Journal of Molecular Structure 1051 (2013) 8694

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Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstruc

Investigation of binding of nanomarkers of uorescein family to bovine

serum albumin at various values of pH: Spectroscopic study
Irina M. Vlasova , Anna A. Kuleshova 1, Alexander A. Vlasov, Alexander M. Saletsky
M.V. Lomonosov Moscow State University, Physical Department Russia, Vorobevi Gori, 119991 Moscow, Russian Federation

h i g h l i g h t s
 The interaction of nanomarkers of uorescein family with BSA is studied.
 There is nanomarker uorescence quenching in BSA solutions.
 There is decrease of nanomarker molecular association degree in BSA solutions.
 The binding of uorescein differs from binding of its halogen-derivatives to BSA.
 The binding of nanomarkers takes place to BSA binding Center I.

a r t i c l e

i n f o

Article history:
Received 21 April 2013
Received in revised form 6 June 2013
Accepted 23 July 2013
Available online 31 July 2013
Keywords:
Fluorescent nanomarker
Bovine serum albumin
Protein binding Centers
Fluorescence analysis
Raman spectroscopy
Absorption spectroscopy

a b s t r a c t
This work is dedicated to investigation of inuence of different values of pH on binding of nanomarkers of
uorescein family (uorescein, erythrosin, eosin and Bengal rose) to bovine serum albumin (BSA). For this
purpose dependences of nanomarkers uorescence, of nanomarkers molecular association, of types of
chemical bonds between BSA and nanomarkers on pH are detected. The red shift of uorescence spectra
and the quenching of uorescence of nanomarkers of uorescein family in BSA solutions are observed.
The decrease of degree of molecular association of nanomarkers in BSA solutions is found out. The dependences of uorescence intensity and dependences of degree of molecular association on pH of halogenderivatives of uorescein differ dramatically from that of uorescein.
2013 Elsevier B.V. All rights reserved.

1. Introduction
The analysis of the mechanism of binding of biomolecules (such
as proteins molecules) with different ligands (such as nanomarkers) is extremely interesting as from the point of view of a biomedicine and pharmaceuticals, so from the point of view of
bionanotechnology: for example, at creation of new drugs and
their tests. The uorescent nanomarkers are widely applied to research structurally-dynamic states of protein molecules [117].
One of the fundamental functions of this protein is the transport
of various substances (drugs, physiological metabolites, and so on).
Thus the research of the binding properties of BSA and their
changes under different factors is extremely important.
Corresponding author. Tel./fax: +7 (495) 9391489.
E-mail addresses: vlasovairina1979@mail.ru (I.M. Vlasova), ak2210rst@
yandex.ru (A.A. Kuleshova), aav1956@mail.ru (A.A. Vlasov), sam@phys.msu.ru
(A.M. Saletsky).
1
Tel./fax: +7 (495) 9391489.
2
Tel.: +7 (495) 9391647; fax: +7 (495) 9391489.
0022-2860/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.07.041

BSA (isoelectric point pI 4.9) is the globular protein of blood

plasma. The structural mobility of molecule of BSA, provided by
unique loop folding of one polypeptide chain of 582 amino acidic
residues, ensures the interaction of molecule of BSA with ligands.
The secondary structure of BSA consists of a-helix segments and
segments of random coil at physiological pH, content of b-sheet
is negligibly. Nowadays the domain model of BSA tertiary structure
is accepted, according to which the molecule of BSA consists of 3
practically identical domains.
BSA is the typical protein of serum albumin family and has similar structure with human serum albumin (HSA) with small differences [1]. BSA and HSA are practically homologies and BSA differ
from HSA by some amino acidic residues. In particular, HSA contains one tryptophan residue Trp 214, and BSA contains two tryptophan residues Trp 135 and Trp 214.
The mechanisms of ligand binding to BSA molecule are determined by availability of specic sites on protein binding Centers
[1]. Some binding effects are caused by electrostatic interactions,
other by covalent interactions. The conformational changes at

I.M. Vlasova et al. / Journal of Molecular Structure 1051 (2013) 8694

binding of ligand can have local nature and can affect only one
binding Center, but can also induce more vast structural changes
of globule that can inuence on metabolism of protein.
The molecular mechanisms of binding of BSA with different ligands still completely are not discovered, and nowadays structure
and physicochemical properties of binding Centers of this protein
are widely studied with the help of uorescent nanomarkers. For
research of binding Centers of BSA, as HSA, and for research of
structurally dynamic state of BSA molecules are widely used uorescent anionic at physiological pH (7.4) nanomarkers, such as
uorescein and its halogen-derivatives erythrosin, eosin and
Bengal rose. Erythrosin is the tetra-iodinated derivative of the uorescein, eosin is the tetra-brominated derivative of the uorescein,
and Bengal rose is the tetra-chlorine-tetra-iodinated derivative of
the uorescein.
Early we investigated processes of molecular association of nanomarkers of uorescein family in HSA solutions [1820] and uorescent characteristics of nanomarkers of uorescein family in HSA
solutions [1922], and we investigated mechanisms of binding of
nanomarkers of uorescein family to HSA [19,2225].
In this work we investigate the inuence of different values of
pH on binding of nanomarkers of uorescent family Bengal rose,
eosin, erythrosin and uorescein to BSA another representative
of family of albumins. The following investigations were done in
this work: steady-state uorescence, molecular association of
nanomarkers of uorescein family in BSA solutions, analysis of
chemical bonds in BSAnanomarker association. The investigations
of characteristics of nanomarkers of uorescein family in BSA solutions allow one to receive the information about mechanisms of
interaction of these nanomarkers with BSA, what can be useful at
research of interaction of drugs with BSA in pharmacology and
medicine.

2. Materials and methods

2.1. Absorption spectroscopy and molecular association of
nanomarkers in BSA solutions
The following buffer solutions were prepared: 0.1 M CH3COOH
KOH (pH 3.05.0) and 0.1 M KH2PO40.1 M NaOH (pH 6.08.0).
On base of buffer solutions were prepared:
(1) solutions of nanomarkers uorescein, erythrosin, eosin
and Bengal rose (350 lM) without BSA at different values
of pH (3.58.0);
(2) solutions of nanomarkers uorescein, erythrosin, eosin
and Bengal rose (350 lM) with BSA (150 lM) at different
values of pH (3.58.0).
Measurements of absorption spectra of nanomarkers in solutions with or without BSA were made on spectrophotometer Lambda 35 (Perkin Elmer). For example, absorption spectra of erythrosin
(30 lM) in solutions (pH 5.0) without BSA (a) and with 150 lM
BSA (b) are shown in Fig. 1.
Prepared samples were placed at room temperature in 1 sm
cuvette. Absorption spectra of nanomarkers in solutions were registered in the range 300700 nm.
2.2. Steady-state uorescence of nanomarkers in BSA solutions
On base of buffer solutions were prepared:
(1) solutions of nanomarkers (3 lM uorescein; 30 lM eosin;
30 lM erythrosin, 3 lM Bengal rose) without BSA at different values of pH (3.58.0);

87

(2) solutions of nanomarkers (3 lM uorescein; 30 lM eosin;

30 lM erythrosin, 3 lM Bengal rose) with BSA (150 lM) at
different values of pH (3.58.0).
In the given work only qualitative inuence of BSA on uorescence of everyone nanomarker is of interest. The quantitative comparison of intensities of uorescence of nanomarkers among
themselves is out our interest. Thus we can use various concentrations of nanomarkers.
Investigations of steady-state uorescence of nanomarkers
were made with the help of spectrouorimeter LS 55 (Perkin Elmer) at room temperature.
Fluorescence of nanomarkers in solutions was excited with the
following wavelengths: (1) uorescein kexc = 440 nm; (2) erythrosin kexc = 530 nm; (3) eosin kexc = 520 nm; and (4) Bengal rose
kexc = 540 nm.
For example, uorescence spectra (kexc = 540 nm) of Bengal rose
(3 lM) in solutions (pH 4.0) without BSA (1) and with 150 lM BSA
(2) are shown in Fig. 2.
2.3. Raman spectroscopy and chemical bonds in BSAnanomarker
association
On base of buffer solutions were prepared:
(1) solutions (pH 3.58.0) without BSA and without
nanomarker;
(2) solutions (pH 3.58.0) with addition of nanomarker uorescein or erythrosin or eosin or Bengal rose (5 lM), but
without BSA;
(3) solutions (pH 3.58.0) with addition of BSA (150 lM), but
without nanomarker;
(4) solutions (pH 3.58.0), containing both nanomarker uorescein or erythrosin or eosin or Bengal rose (5 lM), and
BSA (150 lM). For example, Raman spectrum of 150 lM
BSA solution (pH 4.0) with 5 lM eosin is shown in Fig. 3.
The investigations were carried with the help of Raman spectrometer with Fourier-Transformation EQUINOX 55 (Bruker) with
FRA-106 with InGaAs detector, wavelength of excitation light
was 1064 nm (NdYAG laser). The laser power was 450 mW, and
30 min were used for one sample of solution. The spectral range
was 4003000 cm1, the spectral resolution was 2 cm1. The samples of solutions were placed in the quartz cell at room temperature. The obtained Raman spectra were treated by the program
OPUS 4.2 (Bruker). The Raman peaks, corresponding to chemical
bonds between nanomarkers and BSA, were chosen for investigation among many other peaks.
After registration Raman spectrograms of solutions for each
nanomarker the two difference spectra were composed:

D1 = spectrograms (3)  spectrograms (1),

D2 = spectrograms (4)  spectrograms (2).
Owing to the composition of D1 the contribution of low molecular weight components of buffer solutions is subtracted. The
remaining Raman bands correspond to chemical bonds in native
molecules of BSA.
Owing to the composition of D2 the contribution of low molecular weight components of buffer solutions is subtracted, the contribution of the nanomarker, not bound to protein, also is
subtracted. The remaining Raman bands correspond to chemical
bonds in BSA molecules and also chemical bonds, which have arisen from nanomarker binding to protein.
At comparison of difference spectra (D1 and D2) those Raman
bands were selected, which take place in difference spectrum D2

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I.M. Vlasova et al. / Journal of Molecular Structure 1051 (2013) 8694

Fig. 1. Absorption spectra of erythrosin (30 lM) in solutions (pH 5.0) without BSA (a) and with 150 lM BSA (b).

and not take place in difference spectrum D1. These bands are the
Raman bands, corresponding to binding of each of nanomarker to
BSA molecules. By virtue of extraordinary saturation of selected
spectral range by Raman bands both from different chemical bonds
inside BSA molecules, and from chemical bonds inside each nanomarker, and also by virtue of presence in solutions non-binding
molecules of protein and nanomarker, we have concentrated our
attention on investigation of Raman bands corresponding only to
chemical bonds between molecules of BSA and appropriate
nanomarker.

3. Results and discussion

Fig. 2. Fluorescence spectra of Bengal rose (3 lM) in solutions (pH 4.0) without BSA
(1) and with 150 lM BSA (2).

The molecular nanomarkers uorescein, erythrosin, eosin and

Bengal rose belong to one homologous family of derivatives of uorescein. Eosin is the tetra-brominated derivative of uorescein,
erythrosin is the tetra-iodinated derivative of uorescein, Bengal
rose is the tetra-chlorine-tetra-iodinated derivative of uorescein.
The insert of halogen groups (Cl, Br or I instead of H) in a molecule

Fig. 3. Raman spectrum of 150 lM BSA solution (pH 4.0) with 5 lM eosin.

I.M. Vlasova et al. / Journal of Molecular Structure 1051 (2013) 8694

of uorescein reduces values of pK of its carboxyl (COOH) and hydroxyl (OH) groups and changes characteristics of these markers.
The values of pK for uorescein, erythrosin, eosin and Bengal
rose, cited in the literature, rather strong differ from one work to another, we adhere to the following values (Fig. 4): (1) uorescein
pK(OH)1  5.05.5 and pK(OH)2  6.8, pK(COOH)  8.0; (2)
erythrosin pK(OH)  3.6, pK(COOH)  5.5; (3) eosin pK(OH) 
3.0, pK(COOH)  5.0; and (4) Bengal rose pK(OH)  2.6,
pK(COOH)  4.0.
At values of pH up to 5.05.5 molecules of uorescein are in
weakly positive charged form. At values of pH from 5.05.5 up to
6.8 molecules of uorescein are electrically neutral. At pH 6.8
8.0 molecules of uorescein are in weakly negative form (monoanions). At pH > 8.0 the molecules of uorescein are strongly negatively charged (dianions).
At pH < 3.6 the molecules of erythrosin are electrically neutral.
At pH 3.65.5 molecules of erythrosin are weakly negatively
charged (monoanions). At pH > 5.5 the molecules of erythrosin
are strongly negatively charged (dianions).
At pH < 3.0 molecules of eosin are electrically neutral. At pH
3.05.0 molecules of eosin are weakly negatively charged (monoanions). At pH > 5.0 molecules of eosin are strongly negatively
charged (dianions).
At pH < 2.6 molecules of Bengal rose are electrically neutral. At
pH 2.64.0 molecules of Bengal rose are weakly negatively charged
(monoanions). At pH > 4.0 molecules of Bengal rose are strongly
negatively charged (dianions).
3.1. The molecular association of nanomarkers of uorescein family in
BSA solutions
In the obtained absorption spectra of nanomarkers of uorescein family in solutions with and without BSA, two maximums of
absorption are detected. The long-wave maximum belongs to

89

monomers of nanomarkers, the short-wave maximum to associates (dimers) of nanomarkers. In our work the value of the degree
of molecular association (DMA, 1  X) of all nanomarkers in solutions without and with BSA is determined at different pH. The
DMA of solution of nanomarker is the part, equal to 1  X, of nanomarker molecules in associative (dimer) form, and is determined
by standard procedure as 1  X = Sdim/(Sdim + Smono), where Sdim
the area under the curve of absorption spectrum of dimer, Smono
the area under the curve of absorption spectrum of monomer
(these curves were found as the result of mathematical decomposition of the curve of total absorption spectrum into two Lorentz
curves) [16].
In Figs. 58 the dependences of 1  X of nanomarkers (uorescein, erythrosin, eosin and Bengal rose) on pH are shown. The
dependences of 1  X of nanomarkers on pH are explained by correlation of values of pK of ionized groups (hydroxyl and carboxyl)
of nanomarkers and values of electric charges of nanomarkers at
different pH.
In Fig. 5 are shown the dependences of 1  X of uorescein molecules on pH in solutions without and with BSA. The dependence
on pH of 1  X of uorescein has non-linear nature with a maximum at pH 6.0, at which the molecules of uorescein electrically
are neutral and thus easily form dimers. Under inuence of BSA
the 1  X of uorescein decreases for every value of pH. This is explained by binding of monomers of uorescein to BSA and, accordingly, decreasing of part of free (unbound to protein) uorescein,
capable to form dimers. More strong decrease of 1  X of uorescein in BSA solutions takes place at pH 6.0, this is explained by
strong binding of uorescein to BSA at this value of pH. At increase
of uorescein concentration in solutions without and with BSA
there is increase of 1  X of uorescein.
In Fig. 6 are shown the dependences on pH of 1  X of erythrosin molecules in solutions without and with BSA. At increase of pH
the monotonic decrease of 1  X of erythrosin is seen. Thus with

Fig. 4. Structural formulas of nanomarkers of uorescein family and values of pK of its ionized groups: uorescein (a), erythrosin (b), eosin (c) and Bengal rose (d).

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I.M. Vlasova et al. / Journal of Molecular Structure 1051 (2013) 8694

Fig. 5. Degree of molecular association of uorescein in solutions without BSA (a) and in solutions with 150 lM BSA (b) at different concentration of uorescein: 3 lM (1),
10 lM (2), 20 lM (3), 30 lM (4), and 50 lM (5).

Fig. 6. Degree of molecular association of erythrosin in solutions without BSA (a) and in solutions with 150 lM BSA (b) at different concentration of erythrosin: 3 lM (1),
10 lM (2), 20 lM (3), 30 lM (4), and 50 lM (5).

Fig. 7. Degree of molecular association of eosin in solutions without BSA (a) and in solutions with 150 lM BSA (b) at different concentration of eosin: 3 lM (1), 10 lM (2),
20 lM (3), 30 lM (4), and 50 lM (5).

increase of pH the erythrosin molecules are more negatively

charged and forms dimers more and more weakly. Under inuence
of BSA the value of 1  X of erythrosin decreases for every pH, this
is explained by binding of erythrosin to BSA. At increase of erythrosin concentration in solutions without and with BSA there is increase of 1  X of erythrosin.
In Fig. 7 are shown the dependences on pH of 1  X of eosin
molecules in solutions without and with BSA. At increase of pH
of solution the monotonic decrease of 1  X of eosin is seen. Thus
with increase of pH the eosin molecules forms dimers more and
more weakly (molecules of eosin gain more and more negative
electric charge and thus their mutual attraction becomes more
and more weak). Under inuence of BSA the value of 1  X of eosin
decreases for every pH, this is explained by binding of eosin to BSA.

At increase of eosin concentration in solutions without and with

BSA there is increase of 1  X of eosin.
In Fig. 8 are shown the dependences on pH of 1  X of Bengal
rose molecules in solutions without and with BSA. At increase of
pH of solution the monotonic decrease of 1  X of Bengal rose is
seen. Thus with increase of pH the Bengal rose molecules are more
negatively charged and forms dimers more and more weakly.
Under inuence of BSA the value of 1  X of Bengal rose decreases
for every pH, this is explained by binding of Bengal rose to BSA. At
increase of Bengal rose concentration in solutions without and
with BSA there is increase of 1  X of Bengal rose.
The strongest decreasing of 1  X of halogen-derivatives of
uorescein, induced by BSA, takes place at pH < 5.0 (Figs. 68),
what is explained by most strong binding of negatively charged

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I.M. Vlasova et al. / Journal of Molecular Structure 1051 (2013) 8694

Fig. 8. Degree of molecular association of Bengal rose in solutions without BSA (a) and in solutions with 150 lM BSA (b) at different concentration of Bengal rose: 3 lM (1),
10 lM (2), 20 lM (3), 30 lM (4), and 50 lM (5).

monomers of nanomarkers to positively charged molecules of BSA

at this pH (eosin in monoanion form at 3.0 < pH < 5.0, erythrosin in
neutral form at 3.5 < pH < 3.6 and in monoanion form at
3.6 < pH < 5.5, Bengal rose in monoanion form at 2.6 < pH < 4.0
and dianion form at 4.0 < pH < 5.0).
On changes in value of molecular association of nanomarkers
under inuence of BSA we can conclude, that:
(a) there is the general decrease, induced by BSA, of value of
1  X for all nanomarkers for every value of pH;
(b) the behavior of 1  X vs. pH (with and without BSA) of halogen-derivatives of uorescein (monotonic decrease with
increase of pH) is approximately identical, but differs dramatically from the behavior of 1  X of uorescein (non-linear behavior with maximum approximately at pH 6.0);
(c) the degree of molecular association of erythrosin has intermediate value between degrees of molecular association of
uorescein (greatest values) and eosin (smallest values)
and the degree of molecular association of eosin has intermediate value between degrees of molecular association of
erythrosin (greatest values) and Bengal rose (smallest values).
3.2. Steady-state uorescence of nanomarkers in BSA solutions
The uorescent spectra of nanomarkers of uorescein family in
solutions without and with BSA at various values of pH are registered. In solutions with BSA the maximum of uorescence spectrum of all nanomarkers of uorescein family shifts to red area in
contrast to solutions without protein. For example at pH 5.0 the
maximum of uorescence spectrum of uorescein in solutions
without BSA is at 510 nm and in solutions with BSA at 515 nm,
the maximum of uorescence spectrum of erythrosin in solutions
without BSA is at 550 nm and in solutions with BSA at 566 nm,
the maximum of uorescence spectrum of eosin in solutions without BSA is at 544 nm and in solutions with BSA at 555 nm, the
maximum of uorescence spectrum of Bengal rose in solutions
without BSA is at 563 nm and in solutions with BSA at 572 nm.
In Fig. 9 the dependences of intensities in the maximum of uo

rescence spectra Imax
of nanomarkers on pH of solutions, without
flu
and with BSA, are shown. Due to the chosen values of kexc all these
intensities of uorescence deal with the nanomarkers in their
monomer forms.
In solutions without BSA at increase of pH (Fig. 9) the increase
of Imax
of uorescein is observed that is explained by change of
flu
charge of nanomarker. At pH < 5.5 uorescein is in the poorly positively charged form possessing small ability to uorescence. At
5.5 < pH < 6.8 uorescein molecules are electrically neutral. At
6.8 < pH < 8.0 uorescein is weakly negatively charged and is in

Fig. 9. Intensity in the maximum of uorescence spectra

Imax
flu

of nanomarkers:

3 lM uorescein (1 and 2) at kexc = 440 nm; 30 lM erythrosin (3 and 4) at

kexc = 530 nm; 30 lM eosin (5 and 6) at kexc = 520 nm; 3 lM Bengal rose (7 and 8) at
kexc = 540 nm; in solutions without BSA (1, 3, 5, and 7) and in solutions with 150 lM
BSA (2, 4, 6, and 8).

monoanion form. At pH > 8.0 uorescein is negatively charged

and is in dianion form. Negatively charged forms of uorescein
have larger intensity of uorescence.
In solutions without BSA Imax
flu of erythrosin decreases, and then
leaves on constant value at increase of pH. At pH < 3.6 erythrosin
molecules are electrical neutral. At 3.6 < pH < 5.5 erythrosin is
poorly negatively charged (monoanion) and poorly screened by dipole water molecules, therefore Imax
flu is high. At pH > 5.5 erythrosin
is strongly negatively charged (dianion) what causes its strong
screening by water molecules and, accordingly, decrease of Imax
flu
in comparison with pH < 5.5.
In solutions without BSA the Imax
flu of eosin decreases, and then
leaves on constant value with increase of pH. At pH < 3.0 eosin
molecules are electrically neutral. At 3.0 < pH < 5.0 eosin is poorly
negatively charged (monoanion) and is poorly screened by dipole
water molecules, therefore Imax
flu is high. At pH > 5.0 eosin is strongly
negatively charged (dianion) what causes its strong screening by
water molecules and, accordingly, decrease of Imax
flu in comparison
with pH < 5.0.
In solutions without BSA the Imax
flu of Bengal rose decreases and
then leaves on constant value with increase of pH. At pH < 2.6
Bengal rose molecules are electrically neutral. At 2.6 < pH < 4.0
Bengal rose is poorly negatively charged (monoanion) and is poorly
screened by dipole water molecules, therefore Imax
is high. At
flu
pH > 4.0 Bengal rose is strongly negatively charged (dianion) what
causes its strong screening by water molecules and, accordingly,
decrease of Imax
flu in comparison with pH < 4.0.

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I.M. Vlasova et al. / Journal of Molecular Structure 1051 (2013) 8694

Fig. 10. Dependence of value of quenching of nanomarker uorescence at addition

max
of BSA (Imax
flu in absence of BSA Iflu in presence of BSA) on pH: (1) uorescein, (2)
erythrosin, (3) eosin, and (4) Bengal rose.

The general features for all nanomarkers of uorescein family

are the quenching of nanomarker uorescence at addition of BSA
to solution and the red shift of maximum of uorescence spectrum
of nanomarker in BSA solution. These facts are explained by the
binding of nanomarker to protein.
On quenching of uorescence of uorescein by BSA addition


max
max
one
DImax
flu I flu in absence of BSA  I flu in presence of BSA
can conclude (Fig. 10) that the strongest binding of uorescein to
BSA occurs at pH 5.06.0 (i.e. in that region, where uorescein or
is weakly electrically positive (at 5.0 < pH 5.5) or is electrically
neutral (at 5.5 < pH < 6.0), and the BSA is weakly negatively
charged (pI 4.9)).
On quenching of uorescence of halogen-derivatives of uorescein by BSA addition (Fig. 10) one can conclude that the strongest
binding of halogen-derivatives of uorescein to BSA occurs at
pH < 5.0, what is explained by electric charges of nanomarkers
and BSA: BSA is positive charged, eosin is in monoanion form at
3.0 < pH < 5.0, erythrosin is neutral at 3.5 < pH < 3.6 and in monoanion form at 3.6 < pH < 5.5, Bengal rose is in monoanion form at
2.6 < pH < 4.0 and in dianion form at 4.0 < pH < 5.0.
On the changes of characteristics of steady-state uorescence of
nanomarkers of uorescein family under inuence of BSA we can
conclude, that:
(a) BSA quenches the uorescence of all nanomarkers of uorescein family for all values of pH;

Fig. 11. Dependence of number of Raman peaks corresponding to chemical bonds

between nanomarker and BSA on pH: (1) uorescein and (2) halogen-derivatives of
uorescein (erythrosin, eosin, and Bengal rose).

(b) in solutions with BSA the maximum of the uorescence

spectrum of each nanomarker shifts to red area in contrast
to solutions without protein;
(c) the behavior of Imax
flu vs. pH (without/with BSA) of halogenderivatives of uorescein (erythrosin, eosin and Bengal rose)
are approximately identical, but differs dramatically from
the behavior of Imax
flu vs. pH (without/with BSA) of uorescein.
3.3. The chemical bonds in BSAnanomarker association
As it is well known, changes in Raman bands characterize
changes in chemical bonds. The Raman bands, corresponding to
chemical bonds of nanomarkers to BSA, were registered at different
values of pH (Table 1).
In this work we proposed that the maximal binding of each nanomarker to BSA corresponds to maximal number of Raman bands,
responsible to chemical bonds between nanomarker and BSA.
The maximal binding of uorescein to BSA (maximal number of
Raman peaks) takes place at pH 5.06.0 (Table 1), i.e. in that range,
where the molecules of uorescein are in the neutral form (BSA at
these pH is weakly negatively charged as a whole). At more alkaline values of pH (7.08.0), i.e. in that range, where the uorescein
is negatively charged, the decreasing of number of types of chemical bonds between uorescein and BSA is detected (BSA at these

Table 1
The spectral ranges with Raman bands, corresponding to binding of nanomarker (uorescein, erythrosin, eosin and Bengal rose) to BSA at different values of pH. The sign +
marks the presence of the Raman band in spectrum.
Spectral range (cm1)

11051115
11501160
13801405
15101520
15301545
15501565
15801590
16001610
16951720
17301755

FluoresceinBSA
pH

EosinBSA
pH

ErythrosinBSA
pH

Bengal roseBSA
pH

3.5

4.0

5.0

6.0

7.0

8.0

3.5

4.0

5.0

6.0

7.0

8.0

3.5

4.0

5.0

6.0

7.0

8.0

3.5

4.0

5.0

6.0

7.0

8.0

+
+
+
+

+
+
+
+
+
+

+
+
+

+
+
+
+
+
+

+
+

+
+

+
+

+
+

+
+

+
+

+
+

+
+

+
+

+
+
+
+
+
+
+
+
+
+

+
+
+

+
+

+
+
+
+
+
+
+
+
+
+

+
+
+

+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+

+
+
+

+
+

+
+
+
+
+
+
+
+
+
+

+
+
+

+
+
+

+
+
+
+
+
+
+
+
+
+

+
+
+

+
+
+

+
+
+
+
+
+
+
+
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I.M. Vlasova et al. / Journal of Molecular Structure 1051 (2013) 8694

pH is negatively charged as a whole). But the most small number of

types of chemical bonds between uorescein and BSA is detected
at acid values of pH (3.54.0), i.e. in that range, where the uorescein is positively charged (BSA at these pH is positively charged as
a whole).
The maximal binding of erythrosin to BSA (maximal number of
Raman peaks) takes place at pH 3.55.0 (Table 1), i.e. in that range,
where the erythrosin is weakly negatively charged (pH 3.65.0) or
is electrically neutral (3.53.6), and BSA at these pH is positively
charged as a whole.
The maximal binding of eosin to BSA (maximal number of Raman peaks) takes place at pH 3.55.0 (Table 1), i.e. in that range,
where the eosin is weakly negatively charged (BSA at these values
of pH is positively charged). At pH 5.08.0 the binding of eosin to
BSA decreases due to increase of negative charge of eosin (BSA at
these values of pH is charged negatively).
The maximal binding of Bengal rose to BSA (maximal number of
Raman peaks) takes place at pH 3.55.0 (Table 1), i.e. in that range,
where the Bengal rose is weakly negatively charged in monoanion
form (pH 2.64.0) or is weakly negatively charged in dianion form
(pH 4.05.0), and BSA at these pH is positively charged as a whole.
The dependences of number of types of chemical bonds (number
of Raman bands) between nanomarkers and BSA on pH are shown in
Fig. 11. The dependence of number of types of chemical bonds
between the nanomarker and BSA on pH has similar nature for
halogen-derivatives of uorescein and differs from the uorescein.
The dependence of number of types of chemical bonds between

93

the uorescein and BSA on pH has a non-linear behavior with

maximum at pH 6.0. For halogen-derivatives of uorescein the
dependence of number of types of chemical bonds between the
nanomarker and BSA on pH has monotonic decreasing behavior
with increase of pH.
From the data of Table 1, it is seen, that at binding of these nanomarkers to BSA the Raman bands in identical ranges of spectra are
registered. In Table 2 the decryption of Raman bands, corresponding to binding of nanomarkers to BSA, is shown. The residues of
only polar amino acids of BSA participate in binding of these nanomarkers: the residues of threonine (Thr), serine (Ser), lysine (Lys),
arginine (Arg), glutamine (Gln) and glutaminic acid (Glu).
The mechanism of binding of ligands to BSA molecule is determined by availability of binding Centers on protein. To binding
Centers of small organic molecules, such as uorescein, erythrosin,
eosin and Bengal rose, attribute Centers I and II [1]. On obtained at
decryption of Raman spectra the amino acidic residues of BSA, participating in binding of these nanomarkers, it is established, that
uorescein and its halogen-derivatives bind to Center I of BSA.
On changes in Raman spectra of solutions of BSA with nanomarkers we can conclude, that:
(a) the binding of all nanomarkers of uorescein family takes
place through binding Center I of BSA;
(b) the character of binding of uorescein to BSA differs from
character of binding of its halogen-derivatives to BSA: the
dependence of number of types of chemical bonds between

Table 2
The decryption of Raman bands, corresponding to binding of nanomarkers (uorescein, erythrosin, eosin and Bengal rose) to BSA.
Range (cm1)

Decryption

11051115

Binding of nanomarker through its hydroxyl group with threonine (Thr)

11501160

Binding of nanomarker through its hydroxyl group with serine (Ser)

13801405

Binding of nanomarker through its carboxyl group with lysine (Lys)

15101520

Binding of nanomarker through its hydroxyl group with glutamine (Gln)

15301545

Binding of nanomarker through its hydroxyl group with arginine (Arg)

15501565

Binding of nanomarker through its hydroxyl group with lysine (Lys)

15801590

Binding of nanomarker through its carboxyl group with threonine (Thr)

16001610

Binding of nanomarker through its carboxyl group with serine (Ser)

16951720

Binding of nanomarker through its hydroxyl group with glutaminic acid (Glu)

17301755

Binding of nanomarker through its carboxyl group with glutaminic acid (Glu)

94

I.M. Vlasova et al. / Journal of Molecular Structure 1051 (2013) 8694

the nanomarker and BSA on pH for uorescein has a non-linear behavior with a maximum at pH 6.0 and for halogenderivatives of uorescein has monotonic decreasing behavior with increase of pH.
4. Conclusion
The found differences in the characteristics of nanomarkers of
uorescein family in BSA solution are determined by value of an
electronegativity of atoms of lateral radicals in structural formulas
of nanomarkers. The electronegativity of atoms of lateral radicals
in structural formulas of these nanomarkers inuences on pK of
their ionized groups (carboxyl COOH and hydroxyl OH). The increase of electronegativity is observed in a following direction:
Hydrogen (for uorescein) Iodine (for erythrosin) Bromide
(for eosin) Chlorine and Iodine (for Bengal rose). The presence
in molecule of nanomarker the more electronegative atom results
in strong decreasing of values of pK(COOH) and pK(OH) of these
nanomarkers. The changes of main characteristics of nanomarkers
of uorescein family depending on pH are explained by values of
pK of their ionized groups.
Concerning the processes of molecular association of nanomarkers, it is necessary to mark, that despite of common feature for all
nanomarkers of uorescein family in solutions of BSA (reduction of
1  X), the differences are registered: for uorescein the non-linear
character of dependence of 1  X on pH with maximum at pH 6.0 is
found, and for halogen-derivatives of uorescein (erythrosin, eosin
and Bengal rose) the dependence of 1  X on pH has monotonic
decreasing character at increase of pH. The increase of electronegativity of atoms in nanomarkers results in reduction of 1  X.
Concerning the spectra of steady-state uorescence, it is necessary to mark, that despite of common features of all nanomarkers
of uorescein family in solutions of BSA (quenching of uorescence
and red shift of maximum of uorescence spectrum), the insertion
of the strongly electronegative atoms (Bromide, Chlorine and Iodine) in nanomarker results in change of dependence of uorescence on pH.
Concerning the related changes in chemical bonds in BSAnanomarker association, it is necessary to mark, that despite of the fact
that all nanomarkers of uorescein family are bound to binding
Center I of BSA, the differences are found: for uorescein the
dependence of quantity of chemical bonds on pH has non-linear
behavior with maximum at pH 6.0, and for halogen-derivatives
of uorescein, in molecules of which one the strongly electronegative atoms are entered, this dependence has monotonic decreasing
character with increase of pH.
5. Summary
The insertion of atoms with different electronegativity in structural formulas of nanomarkers of uorescein family allows one to

receive the uorescent nanomarkers with different optical and

molecular characteristics and different character of binding to BSA.
Chemical and electrostatic interactions play the leading role of
between nanomarkers and BSA. The data about binding of uorescent nanomarkers to BSA and about properties of these nanomarkers in BSA solutions allow to receive the information about
mechanisms of interaction of uorescent nanomarkers with BSA,
what can be useful at research of structure and properties of binding Centers (drug-binding Centers) of BSA, and this is of great
importance in medical investigations of binding of drugs to BSA.
The registered regularities in uorescent characteristics, in
molecular association of nanomarkers of uorescein family in
BSA solutions and in binding of nanomarkers to BSA are similar
to the results of data of nanomarkers in HSA solutions received
by us earlier [717] that speaks on accessory of BSA and HSA to
one homologous family of albumins and on identical mechanisms
of interaction of nanomarkers of uorescein family with these
proteins.
References
[1] T. Peters, All about Albumin, Academic Press, New York, 1996.
[2] W. Schmidt, Optical Spectroscopy in Chemistry and Life Sciences, Wiley-VCN
Verlag GmbH & Co. KGaA, 2005.
[3] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum, New York,
London, 1999.
[4] S. Pelet, M. Gratzel, J.E. Moser, J. Phys. Chem. B 107 (2003) 32153224.
[5] L. Birla, B. Prieto, T. Noguel, J. Vigo, A.-C. Ribou, Rev. Roum. Chim. 52 (2007)
639646.
[6] A.A. Waheed, K. Sridhar Rao, P.D. Gupta, Anal. Biochem. 287 (2000) 7379.
[7] I.M. Vlasova, A.Yu. Zemlyansky, A.M. Saletsky, J. Appl. Spectrosc. 73 (2006)
743747.
[8] D. Gao, Y. Tian, F. Liang, D. Jin, Y. Chen, H. Zhang, A. Yu, J. Lumin. 127 (2007)
515522.
[9] B.B. Bhowmik, P. Ganguly, Spectrochim. Acta A 61 (2005) 19972003.
[10] D.E. Buravtcov, I.M. Vlasova, A.M. Saletsky, Photomed. Laser Surg. 26 (2008)
181187.
[11] I.M. Vlasova, A.M. Polyansky, A.M. Saletsky, Laser Phys. Lett. 4 (2007) 390394.
[12] I.M. Vlasova, A.M. Saletsky, Laser Phys. Lett. 5 (2008) 384389.
[13] A. Sulkowska, J. Rownicka, B. Bojko, W. Sulkowski, J. Mol. Struct. 651653
(2003) 133140.
[14] A. Sulkowska, M. Maciazek-Jurczyk, B. Bojko, J. Rownicka, I. Zubik-Skupien, E.
Temba, D. Pentak, W.W. Sulkowski, J. Mol. Struct. 891 (2008) 278283.
[15] Feng Wang, Wei Huang, Zhongxiang Dai, J. Mol. Struct. 875 (2008) 509514.
[16] Zhaolian Yu, Daojin Li, Baoming Ji, Jianjun Chen, J. Mol. Struct. 889 (2008) 422
426.
[17] I.M. Vlasova, A.A. Vlasov, A.M. Saletsky, J. Mol. Struct. 984 (2010) 332338.
[18] E.M. Buharova, I.M. Vlasova, A.M. Saletsky, J. Appl. Spectrosc. 75 (2008) 785
790.
[19] I.M. Vlasova, A.M. Saletsky, J. Mol. Struct. 936 (2009) 220227.
[20] I.M. Vlasova, A.A. Kuleshova, A.I. Panchishin, A.A. Vlasov, J. Mol. Struct. 1016
(2012) 17.
[21] I.M. Vlasova, A.M. Saletsky, Curr. Appl. Phys. 9 (2009) 10271031.
[22] I.M. Vlasova, E.M. Bukharova, A.A. Kuleshova, A.M. Saletsky, Curr. Appl. Phys.
11 (2011) 11261132.
[23] I.M. Vlasova, A.M. Saletsky, Laser Phys. Lett. 5 (2008) 834839.
[24] I.M. Vlasova, A.M. Saletsky, Russ. J. Phys. Chem. A 84 (2010) 10651070.
[25] I.M. Vlasova, A.M. Saletsky, Laser Phys. 20 (2010) 18441848.