubicin hydrochloride and differentconcentrations of CT DNA Cyclic voltammetry of each suchprepared doxorubicin hydrochloride-CT DNA mixture wascarried out and change in cathodic peak current at minus690mV(versus AgAgCl saturated KCl) was used to constructbinding isotherms Cyclic voltammogram of doxorubicinhydrochloride in the absence and presence of differentamounts of CT DNA is shown in Figure 4 Under the sameexperimental conditions cyclic voltammetry of pure DNAshowed that there was neither any cathodic nor anodic peakin the potential range 00 to minus085V clearly indicating thatpure DNA was electrochemically inert in the potential rangeon a glassy carbon electrode Previous studies [25 26 34 35]also showed that CT DNA was electrochemically inactive ata glassy carbon electrode surface


O

O

O

O

O

O O

O

OOO

OH OH

OH

OH

OH OH

OH OH

OH

OH

OH

OH

Ominus

CH3

H

H

HH

HHH

HH3C H3C H3C

O

HOH

Ominus

O∙minus

∙

NH+3

OCH3

H

H

HH

HHH

NH+3

OCH3

H

H

HH

HHH

NH+3

+ 2

Scheme 1 Comproportionation of doxorubicin hydrochloride dianion and doxorubicin hydrochloride to semiquinone

minus08 minus06 minus04 minus02 00minus20

minus15

minus10

minus5

0

5

10

15

20

25

(Dashed line simulation result)

(Solid lines experimental results)

7

1

Potential (V)

Curr

ent (120583

A)

Figure 2 Cyclic voltammograms (solid lines 1ndash7) of doxorubicinhydrochloride in aqueous buffer at pH 74 on glassy carbon elec-trode [Doxorubicin hydrochloride] = 33 120583Mand [NaCl] = 160mMScan rates = 1 Vsminus1 (Curve-1) 05 V sminus1 (Curve-2) 04 Vsminus1 (Curve-3) 03 Vsminus1 (Curve-4) 02 Vsminus1 (Curve-5) 01 Vsminus1 (Curve-6) and005Vsminus1 (Curve-7) temperature = 25∘C Dashed line simulationresult Simulation parameters 119896

119904= 003 cm sminus1 120572 = 05 119864 =

minus0675V (versus AgAgCl saturated KCl 0199V) 1198630= 3 times 10

minus560times10

minus5 10times10minus5 cm2 sminus1 for doxorubicin hydrochloride quinonedianion and semiquinone respectively and electrode area 119860 =01256 cm2 119896

119891= 10 times 10

3 and 119896119887= 4 times 10

2 for the homogeneouschemical reaction (Scheme 1)

To find the reversibility of the reduction of doxorubicinhydrochloride in the presence of DNA a plot of the ratioof the anodic to cathodic peak current (119868pa119868pc) versus thelogarithm of scan rate (log v) (Figure 3) is considered whichshows that the 119868pa119868pc ratio is less than unity for lower scanrates and it is almost unity at higher scan rates [32] clearlysuggesting that the reduction is quasireversible at lower scanrates and it is reversible at higher scan rates However fromFigure 3 it is evident that the reduction of doxorubicinhydrochloride at lower scan rates in the presence of DNAis much more reversible in nature in comparison to thatfound in the absence of DNA It is important to note that

minus08minus12 minus04 0

08

12

04

0

(IpaI

pc)

log V (Vsminus1)

Figure 3 Plot of the ratio of anodic to cathodic peak current(119868pa119868pc) versus the logarithm of scan rate (log v) for the reductionof doxorubicin hydrochloride in aqueous buffer at pH 74 on glassycarbon electrode in the absence (e) and presence (998771) of 20 120583M ofCT DNA

the initial concentration of doxorubicin hydrochloride in theelectrochemical experiments in the absence and presenceof DNA was the same (ie 33 120583M) However owing tothe binding of doxorubicin hydrochloride with DNA theconcentration of free doxorubicin hydrochloride molecules(quinone) is significantly reduced in the presence of DNAwhich means that the chance of the comproportionationreaction between quinone dianion and quinone (Scheme 1)is decreased and as a result the concentration of quinonedianion species is significantly higher in this case This leadsto an increase in the reversibility of the reduction of thecompound in the presence of DNA

With increase in concentration of CT DNA the cathodicpeak current for the reduction of doxorubicin hydrochloridedecreases (Figure 4) It is necessary to mention that such adecrease in the cathodic peak current was due to interactionof doxorubicin hydrochloride with DNA and not because ofany blockage of the electrode surface by an adsorbed layerof DNA To verify this cyclic voltammetry of potassium


minus08 minus06 minus04 minus02 00

minus2

0

2

4

1

6

6

Potential (V)

Curr

ent (120583

A)

Figure 4 Cyclic voltammogram of doxorubicin hydrochloride inthe absence (1) and presence of different CT DNA concentrations199 120583M (2) 5931 120583M (3) 982 120583M (4) 17447 120583M (5) and 23085 120583M(6) Scan rate = 0100Vsminus1 pH = 74 [NaCl] = 0160M andtemperature = 25∘C

ferricyanide was carried out in the absence and presence ofdifferent amounts of CT DNA under similar experimentalconditions It was observed that although the concentrationof CT DNA was increased the cathodic peak current ofpotassium ferricyanide did not decrease indicating that therewas no adsorption of DNA onto the glassy carbon electrodeThe fact that in case of the actual experiment doxorubicinhydrochloride interacted with added DNA for which itseffective concentration in solution decreased was thereforesubstantiated In a previousDNA interaction study Radi et al[35] carried out a similar ferricyanide experiment to establishthat cyclic voltammetric behavior of their studied compoundwas not affected by addition of a very large excess of DNAand that decrease in peak current of the compound was dueto interaction of the compound with DNA and not owing toadsorption

Results of DNA titration using cyclic voltammetry wereanalyzed by themethod of nonlinear fitting [25 26] To do sothe following compound-DNA equilibrium was considered[36ndash39]

119871 + 119863 999445999468 119871119863

119870

119889=

[119871] [119863]

[119871119863]

=

[119871

0] minus [119871119863] [1198630

] minus [119871119863]

119871119863

(2)

where 119871 represents doxorubicin hydrochloride 119863 representsCT DNA and 119871119863 represents the doxorubicin hydrochloride-DNA adduct The dissociation constant (119870

119889) = 1119870 119870

being apparent binding constant of the compound to DNA[1198710] = initial concentration of doxorubicin hydrochloride =

[doxorubicin hydrochloride]0= 33 120583M (which was kept

constant during the titration experiment) [1198630] = [DNA]

= concentration of DNA added to an aliquot If 1198620is the

initial concentration of doxorubicin hydrochloride that is

119862

0= [1198710] = [doxorubicin hydrochloride]

0and 119862

119863= [1198630] =

[DNA] = the concentration of DNA added to an aliquot then

119870

119889=

119862

0minus [119871119863] 119862119863

minus [119871119863]

[119871119863]

(3)

Studies carried out earlier already established the factthat the 910-anthraquinone unit of anthracyclines bindsto DNA by intercalation owing to its planar hydrophobicstructure [40 41] and that doxorubicin hydrochloride-DNAadduct was not electroactive [11 34] Since there was nointerference from doxorubicin hydrochloride-DNA adduct[34] the cathodic peak current (119868pc) was linearly proportionalto the concentration of free that is nonbound doxorubicinhydrochloride (see (1))Using this linear relationship betweencathodic peak current and concentration of doxorubicinhydrochloride and applying the same analogy one applies forabsorption and fluorescence the following parameters weredefined [36ndash39]Δ119868 = (119868pc

0minus 119868pc) = change in cathodic peak current (119868pc)

of doxorubicin hydrochloride at minus690mV (versus AgAgClsaturated KCl) upon each addition of CTDNA for each pointof the titration 119868pc

0 and 119868pc are cathodic peak current ofdoxorubicin hydrochloride at minus690mV (versus AgAgCl sat-urated KCl) in the absence and presence of different amountsof CT DNA respectively Δ119868max is the same parameter whendoxorubicin hydrochloride was totally bound to CT DNATherefore (Δ119868Δ119868max) denotes the fraction of doxorubicinhydrochloride bound to DNA and one gets (Δ119868Δ119868max) C0= [LD] = [DNA-doxorubicin hydrochloride] Therefore itcan be said that at any point of the titration cathodic peakcurrent was due to the contribution of free doxorubicinhydrochloride only Putting the value of [LD] in (3) one gets

119870

119889=

[119862

0minus (Δ119868Δ119868max) 1198620] [119862119863 minus (Δ119868Δ119868max) 1198620]

(Δ119868Δ119868max) 1198620 (4)

119862

0(

Δ119868

Δ119868max)

2

minus (119862

0+ 119862

119863+ 119870

119889) (

Δ119868

Δ119868max) + 119862

119863= 0 (5)

Determination of119870119889using (5) requires the value ofΔ119868max

which was determined by double reciprocal plot (Figure 5)using [25 26]

1

Δ119868

=

1

Δ119868max+

119870

119889

Δ119868max (119862119863 minus 1198620) (6)

This double reciprocal plot (Figure 5) was also used todetermine the apparent dissociation constant (119870

119889) that was

also determined using nonlinear curve fit analysis using (5)The apparent binding constant (119870app = 1119870119889) obtained fromthe double reciprocal plot (see (6) Figure 5) was found to be(166plusmn012)times10

4Mminus1The correlation coefficient for this plotwas observed to be 000037 The parameters are summarizedin Table 2

All the experimental points in the binding isothermswere fitted by least-square analysis The basic assumptionwas that the concentration of doxorubicin hydrochloride was33 120583M (kept constant during titration) CT DNA concen-tration was 10-fold greater than doxorubicin hydrochloride


Table 2 Evaluated electrochemical parameters binding parameters reduced chi square and correlation coefficient for the plots

Diffusion coefficient Double reciprocal plot Nonlinear fit Binding site sizeIntrinsic bindingconstant 1198701015840

(Mminus1)119863

0

(cm2 sminus1)

Reduced chisquare for the

plot119870app (M

minus1)Correlationcoefficient for

the plot119870app (M


the plot119899 (bases)

Reduced chisquares forthe plot

307 times 10minus5 01135 (166 plusmn 012) times 104 000037 (181 plusmn 015) times 104 00022 580 plusmn 040 09849 and08914 (105 plusmn 010) times 105

0050040030020010

07

06

05

04

03

02

1(CD minus C0) (120583Mminus1)

1ΔI

(120583Aminus1)

Figure 5 Double reciprocal plot of doxorubicin hydrochloridemdashCT DNA interaction using cyclic voltammetry [doxorubicinhydrochloride] = 33 120583M pH = 74 [NaCl] = 0160M and temper-ature = 25∘C

Binding stoichiometry or binding site size was determinedfrom the point of intersection of two straight lines obtainedfrom the least-square fit plot of normalized increase ofΔ119868Δ119868max against ratio of concentrations of CT DNA (inbase) to doxorubicin hydrochloride [25 26] that was drawnconsidering points before and after saturation respectively[25 26] The point of intersection of the two straight linesindicates a ratio of the moles of DNA (in bases) to the moleof doxorubicin hydrochloride in which the saturation is justreached that is the number of DNA bases bound per dox-orubicin hydrochloride Figure 6 shows the binding isothermof doxorubicin hydrochloride and CT DNA The correlationcoefficient for this plot was observed as 00022 which isa measure of the extent to which the fitted line matcheswith the experimental data points The apparent bindingconstant (119870app = 1119870119889) was calculated using (5) as describedabove and obtained as (181 plusmn 015) times 104Mminus1 (Table 2) Theapparent binding constant obtained from double reciprocalplot (see (6) Figure 5) was (166 plusmn 012) times 104Mminus1 (Table 2)indicating that there was excellent agreement between thetwo approaches A previous study by Hajian and coworkers[42] showed apparent binding constant for the interactionof doxorubicin hydrochloride-CT DNA in phosphate bufferat pH 74 as (32 plusmn 024) times 104Mminus1 which was also in good

4003002001000

1

08

06

04

02

0

ΔIΔI m

ax

[DNA] (120583M)

Figure 6 Binding isotherm of doxorubicin hydrochloridemdashCTDNA interaction and corresponding nonlinear fit using cyclicvoltammetry [doxorubicin hydrochloride] = 33 120583M pH = 74[NaCl] = 0160M and temperature = 25∘C

agreement with our results The slight deviation between theprevious and present results may be due to different typesof measurements and different reaction media in the twostudies Figure 7 shows the plot of normalized increase ofΔ119868Δ119868max as a function of mole ratio of DNA to doxorubicinhydrochloride and provides the value for the binding sitesize (119899) = 580 plusmn 040 bases that is 290 plusmn 020 basepairs per molecule of doxorubicin hydrochloride (Table 2)The reduced chi square values for the plot of straight lines(Figure 7) before and after the saturation were found tobe 09849 and 08914 respectively which are the measureof compatibility of fitting of the experimental results withthe appropriate equation Knowing 119899 the intrinsic bindingconstant 1198701015840 that is (119870 times 119899) was obtained as (105 plusmn010) times 10

5Mminus1 (Table 2) A previous study had reportedintrinsic binding constant for the interaction of doxorubicinhydrochloride with CT DNA as 27 times 105Mminus1 at pH 74 [43]and binding site size as 31 plusmn 04 base pairs per drugmolecule[44] Thus previous results also justify ours and the methodsof measurements

4 Conclusion

Doxorubicin hydrochloride undergoes two-electronreversible reduction generating a cathodic peak atminus690 plusmn 5mV (versus AgAgCl saturated KCl) with


8 106420

1

08

06

04

02

0

ΔIΔI m

ax

[DNA][Dox Hyd]

Figure 7 Plot of normalized increase of cathodic peak current as afunction of mole ratio of CT DNA to doxorubicin hydrochloride

a corresponding anodic peak at minus640 plusmn 5mV (versusAgAgCl saturated KCl) at pH 74 The formal reductionpotential (119864

12) was found to be minus665 plusmn 5mV (versus

AgAgCl saturated KCl) The reduced species undergoescomproportionation with the neutral molecule to formsemiquinone leading to irreversible reduction at lower scanrate established by simulation Cathodic peak current fordoxorubicin hydrochloride decreased as calf thymus DNAwas gradually increased in solutions containing a fixedconcentration of doxorubicin hydrochloride The decreasein peak current was used to evaluate binding parametersfor the interaction of the drug to CTDNA Intrinsic bindingconstant and bind site size were (105 plusmn 010) times 105Mminus1 and290 plusmn 020 base pairs of DNA per molecule of doxorubicinhydrochloride respectively Our results support earlier data

Abbreviation

CT DNA Calf thymus DNA

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

P S Guin is grateful to the University Grants CommissionNew Delhi India for funding the major research project (Fno 41-2252012 (SR) dated July 18 2012) S Das gratefullyacknowledges the financial support from DST Governmentof West Bengal [794(Sanc)1(10) STPSampT9G-232013] pro-vided in the form of a research project

References

[1] R Ng N Better and M D Green ldquoAnticancer agents andcardiotoxicityrdquo Seminars in Oncology vol 33 no 1 pp 2ndash142006

[2] G Minotti P Menna E Salvatorelli G Cairo and L GiannildquoAnthracyclines molecular advances and pharmacologie devel-opments in antitumor activity and cardiotoxicityrdquo Pharmaco-logical Reviews vol 56 no 2 pp 185ndash229 2004

[3] F Arcamone Doxorubicin Anticancer Antibiotic AcademicPress New York NY USA 1981

[4] V Behal ldquoBioactive products from Streptomycesrdquo Advances inApplied Microbiology vol 47 pp 113ndash156 2000

[5] C E Myers E G Mimnaugh G C Yeh and B K SinhaldquoBiochemical mechanisms of tumor cell kill by the anthracy-clinesrdquo in Anthracycline and Anthracenedione-Based AnticancerAgents J W Lown Ed pp 527ndash569 Elsevier Amsterdam TheNetherlands 1988

[6] L Gianni B Corden and C E Myers ldquoThe biochemical basesof anthracycline toxicity and antitumor actionrdquo in Reviewsin Biochemical Toxicology E Hodgson J R Bend and RM Philipot Eds vol 5 pp 1ndash82 Elsevier Amsterdam TheNetherlands 1983

[7] S E Lipshultz ldquoExposure to anthracyclines during childhoodcauses cardiac injuryrdquo Seminars in Oncology vol 33 no 8 ppS8ndashS14 2006

[8] M Binaschi G Capranico L Dal Bo and F Zunino ldquoRela-tionship between lethal effects and topoisomerase II-mediateddouble-stranded DNA breaks produced by anthracyclines withdifferent sequence specificityrdquoMolecular Pharmacology vol 51no 6 pp 1053ndash1059 1997

[9] D A Gewirtz ldquoA critical evaluation of the mechanisms ofaction proposed for the antitumor effects of the anthracyclineantibiotics adriamycin and daunorubicinrdquo Biochemical Phar-macology vol 57 no 7 pp 727ndash741 1999

[10] G E Kellogg J N Scarsdale and F A Fornari Jr ldquoIdentifi-cation and hydropathic characterization of structural featuresaffecting sequence specificity for doxorubicin intercalation intoDNAdouble-stranded polynucleotidesrdquoNucleic Acids Researchvol 26 no 20 pp 4721ndash4732 1998

[11] H M Zhang and N Q Li ldquoElectrochemical studies of theinteraction of adriamycin to DNArdquo Journal of Pharmaceuticaland Biomedical Analysis vol 22 no 1 pp 67ndash73 2000

[12] J B Chaires ldquoMolecular recognition of DNArdquo in Advances inDNA Sequence-Specific Agents L H Hurley and J B ChairesEds vol 2 pp 141ndash167 CT JAI Press Greenwich UK 1996

[13] A H-J Wang ldquoStructure-activity studies of anthracyclinemdashDNA complexerdquo inMolecular Aspects of Anticancer Drug-DNAInteractions S Neidle and M Waring Eds vol 1 pp 32ndash53CRC Press Boca Raton Fla USA 1993

[14] K StudzianMWasowskaM K Piestrzeniewicz et al ldquoInhibi-tion of RNA synthesis in vitro and cell growth by anthracyclineantibioticsrdquo Neoplasma vol 48 no 5 pp 412ndash418 2001

[15] F Leng and G H Leno ldquoDaunomycin disrupts nuclear assem-bly and the coordinate initiation ofDNA replication in Xenopusegg extractsrdquo Journal of Cellular Biochemistry vol 64 pp 476ndash491 1997

[16] MGigli SMDoglia JMMillot L Valentini andMManfaitldquoQuantitative study of doxorubicin in living cell nuclei bymicrospectrofluorometryrdquo Biochimica et Biophysica Acta vol950 no 1 pp 13ndash20 1988

[17] F Belloc F Lacombe P Dumain et al ldquoIntercalation of anthra-cyclines into living cell DNA analyzed by flow cytometryrdquoCytometry vol 13 no 8 pp 880ndash885 1992

[18] K E vanHoldeChromatin SpringerNewYorkNYUSA 1988


[19] A Rabbani M Iskandar and J Ausio ldquoDaunomycin-inducedunfolding and aggregation of chromatinrdquoThe Journal of Biolog-ical Chemistry vol 274 no 26 pp 18401ndash18406 1999

[20] T Lee T Lau and I Ng ldquoDoxorubicin-induced apoptosis andchemosensitivity in hepatoma cell linesrdquo Cancer Chemotherapyand Pharmacology vol 49 no 1 pp 78ndash86 2002

[21] R Abraham R L Basser and M D Green ldquoA risk-benefitassessment of anthracycline antibiotics in amtineoplastic ther-apyrdquo Drug Safety vol 15 no 6 pp 406ndash429 1996

[22] G P Stathopoulos N A Malamos I Dontas G Deliconstanti-nos D Perrea-Kotsareli and P E Karayannacos ldquoInhibitionof adriamycin cardiotoxicity by 5-fluorouracil a potential freeoxygen radical scavengerrdquoAnticancer Research vol 18 no 6 pp4387ndash4392 1998

[23] G Falcone W Filippelli B Mazzarella et al ldquoCardiotoxicity ofdoxorubicin effects of 21-aminosteroidsrdquo Life Sciences vol 63no 17 pp 1525ndash1532 1998

[24] G F Samelis G P Stathopoulos D Kotsarelis I Dontas CFrangia and P E Karayannacos ldquoDoxorubicin cardiotoxicityand serum lipid increase is prevented by dextrazoxane (ICRF-187)rdquo Anticancer Research vol 18 pp 3305ndash3309 1998

[25] P S Guin and P C Mandal ldquoElectrochemistry and VISspectroscopy on the binding of sodium 1 4-dihydroxy-9 10-anthraquinone-2-sulphonatendashCu(II) complex to calf thymusDNArdquo ChemPlusChem vol 77 pp 361ndash389 2012

[26] P S Guin P Das S Das and P C Mandal ldquoInteractionof calf thymus DNA with the Ni(II) complex of sodium 14-dihydroxy-910-anthraquinone-2-sulphonate a novel methodof analysis using cyclic voltammetryrdquo International Journal ofElectrochemistry vol 2012 Article ID 183745 10 pages 2012

[27] O Warburg and W Christian ldquoIsolation and crystallization ofenolaserdquo Biochemische Zeitschrift vol 310 pp 384ndash421 1942

[28] T Ossowski P Pipka A Liwo and D Jeziorek ldquoElectrochem-ical and UV-spectrophotometric study of oxygen and super-oxide anion radical interaction with anthraquinone derivativesand their radical anionsrdquo Electrochimica Acta vol 45 no 21 pp3581ndash3587 2000

[29] G M Rao J W Lown and J A Plambeck ldquoElectrochemicalstudies of antitumor antibiotics daunorubicin and adriamycinrdquoJournal of the Electrochemical Society vol 125 no 4 pp 534ndash539 1978

[30] C Molinier-Jumel B Malfoy J A Reynaud and G Aubel-Sadron ldquoElectrochemical study of DNA-anthracyclines inter-actionrdquo Biochemical and Biophysical Research Communicationsvol 84 no 2 pp 441ndash449 1978

[31] K Kano T Konse N Nishimura and T Kubota ldquoElectrochem-ical properties of adriamycin adsorbed on a mercury electrodesurfacerdquo Bulletin of the Chemical Society of Japan vol 57 no 9pp 2383ndash2390 1984

[32] P S Guin SDas andPCMandal ldquoElectrochemical Reductionof Sodium 1 4-dihydroxy-9 10-anthraquinone-2-sulphonate inAqueous and aqueous dimethyl formamide mixed solvent acyclic voltammetric studyrdquo International Journal of Electrochem-ical Science vol 3 pp 1016ndash1028 2008

[33] A J Bard and L R Faulkner Electrochemical Methods Funda-mentals and Applications John Wiley amp Sons 2001

[34] J B Hu J Shang and Q L Li ldquoElectrochemical studies ofadriamycin interaction with DNA and determination of DNAat a NIGC ion implantation modified electroderdquo AnalyticalLetters vol 33 no 9 pp 1843ndash1855 2000

[35] A Radi M A El Ries and S Kandil ldquoElectrochemical study ofthe interaction of levofloxacin with DNArdquo Analytica ChimicaActa vol 495 no 1-2 pp 61ndash67 2003

[36] P S Guin S Das and P C Mandal ldquoStudies on the forma-tion of a complex of Cu(II) with sodium 14-dihydroxy-910-anthraquinone-2-sulphonatemdashan analogue of the core unit ofanthracycline anticancer drugs and its interaction with calfthymus DNArdquo Journal of Inorganic Biochemistry vol 103 no12 pp 1702ndash1710 2009

[37] P S Guin S Das and P C Mandal ldquoSodium 1 4-dihydroxy-9 10-anthraquinone- 2-sulphonate interacts with calf thymusDNA in a way that mimics anthracycline antibiotics an electro-chemical and spectroscopic studyrdquo Journal of Physical OrganicChemistry vol 23 no 6 pp 477ndash482 2010

[38] P S Guin S Das and P C Mandal ldquoInteraction of 14-dihydroxy-910-anthraquinone with Calf thymus DNA a com-parison with anthracycline anticancer drugsrdquo Journal of Solu-tion Chemistry vol 40 no 3 pp 492ndash501 2011

[39] P Das P S Guin P C Mandal M Paul S Paul andS Das ldquoCyclic voltammetric studies of 124-trihydroxy-910-anthraquinone its interaction with calf thymus DNA and anti-leukemic activity on MOLT-4 cell lines a comparison withanthracycline anticancer drugsrdquo Journal of Physical OrganicChemistry vol 24 no 9 pp 774ndash785 2011

[40] W Y Li J G Xu X Q Guo Q Z Zhu and Y B ZhaoldquoDetermination of DNA by use of in situ photochemicalfluorescence probe sodium 910-anthraquinone-2-sulfonate as aphotochemical fluorescence proberdquoChemical Journal of ChineseUniversities vol 17 no 11 pp 1706ndash1707 1996

[41] Z Zhu and N Q Li ldquoElectrochemical studies of the interactionof 910-anthraquinone with DNArdquo Microchemical Journal vol59 no 2 pp 294ndash306 1998

[42] R Hajian N Shams and M Mohagheghian ldquoStudy on theinteraction between doxorubicin and deoxyribonucleic acidwith the use of methylene blue as a proberdquo Journal of theBrazilian Chemical Society vol 20 no 8 pp 1399ndash1405 2009

[43] F Frezard andAGarnier-Suillerot ldquoComparison of the bindingof anthracycline derivatives to purified DNA and to cell nucleirdquoBiochimica et Biophysica Acta vol 1036 no 2 pp 121ndash127 1990

[44] F Barcelo J Martorell F Gavilanes and J M Gonzalez-RosldquoEquilibrium binding of daunomycin and adriamycin to calfthymus DNA Temperature and ionic strength dependence ofthermodynamic parametersrdquo Biochemical Pharmacology vol37 no 11 pp 2133ndash2138 1988

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


[12] Intercalation causes a change in the shape of the DNAdouble helix thereby hindering DNA replication and RNAtranscription [12 14 15] Doxorubicin hydrochloride anddaunomycin were observed to accumulate rapidly in livingcells in the nuclei and bind to chromosomal DNA [16 17] Inthe cell nuclear DNA is associated with a variety of proteinsforming a nucleoprotein complex called chromatin [18]Accumulating evidence suggests these drugs interact withchromatin leading to chromatin unfolding and aggregationthat causes a change in structure and function of chromatin[19 20] The structural disruption is likely to interfere withmetabolic processes of DNA (replication and transcription)contributing to apoptosis induced by these drugs in cancercells [19 20]

In addition inside the cell anthracyclines react withcytochrome reductase in the presence of reduced nicoti-namide adenine dinucleotide phosphate (NADPH) to formsemiquinone radical intermediates which participate inoxidation-reduction reactions generating superoxide radi-cal anion (O

2

∙minus) that are converted to hydrogen peroxide

(H2O2) and hydroxyl radical (OH∙) [21 22]The free radicals

induce membrane-lipid peroxidation DNA strand scissionand direct oxidation of purine-pyrimidine bases thiols andamines and also contribute to cardiotoxicity The heart tissuebeing devoid of glutathione peroxidase is unable to convertsuperoxide radicals to oxygen and is therefore handicappedin this regard [23 24] Therefore the study of the redoxbehavior of anthracyclines is an important aspect as it helpsin the understanding of toxicities and therapeutic efficienciesThere are many reports on the redox behavior of these drugscorrelated with biological findings [25 26]The present studyis an attempt to revisit the redox behavior of doxorubicinhydrochloride in aqueous media at physiological pH (74)using cyclic voltammetry trying to understand the mecha-nism by which the molecule copes with reduction

2 Materials and Methods

Doxorubicin hydrochloride [980ndash1000 (HPLC)] was pur-chased from Sigma-Aldrich USA and it was used withoutany purification Since the quinonemoiety is sensitive to lightsolutions were prepared just before experiment and kept inthe dark CT DNA was purchased from Sisco Research Lab-oratory India After dissolution of CT DNA in buffer puritywas checked from absorbance ratio A

260A280

For all solu-tions this ratio was in the range 18 lt A

260A280gt 19 and

provided a good estimate of purity [27] DNA obtained frombiological sources has proteins as contaminants that absorbmore heavily around 280 nm owing to tryptophanwith somecontribution from tyrosine residues that decrease A

260A280

ratio An A260A280

ratio of 18-19 is considered ldquocleanrdquo withregard toDNApurity indicating that no further deproteiniza-tion is required [27] Concentration of DNA (in bases) wasdetermined taking 120598

260= 6600Mminus1 cmminus1 for CT DNA

Analytical grade Hepes buffer (N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid 10mM) (Spectrochem Pvt LtdIndia) was used to maintain a pH of sim74 in all experimentsSodium chloride (AR grade) obtained fromMerck Germanywas used to maintain ionic strength It was also used as

the supporting electrolyte for electrochemical experimentsAll solutions were prepared in triple distilled water Cyclicvoltammetry experiments were done using the conventionalthree-electrode system at 25∘C A glassy carbon electrodeof surface area 01256 cm2 was the working electrode whileplatinum wire and AgAgCl saturated KCl were used asthe counter and reference electrode respectively Experi-ments were carried out using EG amp G Potentiostat Model263A Voltammetric experiments on doxorubicin hydrochlo-ride were done in aqueous buffer (pHsim74) in the pres-ence and absence of CT DNA All experimental solutionswere degassed for 30min with high-purity argon beforecyclic voltammetrywas doneMicroprocessor pHIONmeter(pMX 3000) was used for recording pH of solutions UV-Visspectroscopic studies were carried out using a spectropho-tometer (modelUNICAMUV500)Apair of 10times10mmpathlength quartz cuvettes was used Simulation of electrochem-ical data was done to explain the probable mechanism forthe reactions using esp24b software developed by Dr CarloNervi [28] Simulation was carried out in planar geometryand cathodic currents were taken positive

3 Results and Discussion

31 Electrochemical Reduction of Doxorubicin HydrochlorideIn 15mM Hepes buffer (pHsim74) containing 160mM NaCldoxorubicin hydrochloride (33 120583M) undergoes two-electronreversible reduction generating a cathodic peak at minus690 plusmn5mV (versus AgAgCl saturated KCl) with the correspond-ing anodic peak at minus640 plusmn 5mV (versus AgAgCl saturatedKCl) (Figure 1) at a scan rate of 100mVsminus1 The formalreduction potential (119864

12) was minus665plusmn5mV (versus AgAgCl

saturated KCl) The variation of peak potentials with scanrate is shown in Figure 2 and the corresponding electro-chemical parameters are summarized in Table 1 In Figure 2the cyclic voltammograms were taken in the potential range0 to minus085V since beyond minus085V there is no reductionor oxidation under the experimental condition which isclear from Figure 1 Previous studies [29 30] showed that atacidic pH and neutral pH there were two reduction peaksone reversible two-electron reduction of the quinone unitof doxorubicin hydrochloride to quinone dianion speciesat minus600mV (versus calomel electrode) that is minus644mV(versus AgAgCl saturated KCl) and another irreversiblepeak at much more negative potential owing to hydrogenevolution [31] However the second irreversible reductionpeak vanished at alkaline pH [31 32] Since the presentstudy was carried out at pH 74 that is slightly alkaline thesecond cathodic peak was not observed at more negativepotential which was in accordance with previous results[31 32] A plot of the ratio of the anodic to cathodic peakcurrent (119868pa119868pc) versus the logarithm of scan rate (log v)(Figure 3) shows that the 119868pa119868pc ratio is less than unityin case of lower scan rate while at higher scan rate itis almost unity [32] This suggests that the reduction ofdoxorubicin hydrochloride at physiological pH at lower scanrates was quasireversible while at higher scan rate it wasalmost reversible Using these observations for low and highscan rates a simulation study was performed to understand





12(mV)



119868pc = 04463(119865

3

RT)

12

119899

32119860

0119863

0

12119862

0V12 (1)




0

2

4

6

8

10

Potential (V)

080604020

16

12

8

4

0Cu

rren

t (120583

A)

12 (Vsminus1)12

I pctimes106

(A)




O

O

O

O

O

O O

O

OOO

OH OH

OH

OH

OH OH

OH OH

OH

OH

OH

OH

Ominus

CH3

H

H

HH

HHH

HH3C H3C H3C

O

HOH

Ominus

O∙minus

∙

NH+3

OCH3

H

H

HH

HHH

NH+3

OCH3

H

H

HH

HHH

NH+3

+ 2



minus15

minus10

minus5

0

5

10

15

20

25



7

1

Potential (V)

Curr

ent (120583

A)


119904= 003 cm sminus1 120572 = 05 119864 =


minus560times10


119891= 10 times 10

3 and 119896119887= 4 times 10




08

12

04

0

(IpaI

pc)

log V (Vsminus1)






minus2

0

2

4

1

6

6

Potential (V)

Curr

ent (120583

A)




119871 + 119863 999445999468 119871119863

119870

119889=

[119871] [119863]

[119871119863]

=

[119871

0] minus [119871119863] [1198630

] minus [119871119863]

119871119863

(2)


119889) = 1119870 119870






119862


0and 119862

119863= [1198630] =


119870

119889=

119862

0minus [119871119863] 119862119863

minus [119871119863]

[119871119863]

(3)





119870

119889=

[119862


(Δ119868Δ119868max) 1198620 (4)

119862

0(

Δ119868

Δ119868max)

2

minus (119862

0+ 119862

119863+ 119870

119889) (

Δ119868

Δ119868max) + 119862

119863= 0 (5)



1

Δ119868

=

1

Δ119868max+

119870

119889

Δ119868max (119862119863 minus 1198620) (6)


119889) that was







(Mminus1)119863

0

(cm2 sminus1)


plot119870app (M







0050040030020010

07

06

05

04

03

02


1ΔI

(120583Aminus1)



4003002001000

1

08

06

04

02

0

ΔIΔI m

ax

[DNA] (120583M)




4 Conclusion



8 106420

1

08

06

04

02

0

ΔIΔI m

ax

[DNA][Dox Hyd]





Abbreviation




Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





12(mV)



119868pc = 04463(119865

3

RT)

12

119899

32119860

0119863

0

12119862

0V12 (1)




0

2

4

6

8

10

Potential (V)

080604020

16

12

8

4

0Cu

rren

t (120583

A)

12 (Vsminus1)12

I pctimes106

(A)




O

O

O

O

O

O O

O

OOO

OH OH

OH

OH

OH OH

OH OH

OH

OH

OH

OH

Ominus

CH3

H

H

HH

HHH

HH3C H3C H3C

O

HOH

Ominus

O∙minus

∙

NH+3

OCH3

H

H

HH

HHH

NH+3

OCH3

H

H

HH

HHH

NH+3

+ 2



minus15

minus10

minus5

0

5

10

15

20

25



7

1

Potential (V)

Curr

ent (120583

A)


119904= 003 cm sminus1 120572 = 05 119864 =


minus560times10


119891= 10 times 10

3 and 119896119887= 4 times 10




08

12

04

0

(IpaI

pc)

log V (Vsminus1)






minus2

0

2

4

1

6

6

Potential (V)

Curr

ent (120583

A)




119871 + 119863 999445999468 119871119863

119870

119889=

[119871] [119863]

[119871119863]

=

[119871

0] minus [119871119863] [1198630

] minus [119871119863]

119871119863

(2)


119889) = 1119870 119870






119862


0and 119862

119863= [1198630] =


119870

119889=

119862

0minus [119871119863] 119862119863

minus [119871119863]

[119871119863]

(3)





119870

119889=

[119862


(Δ119868Δ119868max) 1198620 (4)

119862

0(

Δ119868

Δ119868max)

2

minus (119862

0+ 119862

119863+ 119870

119889) (

Δ119868

Δ119868max) + 119862

119863= 0 (5)



1

Δ119868

=

1

Δ119868max+

119870

119889

Δ119868max (119862119863 minus 1198620) (6)


119889) that was







(Mminus1)119863

0

(cm2 sminus1)


plot119870app (M







0050040030020010

07

06

05

04

03

02


1ΔI

(120583Aminus1)



4003002001000

1

08

06

04

02

0

ΔIΔI m

ax

[DNA] (120583M)




4 Conclusion



8 106420

1

08

06

04

02

0

ΔIΔI m

ax

[DNA][Dox Hyd]





Abbreviation




Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


O

O

O

O

O

O O

O

OOO

OH OH

OH

OH

OH OH

OH OH

OH

OH

OH

OH

Ominus

CH3

H

H

HH

HHH

HH3C H3C H3C

O

HOH

Ominus

O∙minus

∙

NH+3

OCH3

H

H

HH

HHH

NH+3

OCH3

H

H

HH

HHH

NH+3

+ 2



minus15

minus10

minus5

0

5

10

15

20

25



7

1

Potential (V)

Curr

ent (120583

A)


119904= 003 cm sminus1 120572 = 05 119864 =


minus560times10


119891= 10 times 10

3 and 119896119887= 4 times 10




08

12

04

0

(IpaI

pc)

log V (Vsminus1)






minus2

0

2

4

1

6

6

Potential (V)

Curr

ent (120583

A)




119871 + 119863 999445999468 119871119863

119870

119889=

[119871] [119863]

[119871119863]

=

[119871

0] minus [119871119863] [1198630

] minus [119871119863]

119871119863

(2)


119889) = 1119870 119870






119862


0and 119862

119863= [1198630] =


119870

119889=

119862

0minus [119871119863] 119862119863

minus [119871119863]

[119871119863]

(3)





119870

119889=

[119862


(Δ119868Δ119868max) 1198620 (4)

119862

0(

Δ119868

Δ119868max)

2

minus (119862

0+ 119862

119863+ 119870

119889) (

Δ119868

Δ119868max) + 119862

119863= 0 (5)



1

Δ119868

=

1

Δ119868max+

119870

119889

Δ119868max (119862119863 minus 1198620) (6)


119889) that was







(Mminus1)119863

0

(cm2 sminus1)


plot119870app (M







0050040030020010

07

06

05

04

03

02


1ΔI

(120583Aminus1)



4003002001000

1

08

06

04

02

0

ΔIΔI m

ax

[DNA] (120583M)




4 Conclusion



8 106420

1

08

06

04

02

0

ΔIΔI m

ax

[DNA][Dox Hyd]





Abbreviation




Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



minus2

0

2

4

1

6

6

Potential (V)

Curr

ent (120583

A)




119871 + 119863 999445999468 119871119863

119870

119889=

[119871] [119863]

[119871119863]

=

[119871

0] minus [119871119863] [1198630

] minus [119871119863]

119871119863

(2)


119889) = 1119870 119870






119862


0and 119862

119863= [1198630] =


119870

119889=

119862

0minus [119871119863] 119862119863

minus [119871119863]

[119871119863]

(3)





119870

119889=

[119862


(Δ119868Δ119868max) 1198620 (4)

119862

0(

Δ119868

Δ119868max)

2

minus (119862

0+ 119862

119863+ 119870

119889) (

Δ119868

Δ119868max) + 119862

119863= 0 (5)



1

Δ119868

=

1

Δ119868max+

119870

119889

Δ119868max (119862119863 minus 1198620) (6)


119889) that was







(Mminus1)119863

0

(cm2 sminus1)


plot119870app (M







0050040030020010

07

06

05

04

03

02


1ΔI

(120583Aminus1)



4003002001000

1

08

06

04

02

0

ΔIΔI m

ax

[DNA] (120583M)




4 Conclusion



8 106420

1

08

06

04

02

0

ΔIΔI m

ax

[DNA][Dox Hyd]





Abbreviation




Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




(Mminus1)119863

0

(cm2 sminus1)


plot119870app (M







0050040030020010

07

06

05

04

03

02


1ΔI

(120583Aminus1)



4003002001000

1

08

06

04

02

0

ΔIΔI m

ax

[DNA] (120583M)




4 Conclusion



8 106420

1

08

06

04

02

0

ΔIΔI m

ax

[DNA][Dox Hyd]





Abbreviation




Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


8 106420

1

08

06

04

02

0

ΔIΔI m

ax

[DNA][Dox Hyd]





Abbreviation




Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article exploration of electrochemical intermediates ...research article exploration of...

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