PhotonicsandOptoelectronics(P&O)Volume3,2014www.jpojournal.

org
doi:10.14355/jpo.2014.0301.03

TheRelationshipbetweentheStructureand
theBindingtoBovineSerumAlbuminof
SomeStyrylcyanineDyes
EldarN.Kurtaliev1,NegmatNizomov1,GairatKhodjayev2*
Physicdepartment,Samarkandstateuniversity,UniversityBlvd.,15,140104Samarkand,Uzbekistan

Samarkandagricultureinstitute,M.Ulugbekstr.,77,140103Samarkand,Uzbekistan.

kurtaliev@rambler.ru;1nnizamov@yandex.ru;*2gayrat_kh@mail.ru

Received7June2013;Revised24September2013;Accepted27September2014;PublishedJanuary2014
2014ScienceandEngineeringPublishingCompany

Abstract
Spectralfluorescent characteristics of styrylcyanine dye Sbt
((E)2(4(dimethylamino) styryl)3methylbenzo [d] thiazol
3ium iodide) and its derivatives in aqueous solutions with
and without the presence of bovine serum albumin (BSA)
werestudied.InthepresenceofBSAforallstudieddyesin
the absorption spectra, we observed an increase in the
absorptionintensity,asmallbroadening,andanewbandof
the shorter wavelengths with max=412 nm. The intensity of
the fluorescence emission of the baseband of the studied
dyesinthepresenceofBSAincreasedfrom1.1to4.6times.
Thebindingconstant(K)andthenumberofbindingsites(N)
of the studied dyes with BSA were determined. The
dependenceofthebindingconstantwithBSAonthedipole
moment of the dye molecules was determined, which
indicated that besides electrostatic attraction forces,
hydrophobic interactions are significant between styrylcya
ninedyeswithBSAmolecules.Itwasdemonstratedthatthe
aggregationofdyesinfluencestheinteractionsofdyeswith
BSA.
Keywords
Styrylcyanine Dyes; Fluorescence; Electronic Absorption Spectra;
BovineSerumAlbumin;theBindingConstant

Introduction
Organic dyes are used in quantum electronics
(Svetlichnyi, et al., 2009; Bezrodnyi, et al., 2009),
analytical chemistry (Tian, etal.,2012), photodynamic
therapy (Yukruk, et al., 2005), tissue optics (Smith, et
al., 2007) and in biomedical research for the detection
of nucleic acids (Timtcheva, et al., 2000). In order to
expand the area of their application, research and
synthesis of new organic dyes is necessary. In
particular, the search for new fluorescent probes and
labels is stimulated by the growing volume of

biomedical research and diagnosis of diseases

(Khodjaev,etal.,2007;Matiukas,etal.,2007).
Various photophysical and photochemical properties
of the fluorophores, such as quantum yield (Al
Shamiri, etal., 2009), the fluorescence lifetime (Lee, et
al., 2008), photostability (Song, et al., 2009) and pH
dependence (Mazieres, et al., 2009) define their
applicabilityarea.Forinvivomeasurements,theorganic
dyes of different classes are usedtriphenylmethane
(Guo, et al., 2009), xanthene (Delgadillo, et al., 2010)
and polymethine (Tatikolov, 2012). Recently,
styrylcyanine dyes gained popularity and are widely
used as fluorescent probes and labels (Krieg, et al.,
2011; Wuskell, et al., 2006; Kurtaliev, et al., 2009).
Fluorescent probes that are used in medicine and
biologyvaryinstructureandhavespecificcharacteris
tics during the interaction and binding with various
biomacromolecules which then manifest themselves
differently in the absorption and fluorescence spectra
and subsequently affect the photophysical properties
of the probes. Therefore, the study of interaction of
new synthetic styrylcyanine dyes with biomacro
molecules is of great scientific and practical interest,
with priority placed on their further application as
probes in biomedical research as well as for targeted
synthesis of organic dyes with specified spectral
fluorescentproperties.
Inthispaper,wepresenttheresultsofastudyonthe
relationship between the structure and bonding of
styrylcyaninedyeSbtanditsderivativeswithBSA.
Methods and Experiment
Thestructuralformulasofthestudiedcompoundsare

15

www.jpojournal.orgPhotonicsandOptoelectronics(P&O)Volume3,2014

displayedinTable1.Thesynthesisofdyesandtestof
their purity were carried out at the Institute of
MolecularBiologyoftheNationalAcademyofScience
of Ukraine according to the methods described in
(Kovalska, etal., 2005; Tokar, etal., 2006; Kovalska,et
al., 2004). The electronic absorption spectra were
measured with the help of a Specord 50 SA
(Analytikjena, Germany) spectrophotometer which
allowsmeasurementswithanaccuracyof(+/0.003D),
resolutionof(0.3nm)intherangeof1901100nmand
automaticregistrationoflightscattering.Fluorescence
spectraweremeasuredwiththehelpofanexperimental
fluorescence measurement setup assembled on the
basisofthemonochromatorMDR12(LOMO,Russia)
with automatic correction and PC output, and a
photomultiplier tube FEU100 (Russia). Excitation
lightsourceswerehighbrightnessLEDsofblueglow
with light filters SZS22 and SZS23 (=450 nm), SZS8
andSZS20(=485nm).BSA(MedpreparatKonotop,
Ukraine)wasusedasaprotein.Asasolventweused
double distilled water (pH=7.1). The pH values were
measured on the pHmeter Seven Easy(Mettler
Toledo, Switzerland). Preparation of stock dye
solutions was performed by volumeweight method.
Toobtainstocksolutionswithaspecifiedconcentration,
dye samples were weighed on microanalytical scales
AB135S/FACT(MettlerToledo,Switzerland)withan
accuracy of 0.01 mg. Operational concentrations 105
106Mofsolutionswerepreparedbydilutingthestock
solution. To avoid reabsorption, fluorescence
measurementswerecarriedoutwiththinlayersofthe
solutions, in which the absorption of the excitation
light did not exceed ~5%. Depending on the
concentrationofthesolution,specialquartzcellswith
thickness of the layerin the range of 0.150 mm were
used. The values of concentrations of aqueous
solutionsofBSAweredeterminedspectrophotometrically
bytheabsorptionintensity.TheconcentrationofBSA
(p)isdefinedbytheformula:p=1.45D2800.74D260
(inmg/ml),whereD280andD260opticaldensityofBSA
solution at the wavelengths of the absorption 280 nm
and260 nm (Gerhard, 1981). To determine the values
ofthebindingconstant(K)andthenumberofbinding
sites (N) dyes with BSA, the titration was performed
by the method of Scatchard (Dobretsov, 1989).
TitrationofdyewithBSAwasconductedataconstant
concentration of dye by serial dilution of the stock
concentrationofBSA.ReversetitrationofBSAbydye
solutionwasconductedataconstantconcentrationof
BSA by adding of 20, 40, 60, 80 and 100 l of dye
aqueoussolutionto3mloftheBSAsolution.

16

TABLE1STRUCTURALFORMULASOFTHESTUDIEDDYES.

Dye

Structuralformulas

Chemicalname
(E)2(4(dimethyl
amino)styryl)3
methylbenzo[d]
thiazol3iumiodide

(E)6acetamido2(4
(dimethylamino)styryl)3
methylbenzo

[d]thiazol3ium

Sbt
N+

I-

S5

S6

(E)2(4(dimethyl
amino)styryl)3ethyl4
phenylthiazol3ium
perchlorate

N
+

O
O Cl O
O

(E)3(4((4(dimethyl
amino)phenyl)
dimethylammonio)butyl)
2(4(dimethylamino)
styryl)benzo[d]thiazol3
iumiodide
(E)2(4(dimethylamino)
styryl)3(3(trimethyl
ammonio)propyl)
benzo[d]thiazol3ium
iodide

S
I

S12

I
+

S20

N
+

(E)2(4(dimethylamino)
styryl)3(3(pyridinium
1yl)propyl)benzo[d]
thiazol3ium

S34
+

S
N

(E)2(4(dimethylamino)
styryl)3(4(trimethyl
ammonio)butyl)benzo
[d]thiazol3ium

S36
+

S38

O
I

S
N

D
165

D
166

D
174

(E)2(4(dimethylamino)
styryl)3methyl6
(2(trimethylammonio)
acetamido)benzo[d]
thiazol3iumiodide
(E)3(4(dimethyl(6
(trimethylammonio)
hexyl)ammonio)butyl)
2(4(dimethylamino)
styryl)benzo[d]
thiazol3iumiodide
(E)3(4(dimethyl(2
(trimethylammonio)ethyl)
ammonio)butyl)2(4
(dimethylamino)styryl)
benzo[d]thiazol3ium
(E)2(4(dimethylamino)
styryl)3(3(triethyl
ammonio)propyl)
benzo[d]thiazole
3iumbromide

FluorescenceintensitiesofdyeinasolutionofBSA(I1)
and water (I0) were measured at a wavelength of
maximum. I1 was determined by subtracting of I0

PhotonicsandOptoelectronics(P&O)Volume3,2014www.jpojournal.org

tothemonomerformofdyemolecules.Thefollowing
main spectralfluorescent characteristics of dyes in
monomeric form were determined based on the
experimentalmeasurementdataforaqueoussolutions
of the studied dyes in accordance to calculation
procedures described in (Nizomov, et al., 2006):
extinction coefficient (), oscillator strength (fe),
radiativelifetimeoftheexcitedstate(),thefrequency
ofpurelyelectronictransition(00)andStokesshift(SS)
(displayedinTable2).Thequantumyieldsofaqueous
solutions of the studied dyes are very low,
approximatelyintherangeof0.010.02%.

from I1. The values of Iun=1/y0C and KCI=x0 were

determinedonthegraphofdependencyof1/I1from
1/p as intersections with 1/p(x) and 1/I1(y) axises.
Fluorescence intensity of dye in solutions of BSA (I2)
wasdeterminedinmaximum.BasedonI2andI0,their
difference I2 was calculated. Then the concentration
ofboundprober=I2/Ispandtherelativeconcentration
oftheboundprober/c=I2/(cIspI2)werecalculated
where c is the total concentration of the dye and r is
the concentration of bound probe. The binding
constant of the probe Kc=y0/x0 and the quantity of
binding sites to the BSA N=x0/p were identified from
the graphical dependence of r/c against r, where y0 is
the ordinate of intersection with r/c axis and x0 is the
abscissaofintersectionwiththeraxis.Thevalueofthe
possiblesystematicerrordoesnotexceed12%,andit
is related to the inaccuracy of graduation counting
marksanddifferentwettingofwallsofthemeasuring
utensils, as well as identifying the main spectral
luminescentpropertiesandparametersofthebinding.
All measurements were performed at a room
temperature (297K). For ease of comparison, the
presented absorption and fluorescence spectra were
normalized to unity. The calculation of the dipole
momentandthechargedistributiononthedyeatoms
wasperformedduringthequantumchemicalcalculation
ofthestructureofthedyemoleculesbysemiempirical
method AM1 with the help of the MOPAC 2009
softwarepackage(http://www.openmopac.net)witha
standard set of parameters (Stewart, 1990). All
calculationswere performed for isolated molecules in
a vacuum. Previously, we made optimization of the
geometry of the molecules using a restricted Hartree
Fock method and PolakRibier algorithm with
accuracy 0.001 kcal/(mole) and by taking into
accountthedifferentvariantsofinitialconformations.
Optimizationwastobefinishedwhenthermsenergy
gradient was less than 1 kcal/mol/. Among the
resulting outcomes, we selected only those with
complete final energies that are higher than the most
minimal to no more than 1 kkal/mol, representing
approximately1.7kTattheroomtemperature.

TABLE2SPECTRALFLUORESTSENTCHARACTERISTICSOFAQUEOUS
SOLUTIONSOFTHESTUDIEDDYES.

Dye
Sbt
Sbt+BSA
S5
S5+BSA
S6
S6+BSA
S12
S12+BSA
S20
S20+BSA
S34
S34+BSA
S36
S36+BSA
S38
S38+BSA
D165
D165+BSA
D166
D166+BSA
D174
D174+BSA

(nm)
511
512
513
508
469
461
529
528
531
533
533
533
522
522
523
509
523
520
524
519
527
531

,lmole1
cm1)
(nm)
596
23500
598
19200
589
47700
582
40800
584
6100
572
9600
598
9000
594
9500
600
48400
591
51800
599
50500
595
50500
597
38600
593
35100
598
32400
591
27900
604
49300
602
41800
598
51900
593
47600
598
59000
602
49200

f
0.27
0.33
0.90
0.84
0.15
0.40
0.15
0.18
0.80
0.97
0.87
0.95
0.69
0.77
0.62
0.65
0.95
0.99
0.93
0.98
0.94
0.99

, 00
SS
(ns) (cm1) (cm1)
0.1
0.2
4.7
4.8
0.2
0.04
0.3
0.24
4.6
3.4
5.3
4.8
6.5
5.9
7.2
6.4
4.6
4.4
4.6
3.8
3.7
3.1

17480
17500
17820
17850
18350
18870
17450
17640
17730
17820
17360
17390
17540
17600
17510
17640
17330
17290
17530
17590
17470
17710

2790
2808
2515
2502
4198
4209
2181
2104
2165
1841
2067
1955
2406
2293
2398
2725
2564
2619
2361
2404
2252
2221

TheSbtdyeisamonomer,andS5,S20,S34,S36,S
38, D165, D166 and D174 dyes are its derivatives,
distinguished from Sbt by different substituents in
theirstructures.Forexample,S5andS38dyesdiffer
from the Sbt dye as their structure contains
methylacetamide (S5) and trimethyl2(methylamino)
2oxoethan1aminium (S38) groups, which can lead
toabathochromicshiftoftheabsorptionmaximumof
2 and 12 nm, and an increase of the extinction
coefficientof1.4and2times,respectively.Introduction
of toluidine and the methyl group (S6 dye) to the
cyclopentane fragment, and the replacement of anion
oftheiodineatombytheanionofperchlorateleadsto
hypsochromicshiftofthemaximumofabsorptionand
fluorescence at 42 nm and 12 nm, respectively, and
fourtime decrease in the extinction coefficient as

Results and Discussion

The concentration dependence of the absorption and
fluorescencespectraoftheselecteddyesinwaterwas
studied. It was found that in 106105M concentration
range for Sbt, S5, S20, S34, S36, S38, D165, D166
andD174dyes,andin106103Mconcentrationrange
for S6, S12 dyes, the shape of absorption and
fluorescence spectra remain constant and they belong

abs. ,
fl . ,
max
max

17

www.jpojournal.orgPhotonicsandOptoelectronics(P&O)Volume3,2014

Study of the interaction of the presented compounds

to BSA demonstrated that with the increase
concentration of BSA, the increase in the intensity of
the absorption and fluorescence is observed (Fig. 1).
Figure 1 shows that as the concentration of BSA in
aqueous solution S6 dye increases, a twotime
increase of the intensity of the absorption and
fluorescencespectraisobserved.Inaddition,withthe
increasing the content of BSA, a new band with
max=412nmisobservedinshortwavelengthside,and

18

1.2

1.2
b)

a)
1

0.8

5
4
3

0.6

0.4

0.2

0
340

1
Fluorescence intensity (a.u)

HypsochromicshiftintheabsorptionspectraofdyeS
6 is explained by the difference in the basicity of
heterocycles on the end and the fact that a rotation,
decreasing steric hindrance, is around the bond
betweenthiazoleringandbenzenering.Formationof
a nonplanar molecule leads to a reduction of the
dipole moment as will be shown below, and hence
reduces the intensity of absorption. Introduction of
polymethylene chains of different lengths to the
structureofthedyemolecules,andpresenceofvariety
ofsubstituentsandeffectorgroups(S12,S20,S34,S
36, D165, D166 and D174 dyes) results in a
bathochromicshiftoftheabsorptionmaximumon11
20 nm and an increase in the extinction coefficient of
1.62.5 times. The presence of dimethylanyline group
in the structure of S12 dye leads to a bathochromic
shiftoftheabsorptionspectraof7nmandadecrease
intheextinctioncoefficientof4timeascomparedwith
theS36dye.ComparisonofS20,S34andD174dyes
shows that the replacement of trimethylamine group
(S20) with pyridinium (S34) leads to a slight
bathochromic shift and an increase in the extinction
coefficient. The introduction of additional methyl
groupsand the replacement of the anion iodine atom
with atom of bromine (D174) result in a
hypsochromic shift of 4 nm and an increase in the
extinction factor correction by 20% as compared with
theS20dye.ComparisonofD166dyewithS36dye
shows that the introduction of trimethylamine group
to the structure of the chromophore dye leads to an
increase in the extinction coefficient of an aqueous
solution of D166 dye to 1.5 times as compared with
the S36 dye. Replacing pyridinium group in the S34
dyewiththetrimethylammonium(S36dye)leadstoa
hypsochromic shift of 11 nm and a decrease in the
molarextinctioncoefficient.However,fortheS20dye,
shortening of the polymethylene chain connecting
trimethylammoniumgroupwiththenitrogenatomto
one link increases the molar extinction coefficient of
25%ascomparedwiththeS36dye.

hypsochromic shift of about 1012 nm is observed in

thefluorescencespectra.Thebroadeningandincrease
of the intensity of the main absorption band with
max=461 nm and the appearance of a band with
max=412 nm in the dye S6, is possibly due to the
formation of aggregates of the dye molecules. By
increasingtheamountofprotein,theconcentrationof
bindingsitesincreases(fordyeS6,ithasamaximum
value), thereby the dye molecule are dispersed by
binding sites. A similar pattern is observed with the
increasing concentration of BSA in thesolutionin the
absorptionandfluorescencespectrafortheremaining
ofthestudieddyes(Table2).

Optical density (a.u)

comparedwiththeSbtdye.

0.8

4
3
2

0.6

1
0.4

0.2

390

440

490

540

Wavelength, nm

0
490

590

540

590

Wavelength, nm

640

FIG.1THESPECTRAOFABSORPTION(a)AND
FLUORESCENCE(B)OFAQUEOUSSOLUTIONSOFDYES6
(=105M)WITHTHEADDITIONOFBSA:10,21.6106,33.2106,
46.5106,51.3105.

Theincreaseinfluorescenceintensitycanbeexplained
by the interaction of the dye molecules to BSA in the
excitedstateincreases,andleadstoastrengtheningof
a flat configuration of monomer dye molecules
bonded with the BSA, as well as increasing of the
rigidity of the dye molecules in complex formation
with BSA. Transport of low molecular weight
substances is one of the main functions of BSA
(Lakovicz, 2002). Regulation of this process,
depending on the content of modulating substances,
occursduetothechangesinthebindingpropertiesof
albumin.AvailabilityoffreebindingsitesontheBSA
molecules in relation to a particular connection, and
the extent of their binding capacity determine the
functionalpropertiesofalbumin.
Bindingparametersofthedyewithbioobjectserveas
aquantitativemeasureoftheinteractionofdyeswith
biological objects: the binding constant (K) and the
concentrationofbindingsites(N).Bindingparameters
of the studied dye molecule with BSA were
determined by titration method of the dye solutions
with BSA solutions, and, conversely, BSA solutions
with dye solutions (Table 3). Table3shows thatS12,

PhotonicsandOptoelectronics(P&O)Volume3,2014www.jpojournal.org

D174,Sbt,S5andS38dyeshavethehighestbinding
constants, and S34 and S6 dyes have the lowest
bindingconstants.S12dyehasthestrongestbinding,
while the S34 and S6 dyes have 1379 times and 727
times lower binding, respectively, compared with the
S12.

the nitrogen atoms in the trimethylamine part of the

dye, equals to 0.24. The low value of the binding
constants ofdye S6 canbe explainedby the fact that
the introduction of toluidine into the structure of the
dye creates a strong obstacle in the interaction with
BSA. Availability spatial complications upon binding
to protein leads to that dye S6 is unsuitable as
fluorescentprobeproteins.Introductionofpyridinium
group into the structure S34 dye leads to increased
fluorescence,withsimultaneousweakeningofbinding
ofthedyemoleculestoBSA.Conversely,replacement
ofpyridiniumgroupbytrimethylamoniuminthedye
S36 increases the binding constants by one order of
magnitude, with simultaneous 4.2 fold increase in
fluorescence. Increasing the length of the poly
methylenechaininthestructureofthemoleculesofD
165 and D166 dyes rises the binding constant at 35
timesand51times,respectively,comparedwiththeS
34dye.

TABLE3BINDINGPARAMETERSOFTHESTUDIEDDYESWITHBSA.

Dye
Sbt
S5
S6
S12
S20
S34
S36
S38
D165
D166
D174

Concentration,
Dye
BSA
2.5105
4.6106
1.0105
2.8105
1.0105
1.3105
1.0105
1.3105
5.0106
1.2105
1.0105
3.0105
5
1.010
3.2104
5
1.010
1.3105
1.0105
2.7106
5.0106
2.4105
1.0105
5.1106

,1

4.8105
3.5105
5.8102
8.0105
8.6104
1.1103
1.5104
2.4105
3.9104
5.6104
7.6105

6.0
0.5
14.0
1.5
0.58
4.7
0.6
3.8
1.3
0.7
2.7

IBSA/
Iwater
3.5
1.1
2.1
4.1
3.6
2.5
4.2
3.1
1.4
4.6
2.3

In S20 dye, the presence of the shorter connecting

polymethylene chain than in S36 dye leads to a
decreaseoffluorescenceintensityinBSAsolutionand
to an increase in the binding constants of 5.7fold.
Introductionofanoxygenatom(dyesS38andS5)to
the structure of molecules enhances the interaction of
dyeswithBSAandincreaseofthebindingconstantin
218timesand318times,respectively,comparedtothe
S34dye.Inaddition,thecalculateddistributionofthe
charge on the oxygen atoms has negative values and
equalto0.32and0.33,respectively.Asimilarpattern
isobservedwiththeintroductionofthemethylgroups
intothestructureoftheD174dyeandreplacementof
the anionatom of iodine with the atom of bromine,
which leads to an increase of the binding constant in
690 times in comparison with the dye S34. The
concentration of binding sites is a measure of N
limiting the concentration of bound probe in the
protein.ThenumberofbindingsitesfordyeS5,S20,
S36andD166islessthanunity.Thismaybedueto
the fact that large protein concentration chosen for
these dyes on the other side of positive cooperative
binding(KincreaseswithdecreasingN).

FIG.2THEORIENTATIONOFTHEAXESOFCOORDINATES,
BONDLENGTHSANDTHEDISTRIBUTIONOFCHARGESON
THEATOMSOFTHEDYES12.

FluorescenceofS12andS20dyesolutionswithBSA
increased approximately 4 times. Inclusion of the
benzeneringandaneffectorgroupinstructureofthe
molecule S12 dye apparently increases the negative
charge of the trimethylamine part of the dye, thus
increasing the attraction of the dye molecules to the
positively charged areas of BSA. The presence of the
twogroupsoftrimethylamineinthedyestructurealso
enhances the interaction of the dye molecule and the
positively charged areas of BSA. This assumption is
confirmed by the performed calculation of the charge
distribution,whichshowsthatthevalueofachargeon

Thestructureofthedyemoleculesandthepeculiarity
of the binding sites of the protein are determining
factors of the interaction of protein and dye. It is
knownthatnegativelychargedgroupsofthefirstand
seconddomainsofBSAarefoundinsidetheglobules,
whilethepositivelychargedarefoundonthesurface
of the globule (Luik, et al., 1984). Binding between
dyesandproteinscanoccurduetobothhydrophobic
interactions,andelectrostaticinteractions.Itshouldbe

19

www.jpojournal.orgPhotonicsandOptoelectronics(P&O)Volume3,2014

Moreover, the relatively small values of the binding

constantscanbeexplainedbythefactthatatdifferent
ratiosofconcentrationofBSAandthedye,molecules
of dye tend to form nonfluorescent complexes
between dye and BSA. Ability to bind dyes depends
on the concentration of the protein. Decline of the
fluorescence intensity during the interaction of BSA
with dyes is due not only to the formation of non
fluorescentdyecomplexes,butpossiblyduetochange
oftheconformationofalbumin.
Forthesuccessfuluseofdyesasaprobeitisnecessary
thattheydonotdamagethestructureandfunctionof
proteins during binding. Protein conformational
changes inevitably occur at the point of the probe
location. The higher the concentration of the dye, the
more is the damaging activity. Therefore, during
higher degree binding, dyes can significantly change
theconformationofalbumin.Hence,optimalchoiceof
thedyeconcentrationandproteinisimportant.Asan
example, Figure 3 shows a plot of the dependence of
the fluorescence intensity (F/F0) on the ratio of the
concentration of the dye to the concentration of BSA
(C/P). Sbt, S12, S36, D165, and D166 dyes when
bound to BSA increase the intensity of fluorescence
(F/F0)in3.5,4.1,4.2,3.1,1.4and4.6times,respectively,
with equal ratio of the concentration of the dye/
proteinconcentration(C/P)1:5(Fig.3,curves13,5,6).
A similar pattern is observed for the S38 dye in
bindingtoBSA:fluorescenceintensity(F/F0)ofwhich
isincreasedby3.1timeswithequalratioof1:2.5(Fig.3,
curve4).Afurtherincreaseintheamountofdyeleads
to a decrease in fluorescence intensity. Such pattern
can be explained by static fluorescence quenching,

20

which is related to the formation of nonfluorescent

complexes in the ground state between the dye
molecules and BSA, or between the dye molecules
directly with each other, by means of the solvent
molecules(dimersorlargeraggregates)withincreasing
concentrationofthedye.
1.2
F/F0
1
Fluorescence intensity (a.u)

notedthatduringthenoncovalentbindingofthedye
with the protein, the dye chooses binding sites can
respond to changes not only in a certain fragment of
theprotein,butalsoinotherplaceswherethebinding
sites may occur, and it can be redistributed. Active
groupsBSAsurroundedbyhydrophobicregionsform
the binding sites capable of interacting with dye
molecules.Thepresenceofthevariouseffectorgroups
in the structure of molecules ofS20, D166, D165, S
36, S34, andS6 dyes canspatially hinder interaction
of dyes with BSA. Therefore, it can be assumed that
thebindingtoBSAisduetotheelectrostaticattraction
betweenthenegativelychargedoxygenatoms(dyesS
5andS38),thenitrogenatomsinthetrimethylamine
group(S12,D174,Sbt,S20,S38,D166,D165andS
36) and positively charged globules located on the
surfaceoftheprotein.

0.8

5
1

0.6
6
0.4

3
2,4

0.2
0
0

0.2
0.4
0.6
0.8
Ratio concentration of dye to
the concentration of BSA

1
/P

FIG.3DEPENDENCEOFRELATIVEFLUORESCENCE
INTENSITYRATIOSOFDYEONTHECONCENTRATIONOF
BSAINWATER:1Sbt(9.3106M),2S12(8.9106M),3S36(6.510
6M),4S38(8.6106M),5165(5.1106M),6D166(4.2106M).

Large dye concentration increases the degree of

complexation of the dye molecules, and this in turn
resultsinadecreasetothebindingcapacityofBSA.In
other words, the proteins in solutions with dyes is a
competition:bindingprocessesbinddyemoleculesto
each other on one side and bind the dye molecules
with another molecule of BSA. Such a pattern is
detectedforderivativesrhodaminedyesuponbinding
to serum albumin (Nizomov, et al., 2006). Since the
formationofthecomplexoccursinthegroundstate,it
canleadtochangesintheabsorptionspectrumofthe
dye, which is observed in the experiment (Figure 3
andTable3).Electrostaticforcesplayessentialrolein
the formation of molecular aggregates, as well as in
theinteractionofthedyewiththeproteins(Patonay,et
al.,2004;Nizamov,etal.,2011).Totestthisassumption,
wecalculatedthedipolemomentofthestudieddyes.
Presented dependence of the binding constant on the
dipole moment of the dye molecules (Fig. 4) shows
that there is a correlation between the binding
constant of dyes with BSA and their molecular
structure,inparticular,withitselectrostaticfield.The
growthinrowofdyesS6,S20andS38(arrow1)and
thegrowthofthebindingconstantintheseriesS6,S5,
Sbt, D174 and S12 (arrow 2) indicates that between
dye and BSA molecules, in addition of electrostatic
attraction, there are significant hydrophobic interac

PhotonicsandOptoelectronics(P&O)Volume3,2014www.jpojournal.org

tions. Increasing fluorescence intensity and the

fluorescence hypsochromic shift suggest that the
binding of a protein fluorophore is flagged for
hydrophobic entourage. For S6, S20 and S38 dyes
increased protein binding correlates with increasing
charge and dipole moment of the ground state.
Increase of binding protein with S5 and Sbt dyes
leads to more dependence on hydrophobic interac
tions, whereas for the dye D174 and S12, the
dependence is on the total charge of the molecule.
Reduction of chain thrimetylamonium group D166
dyeleadstoamorerigidconformationandincreased
fluorescenceismorethan3timescomparedtoD165.
Thus, it can be argued that electrostatic attraction,
combined with a hydrophobic interaction, underlies
the interaction of the dye molecules with BSA. In
addition, it is suggested that there is a hydrophobic
cavity close to the positively charged site of the BSA
molecule, which can be penetrated by the dye
molecules.

However, the BSA molecule has other binding sites,

such as uncharged hydrophobic. In fact, with which
BSAbindsthedyecenter,itisstronglyinfluencedby
thenumberofdeputies,andparticularlytheirlocation.
Apparently, this is connected with such a significant
differencebetweenthevaluesofthebindingconstant.
Arrow3(Fig.4)showsthatthereisanincreaseofthe
electrostatic field of the dye molecules in S6, S34, S
36,D165andD166seriesofdyes,wherethevaluesof
bindingconstantofS34,S36,D165andD166dyes
are similar. Such dependence may indicate that the
molecules of these dyes tend to form aggregates or
dimers. The detected dependence of the dimerization
and aggregation processes of studied dyes on the
moleculeselectrostaticfield,representedbyintegrally
characterized dipole moment projections on the
coordinate axes (Fig. 5) serves as the confirmation of
thesefindings.
45
40

S-12
Dipole moment [] (in Debye)

7
Binding constant (x10 5)

35

D-174
2

6
5 Sbt
4

S-5

S-38

25
20

S-36
15

S-20
S-34

10

D-174
S-12

S-20

S-6 S-34

0
0

D-165

S-36
20

40

S-38
aggregation

Sbt, S-5
S-6

D-166

Dipole moment (in Debye)

12

16

20

Dipole moment [] ( in Debye)

60

FIG.5THEDEGREEOFFORMATIONAGGREGATEOFTHE
DIPOLEMOMENTOFTHEMOLECULES.

FIG.4DEPENDENCEOFBINDINGCONSTANTWITHBSAON
THEDIPOLEMOMENTOFDYEMOLECULES.

Average values of the absolute values of the dipole

moment and its projections along the coordinate axes
forthemoleculesofstudiedcompoundsaredisplayed
inTable4.

Thesameconclusionwasreachedbyauthors(Welder,
et al., 2003; Sophianopoulos, et al., 1997) who studied
noncovalent interaction between symmetric and
asymmetric squaraine dyes with BSA, which
demonstrated that the hydrophobicity of the dye
molecules strongly affects their ability to bind to the
protein, and the binding occurs in the hydrophobic
cavitylocatedclosetothepositivelychargedsiteofthe
BSA. The electrostatic and hydrophobic nature of the
bindingproteinmoleculesisindicatedbythefactthat
surfaceactive compounds (surfactants) have an
influence on this interaction. This detergent nonpolar
moiety will bind to hydrophobic protein adsorption
centers and thus inhibit their subsequent interaction
with the dye molecules (Nizomov, et al., 2006).

30

dimerization

D-165
D-166

TABLE4THECALCULATEDDIPOLEMOMENTSOFSTUDIEDDYES.

Dye
Sbt
S5
S6
S12
S20
S34
S36
S38
D165
D166
D174

Charge
1
1
1
2
2
1
1
2
3
3
2

[Dx]
1.6
1.2
0.5
0.7
1.0
0.4
1.7
20.3
6.2
6.7
0.9

[Dy]
1.8
2.3
0.4
6.6
13.1
12.1
16.2
5.0
39.0
38.2
9.9

[Dz]
0.2
0.1
0.1
0.03
9.9
9.8
10.7
0.1
16.1
13.7
6.8

D
2.4
2.6
0.7
6.6
16.5
15.6
19.5
20.9
42.6
41.2
12.1

21

www.jpojournal.orgPhotonicsandOptoelectronics(P&O)Volume3,2014

22

wherethesolventmoleculesactasaconnectingbridge
between dye molecules through hydrogen bonding.
The processes of aggregates formation with the
increase in concentration of nonfluorescent dyes are
widespreadamongseriesoforganicdyes,inparticular
for squaraine (Nizamov, etal., 2009), cyanine (Viteva,
et al., 2007), rhodamine (Nizomov, 1997) and styryl
(Kurtaliev, Nizomov and Rahimov, 2011; Kurtaliev,
2011)dyes.
1,2

1,2
b)

a)
1
3

0,8

0,6

0,4

0,2

1
Fluorescence intensity (a.u)

1
Optical density (a.u)

Charges on the atoms of the substituents make a

fundamentalcontributiontothedipolemomentofthe
moleculessothatthedipolemomentisorientedalong
theYaxisandnaturalgrowthofthedipolemomentin
S6,S5,Sbt,S12,D174,S34,S20,S36,D166andD
165dyesisobserved.AsseenfromFigure5,thedyeS
38 oriented dipole moment along the axis X has a
tendency to form aggregates, while dyes S6, S5, D
174, S34 and D165 practically do not exhibit
predisposition aggregation, and are inclined to
dimerization.The ability to form different aggregates
and dimmers is explained by the structure of their
chromophores, namely significant dipole moments,
the alternation of opposite charges, and planar
structure of the chromophore, which causes the high
energy of the intermolecular interactions (Ishchenko,
1994).Thisassumptionisconfirmedbythestudyofthe
concentration dependence of the absorption and
fluorescencespectrapresentcompoundsinthewater,
aswellasabinarymixtureofwater+dioxane.Itshould
be noted that the preparation of solutions in binary
solventwascarriedoutsothattheconcentrationofthe
dye remained constant, while the ratio of binary
solvent change. Found that increasing the concentra
tionofSbt,S5,S20,S34,S36,S38,D165,D166and
D174dyesinaqueoussolutionincreases,anewband
located on the side of short wavelength is observed,
starting from the concentration of 104 M (Fig. 6).
Observed maximum in the absorption spectrum of a
dilutesolutionwithmax=523nm(Fig.6,curve1)refers
to the monomeric form of the dye D165. As the
concentration of the dye in the absorption spectrum
increases, a slight drop in the intensity of the band
with max=523 nm, and a new band with max=504 nm
are observed in the short wavelengths side (Fig. 6,
curve 3). At the same time, with the increase of
concentrationofthedye,thedeclineintheintensityof
light, that is, the concentration quenching of
fluorescenceisobservedinthefluorescencespectra.A
similar pattern in the absorption spectra and
fluorescenceofS5,S20,S34,S36,S38,D165,D166
and D174 dyes are observed with the increasing
concentrationofdyeinaqueoussolution.Forexample,
for S36, S5, D174, D166 and S34 dyes, with the
increasing concentration of dyes, position of the
absorption spectrum maximum hypsochromically
shiftsto15nm,31nm,11nmand17nm,respectively.
Observed phenomena in the absorption and
fluorescence spectra in aqueous solutions can be
explainedbyformationofnonluminescentaggregates
of dye molecules with solvent molecules (water),

0,8

0,6

Wavelength, nm

0,4

0,2

0
350 400 450 500 550 600 650

0
560

585 610 635

Wavelength, nm

FIG.6CONCENTRATIONDEPENDENCEOFABSORPTION(A)
ANDFLUORESCENCE(B)SPECTRAOFDYED165INWATER:1
105,2104,103

In the absorption and fluorescence spectra S38 dye

(c=105 M) at the transition from water to water
dioxane solution of bathochromic shift on 16 nm and
increasingtheabsorptionandfluorescenceintensityis
observed.(Fig.7,curve2).Atthesametime,theintensity
of fluorescence increases up to a certain volume
fractions(13%water+87%dioxane).Thefurtherincrease
ofthecontentdioxane(7%water+93%dioxane)leadsto
the emergence of shortwavelength band with max=
457nm; simultaneously, there is a decline in the
intensityofthebandmax=542nm.Atthesametime,the
intensityoffluorescencedecreasesproportionaltothe
increase amount of dioxane. At the volume fraction,
1% water adding with 99% dioxane in the absorption
spectraisobservedwithmax=441nm,andthesolution
haspracticallynofluorescenceability(Fig.7,curve7).
The observed phenomena in the absorption and
fluorescence spectra of waterdioxane solution can be
explainedbythefactthatatasmallfractionofdioxane
content in the solution solvation processes are
observed that lead to batahromic shifts of the
absorption and increase the intensity of fluorescence.
Solvation may in turn result in the formation of
aggregates ionic separated solvent or outersphere
complexes(Shakhverdov,etal.,1999).Inthiscase,the
cation is actually surrounded by solvent molecules

PhotonicsandOptoelectronics(P&O)Volume3,2014www.jpojournal.org

ACKNOWLEDGEMENTS

and visibly is separated from the anion. Further

increase in dioxane solution leads to the fact that
formed ionic aggregates unite scheme K+AK+A
K+A and form Haggregates, do not have
fluorescentability.

WegreatfullyacknowledgeProf.S.M.Yarmolukofthe
Institute of Molecular Biology and Genetics, NAS
Ukraine for the dye samples. This work was
supportedbygrantU3104(STCU,Ukraine).

Thus, for the synthesis of fluorescent probes with

higher protein sensitivity on the basis of styryl dyes,
the presence of the methyl and effector groups is
essential. This conclusion has been confirmed by the
fact that the binding constant of the S34 dye with a
benzene ring attached by polymethylene chain is
significantlylower,comparedtodyeswithmethyl(D
174, S38) and effector groups (S12) which facilitate
thebindingofthesedyestothesurfaceoftheprotein.
1.2

0.8

1
0.6

0.4

6
0.2

0
370 420 470 520 570 620
Wavelength, nm

Structure

and

spectralluminescent

1994.(inRussian).
A.I. Luik, V.D. Lukyanchik, Serum albumin, and
transportation of poisons, Medicine, Moskow. 1984. (in
Russian).
A.J. Sophianopoulos, J.Lipowsky, N.Narayanan, G.Patonay,

Association of NearInfrared Dyes with Bovine Serum

Albumin.Appl.Spectr.vol.51(10),pp.15111515,1997.

0.8
5
0.6

A.Matiukas, B.G.Mitrea, M.Qin, A.M.Pertsov, A.G.Shvedko,

M.D.Warren, A.V.Zaitsev, J.P.Wuskell, M.Wei, J.Watras,

L.M.Loew, Nearinfrared voltagesensitive fluorescent

0.4

0.2

0
540

dyes optimized for optical mapping in bloodperfused

1
7

myocardium, Heart Rhythm, vol.4(11), pp.14411451,

2007.

565

590

615

640

A.S. Tatikolov. Polymethine dyes as spectralfluorescent

Wavelength, nm

probes for biomacromolecules. J.Photochem. Photobiol.

FIG.7THEABSORPTION(a)ANDFLUORESCENCE(b)SPECTRA
OFS38DYE(=105M)ATMEASUREADDITIONDIOXANE:1
AQUEOUSSOLUTION,250%374%487%590%,693%,799%
DIOXANE.

C:PhotochemistryReviews.,vol.13(1),pp.5590,2012.
B.Song,Q.Zhang,W.H.Ma,X.J.Peng,X.M.Fu,B.Sh.Wang,
The synthesis and photostability of novel squarylium

Conclusion

indocyaninedyes.DyesandPigm.,vol.82(3),pp.396400,
2009.

It was established that for aqueous solutions of all

studieddyesinthepresenceofBSA,anincreaseinthe
absorption capacity and a slight broadening and the
appearanceofanewbandoftheshorterwavelengths
with max=412 nm were observed. In the presence of
BSA, the emission intensity of the main fluorescence
bandinallstudieddyesincreasedfrom1.1to4.6times.
Main spectralfluorescent characteristics, the binding
constant(K)andthenumberofbindingsites(N)were
identifiedforthedyemoleculesthatareinmonomeric
form when bound to BSA. The dependence of the
binding constant on the dipole moment of the dye
molecules showed that in addition to the electrostatic
attractionbetweenthemoleculesofstudieddyeswith
BSA,therearesignificanthydrophobicinteractions.It
wasshownthatprocessesofformationofdimersand
aggregatesofthedyemoleculesmaycontributetothe
interactionofthelatterwiththeBSA.

Ishchenko,

properties of polymethine dyes, Naukova dumka, Kiev.

b)
2
3

Optical density (a.u)

A.A.

1.2

Fluoresccence intensity (a.u)

a)

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