Study on the interaction of sodium morin-5-sulfonate with bovine serum albumin by spectroscopic techniques

Article abstract of DOI:10.1016/j.saa.2011.10.023

In the present investigation, an attempt has been made to study the interaction of sodium morin-5-sulfonate (NaMSA) with the transport proteins, bovine serum albumin (BSA) employing UV-vis, fluorometric and circular dichroism (CD) techniques. The experime

Full text of DOI:10.1016/j.saa.2011.10.023

Spectrochimica Acta Part A 86 (2012) 191–195
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Study on the interaction of sodium morin-5-sulfonate with bovine serum
albumin by spectroscopic techniques
Nahid Shahabadi∗, Mahnaz Mohammadpour
Department of Chemistry, Faculty of Science, Razi University, Kermanshah, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present investigation, an attempt has been made to study the interaction of sodium morin-
5-sulfonate (NaMSA) with the transport proteins, bovine serum albumin (BSA) employing UV-vis,
fluorometric and circular dichroism (CD) techniques. The experimental results indicated that the quench-
ing mechanism of BSA by the compound was a static procedure. Various binding parameters were
evaluated. The negative value of ꢀH, positive value of ꢀS and the negative value of ꢀG indicated that
electrostatic interactions and hydrogen bonding play major roles in the binding of the NaMSA and BSA.
Based on the Forster’s theory of non-radiation energy transfer, the binding distance, r, between the donor
(BSA) and acceptor (NaMSA) was evaluated. The results of CD and UV-vis spectroscopy showed that the
binding of this complex to BSA induces some conformational changes in BSA.
Received 9 September 2011
Received in revised form 13 October 2011
Accepted 14 October 2011
Keywords:
BSA
Morin
Fluorescence
Circular dichroism
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
constituted of 582 amino acid residues and based on the distri-
bution of the disulphide bridges and of the amino acid sequence,
Flavonol morin belongs to the flavonoid family and is an impor-
tant plant pigment in herbal medicine. Recent interest in flavonols
stems from their broad-spectrum pharmacological and therapeutic
activities. The basic structure of flavonols is usually characterized
by two aromatic rings, ring A and ring B, which are joined by a
three-carbon linked ␥-pyrone ring (ring C), forming a C6–C3–C6
skeleton unity where polar groups, usually hydroxyl, methoxyl or
glycosyl appended at various positions. (Fig. 1a, [1]).
it seems possible to regard BSA as composed of three homolo-
gous domains linked together. The domains can all be subdivided
into two sub-domains. The binding sites of BSA for endogenous
and exogenous ligands may be in these domains and the princi-
pal regions of drugs binding sites of albumin are often located in
hydrophobic cavities in sub-domains IIA and IIIA. So called sites I
and II are located in subdomain IIA and IIIA of albumin, respectively.
Many ligands bind specifically to serum albumin. As proposed by
Kragh-Hansen [5], there are at least six binding regions, one or two
high affinity binding sites (primary sites) and a number of sites with
lower affinity [6].
In this study, we have reported the synthesis of NaMSA water-
soluble compound and the binding properties of this compound
with BSA has been carried out using different instrumental meth-
ods and the binding mode is discussed. Some techniques commonly
used to detect interaction between drugs and serum albumin
includes fluorescence, UV-vis, and circular dichroism techniques
[7].
Morin (3,5,7,2ꢀ,4ꢀ-pentahydroxyflavone) is found in almonds,
milk, fig and other moraceae which are used as dietary agents and
also as herbal medicines [2]. A light yellowish pigment found in
the wood of old fustic Chlorophora tinctori [3]. Morin hydrate is
known to have a broad pharmacological activity, such as antitu-
mour, antioxidation, cardiovascular protection and possibly even
protective effects against chronic diseases. Each component of
flavonols has its specific biological function and activity, and the
pharmacological actions of morin are different from those of other
flavonols. Morin exerts its functions usually by regulating the acti-
vation or modifying the structure of enzymes [1].
The interaction between bio-macromolecules and flavonoids
has attracted great interest among researchers for several decades
[4]. In this paper, bovine serum albumin (BSA) was selected as
the protein model because of its low cost, ready availability, and
unusual ligand-binding properties. Bovine serum albumin (BSA) is
2. Experimental
2.1. Materials
Bovine serum albumin (BSA) was purchased from
Sigma–Aldrich. All BSA solutions were prepared in the Tris–HCl
0.05 M and 0.1 M NaCl buffer to keep pH value constant (pH 7.40),
and to maintain the ionic strength of all solutions in experiments.
BSA solutions stocked in the dark at 4 ◦C. Morin was purchased
∗
Corresponding author. Tel.: +98 831 8360795; fax: +98 831 8360795.
E-mail address: nahidshahabadi@yahoo.com (N. Shahabadi).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.10.023

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N. Shahabadi, M. Mohammadpour / Spectrochimica Acta Part A 86 (2012) 191–195
2.2.3. Fluorescence spectra
All fluorescence spectra were recorded with a LS-55 Spectroflu-
orimeter (Perkin-Elmer corporate, UK) equipped with quartz cells
(1.0 cm) and a thermostat bath. The maximal fluorescence emission
of BSA at ꢁex = 289 nm was located at 346 nm.
Fluorescence spectra were recorded from 300 to 500 nm with
the excitation wavelength at 289 nm. First 3 mL of solution con-
taining constant concentration of BSA (6 × 10−6) was titrated by
the successive addition of NaMSA solution (concentration was var-
ied from 0 to 20 × 10−6 mol L−1). Titration was done manually
via a microsyringe and the fluorescence intensity of BSA in the
absence and the presence of the NaMSA was measured. All experi-
ments were measured at three temperatures (295, 298, and 303 K).
For quenching property, the mechanism can be described by the
Stern–Volmer equation (Eq. (3)).
F0
= 1 + Ksv[Q]
(3)
F
where F0 and F are the fluorescence intensities in the absence and
presence of a quencher, KSV is the Stern–Volmer quenching con-
stant and [Q] is the concentration of a quencher. The plot of F0/F vs.
[Q] shows the value of KSV (KSV = Kqꢂ0).where Kq is the quenching
rate constant and ꢂ0 is the fluorescence lifetime of protein in the
absence of quencher, the value of ꢂ0 is considered to be 10−8 s [9].
Fig. 1. (a) The structure of 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-
one (morin); (b) the basic structure of the sodium salt of morin-5ꢀ-sulfonic acid
(NaMSA).
2.3. Synthesis
2.3.1. Synthesis of NaMSA
Sulfonate derivative of morin, sodium morin-5-sulfonat
(NaMSA) was obtained by the method described [10].
from Merck, dry EtOH, DMSO, methanol and H2SO4 were obtained
from Fluka Company. Doubly distilled water was used to prepare
the buffers.
40 mL of concentrated sulfuric acid was added to 10 g of morin.
The mixture was heated for 4 h at 80 ◦C with occasional stirring. The
pH of the cold reaction mixture was adjusted to 3 with 20% aqueous
NaOH, and then 50 mL of saturated aqueous NaCl was added. The
precipitated yellow solid was separated and the isolated yield was
50%. Elemental analysis gave the formula C15H9O10SNa·2H2O.
2.2. Methods
2.2.1. UV-spectrophotometry
Absorbance spectra were recorded using an hp spectropho-
tometer (agilent 8453), equipped with
a thermostated bath
3. Results and discussion
(Huber polysat cc1). Absorption titration experiments were car-
ried out by keeping the concentration of BSA constant (3 × 10−5 M)
while varying the NaMSA concentration from 0 to 4.5 × 10−5 M
(ri = [morin]/[BSA] = 0.0, 0.2, 0.4, 0.6, 0.8, 1, 1.2,1.5). Absorbance val-
ues were recorded after each successive addition of BSA solution
and equilibration (ca. 10 min).
3.1. General
In this study, we eliminated the inner filter effect for all of the flu-
orescence results to obtain accurate data. For the BSA–drug system,
the inner filter effect is caused by the absorption of the excita-
tion and emission wavelengths by drug and BSA in fluorescence
experiments, which change the intensity of fluorescence spectra
of BSA, affecting the binding parameters calculated from the fluo-
rescence data [11]. The fluorescence intensities were corrected for
absorption of exciting light and reabsorption of the emitted light
to decrease the inner filter effect using the relationship [12]:
2.2.2. CD studies
Circular dichroism (CD) measurements were recorded on a
JASCO (J-810) spectropolarimeter (between 200 and 250 nm and
cell length path was 0.1 cm) by keeping the concentration of BSA
constant (3 × 10−6 M) while varying the NaMSA concentration from
0 to 6 × 10−6 M (ri = [morin]/[BSA]) = 0.0, 0.3, 0.5, 1, 1.5, 2). The CD
results were expressed in terms of mean residue ellipticity (MRE)
in degcm2 dmol−1 according to the following equation:
+A /2)
em
FCOR = Fobs × e(A
(4)
ex
where Fcor and Fobs are the fluorescence intensities corrected and
observed, respectively, and Aex and Aem are the absorption of the
systems at the excitation and the emission wavelength, respec-
tively. The intensity of fluorescence used in this paper is the
corrected fluorescence intensity
Fluorescence spectroscopy has been usually used in the study
of molecular interactions between ligands and proteins, mainly
due to the high sensitivity of this methodological approach and
the variety of parameters related with the interaction molecular
that can be obtained. Fluorescence quenching refers to any pro-
cess which decreases the fluorescence intensity of a fluorophore. A
variety of molecular interactions can result in fluorescence quench-
ing, including excited-state reactions, molecular rearrangements,
energy transfer, ground-state complex formation and collisional
observed CD (mdeg)
MRE =
(1)
Cpnl × 10
where Cp is the molar concentration of the protein, n the number
of amino acid residues (583 for BSA) and l the path-length (0.1 cm).
The ␣-helical contents of free and combined BSA were calculated
from MRE values at 208 nm using the Eq. (2):
−MRE280 − 4000
˛-helix(%) =
(2)
33, 000 − 4000
The observed MRE value at 208 nm, MRE208, of ␤-form and ran-
dom coil conformation cross in total and a pure ␣-helix were 4000
and 33000, respectively. From the above equation, the ␣-helicity in
the secondary structure of BSA was determined [8].

N. Shahabadi, M. Mohammadpour / Spectrochimica Acta Part A 86 (2012) 191–195
193
Fig. 2. Fluorescence spectra of BSA in the absence and the presence of NaMSA, [BSA] = 6 × 10−6 M and [NaMSA] = 0, 2, 4, 6, 10, 15, 20 × 10−6, ꢁex = 289 nm and ꢁem = 346 nm,
silt width = 10 nm. Insets in: Stern–Volmer curve for the binding of NaMSA with BSA.
quenching [13]. The maximal fluorescence emission of BSA at
ꢁex = 289 nm was located at 346 nm (Fig. 2) The fluorescence inten-
sity of BSA decreased in the presence of NaMSA and the maximum
emission wavelengths was shifted from 346 to 345.07 nm. This sug-
gested that the microenvironment around BSA was changed after
the addition of the compound. The results indicate that there is
strong interaction and energy transfer between BSA and sulfonated
morin [14].
determined according to the method described by Jiang et al. [17]
using the following equation:
ꢀ
ꢁ
F0 − F
log
= log Kb + n log[Q]
(5)
F
where, in the present case, Kb is the binding constant for the NaMSA
with protein interaction and n is the number of binding sites per
albumin molecule, which can be determined by the slope and the
intercept of the double logarithm regression curve of log(F0 − F/F)
versus log [Q] based on the Eq. (5) (Fig. 3 and Table 1). The correla-
tion coefficients are larger than 0.9, indicating that the assumption
underlying the deviation of Eq. (5) is satisfactory. The value of n
at the experimental condition are approximately equal to 1, which
indicates that there is just one single binding site in BSA for the
compound.
3.2. Quenching constants
Fluorescence quenching is the decrease of the quantum yield
of fluorescence from a fluorophore induced by a variety of molecu-
lar interactions with a quencher molecule. Fluorescence quenching
can occur via different mechanisms, which are usually classified as
either static quenching or dynamic quenching. Static quenching,
resulting from the formation of a ground-state complex between
the fluorophore and a quencher, dynamic quenching, resulting from
collisional encounters between the fluorophore and quencher. In
both cases, molecular contact is required between the fluorophore
and the quencher for fluorescence quenching to occur [15].
Fig. 2 displays the Stern–Volmer plots of the quenching of BSA
fluorescence by NaMSA. The data are shown in Table 1. Using the
approximate fluorescence lifetime of BSA (10 ns) [16], the bimolec-
ular quenching constant (kq) was determined from Kq = KSV/ꢂ0. The
values of Kq obtained for the binding of BSA to the compound are
of the order of 1013 L mol −1 s−1 (Table 1). The quench constants are
greater than those in the biopolymer (2.0 × 1010 L mol−1 s−1) by the
maximum scatter collision mechanism. This means that the possi-
ble quenching mechanism of fluorescence of BSA by NaMSA is not
initiated by dynamic collision but from the formation of a complex.
The quenching procedure can be further confirmed by tempera-
ture dependence of the quenching: KSV values decrease with an
increase in temperature for static quenching and the reverse will
be observed for dynamic quenching [16]. From Table 1, it can be
found that the Ksv decreasing with increasing temperature. So the
quenching is a static quenching procedure [16].
In order to elucidate the interaction of the NaMSA with BSA,
the thermodynamic parameters were calculated. The plot of log Kb
versus 1/T (Eq. (6)), where Kb is the binding constant at the cor-
responding temperatures and R is the gas constant, allows the
determination of enthalpy change (ꢀH) and entropy change (ꢀS).
If the temperature does not vary significantly, the enthalpy change
(ꢀH) can be regarded as a constant. Based on the binding con-
stants at different temperatures, the free energy change (ꢀG) can
be estimated by Eq. (7) (Table 1).
−ꢀH
ꢀS
Ln Kb
=
+
(6)
(7)
RT
R
ꢀG = ꢀH − TꢀS
3.3. Binding constants and the number of binding sites
For the static quenching interaction, if it is assumed that there
are similar and independent binding sites in the biomolecule, the
binding constant (Kb) and the number of binding sites (n) can be
Fig. 3. Determination of NaMSA–BSA binding constant and number of binding sites
on BSA at 298 K.

194
N. Shahabadi, M. Mohammadpour / Spectrochimica Acta Part A 86 (2012) 191–195
Table 1
The quenching constants (Ksv) (Kq), binding constants (Kb), number of binding sites (n) and relative thermodynamic parameters of the BSA–NaMSA system at different
temperatures.
T (K)
KSV × 10−4 (L mol−1
)
Kq × 1012 (L mol− s−1
)
Kb × 103 (mol L−1
)
n
ꢀS (J mol−1 K−1
)
ꢀH (kJ mol−1
−7.28
)
ꢀG (kJ mol−1
)
293
298
303
1.30
1.23
1.08
1.3
1.23
1.08
2.37
2.25
2.16
0.98
0.94
0.95
−11.65
−11.85
−12.05
39.73
Ross and Subramanian [18] have characterized the sign and
magnitude of the thermodynamic parameter associated with var-
ious individual kinds of interaction that may take place in protein
association process, which can be easily concluded as: (a) ꢀH > 0
and ꢀS > 0, hydrophobic force; (b) ꢀH < 0 and ꢀS < 0, van der Waals
force and hydrogen bond; (c) ꢀH < 0 and ꢀS > 0, electrostatic inter-
actions. The negative ꢀH value is frequently taken as evidence for
hydrogen bond in the binding interaction [19]. Thus, from the ther-
modynamic characteristics summarized above, the negative ꢀH
and positive ꢀS values indicate that electrostatic force and hydro-
gen bond played major roles in the NaMSA–BSA binding reaction
and contributed to the stability of the complex.
where R0 is the critical distance when the transfer efficiency, E, is
50%. It can be estimated from Eq. (9):
R06 = 8.8 × 10−25K2n−4J˚
(9)
where K2 is the spatial orientation factor of the dipole, n the refrac-
tive index of the medium, ˚ the fluorescence quantum yield of
the donor, J the overlap integral of the fluorescence emission spec-
trum of the donor and the absorption spectrum of the acceptor.
Therefore, Eq. (10)
˙F0(ꢁ)ε(ꢁ)ꢁ4ꢀꢁ
J =
(10)
F(ꢁ)ꢀꢁ
where F(ꢁ) is the fluorescence intensity of the fluorescent donor at
wavelength ꢁ, and ε(ꢁ) is the molar absorptivity of the acceptor at
wavelength ꢁ. J can be obtained by integrating the overlap in the
spectra (Fig. 4). The required constants in the above equations for
BSA are: k2 = 2/3, ꢁ = 0.118, n = 1.336 [21] and with these values, it
was found that r = 2.38 nm for NaMSA, the donor–acceptor distance,
r < 8 indicates that the energy transfer from BSA to NaMSA occurs
with high possibility [22].
3.4. Energy transfer between NaMSA and BSA
The spectral studies suggested that NaMSA form a complex
with BSA. To get much more information about NaMSA–BSA sys-
tem attention was focused on the aspect of energy transfer in the
system. Generally speaking, fluorescence resonance energy trans-
fer occurs when the emission spectrum of a fluorophore overlaps
the absorption spectrum of another molecule. Fig. 4 shows the
spectral overlap between absorption spectrum of NaMSA with fluo-
rescence spectrum of BSA in the wavelength range of 300–500 nm.
The importance of the Förster resonance energy transfer in bio-
chemistry is that the efficiency of transfer can be used to evaluate
the distance between the ligand and the tryptophan residues
responsible of the natural intrinsic fluorescence of the protein [20].
According to Förster’s non-radiative energy transfer theory, the
energy transfer between the excited molecule and its neighbours
will occur under the following conditions: (i) the donor must fluo-
resce; (ii) the fluorescence emission spectrum of the donor and the
UV–vis absorbance spectrum of the acceptor must overlap signifi-
cantly; and (iii) the distance between the donor and the acceptor
must be lower than approximately 8 nm. The distance, r, between
the acceptor (NaMSA) and the donor (BSA) was calculated as fol-
lows: [21].
3.5. Conformation investigation
3.5.1. UV-vis spectroscopy
Spectroscopy techniques can be used to explore the structural
changes of protein and to investigate protein–ligand complex for-
mation. BSA has two absorption peaks. The strong absorption peak
at about 208 nm reflects the framework conformation of the pro-
tein. The weak absorption peak at about 279 nm appears to be due
to the aromatic amino acids (Trp, Tyr, and Phe) [9]. It is well known
that the absorption of a chromophore is shifted in directions and
magnitudes that depend on whether it is transferred to a more
hydrophilic or more hydrophobic environment. These shifts are
*
ascribed to a change of ␲–␲ transition brought about by changes
in the polarizability of the solvent [23].
In order to obtain more information on NaMSA–BSA interaction,
UV–vis absorption spectrum of system was recorded at 298 K. It
was observed that the absorption of BSA increased upon the addi-
tion of drug (Fig. 5). The maximum peak position of NaMSA–BSA
was shifted slightly towards longer wavelength region. This indi-
cated the change in polarity around the tryptophan residue and the
change in peptide strand of BSA molecules and hence the change
in hydrophobicity. So, the binding of NaMSA to protein molecule
might lead to change in protein conformation [24].
R06
R06
F0 − F
E =
+ r6
=
(8)
F0
3.5.2. Circular dichroism
CD is a sensitive technique for monitoring conformational
changes of protein upon interaction with small molecules. Bovine
serum albumin has a high percentage of ␣-helical structure which
shows characteristic strong double minimum signals at 222 and
208 nm [8]. The reasonable explanation may be that the negative
peaks between 208 and 222 nm are both contributed to n–␲* trans-
fer for the peptide bond of ␣-helix [25]. The CD spectra of BSA in the
absence and the presence of NaMSA are shown in Fig. 6. The addi-
tion of increasing amount of NaMSA causes the distortion in the CD
spectra. This indicates the disordered form of ␣-helical structure.
Fig. 4. Spectral overlap of NaMSA absorption with BSA fluorescence.

N. Shahabadi, M. Mohammadpour / Spectrochimica Acta Part A 86 (2012) 191–195
195
4. Conclusion
In this paper, we studied the interaction of the NaMSA with
BSA via fluorescence, UV–vis and CD spectrum. The results of
fluorescence, UV–vis and CD spectra indicate that the secondary
structure of BSA molecules changes in the presence of NaMSA.
The experimental results also indicate that the quenching of flu-
orescence of BSA by NaMSA is a static quenching process. The
negative ꢀH and positive ꢀS values indicate that electrostatic
force and hydrogen bond played major roles in the NaMSA–BSA
binding reaction and contributed to the stability of the com-
plex.
This work provides some valuable information for the trans-
portation and distribution of NaMSA in vivo, and is helpful for
clarifying toxicity and dynamics of NaMSA.
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The percentage of helicity of BSA is 87.8% in pure BSA (Fig. 6) and
in the presence of NaMSA it becomes 95.6% which shows that bind-
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can be deduced that the ␣-helical structure is affected probably
due to insertion of some of NaMSA residues into the hydropho-
bic surface of BSA. So when NaMSA bounds to BSA the ␣-helicity
increases. This observation strongly indicates that the binding of
NaMSA to BSA induces some conformational changes in BSA but
the protein retains its secondary structure and helicity when inter-
act with NaMSA. This phenomenon is important for biomedical
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