Спектрофотометрическое исследование механизмов связывания аналогов цитидина и бромистого этидия с ДНК

Для выяснения механизмов связывания цитидина и его биологически активных производных с ДНК исследовано их взаимодействие с ДНК в присутствии интеркалятора бромистого этидия (ЭБ). Проведен спектрофотометрический анализ электронных спектров поглощения смесей ЭБ–ДНК в присутствии цитидина и его аналого...

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Datum:	2007
Hauptverfasser:	Ермак, Е.Л., Круглова, Е.Б., Пальчиковская, Л.И., Алексеева, И.В.
Format:	Artikel
Sprache:	Russian
Veröffentlicht:	Інститут молекулярної біології і генетики НАН України 2007
Schriftenreihe:	Біополімери і клітина
Schlagworte:	Молекулярна біофізика
Online Zugang:	https://nasplib.isofts.kiev.ua/handle/123456789/157523
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Zitieren:	Спектрофотометрическое исследование механизмов связывания аналогов цитидина и бромистого этидия с ДНК / Е.Л. Ермак, Е.Б. Круглова, Л.И. Пальчиковская, И.В. Алексеева // Біополімери і клітина. — 2007. — Т. 23, № 6. — С. 529-537. — Бібліогр.: 32 назв. — рос., англ.

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spelling	nasplib_isofts_kiev_ua-123456789-1575232025-02-09T11:34:32Z Спектрофотометрическое исследование механизмов связывания аналогов цитидина и бромистого этидия с ДНК Спектрофотометричне дослідження механізмів зв’язування аналогів цитидину і бромистого етидію з ДНК Spectrophotometrical study of mechanisms of cytidine analogues and ethidium bromide binding with DNA Ермак, Е.Л. Круглова, Е.Б. Пальчиковская, Л.И. Алексеева, И.В. Молекулярна біофізика Для выяснения механизмов связывания цитидина и его биологически активных производных с ДНК исследовано их взаимодействие с ДНК в присутствии интеркалятора бромистого этидия (ЭБ). Проведен спектрофотометрический анализ электронных спектров поглощения смесей ЭБ–ДНК в присутствии цитидина и его аналогов в широкой области длин волн и концентраций ДНК. Аналоги цитидина, содержащие дополнительный атом азота в гетероцикле (6AZC, AZAfur и AZAxyl), конкурируют с ЭБ за места связывания на ДНК. Константы ассоциации и параметры мест связывания образующихся комплексов для этих производных рассчитаны с помощью программ оптимизации спектров поглощения смесей ЭБ–ДНК–нуклеозид COMP и DALSMOD. Немодифицированные по цитозиновому кольцу нуклеозиды (цитидин и Ara-C) не являются конкурентами ЭБ за места связывания на ДНК, однако в их присутствии изменяются концентрационные зависимости кривых титрования ЭБ в области низких концентраций ДНК. Это можно объяснить влиянием упомянутых нуклеозидов на структурные или конформационные изменения матрицы ДНК в присутствии ЭБ в области низких значений P/DЭБ, где P/DЭБ – отношение общих концентраций ДНК и ЭБ. Для з’ясування механізмів зв’язування цитидину і його біологічно активних похідних з ДНК досліджено їхню взаємодію з ДНК у присутності інтеркалятора бромистого етидію (ЕБ). Здійснено детальний спектрофотометричний аналіз електронних спектрів поглинання сумішей ЕБ–ДНК за присутності цитидину і його аналогів у широкій області довжин хвиль і концентрацій ДНК. Аналоги цитидину, що містять додатковий атом азоту у гетероциклі (6AZC, AZAfur та AZAxyl), конкурують з ЕБ за місця зв’язування на ДНК. Константи асоціації і величини місць зв’язування утворюваних комплексів для цих похідних розраховано нами за допомогою програм оптимізації спектрів поглинання сумішей ЕБ–ДНК–нуклеозид COMP та DALSMOD. Немодифіковані по цитозиновому кільцю нуклеозиди (цитидин та Ara-C) не є конкурентами ЕБ за місця зв’язування на ДНК, але у їхній присутності змінюється характер концентраційних залежностей кривих титрування в області низьких концентрацій ДНК. Це можна пояснити впливом зазначених нуклеозидів на структурні та конформаційні зміни матриці ДНК за присутності ЕБ в області низьких значень P/DЕБ, де P/DEБ – відношення загальних концентрацій ДНК та EБ. To study the mechanisms of cytidine and its biologically active analogues binding to DNA we analyzed the binding of these ligands to the DNA in the presence of well-known intercalator ethidium bromide (EtBr). Thereto, we carried out the detailed spectrophotometric research of EtBr-DNA mixtures absorption in the presence of cytidine and its analogues in the wide range of wavelengths and DNA concentrations. Cytidine derivatives containing azagroup in the cytosine ring (6AZC, AZAfur, and AZAxyl) compete with EtBr for the DNA binding sites. The binding constants and binding site sizes of the ligand-DNA complexes were calculated via absorption spectra optimization programs COMP and DALSMOD. Unmodified in cytosine ring ligands (cytidine and Ara-C) do not compete with EtBr for the DNA binding sites, however they contribute to the change of concentration dependencies of titration curves in the region of low DNA concentrations. 2007 Article Спектрофотометрическое исследование механизмов связывания аналогов цитидина и бромистого этидия с ДНК / Е.Л. Ермак, Е.Б. Круглова, Л.И. Пальчиковская, И.В. Алексеева // Біополімери і клітина. — 2007. — Т. 23, № 6. — С. 529-537. — Бібліогр.: 32 назв. — рос., англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000788 https://nasplib.isofts.kiev.ua/handle/123456789/157523 577.323:539.6.199 ru Біополімери і клітина application/pdf application/pdf Інститут молекулярної біології і генетики НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	Russian
topic	Молекулярна біофізика Молекулярна біофізика
spellingShingle	Молекулярна біофізика Молекулярна біофізика Ермак, Е.Л. Круглова, Е.Б. Пальчиковская, Л.И. Алексеева, И.В. Спектрофотометрическое исследование механизмов связывания аналогов цитидина и бромистого этидия с ДНК Біополімери і клітина
description	Для выяснения механизмов связывания цитидина и его биологически активных производных с ДНК исследовано их взаимодействие с ДНК в присутствии интеркалятора бромистого этидия (ЭБ). Проведен спектрофотометрический анализ электронных спектров поглощения смесей ЭБ–ДНК в присутствии цитидина и его аналогов в широкой области длин волн и концентраций ДНК. Аналоги цитидина, содержащие дополнительный атом азота в гетероцикле (6AZC, AZAfur и AZAxyl), конкурируют с ЭБ за места связывания на ДНК. Константы ассоциации и параметры мест связывания образующихся комплексов для этих производных рассчитаны с помощью программ оптимизации спектров поглощения смесей ЭБ–ДНК–нуклеозид COMP и DALSMOD. Немодифицированные по цитозиновому кольцу нуклеозиды (цитидин и Ara-C) не являются конкурентами ЭБ за места связывания на ДНК, однако в их присутствии изменяются концентрационные зависимости кривых титрования ЭБ в области низких концентраций ДНК. Это можно объяснить влиянием упомянутых нуклеозидов на структурные или конформационные изменения матрицы ДНК в присутствии ЭБ в области низких значений P/DЭБ, где P/DЭБ – отношение общих концентраций ДНК и ЭБ.
format	Article
author	Ермак, Е.Л. Круглова, Е.Б. Пальчиковская, Л.И. Алексеева, И.В.
author_facet	Ермак, Е.Л. Круглова, Е.Б. Пальчиковская, Л.И. Алексеева, И.В.
author_sort	Ермак, Е.Л.
title	Спектрофотометрическое исследование механизмов связывания аналогов цитидина и бромистого этидия с ДНК
title_short	Спектрофотометрическое исследование механизмов связывания аналогов цитидина и бромистого этидия с ДНК
title_full	Спектрофотометрическое исследование механизмов связывания аналогов цитидина и бромистого этидия с ДНК
title_fullStr	Спектрофотометрическое исследование механизмов связывания аналогов цитидина и бромистого этидия с ДНК
title_full_unstemmed	Спектрофотометрическое исследование механизмов связывания аналогов цитидина и бромистого этидия с ДНК
title_sort	спектрофотометрическое исследование механизмов связывания аналогов цитидина и бромистого этидия с днк
publisher	Інститут молекулярної біології і генетики НАН України
publishDate	2007
topic_facet	Молекулярна біофізика
url	https://nasplib.isofts.kiev.ua/handle/123456789/157523
citation_txt	Спектрофотометрическое исследование механизмов связывания аналогов цитидина и бромистого этидия с ДНК / Е.Л. Ермак, Е.Б. Круглова, Л.И. Пальчиковская, И.В. Алексеева // Біополімери і клітина. — 2007. — Т. 23, № 6. — С. 529-537. — Бібліогр.: 32 назв. — рос., англ.
series	Біополімери і клітина
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fulltext	Spectrophotometrical study of mech a nisms of cytidin an a logues and ethidium bro mide bind ing with DNA Ie.L. Iermak 1,2, O.B. Kruglova 2, L.H. Palchykovska3 , I.V. Alexeeva3 1V.N.Karasin Kharkiv Na tional Uni ver sity, 4 Svoboda sq., Kharkiv, Ukraine, 61077 2In sti tute for Radiophysics and Elec tron ics NAS of Ukraine, 12 Acad. Proskura Str., Kharkiv, Ukraine, 61085 3In sti tute of Mo lec u lar Bi ol ogy and Ge net ics NAS of Ukraine, 150 Zabolotny Str., Kyiv, Ukraine, 03143 e_ermak@ire.kharkov.ua To study the mech a nisms of cytidine and its bi o log i cally ac tive an a logues bind ing to DNA we an a lyzed the bind ing of these lig ands to DNA in the pres ence of well-known intercalator ethidium bro mide (EtBr). Thereto, we have car ried out the de tailed spec tro pho to met ric re search of EtBr-DNA mix tures ab sorp tion in the pres ence of cytidine and its an a logues in the wide range of wave lengths and DNA con cen tra tions. Cytidine de riv a tives con tain ing aza group in the cy to sine ring (6AZC, AZAfur and AZAxyl) com pete with EtBr for the DNA bind ing sites. The bind ing con stants and bind ing site sizes of ligand-DNA com plexes were cal cu lated via ab sorp tion spec tra op ti mi za tion pro grams COMP and DALSMOD. Un mod i fied in the cy to - sine ring lig ands (cytidine and Ara-C) do not com pete with EtBr for the DNA bind ing sites, how ever, they con trib ute to the change in con cen tra tion de pend en cies of ti tra tion curves in the re gion of low DNA con cen - tra tions. This phe nom e non can be ex plained by the cytidine and Ara-C in flu ence on the DNA con for ma tion in the pres ence of EtBr at low P/DEtBr val ues, where P/DEtBr is the phos phate/EtBr ra tio. Key words: cytidine an a logues, DNA, mod els of bind ing, spectrophotometry, ethidium bro mide. In tro duc tion. Nu cleo sides rep re sent the class of bi o - log i cally ac tive agents, in ter act ing with DNA and in - flu enc ing its func tion in cells. Many rep re sen ta tives of this group are the phar ma ceu ti cal med i ca tions of gene-di rected ac tion, widely used in the antitumour ther apy of leu ke mia and other dis eases [1-5]. In ter ac - tion of ther a peu ti cally ac tive nu cleo sides with DNA in cell causes in hi bi tion of nu cleic acid syn the sis and apoptosis [6]. For ex am ple, one of the most ef fec tive drugs used in the treat ment of leu ke mia, namely, cy to - sine arabinoside (Ara-C), is phosphorylated in side cells by their en zymes to cytotoxic form of cy to sine arabinoside triphosphate and is in cor po rated into DNA by DNA poly mer ase [7, 8]. Af ter Ara-C in cor po ra tion into DNA, fur ther DNA syn the sis is in hib ited, what leads to the cell death. An other cytidine (Cyd) an a - logue 6-azacytidine has antitumour and an ti vi ral ac tiv - ity [9-12]. Un like Ara-C, there are no ex act data on 6AZC bi o log i cal ac tion. In lit er a ture there is only de - scrip tion of in di rect in flu ence of 6AZC on vi rus re pro - duc tion in hi bi tion which re sults in de crease of vi rus DNA and polypeptide syn the sis [9, 10]. In hi bi tion of 529 ISSN 0233-7657. Biopolymers and cell. 2007. vol. 23. N 6. Translated from Ukrainian. © Ie.L. IERMAK, O.B. KRUGLOVA, L.H. PALCHYKOVSKA, I.V. ALEXEEVA, 2007 tu mor growth is also as sumed to be as so ci ated with de - crease of macromolecules syn the sis [11, 12]. Thus, the mech a nism of 6AZC in flu ence on DNA biosynthesis and 6AZC in ter ac tion with DNA re mains un known. In ad di tion to in cor po ra tion of nu cleo sides into DNA mol e cule with cell en zymes it is also pos si - ble that they in ter ca late into DNA or have the groove bind ing. Nev er the less, the mo lec u lar mech a nisms of their in ter ac tion with DNA still re main un clear. In this re spect, it is in ter est ing to study pos si ble mech a nisms of in ter ac tion of cytidine an a logues with DNA us ing new the o ret i cal (mod el ing of the pro cesses) and ex - per i men tal (com pet i tive bind ing) ap proaches. One of the in for ma tive meth ods of study on the ligand-DNA in ter ac tions is spectrophotometry which al lows not only de tect ing the ligand-DNA com plex for ma tion and but also re veal ing the char ac ter is tics of pos si ble mech a nisms of such in ter ac tion by ap pro pri - ate mod el ling ap proaches [13, 14]. As ab sorp tion spec tra of nu cleo sides are very sim - i lar to the nu cleic ac ids ab sorp tion spec tra, we have used the method of study of their com pet i tive bind ing to DNA in the pres ence of col ored la bel. We used ethidium bro mide (EtBr) as la bel-ligand, ab sorb ing in the VIS spec tra re gion. Ma te ri als and Meth ods. Com mer cial calf thy mus DNA has been pur chased from Serva. Ethidium bro - mide has been pur chased from Fluka (Swit zer land). Cytidine and its an a logues (cy to sine arabinoside (Ara-C), 6-azacytosine ribofuranoside (6AZC), 6-azacytosine tetrahydrofuril (AZAfur), 6-azacytosine xylofuranoside (AZAxyl)) have been syn the sized in the In sti tute of Mo lec u lar Bi ol ogy and Ge net ics NAS of Ukraine. Con cen tra tions of nu cleo sides have been de - ter mined by the weight method. Chem i cal struc tures of the nu cleo sides stud ied are shown in Fig.1. Con cen tra tions of DNA (Cp, moles of phos phates) and EtBr (CD) have been de ter mined by ab sorp tion spec - tros copy us ing mo lar ex tinc tion co ef fi cients at ab sorp - tion max i mum: e260 = 6.4´103 Ì-1 cm-1 [15] for calf thy - mus DNA and e480 = 5.85´103 M-1 cm-1 for EtBr [15]. All ab sorp tion mea sure ments have been car ried out on Specord M 40 (Ger many) spectrophotometer in ther mo - static quartz cells, hav ing light paths of 10 and 20 mm. Complexation stud ies have been car ried out in phos phate buffer (2.5´10-2Ì KH2PO4, 2.5´10-2Ì Na2HPO4, pH 6.86). P/D is phos phate/dye ra tio. All prep a ra tions have been used with out fur ther pu ri fi ca tion. As there was a pos si bil ity that cytidine and its an a - logues could make heteroassoñiates with EtBr, we have checked their in flu ence on the EtBr spec trum in the ab - sence of DNA. We have found that these nu cleo sides do not in flu ence EtBr spec trum and there fore there is no heteroassociation be tween these lig ands. Cal cu la tion of bind ing pa ram e ters of nucleoside-DNA com plexes (bind ing con stants and bind ing site sizes) has been per formed ac cord ing to two dif fer ent mod els of bind ing. In Model 1 only one type of nucleoside-DNA and EtBr-DNA com plexes is pre sumed. Val ues of lig ands bind ing sites n1 and n2 are al lowed to vary in a wide re - gion of val ues. Equi lib rium con cen tra tions of free and bound lig ands for ev ery mix ture with to tal con cen tra - IERMAK Ie. L. ET AL. 530 Fig.1 Chem i cal struc tures of cy to sine (Cyt) (1), cy to sine arabinoside (Ara-C) (2), 6-azacytosine ribofuranoside (6AZC) (3), 6-azacytosine tetrahydrofuril (AZAfur) (4), 6-azacytosine xylofuranoside (AZAxyl) (5) tions of lig ands and DNA CDi, Cpi, re spec tively, are cal - cu lated ac cord ing to equations (1)-(4): R m K R n R n R n R n R n1 1 1 1 1 2 2 1 1 2 2 1 1 1= - × - × - × - × + ´[ ] ´ - × - × +( );1 1 1 2 2 R n R n R (1) R m K R n R n R n R n R n2 2 2 1 1 2 2 1 1 2 2 1 1 2= - × - × - × - × + ´[ ] ´ - × - × +( );1 1 1 2 2 R n R n R (2) C m R C D P1 1 1 = + × ; (3) C m R C D P2 2 2 = + × . (4) Equa tions (1) and (2) de scribe the com pet i tive bind ing of two lig ands to DNA [17]. Equa tions (3) and (4) rep re sent the law of mass bal ance for lig ands. The fol low ing des ig na tions have been used in equa - tions (1) – (4): m1 and m2 are the equi lib rium con cen tra - tions of free lig ands, where in dex 1 is used for nu cleo - sides and in dex 2 for EtBr, R1 è R2 – are the shares of bound lig ands de ter mined as quo tient from the di vi sion of cor re spond ing com plexes con cen tra tions to CP, Ê1, Ê2 – are the bind ing con stants for lig ands 1 and 2 on bind ing sites n1 and n2 DNA bases per ligand, R is the sum (R1 + R2). In Model 2 bind ing of nucleoside to DNA is de - scribed with the as sump tion on the fixed value of bind - ing site size n1=1 (one nucleoside per one DNA phos - phate), when EtBr binds to DNA in a co op er a tive man - ner, i.e. w value is vary ing (w is co op er a tive pa ram e ter which char ac ter izes the prob a bil ity of ligand bind ing to the ad ja cent bind ing sites). Such model al lows tak ing into ac count changes in the spec trum of the bound EtBr when it does not have neigh bours (high P/DEtBr val ues) or when it may have neigh bours (low P/DEtBr val ues). To cal cu late the equi lib rium con cen tra tions of EtBr-DNA-nucleoside mix ture in this case we use the equa tion sys tem, anal o gous to the one de scribed above but in stead of equa tion (2) the McGhee and von Hippel equa tion for co op er a tive bind ing [18] is in tro duced. Such bind ing model was de scribed by us in [19]. The R1CP value is the sum of con cen tra tions of monomerically bound lig ands (with out neigh bours) and lig ands hav ing neigh bours. The con cen tra tion of the monomerically bound ligand is cal cu lated us ing the fol low ing re la tion Cm.b. = (R1 - g )2 / R1 ´ CD [17]. Such sep a ra tion of con cen tra tions is nec es sary be cause we as sume that monomerically bound lig ands and lig ands hav ing neigh bours can have dif fer ent ab sorp tion spec - tra. Cal cu la tion of the equi lib rium com po si tion of mix - tures and bind ing con stant val ues ac cord ing to Mod els 1 and 2 was car ried out via COMP [20] and DALSMOD [21, 22] op ti mi za tion pro grams, re spec - tively. COMP and DALSMOD op ti mi za tion pro grams have been de vel oped as new ver sions of the orig i nal DALS pro gram [23] by chang ing the pro ce dure of equi - lib rium con cen tra tion cal cu la tion. In COMP op ti mi za - tion pro gram the equa tions (1) – (4) are used in or der to cal cu late the equi lib rium con cen tra tions in the study on complexation of lig ands (or drugs) with poly meric ma - tri ces [19]. De tailed de scrip tion of DALSMOD op ti mi - za tion pro gram is given in [20, 21]. In these pro grams op ti mal val ues of mo lar ex tinc tion co ef fi cients, bind ing con stants and bind ing site sizes for each type of com - plex are cal cu lated through minimization of the sum of squares of de vi a tions of ex per i men tal ab sorp tions Aij 0 from cal cu lated ones Aij, in wide wave length and con - cen tra tion ranges. The val ues of ab sorp tion Aij are cal cu lated ac cord - ing to the Beer’s law: A l C ij jk ki k = × ×å e , where ejk is the mo lar ex tinc tion co ef fi cient of kth com - po nent in jth wave length, and cki is the equi lib rium con - cen tra tion of cor re spond ing com po nent in ev ery ith mix ture, l is the op ti cal path length. The op ti mi za tion pro ce dure is ter mi nated when fur - ther it er a tions of op ti mized pa ram e ters (Êi and eij) do not im prove the value of op ti mized func tion for each model tested. At the end of op ti mi za tion pro cess the val ues of both Q and Qlim Hamiltonian fac tors [23] are cal cu lated: Q A A A ij ij ij ijij = - åå{( ) / ) } ;/0 2 0 2 1 2 (5) Q e A ij ij ijij lim /{( ) / ) } ,= åå 2 0 2 1 2 (6) 531 SPECTROPHOTOMETRICAL STUDY OF CYTIDIN AN A LOGUES BIND ING WITH DNA where eij is the de vi a tion of absorbance in the ith mix ture cor rected to the 1% er ror in the to tal com po nent con - cen tra tions and the 0.005 op ti cal unit er ror in the mea - sure ment of ab sorp tion. Q and Qlim char ac ter ize the cor - re spon dence of the bind ing model to the ex per i men tal data. The se lected model (and cor re spond ing bind ing pa ram e ters n1, n2 and w) sat isfy the ex per i men tal ab - sorp tion data if Q<Qlim. Re sults and Dis cus sion. Fig.2, a shows the ab sorp - tion spec tra of EtBr-DNA mix tures in wide DNA con - cen tra tion range ((0 ¸ 2.5)·10-3 M). It can be seen that not all the spec tra of the stud ied P/DEtBr re gion go through the isobestic points (l = 390 nm and l = 510 nm). This is the ev i dence of for ma tion of more than one EtBr-DNA com plex. For ma tion of sev eral types of EtBr-DNA com plexes is con firmed by other au thors [24-26]. At the same time, many au thors in sist that at low P/DEtBr val ues when EtBr mol e cules bind closely to each other, the B>A conformational tran si tion of DNA mol e cule can be ob served [27-31]. As many au thors say that EtBr in ter ca lates into DNA, we can as sume that there is in ter ca la tion both in the ad ja cent base pairs and in ter ca la tion ac cord ing to the law of ex clu sion of the near est neigh bour. Ab sorp tion spec tra of these com - plexes are dif fer ent. This re sults in ap pear ance of the pseudoisobestic points on the EtBr-DNA ab sorp tion spec tra. Also ti tra tion curves of EtBr-DNA mix tures could have stages be cause of dif fer ent mo lar ex tinc tion co ef fi cients of com plexes (Fig.3, a, curves 1 and 3). Let us con sider the way cytidine and its an a logues ex ert in flu ence on EtBr bind ing to DNA. The EtBr-DNA ab sorp tion spec tra change weakly in the pres ence of nu cleo sides (Fig.2, b) but these changes can be clearly seen in the ti tra tion curves of EtBr-DNA mix tures in the ab sence and in the pres ence of these com pet ing lig ands (Fig. 3-5). All fig ures show ti tra tion curves at l = 340 nm. Spec tral changes at this wave length, be long ing to the sec ond ab sorp tion band with the ab sorp tion max i mum at l = 270 nm, are more sig nif i cant than in an other ab - sorp tion max i mum at l=480 nm. One can see from the fig ures that dif fer ent nu cleo sides cause dif fer ent changes of EtBr-DNA mix tures ti tra tion curves. Thus, in the pres ence of un mod i fied cy to sine an a logues (cytidine and AraC) (Fig.3), the stage on the EtBr-DNA-nucleoside ti tra tion curve be comes more ev i dent. But fur ther in crease of P/DEtBr value dif fer - ences in ti tra tion curves in the ab sence and in the pres - ence of these nu cleo tides is lower. One can as sume that these de riv a tives are not di rect com pet i tors of EtBr dur - ing its bind ing to DNA but in stead they change DNA hydration en vi ron ment and main tain DNA mol e cule in 532 IERMAK Ie. L. ET AL. Fig.2 Ab sorp tion spec tra of EtBr-DNA mix tures at Ñ EtBr = 1.145 ´ 10-4 , P/D = 0 (1), 1.0 (2), 2.0 (3), 5.0 (4), 21.7 (5) (a), EtBr-DNA-6AZC mix tures at Ñ EtBr = 9.865 ´ 10-5 Ì, Ñ 6AZC = 1.124 ´ 10-4 Ì, P/D = 0 (1), 1.1 (2), 2.7 (3), 7.3 (4), 13.4 (5) (b). A-like con for ma tion. As none of the mod els de scribed re veals conformational changes of DNA in the bind ing pro cess, we can not cal cu late the bind ing pa ram e ters of these de riv a tives with DNA. Nev er the less, we can as - sume that cytidine and AraC in flu ence EtBr bind ing to DNA in a dif fer ent way, for ex am ple, they can in ter act with DNA func tional groups which are not in volved in bind ing with EtBr but thus pro duce steri cal hin drance for EtBr bind ing to DNA mol e cule. As seen from Fig. 4 and 5, mod i fied cytidine an a - logues (6AZC, AZAfur and AZAxyl),hav ing aza group in cy to sine ring, com pete with EtBr for DNA bind ing sites. This is ev i dent from the in crease of the mix ture ab sorp tion caused by DNA con cen tra tion rise. Also stages on EtBr-DNA ti tra tion curves in the pres ence of these lig ands de crease, i.e. 6AZC, AZAfur, and AZAxyl bind to DNA in B-con for ma tion. Bind ing pa - ram e ters of com plexes of these lig ands with DNA in the pres ence of EtBr is cal cu lated us ing the mod els of bind - ing de scribed above. We have cal cu lated bind ing pa ram e ters of 6AZC, AZAfur and AZAxyl com plexes with DNA us - ing the Model 1 and COMP op ti mi za tion pro gram. As mo lar ex tinc tion co ef fi cients of the two EtBr com - plexes with DNA are sim i lar and in the pres ence of com pet ing nu cleo sides the amount of EtBr ag gre gates on DNA is de creased, in this model we as sume that EtBr forms only one type of com plexes with DNA. Val - ues of bind ing con stants and bind ing site sizes are shown in Table 1. It can be seen from Ta ble 1 that Model 1 is suf fi - cient to de scribe spec tral changes which take place in nu cle o tide-DNA-EtBr sys tems, as Q < Qlim in all cases. Also it is ev i dent that bind ing site sizes for all nu cleo - sides are ap prox i mately one DNA base per one nucleoside mol e cule. As EtBr forms two com plexes with DNA with two dif fer ent mo lar ex tinc tion co ef fi cients we have used Model 2 which takes this fact into ac count, as sign ing n1 = 1. Val ues of bind ing pa ram e ters for nu cleo sides cal - cu lated via Model 2 us ing DALSMOD op ti mi za tion pro gram are shown in Table 2. Model 2 is also suf fi cient to de scribe the nu cle o - tide-DNA-EtBr sys tem, as in all cases Q < Qlim. It can be seen that bind ing con stants of 6AZC-DNA and AZAxyl-DNA com plexes, cal cu lated via two dif fer ent bind ing mod els, co in cide. This fact ev i dences that 6AZC and AZAxyl are not sen si tive to dif fer ences of EtBr-DNA com plexes mo lar ex tinc tion co ef fi cients as they are com pet i tors of only sec ond type of com plexes. 533 SPECTROPHOTOMETRICAL STUDY OF CYTIDIN AN A LOGUES BIND ING WITH DNA Fig.3 Con cen tra tion de pend en cies of the ab sorp tion of EtBr-DNA mix tures (C EtBr = 1.145 ́ 10-4) (1, à, b) and EtBr-DNA-AraC (C EtBr = 4.7 ́ 10-5 , C AraC = 4.48 ´ 10-4) (2) (à) and EtBr-DNA-Cyt mix tures (C EtBr = 4.9 ´ 10-5, C Cyt = 1.3 ´ 10-4) (2) (b) at l=340 nm of P/D EtBr Dif fer ences in bind ing con stants of AZAfur-DNA com - plex are prob a bly re lated to the fact that in Model 1 for - ma tion of two EtBr-DNA com plexes with dif fer ent mo - lar ex tinc tion co ef fi cients is not taken into ac count, but AZAfur is a com pet i tor of both EtBr-DNA com plexes. Also it is sig nif i cant that bind ing con stants of lig ands with dif fer ent OH-groups po si tion in furanose ring vary con sid er ably, i.e. both struc ture and con for ma tion of glycoside frag ments are es sen tial for bind ing of these lig ands to DNA. As bind ing site sizes for 6AZC, AZAfur and AZAxyl com plexes with DNA are ap prox i mately one DNA base per ligand mol e cule, these nu cleo sides do not show AT- or GC- bind ing spec i fic ity and do not in - ter ca late into DNA be cause about 4-5 DNA bases per ligand are needed for the intercalative com plex. There - fore, one can as sume that 6AZC, AZAfur, and AZAxyl can in ter act with DNA by their azo groups (N-6 atom of threeazine bases) and DNA aminogroups. EtBr also in - ter acts with the DNA aminogroups while in ter ca lat ing into DNA [32]. Con clu sions.De tailed spec tro pho to met ric study of cytidine and its an a logues bind ing to DNA has shown that EtBr can be used as col ored la bel in spec tro pho to - met ric ab sorp tion stud ies in VIS re gion of spec tra. Anal y sis of nucleoside-DNA-EtBr ti tra tion curves has shown that un mod i fied cytidine an a logues (cytidine and AraC) do not com pete with EtBr for DNA bind ing sites. On the other hand, these nu cleo sides in flu ence EtBr bind ing to DNA at low P/DEtBr val ues. Mod i fied cytidine an a logues (6AZC, AZAfur and AZAxyl) com - pete with EtBr for DNA bind ing sites at high P/DEtBr val ues. 534 IERMAK Ie. L. ET AL. Fig.4 Con cen tra tion de pend en cies of the ab sorp tion of EtBr-DNA mix tures (C EtBr = 1.145 ´ 10-4) (1 a, b) and EtBr-DNA-AZAfur mix tures (C EtBr = 7.77 ́ 10-5, C AZAfur = 3.01 ́ 10-5) (2) (à) and EtBr-DNA-6AZC (C EtBr = 7.54 ́ 10-5, C 6AZC = 6.92 ́ 10-4) (2) (b) at l = 340 nm of P/D EtBr . In - sert on Fig.4, à: Con cen tra tion de pend en cies of the ab sorp tion of EtBr-DNA mix tures (C EtBr = 1.145 ́ 10-4) (1) and EtBr-DNA-AZAfur mix - Fig.5 Con cen tra tion de pend en cies of the ab sorp tion of EtBr-DNA mix tures (C EtBr = 1.145 ´ 10-4) (1) and EtBr-DNA-AZAxyl mix tures (C EtBr = 1.28 ́ 10-4, C AZAxyl = 3.51 ́ 10-4) (2) at l = 340 nm of P/D EtBr . We can as sume that both azagroups of these nu cleo - sides and their glycoside frag ment in ter act with DNA. It is shown that pro posed sys tem of equa tions (1) – (4) as well as the model of com pet i tive bind ing pro posed by Nechipurenko [17] and COMP op ti mi za tion pro - gram [20] can be used for cal cu la tion of bind ing pa ram - e ters of lig ands, com pet ing for DNA bind ing sites. Bind ing con stants of 6AZC and AZAxyl com plexes with DNA cal cu lated via two dif fer ent mod els of bind - ing co in cide very well. Bind ing con stants of com plex of AZAfur which does not have OH-groups in furanose ring with DNA cal cu lated via two dif fer ent mod els of bind ing do not co in cide. This could be re lated to the fact that EtBr forms two com plexes with DNA and this is not taken into ac count in Model 1. Bind ing con stants of 6AZC and AZAfur com plexes with DNA have much higher val ues than for those for AZAxyl, hence nucleoside sugar struc ture and con for ma tion in flu ence greatly their abil ity to bind to DNA. Å. Ë. Åðìàê, Å. Á. Êðóãëîâà, Ë. È. Ïàëü÷èêîâñêàÿ, È. Â. Àëåêñååâà Ñïåêòðîôîòîìåòðè÷åñêîå èññëåäîâàíèå ìåõàíèçìîâ ñâÿçûâàíèÿ àíàëîãîâ öèòèäèíà è áðîìèñòîãî ýòèäèÿ ñ ÄÍÊ Ðåçþìå Äëÿ âûÿñíåíèÿ ìåõàíèçìîâ ñâÿçûâàíèÿ öèòèäèíà è åãî áèîëîãè÷åñêè àêòèâíûõ ïðîèçâîäíûõ ñ ÄÍÊ èññëåäîâàíî èõ âçàèìîäåéñòâèå ñ ÄÍÊ â ïðèñóòñòâèè èíòåðêàëÿòîðà áðîìèñòîãî ýòèäèÿ (ÝÁ). Ïðîâåäåí ñïåêòðîôîòîìåòðè÷åñêèé àíàëèç ýëåêòðîííûõ ñïåêòðîâ ïîãëîùåíèÿ ñìåñåé ÝÁ–ÄÍÊ â ïðèñóòñòâèè öèòèäèíà è åãî àíàëîãîâ â øèðîêîé îáëàñòè äëèí âîëí è êîíöåíòðàöèé ÄÍÊ. Àíàëîãè öèòèäèíà, ñîäåðæàùèå äîïîëíèòåëüíûé àòîì àçîòà â ãåòåðîöèêëå (6AZC, AZAfur è AZAxyl), êîíêóðèðóþò ñ ÝÁ çà ìåñòà ñâÿçûâàíèÿ íà ÄÍÊ. Êîíñòàíòû àññîöèàöèè è ïàðàìåòðû ìåñò ñâÿçûâàíèÿ îáðàçóþùèõñÿ êîìïëåêñîâ äëÿ ýòèõ ïðîèçâîäíûõ ðàññ÷èòàíû ñ ïîìîùüþ ïðîãðàìì îïòèìèçàöèè ñïåêòðîâ ïîãëîùåíèÿ ñìåñåé ÝÁ–ÄÍÊ–íóêëåîçèä COMP è DALSMOD. Íåìîäèôèöèðîâàííûå ïî öèòîçèíîâîìó êîëüöó íóêëåîçèäû (öèòèäèí è Ara-C) íå ÿâëÿþòñÿ êîíêóðåíòàìè ÝÁ çà ìåñòà ñâÿçûâàíèÿ íà ÄÍÊ, îäíàêî â èõ ïðèñóòñòâèè èçìåíÿþòñÿ êîíöåíòðàöèîííûå çàâèñèìîñòè êðèâûõ òèòðîâàíèÿ ÝÁ â îáëàñòè íèçêèõ êîíöåíòðàöèé ÄÍÊ. Ýòî ìîæíî îáúÿñíèòü âëèÿíèåì óïîìÿíóòûõ íóêëåîçèäîâ íà ñòðóêòóðíûå èëè êîíôîðìàöèîííûå èçìåíåíèÿ ìàòðèöû ÄÍÊ â ïðèñóòñòâèè ÝÁ â îáëàñòè íèçêèõ çíà÷åíèé P/DÝÁ, ãäå P/DÝÁ – îòíîøåíèå îáùèõ êîíöåíòðàöèé ÄÍÊ è ÝÁ. 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