)JOEBXJ1VCMJTIJOH$PSQPSBUJPO *OUFSOBUJPOBM ...downloads.hindawi.com/archive/2015/586231.pdfBehavior...

Research ArticleBehavior of Ethidium Bromide-Hoechst 33258-DNA andEthidium Bromide-Methylene Blue-DNA Triple Systems bymeans of UV Melting

Poghos O Vardevanyan Ara P Antonyan Marine A ParsadanyanMariam A Shahinyan and Gayane A Melkonyan

Department of Biophysics Faculty of Biology Yerevan State University A Manoogian 1 0025 Yerevan Armenia

Correspondence should be addressed to Poghos O Vardevanyan biophys depmailru

Received 23 September 2015 Revised 17 November 2015 Accepted 19 November 2015

Academic Editor Hicham Fenniri

Copyright copy 2015 Poghos O Vardevanyan et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The study of EtBr and H33258 interaction as well as EtBr and MB interaction with DNA has been carried out It was revealed thatat joint interaction the effect of two ligands on the change of melting thermodynamic parameters of EtBr-DNA-H33258 or EtBr-DNA-MB complexes is not an addition of separate interaction influences It was shown that at joint binding of EtBr and MB withDNA competition occurs while in the case of EtBr and H33258 the mutual strengthening of stabilizing effect of each of them onDNA double-stranded structure mainly takes place

1 Introduction

Noncovalent binding of different compounds (ligands) withDNA is an object of numerous studies since these substancespossess high biological activity and influence on many vitallyimportant processes occurring in the cells Some ligands aremutagens (particularly phenanthridine dye ethidium bro-mide (EtBr)) and transcription inhibitors (EtBr actinomycinand other antibiotics) which is conditioned by the ability ofthese compounds to form slowly dissociating complexes andas a consequence of this DNA untwisting is obstructed [1ndash7]

According to themain interactionmechanismwithDNAligands are divided into intercalators and groove bindingcompounds EtBr and methylene blue (MB) represent aspecial interest as intercalators Hoechst 33258 (H33258) asa groove binding ligand EtBr is a strong mutagen as well astranscription inhibitor [3 8 9]MB is applied to the treatmentof different diseases including malaria and different types oftumors [10ndash16] It was also revealed that MB may suppressviruses of AIDS and hepatitis B and hepatitis C in humanblood plasma [10ndash16] H33258 is a ligand specifically bindingto AT sequences in DNAminor grooveMoreover this ligand

is also used in medicine particularly as an antihelminthicagent [5 17ndash20]

Studies of DNA binding with these ligands showed manyaspects of such interactions [6ndash9 21] It was shown that manyligands bind to DNA mainly by several modes (multimodalligands) [8 11 20] Thus for EtBr and actinomycin D threemodes of binding with double-stranded (ds-) DNA andtwo modes with single-stranded (ss-) DNA were revealed[7 8] Nevertheless there are few researches dedicated tothe studies of simultaneous interaction of two differentligands with DNA despite the fact that DNA is surroundedby numerous compounds and many of them may at thesame time form complexes with it in the cells There aretheoretical studies where different models of formation ofseveral types of complexes of the same ligand with DNAare discussed moreover these models cannot be referredto joint interaction of DNA with different ligands that bindby various mechanisms From this point of view thosestudies may be significantly relevant in which the jointinteraction of two different ligands (either two intercalatorsand two groove binding ligands or one intercalator and onegroove binding) with DNA is investigated for nowadays new

Hindawi Publishing CorporationInternational Journal of SpectroscopyVolume 2015 Article ID 586231 5 pageshttpdxdoiorg1011552015586231

2 International Journal of Spectroscopy

multicomponent preparations having drug destination aresynthesized

The goal of the present work is to study the joint bindingof two intercalators EtBr and MB as well as EtBr and groovebinding compound H33258 with DNA

2 Materials and Methods

In the work the following preparations were used calfthymusDNA (ldquoSigmardquo USA)MB (ldquoAldrichrdquo USA) H33258(ldquoSigmardquo USA) and EtBr (ldquoServardquo Germany) All prepara-tions were used without additional purification in that theywere ultrapure Concentration of the used preparations wasdetermined by absorption method applying the followingmolar absorption coefficients 120576

260= 6600Mminus1 cmminus1 for calf

thymus DNA [22] 120576664= 76000Mminus1 cmminus1 forMB [16] 120576

343=

42000Mminus1 cmminus1 for H33258 [23] and 120576480= 5600Mminus1 cmminus1

for EtBr [24] Experiments were carried out at 120583 = 002Mionic strength of solution and pH asymp 70 The concentration ofDNA was equal to 76sdot10minus5M

The melting of DNA complexes with ligands as well asspectrophotometric measurements of absorption of prepa-ration solutions was carried out on PYE Unicam-SP8-100spectrophotometer (England) The heating of solutions ofcomplexes was carried out by program device SP 876 Series 2For spectrophotometric measurements quartz cuvettes withhermetically closed Teflon stoppers with 3mL volume and1 cm optic pathway length were used Melting was carried outat 120582 = 260 nm wavelength corresponding to DNA maximalabsorption Absorption values of complexes at melting weredisplayed on PC monitor through a program elaborated inLabVIEW medium The melting curves of complexes wereconstructed as described in [6]

DNA complexes with ligands were prepared according to119903 = [119862][119875] concentration ratio where119862 is the concentrationof one ligand (EtBr H33258 or MB) or two ligands (EtBr-MB or EtBr-H33258) and 119875 is the concentration of DNAphosphate groups Values of 119903 were changed in 0 lt 119903 le033 interval In the case of joint binding of two ligands withDNA concentration of each ligand was twice less to providesimilarity of values of 119903 with those corresponding to DNAcomplexes with the mentioned ligands

3 Results and Discussion

Among biologically active compounds interacting with DNAand significantly influencing its structural-functional charac-teristics EtBr takes a special place binding to DNA both invivo and in vitro and inhibiting replication and transcriptionprocesses [2ndash4 6]This ligand is a classical intercalator and anappropriate object formodelling ofmolecularmechanisms ofinteraction of different substances with DNAThe theoreticalmodel of DNA helix-coil transition in complex with EtBrwas elaborated which permits calculating heat value (Δ119867)throughout dependence of temperature (119879

119898) and melting

interval width (Δ119879) changes on ligand concentration as wellas carrying out thermodynamic analysis of DNA formedcomplexes with noncovalently binding ligands Comparisonof theory with experiment allows stating that EtBr forms

three types of complexes with ds-DNA intercalative semi-intercalative and electrostatic [2ndash4 6ndash9] Moreover thesemechanisms of EtBr binding to DNA are universal Particu-larly it was shown that EtBr intercalation into DNA dependson ionic strength and pH of solution or other external factors[8 25 26]

In the case of MB two (semi-intercalative and electro-static) modes of binding to DNA at 002M ionic strengthof solution and three (intercalative semi-intercalative andelectrostatic) modes at 0002M ionic strength have beenrevealed [25] which indicates that MB binding mechanismswith DNA are not universal (despite the fact that bothligands are intercalators) From this point of view the bindingpeculiarities of these ligandswithDNAat joint interaction areof special interest In the case of H33258 two binding modesstrong and weak were found out Moreover at relativelyhigh ionic strengths of solution (120583 gt 0004M) AT-specific(strong) and electrostatic modes of binding are shown at120583 le 0004M intercalative (strong) and electrostatic [5]Therefore in the case of MB andH33258 at least two bindingmodes with DNA are revealed one of which is electrostaticDisplaying of this mode practically does not depend onsolution ionic strength the other mode is specific and itsdisplaying depends on solution ionic strength

These properties of the mentioned ligands may havepractical value at both separate and joint interaction withDNA For this aim we have chosen two systems ofmulticom-ponent complexes EtBr-DNA-H33258 and EtBr-DNA-MBwhich were studied by the melting method and the changesof helix-coil transition parametersmdashdependencies of 120575(1119879

119898)

(120575(1119879119898) = 1119879

0minus 1119879

119898 where 119879

0and 119879

119898are melting

temperatures of DNA and DNA-ligand complexes resp) and120575(Δ119879119879

119898

2) (120575(Δ119879119879

119898

2) = Δ119879

01198790

2minus Δ119879119879

119898

2 where Δ1198790

and Δ119879 are melting interval widths of DNA and DNA-ligandcomplexes resp) on 119903 were obtained [27]

Dependence curves of 120575(1119879119898) on 119903 of EtBr-DNA-

H33258 complexes (curve 1) additive curve obtained throughsummation of 120575119879

119898values of DNA-EtBr and DNA-H33258

(curve 2) EtBr-DNA-MB (curve 3) complexes and additivecurve obtained through summation of values of 120575(1119879

119898) for

DNA-EtBr and DNA-MB (curve 4) complexes are presentedin Figure 1 Values of 120575(1119879

119898) and its dependence curves

on 119903 in the case of DNA-EtBr DNA-H33258 and DNA-MBcomplexes are presented in [5 8 25] As it is obvious fromFigure 1 curves 1 and 2 increasing in the whole interval ofchange of 119903 coincide with each other Moreover at jointinteraction of EtBr and H33258 with DNA values of 120575119879

119898

are higher (curve 1) depending on 119903 than in the case ofsummation of 120575119879

119898values (curve 2)

This experimental result indicates that stabilizing effect ofEtBr and H33258 on DNA double-stranded structure is not asimple sum of separate effects of these ligands It is importantthat values of 120575(1119879

119898) at joint binding of the mentioned

ligands are higher than those of their separate binding Moreinteresting result is obtained for EtBr-DNA-MB complexes(Figure 1 curves 3 and 4) Comparison of curve 3 in Figure 1with curves obtained for separate complexes of DNA-EtBrand DNA-MB shows that at joint interaction of these

International Journal of Spectroscopy 3

024681012141618

0

3

4

12

005 01 015 02 025 03 035r

120575(1Tm)10minus5

Figure 1 Curves of 120575(1119879119898) dependence on 119903 of EtBr-DNA-H33258

complexes (curve 1) additive curve obtained via summation of120575(1119879

119898) values of DNA-EtBr and DNA-H33258 (curve 2) EtBr-

DNA-MB (curve 3) complexes and additive curve obtained viasummation of 120575(1119879

119898) values of DNA-EtBr and DNA-MB (curve

4) complexes Concentration of DNA was equal to 76sdot10minus5M

ligands with DNA most probably competition for bindingsites emerges in consequence of which an area on curve3 (Figure 1) corresponding to change of 120575(1119879

119898) in 01 le

119903 le 02 interval appeared In the mentioned interval ofchange of 119903 120575(1119879

119898) practically remains constant while at

separate binding of EtBr and MB with DNA it increases inall interval of 119903 change These results indicate that at 002Mionic strength of solution at which EtBr andMB bind to DNAby intercalation mechanism H33258mdashAT specifically thecompetition is formed only between intercalators while EtBrand H33258 mutually strengthen the stabilizing effect of eachother Maintenance of this conclusion is that EtBr bindingconstant value (119870) with DNA by intercalation mechanismis higher by an order than value of 119870 at MB binding [7 8]Moreover value of119870 at EtBr binding intercalationmode withDNA is lower by two orders than value of 119870 in the case ofH33258 AT-specific binding to DNA [5 8]

Dependence curves of 120575(Δ119879119879119898

2) on 119903 of EtBr-DNA-

H33258 complexes (curve 1) additive curve obtained throughsummation of 120575Δ119879 values of DNA-EtBr and DNA-H33258(curve 2) EtBr-DNA-MB (curve 3) complexes and additivecurve obtained through summation of 120575Δ119879 values for DNA-EtBr and DNA-MB (curve 4) complexes are presented inFigure 2

Values of 120575(Δ119879119879119898

2) dependence on 119903 for DNA-EtBr

DNA-H33258 and DNA-MB complexes are presented in [58 25] It is obvious from Figure 2 that Δ119879 change at jointbinding of EtBr and H33258 with DNA is not sum alterationof this parameter (curves 1 and 2 in Figure 2) Moreover atlow concentrations of both parameters (in 0 lt 119903 le 004)EtBr effect on melting interval width of EtBr-DNA-H33258complex prevailed in 004 lt 119903 le 01 interval H33258effect starts prevailing and in 01 lt 119903 le 033 interval EtBreffect on Δ119879 strengthensThis fact is proved by data obtainedvia summation of corresponding dependencies of 120575(Δ119879119879

119898

2)

on 119903 (curves are not presented) for separate binding of thementioned ligands with DNA as a result of which curve 2 isformed which does not coincide with curve 1 (Figure 2)

0

2

4

6

8

0 005 01 015 02 025 03 035minus2

minus4

minus6

120575(Δ

TT

m2)10minus5

r

12

3

4

Figure 2 Curves of 120575(Δ119879119879119898

2) dependence on 119903 of EtBr-DNA-

H33258 complexes (curve 1) additive curve obtained via summationof 120575(Δ119879119879

119898

2) values of DNA-EtBr and DNA-H33258 (curve 2)

EtBr-DNA-MB (curve 3) complexes and additive curve obtained viasummation of 120575(Δ119879119879

119898

2) values of DNA-EtBr andDNA-MB (curve


As it is obvious from Figure 2 curve 3 which presentschange of 120575(Δ119879119879

119898

2) on 119903 at joint interaction of EtBr and

MB with DNA consists of two bell-like regions It is theresult of competition of EtBr and MB binding to DNABell-like dependence of 120575Δ119879 on 119903 is inherent for EtBr[7] which is conditioned by several simultaneous bindingmodes of this ligand with DNA [6] In the case of MB at002M ionic strength of solution two binding modes aredisplayed [25 28] Consequently at enhancement of EtBr andMB concentrations redistribution of EtBr and MB boundmolecules (displacement of MB bound molecules by EtBrmolecules) occurs during melting Moreover this effect ismore pronounced at relatively high concentrations of bothligandswhenDNAmolecules aremostly saturated by ligandsIt is obvious from Figure 2 that additive curve 4 radicallydiffers from curve 3 which proves the fact of the presenceof competition at joint interaction of two intercalators withDNA

4 Conclusion

Thus the obtained data indicate that at joint interactionof two different ligands with DNA their effect on meltingthermodynamic parameters of complexes is not the sumof separate effects of these ligands Moreover comparisonof obtained data with theoretical ones discussed in [29]indicates that joint interaction of two intercalators (EtBr andMB) or intercalator (EtBr) and nonintercalator (H33258)withDNA does not refer to one ligand interaction with DNA bytwo differentmodes Furthermore the obtained results revealthe competition at interaction of two different intercalatorswith DNA which is not found out at interaction of one ofthem by several modes It is ultimately interesting and impor-tant to highlight that at joint interaction of EtBr and H33258with DNA mutual strengthening of stabilizing effect of eachof them on double-stranded structure of DNA is revealedThese data may have practical significance at the screening ofnew preparations binding to DNA and influencing its variouscharacteristics These data are especially important because


they may be applied in therapy of drug preparations sincethe majority of them at their separate effect with high doseshave undesirable by-effects on cells From this point of viewthe joint application of different compounds having similarimpact on various characteristics of DNAmay give a positiveeffect in low doses (in the cases of both EtBr and H33258) orsuppress the effect of one of them by the other (as in the caseof MB and EtBr)

Conflict of Interests

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

References

[1] S Neible and M J Waving Molecular Aspects of Anti-CancerDrug Action Macmillan Publishers London UK 1993

[2] J B Chaires ldquoA thermodynamic signature for drug-DNAbindingmoderdquoArchives of biochemistry and biophysics vol 453no 1 pp 26ndash31 2006

[3] A N Lane and T C Jenkins ldquoThermodynamics of nucleicacids and their interactions with ligandsrdquo Quarterly Reviews ofBiophysics vol 33 no 3 pp 255ndash306 2000

[4] R R Monaco ldquoA novel major groove binding site in B formDNA for ethidium cationrdquo Journal of Biomolecular Structureand Dynamics vol 25 no 2 pp 119ndash125 2007

[5] P O Vardevanyan A P Antonyan M A Parsadanyan K VPirumyan A M Muradyan and A T Karapetian ldquoInfluenceof ionic strength on Hoechst 33258 binding with DNArdquo Journalof Biomolecular Structure and Dynamics vol 25 no 6 pp 641ndash646 2008

[6] P O Vardevanyan A P Antonyan G A Manukyan andA T Karapetyan ldquoStudy of ethidium bromide interactionpeculiarities with DNArdquo Experimental andMolecular Medicinevol 33 no 4 pp 205ndash208 2001

[7] R M Wadkins E A Jares-Erijman R Klement A Rudigerand T M Jovin ldquoActinomycin D binding to single-strandedDNA sequence specificity and hemi-intercalation model fromfluorescence and 1H NMR spectroscopyrdquo Journal of MolecularBiology vol 262 no 1 pp 53ndash68 1996

[8] P O Vardevanyan A P Antonyan M A Parsadanyan HG Davtyan and A T Karapetyan ldquoThe binding of ethidiumbromide with DNA interaction with single- and double-stranded structuresrdquo Experimental andMolecularMedicine vol35 no 6 pp 527ndash533 2003

[9] A N Veselkov S F Baranovkii L N Dymant et al ldquoComplex-formation of ethidium bromide with single-stranded non-complementar deoxitetranucleotide 51015840-d(ApApGpC)rdquo Molecu-lar Biology vol 31 no 2 pp 263ndash273 1997

[10] S Nafisi A A Saboury N Keramat J-F Neault and H-ATajmir-Riahi ldquoStability and structural features of DNA interca-lation with ethidium bromide acridine orange and methylenebluerdquo Journal of Molecular Structure vol 827 no 1ndash3 pp 35ndash432007

[11] M Hossain P Giri and G S Kumar ldquoDNA intercalation byquinacrine and methylene blue a comparative binding andthermodynamic characterization studyrdquo DNA and Cell Biologyvol 27 no 2 pp 81ndash90 2008

[12] R Hajian N Shams and M Mohagheghian ldquoStudy on theinteraction between doxorubicin and deoxyribonucleic acid

with the use of methylene blue as a proberdquo Journal of theBrazilian Chemical Society vol 20 no 8 pp 1399ndash1405 2009

[13] C Tong Z Hu and J Wu ldquoInteraction between methyleneblue and calfthymus deoxyribonucleic acid by spectroscopictechnologiesrdquo Journal of Fluorescence vol 20 no 1 pp 261ndash2672010

[14] R Rohs H Sklenar R Lavery and B Roder ldquoMethylene bluebinding toDNAwith alternating GC base sequence amodelingstudyrdquo Journal of the American Chemical Society vol 122 no 12pp 2860ndash2866 2000

[15] R Rohs and H Sklenar ldquoMethylene blue binding to DNAwith alternating AT base sequence minor groove binding isfavored over intercalationrdquo Journal of Biomolecular Structureand Dynamics vol 21 no 5 pp 699ndash711 2004

[16] M Hossain and G S Kumar ldquoDNA intercalation of methyleneblue and quinacrine new insights into base and sequencespecificity from structural and thermodynamic studies withpolynucleotidesrdquo Molecular BioSystems vol 5 no 11 pp 1311ndash1322 2009

[17] A Abu-Daya PM Brown and K R Fox ldquoDNA sequence pref-erences of several AT-selective minor groove binding ligandsrdquoNucleic Acids Research vol 23 no 17 pp 3385ndash3392 1995

[18] F Han N Taulier and T V Chalikian ldquoAssociation of theminor groove binding drug Hoechst 33258 with d(CGC-GAATTCGCG)

2 volumetric calorimetric and spectroscopic

characterizationsrdquo Biochemistry vol 44 no 28 pp 9785ndash97942005

[19] J A Parkinson J Barber K T Douglas J Rosamond and DSharples ldquoMinor-groove recognition of the self-complemen-tary duplex d(CGCGAATTCGCG)2 by Hoechst 33258 a high-field NMR studyrdquo Biochemistry vol 29 no 44 pp 10181ndash101901990

[20] K Steinmetzer and K-E Reinert ldquoMultimode interaction ofHoechst 33258 with eukaryotic DNA Quantitative analysisof the DNA conformational changesrdquo Journal of BiomolecularStructure and Dynamics vol 15 no 4 pp 779ndash791 1998

[21] M Hossain and G S Suresh Kumar ldquoThermodynamic profilesof the DNA binding of benzophenanthridines sanguinarineand ethidium a comparative study with sequence specificpolynucleotidesrdquo Journal of Chemical Thermodynamics vol 42no 10 pp 1273ndash1280 2010

[22] B Norden and F Tjerneld ldquoStructure of methylene blue-DNA complexes studied by linear and circular dichroismspectroscopyrdquo Biopolymers vol 21 no 9 pp 1713ndash1734 1982

[23] M V Mikhailov A S Zasedatelev and G V Durskii ldquoMecha-nism of AT base pairs recognition by molecules of dye lsquoHoechst33258rsquordquo Molekuliarnaia Biologiia vol 15 no 3 pp 690ndash7051981

[24] M JWaring ldquoThe effects of antimicrobial agents on ribonucleicacid polymeraserdquoMolecular Pharmacology vol 1 no 1 pp 1ndash131965

[25] P O Vardevanyan A P Antonyan L A Hambardzumyan MA Shahinyan and A T Karapetian ldquoThermodynamic analysisof DNA complexes withmethylene blue ethidium bromide andHoechst 33258rdquoBiopolymers and Cell vol 29 no 6 pp 515ndash5202013

[26] A P Antonyan ldquoAcidic denaturation of ethidium bromidecomplexes with DNArdquo Biological Journal of Armenia vol 65no 2 pp 31ndash36 2013

[27] A T Karapetian N M Mehrabian G A Terzikian et alldquoTheoretical treatment of melting of complexes of DNA with


ligands having several types of binding sites on helical andsingle-stranded DNArdquo Journal of Biomolecular Structure andDynamics vol 14 no 2 pp 275ndash283 1996

[28] P O Vardevanyan A P Antonyan M A Parsadanyan MA Shahinyan and L A Hambardzumyan ldquoMechanisms forbinding between methylene Blue and DNArdquo Journal of AppliedSpectroscopy vol 80 no 4 pp 595ndash599 2013

[29] A T Karapetian Z A Grigoryan Y S Mamasakhlisov MV Minasyants and P O Vardevanyan ldquoTheoretical treatmentof helix-coil transition of complexes DNA with two differ-ent ligands having different binding parametersrdquo Journal ofBiomolecular Structure and Dynamics pp 1ndash5 2015

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


multicomponent preparations having drug destination aresynthesized

The goal of the present work is to study the joint bindingof two intercalators EtBr and MB as well as EtBr and groovebinding compound H33258 with DNA

2 Materials and Methods

In the work the following preparations were used calfthymusDNA (ldquoSigmardquo USA)MB (ldquoAldrichrdquo USA) H33258(ldquoSigmardquo USA) and EtBr (ldquoServardquo Germany) All prepara-tions were used without additional purification in that theywere ultrapure Concentration of the used preparations wasdetermined by absorption method applying the followingmolar absorption coefficients 120576

260= 6600Mminus1 cmminus1 for calf

thymus DNA [22] 120576664= 76000Mminus1 cmminus1 forMB [16] 120576

343=

42000Mminus1 cmminus1 for H33258 [23] and 120576480= 5600Mminus1 cmminus1

for EtBr [24] Experiments were carried out at 120583 = 002Mionic strength of solution and pH asymp 70 The concentration ofDNA was equal to 76sdot10minus5M

The melting of DNA complexes with ligands as well asspectrophotometric measurements of absorption of prepa-ration solutions was carried out on PYE Unicam-SP8-100spectrophotometer (England) The heating of solutions ofcomplexes was carried out by program device SP 876 Series 2For spectrophotometric measurements quartz cuvettes withhermetically closed Teflon stoppers with 3mL volume and1 cm optic pathway length were used Melting was carried outat 120582 = 260 nm wavelength corresponding to DNA maximalabsorption Absorption values of complexes at melting weredisplayed on PC monitor through a program elaborated inLabVIEW medium The melting curves of complexes wereconstructed as described in [6]

DNA complexes with ligands were prepared according to119903 = [119862][119875] concentration ratio where119862 is the concentrationof one ligand (EtBr H33258 or MB) or two ligands (EtBr-MB or EtBr-H33258) and 119875 is the concentration of DNAphosphate groups Values of 119903 were changed in 0 lt 119903 le033 interval In the case of joint binding of two ligands withDNA concentration of each ligand was twice less to providesimilarity of values of 119903 with those corresponding to DNAcomplexes with the mentioned ligands

3 Results and Discussion

Among biologically active compounds interacting with DNAand significantly influencing its structural-functional charac-teristics EtBr takes a special place binding to DNA both invivo and in vitro and inhibiting replication and transcriptionprocesses [2ndash4 6]This ligand is a classical intercalator and anappropriate object formodelling ofmolecularmechanisms ofinteraction of different substances with DNAThe theoreticalmodel of DNA helix-coil transition in complex with EtBrwas elaborated which permits calculating heat value (Δ119867)throughout dependence of temperature (119879

119898) and melting

interval width (Δ119879) changes on ligand concentration as wellas carrying out thermodynamic analysis of DNA formedcomplexes with noncovalently binding ligands Comparisonof theory with experiment allows stating that EtBr forms

three types of complexes with ds-DNA intercalative semi-intercalative and electrostatic [2ndash4 6ndash9] Moreover thesemechanisms of EtBr binding to DNA are universal Particu-larly it was shown that EtBr intercalation into DNA dependson ionic strength and pH of solution or other external factors[8 25 26]

In the case of MB two (semi-intercalative and electro-static) modes of binding to DNA at 002M ionic strengthof solution and three (intercalative semi-intercalative andelectrostatic) modes at 0002M ionic strength have beenrevealed [25] which indicates that MB binding mechanismswith DNA are not universal (despite the fact that bothligands are intercalators) From this point of view the bindingpeculiarities of these ligandswithDNAat joint interaction areof special interest In the case of H33258 two binding modesstrong and weak were found out Moreover at relativelyhigh ionic strengths of solution (120583 gt 0004M) AT-specific(strong) and electrostatic modes of binding are shown at120583 le 0004M intercalative (strong) and electrostatic [5]Therefore in the case of MB andH33258 at least two bindingmodes with DNA are revealed one of which is electrostaticDisplaying of this mode practically does not depend onsolution ionic strength the other mode is specific and itsdisplaying depends on solution ionic strength

These properties of the mentioned ligands may havepractical value at both separate and joint interaction withDNA For this aim we have chosen two systems ofmulticom-ponent complexes EtBr-DNA-H33258 and EtBr-DNA-MBwhich were studied by the melting method and the changesof helix-coil transition parametersmdashdependencies of 120575(1119879

119898)

(120575(1119879119898) = 1119879

0minus 1119879

119898 where 119879

0and 119879

119898are melting

temperatures of DNA and DNA-ligand complexes resp) and120575(Δ119879119879

119898

2) (120575(Δ119879119879

119898

2) = Δ119879

01198790

2minus Δ119879119879

119898

2 where Δ1198790

and Δ119879 are melting interval widths of DNA and DNA-ligandcomplexes resp) on 119903 were obtained [27]

Dependence curves of 120575(1119879119898) on 119903 of EtBr-DNA-

H33258 complexes (curve 1) additive curve obtained throughsummation of 120575119879

119898values of DNA-EtBr and DNA-H33258

(curve 2) EtBr-DNA-MB (curve 3) complexes and additivecurve obtained through summation of values of 120575(1119879

119898) for

DNA-EtBr and DNA-MB (curve 4) complexes are presentedin Figure 1 Values of 120575(1119879

119898) and its dependence curves

on 119903 in the case of DNA-EtBr DNA-H33258 and DNA-MBcomplexes are presented in [5 8 25] As it is obvious fromFigure 1 curves 1 and 2 increasing in the whole interval ofchange of 119903 coincide with each other Moreover at jointinteraction of EtBr and H33258 with DNA values of 120575119879

119898

are higher (curve 1) depending on 119903 than in the case ofsummation of 120575119879

119898values (curve 2)

This experimental result indicates that stabilizing effect ofEtBr and H33258 on DNA double-stranded structure is not asimple sum of separate effects of these ligands It is importantthat values of 120575(1119879

119898) at joint binding of the mentioned

ligands are higher than those of their separate binding Moreinteresting result is obtained for EtBr-DNA-MB complexes(Figure 1 curves 3 and 4) Comparison of curve 3 in Figure 1with curves obtained for separate complexes of DNA-EtBrand DNA-MB shows that at joint interaction of these


024681012141618

0

3

4

12

005 01 015 02 025 03 035r

120575(1Tm)10minus5








119898) in 01 le







Values of 120575(Δ119879119879119898



119898

2)


0

2

4

6

8

0 005 01 015 02 025 03 035minus2

minus4

minus6

120575(Δ

TT

m2)10minus5

r

12

3

4




119898



119898




119898



4 Conclusion






References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


024681012141618

0

3

4

12

005 01 015 02 025 03 035r

120575(1Tm)10minus5








119898) in 01 le







Values of 120575(Δ119879119879119898



119898

2)


0

2

4

6

8

0 005 01 015 02 025 03 035minus2

minus4

minus6

120575(Δ

TT

m2)10minus5

r

12

3

4




119898



119898




119898



4 Conclusion






References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of














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