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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| Дата: | 2007 |
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| Мова: | Russian |
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Інститут молекулярної біології і генетики НАН України
2007
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| Назва видання: | Біополімери і клітина |
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| Цитувати: | 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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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 |
| work_keys_str_mv |
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2025-11-25T22:13:12Z |
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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.
Êëþ÷åâûå ñëîâà: àíàëîãè öèòèäèíà, ÄÍÊ, ìîäåëè
ñâÿçûâàíèÿ, ñïåêòðîôîòîìåòðèÿ, áðîìèñòûé ýòèäèé.
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535
SPECTROPHOTOMETRICAL STUDY OF CYTIDIN AN A LOGUES BIND ING WITH DNA
Parameter 6AZC AZAfur AZAxyl
Model 1
K1, M
–1
(1,4 ± 0,5)× 104 (2,5 ± 0,3)× 104 (9,5 ± 0,5)× 1024
n1 1,1 ± 0,1 1,2 ± 0,1 1,1 ± 0,1
n2 2,9 ± 0,1 3,0 ± 0,4 2,5 ± 0,1
Q 0,027 0,024 0,018
Qlim 0,031 0,035 0,030
Model 2
K1, M
–1
(1,3 ± 0,3)× 104 (8,3 ± 1,3)× 103 (8,7 ± 1,5)× 102
n1 1,0 1,0 1,0
n21 2,8 ± 0,1 2,9 ± 0,1 2,9 ± 0,1
Q 0,027 0,023 0,013
Qlim 0,036 0,031 0,033
Bind ing pa ram e ters of nucleoside com plexes with DNA cal cu lated
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DALSMOD op ti mi za tion pro gram us ing Model 2
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