Spectroscopic and ITC studies of binding of Ferulic acid with BSA

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Thermochimica Acta 548 (2012) 56–64
Contents lists available at SciVerse ScienceDirect
Thermochimica Acta
journal homepage: www.elsevier.com/locate/tca
Spectroscopic and isothermal titration calorimetry studies of binding interaction
of ferulic acid with bovine serum albumin
Himanshu Ojhaa
, Krishnanand Mishraa
, Mohammad I. Hassanb
, Nabo K. Chaudhurya,∗
a
Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Delhi 110054, India
b
Center for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India
a r t i c l e i n f o
Article history:
Received 23 May 2012
Received in revised form 23 July 2012
Accepted 5 August 2012
Available online 17 August 2012
Keywords:
Bovine serum albumin
Ferulic acid
Anisotropy
ITC
Circular dichroism
Fluorescence lifetime
a b s t r a c t
The binding characteristics of ferulic acid (FA) with bovine serum albumin (BSA) were investigated
using fluorescence, circular dichroism (CD) and isothermal titration calorimetry (ITC) techniques at pH
7.4. Fluorescence technique on basis of quenching method determined one class of binding site with
binding constant (40.14 ± 0.02) × 104
(mol−1
dm3
) at 298 K. Fluorescence anisotropy and lifetime study
indicated significant microenvironment changes around FA upon interaction with BSA suggesting bind-
ing. The circular dichroism data indicated changes in the secondary structure of BSA upon binding with
FA thus confirming the role of non-covalent forces and also suggested that thermal stability of BSA
increases upon binding with FA. ITC data suggest that FA binds to BSA at two sites with high affinity
and binding forces are mainly electrostatic and hydrogen bonding. Inconsistency between calorimetric
enthalpy and Van’t Hoff enthalpy indicated appreciable conformational change in BSA upon binding
of FA.
© 2012 Published by Elsevier B.V.
1. Introduction
Ferulic acid (FA) [3-(4-hydroxy-3-methoxy-phenyl) prop-2-
enoic acid] (Fig. 1) is a natural antioxidant commonly found in
fruits and vegetables including tomato, sweet corn and rice bran,
etc. FA has a wide range of therapeutic effects against diseases,
such as cancer, diabetes, cardiovascular, neurodegerative disorder,
and antiaging owing to its antioxidant properties [1–4]. Ogiwara
et al. [5] have shown scavenging capacity of FA for nitric oxide
(NO), superoxide e(O2
−) and hydroxyl (OH) radicals, while Itagaki
et al. [6] have reported a comparative study on in vitro and in vivo
antioxidant properties of FA with other natural oxidation inhibitors.
Khanduja et al. [7] demonstrated the antiapop-
totic potential of FA in normal human peripheral blood
mononuclear cells. Chemopreventive potential of FA in 7,12-
dimethylbenz[a]anthracene-induced mammary carcinogenesis
in Sprague-Dawley rats decreases the yield of DNA strands
and breaks in murine peripheral blood leukocytes both in pre-
irradiation and post-irradiation administration of FA lowering of
gamma radiation-induced dicentric aberration on bone marrow,
∗ Corresponding author at: Division of Radiation Biosciences, Institute of Nuclear
Medicine and Allied Sciences, Brig S K Mazumdar Road, Timarpur, Delhi 110054,
India. Tel.: +91 11 23905131; fax: +91 11 23919509.
E-mail address: nkcinmas@rediffmail.com (N.K. Chaudhury).
and lipid peroxidation in cultured lymphocytes [8–10]; all of which
thus suggested the potential of FA as radioprotector.
The focus of our research is to develop a mechanistic based
approach to counter radiation effects in different models and even-
tually evaluate the radioprotective efficacy in animal model. The
group is engaged in studies on cellular radiation responses with
various potential molecules [11–13]. Since FA has shown radical
scavenging and other therapeutic potentials, FA is the molecule of
interest for us to develop as a radioprotector and/or mitigator of
radiation counter measure agents.
It is known that the free concentration, bio-distribution and
metabolism of a therapeutic molecule are strongly affected by
protein–ligand (small molecule) interactions in the blood stream.
Protein–ligand interaction may also influence the drug stability and
toxicity of drug in systemic levels. Most of the organic compounds
in the blood stream are mainly transported by serum proteins while
only a minor fraction is dissolved in the serum. Albumin is the most
abundant protein in serum and it accounts for about 60% of its
total protein content. Besides, the major soluble protein constituent
of the circulatory system, serum albumin has many physiologi-
cal functions and plays a key role in the transport of endogenous
and exogenous ligands [14]. Ligands are bound to serum albu-
min with a high affinity in the process of transportation and this
ligand–protein interaction results in a stable protein–ligand com-
plex which may influence the bioavailability and efficacy of the
drug in vivo [15,16]. Consequently, it is important to study the
interaction of a therapeutic molecule with serum albumin.
0040-6031/$ – see front matter © 2012 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.tca.2012.08.016

H. Ojha et al. / Thermochimica Acta 548 (2012) 56–64 57
Fig. 1. Molecular structure of FA.
Earlier studies (Table 1) have reported FA binding to BSA [17–20]
but these studies have investigated binding mechanism only in
terms of conformational aspects and assessed binding parameters
using techniques, fluorescence, Hummel Dryer (HD) size exclusion
chromatography, affinity capillary electrophoresis (ACE), and sur-
face plasmon resonance (SPR). It was observed that these studies
did not focus the on the energetics of FA–protein interactions.
As the binding parameters, especially binding constant
(K) reported for FA–BSA binding, were found to vary from
4.1 ± 0.07 × 103 mol−1 dm3 to 5.6 × 104 mol−1 dm3 with single
class of binding site for FA, this variation in binding parameters
is significant and needs attention. The analysis of findings from
these studies suggests that the different experimental conditions
are employed and also limitations of each method are important
factors contributing variations in values of binding parameters. One
of such problems was that of different buffers and/or water for
protein, and different solvents were used to prepare FA solutions.
In the present study we have made an attempt to compare
binding parameters and thermodynamical parameters obtained
through fluorescence tryptophan quenching method with isother-
mal titration calorimetric (ITC) measurements. All studies in
the present work have been performed in phosphate buffer at
physiological pH. The results will contribute toward develop-
ing the understanding of binding interaction both in terms of
conformational and energetics aspects for various researchers
engaged in thermodynamics for molecular interactions and drug
developments.
2. Experimental
2.1. Material and methods
Bovine serum albumin (BSA, Fraction V, approximately 99%;
protease free and essentially ␥-globumin free) and ferulic acid
(FA) were purchased from Sigma Chemical, USA. Sodium chloride,
monobasic phosphate and dibasic phosphate were purchased from
Merck (India). The water used for preparation of solutions was
18 M MQ grade derived from Millipore water system (model Elix
3, Millipore Corp., USA).
The protein stock solution was prepared in 10 × 10−3 mol dm−3
phosphate buffer (pH 7.4) with 0.15 mol dm−3 NaCl and dialyzed
overnight at 277 K with three changes in buffer solution. The con-
centration of BSA was determined on UV–Visible spectrophotome-
ter (Cary Bio100, Varian, Australia), using extinction coefficient of
E1%
280 nm
6.8 [21]. Stock solution of FA (1 × 10−3 mol dm−3) was pre-
pared in the same phosphate buffer (pH 7.4) and the concentration
was assessed spectrophotometrically using the molar extinction
coefficient of E310 nm = 18,000 mol−1 dm3 cm−1.
2.2. Fluorescence spectroscopy
Fluorescence steady state measurements were performed on
a spectrofluorimeter (FS920 Edinburgh Instruments, UK) with
standard 3.5 ml quartz cell. The excitation source was xenon lamp
(450 W) and sample chamber was equipped with Peltier acces-
sory. Scanning parameters for all measurements were optimized
with slit width 4.0 nm for excitation and emission, dwell time
0.2 s and wavelength step 0.5 nm. The protein concentration was
kept at 10 × 10−6 mol dm−3 and FA concentration was added up
to 52 × 10−6 mol dm−3 for titration. The excitation wavelength
was set at 295 nm to selectively excite the tryptophan residues.
The emission spectra were recorded in the wavelength range of
310–450 nm at a scan rate of 100 nm min−1.
The spectral data thus obtained were analyzed with
Stern–Vomer equation:
F0
F
= 1 + Ksv[FA] (1)
where F0 and F are the fluorescence intensities of BSA at 336 nm
in the absence and presence of the quencher molecule FA,
Table 1
Summary of literature reports on binding of FA with BSA based on various techniques.
S. no. Parameters Techniques used References
K (mol−1
dm3
) n
1 4.01 ± 0.07 × 103
3.6 ± 0.1 Hummel Dryer (HD)
Size exclusion chromatography
[17]
2 1.793 × 104
0.99 Fluorescence
(Tryptophan quenching method)
[20]
3 5.56 ± 0.25 × 104
nda
Fluorescence
[18]
4 26.1 × 104
nd Affinity capillary electrophoresis
(Mobility shift method)
[19]
5.6 × 104
nd Affinity capillary electrophoresis
(Mobility ratio method)
5.1 × 104
nd Surface plasmon resonance
5 5.09 × 104
nd Affinity capillary electrophoresis [32]
2.49 ± 0.0003 × 104
0.91 ± 0.08 Fluorescence
a
Not determined.

58 H. Ojha et al. / Thermochimica Acta 548 (2012) 56–64
respectively. Ksv is the Stern–Volmer quenching constant and [FA]
is the concentration of the quencher FA.
The static and dynamic quenching constants were separately
estimated on the basis of modified Stern–Volmer equation:
F0
F
= (1 + Kq[FA])eV[FA]
(2)
where Kq is the collisional quenching constant and V is the static
quenching constant.
The binding constants were calculated according to the double
logarithm equation [22]
log(F0 − F)
F
= log K + n log[FA] (3)
where K is the binding constant and n is the number of binding
sites.
The fluorescence measurements were carried out at 4 different
temperatures 295, 298, 305 and 310 K. Thermodynamical param-
eters, change in enthalpy ( H) and change in entropy ( S) were
obtained based on Van’t Hoff equation:
log K = −
H
2.303RT
+
S
2.303R
(4)
where K is the binding constant at the particular temperature and
R is the gas constant. H and S are calculated from the slope and
intercept of the plot log K versus 1/T, respectively. The free energy
change ( G) was determined from the relation:
G = H − T S (5)
2.3. Fluorescence anisotropy measurements
Fluorescence anisotropy measurements were performed
on spectrofluorimeter model FS900CDT by using a pair of
Glan–Thompson prisms in parallel and perpendicular directions,
viz. IVV, IVH, IHV and IHH and the anisotropy values r were calculated
by using these intensity files in anisotropy calculation mode
provided in the software by the following equation.
r =
GIVV − IVH
GIVV + 2IVH
(6)
where G is the correction factor obtained from the anisotropy soft-
ware provided in the instrument.
Fluorescence anisotropy values of FA (15 × 10−6 mol dm−3)
alone as well as in the presence of BSA (15 × 10−6 mol dm−3) were
obtained by recording emission scan for FA in all the four directions
as mentioned above. The measurement of temperature was done
at room temperature and the excitation wavelength and emission
wavelength were kept at 310 nm and 420 nm respectively.
2.4. Fluorescence lifetime measurements
Fluorescence decays were recorded using standard time cor-
related single photon counting (TCSPC) method with the help of
a customized integrated steady state spectrofluorimeter model
FS900CDT and fluorescence lifetime instrument model FL900CDT
(Edinburgh Analytical Instruments UK). The excitation source was
nanosecond flash lamp (model nF900) filled with low H2 gas pres-
sure of 0.4 bar operating at frequency 40 kHz. Silt width for both
excitation and emission monochromators was kept fully open. The
intensity decay curves were obtained at emission maximum and
fitted as sum of exponentials as
It = I0 Ai exp −
t
i
(7)
where i and Ai are representing the fluorescence lifetime and
pre-exponential factor for ith decay component. The detailed pro-
cedures of measurement and analysis of decay parameters are
discussed elsewhere [23]. The excited state lifetime values were
calculated by deconvoluting the intensity decay profiles. Multiple-
exponential fittings are more precisely applied along with the
instrument response function of the excitation wavelength in
the deconvolution process. The instrument response function was
recorded with Ludox as scattering medium. The analysis of the fit-
ted data was tested using statistical parameters provided in the
analysis software.
Fluorescence lifetime measurements of FA
(15 × 10−6 mol dm−3) in the presence and absence of BSA
(15 × 10−6 mol dm−3) were recorded by fixing 310 nm as the
excitation wavelength and 420 nm as the emission wavelength. All
the lifetime measurements were carried out at room temperature.
2.5. Circular dichroism (CD) spectroscopy
The alterations in the secondary and tertiary structure of the
protein in the presence of the FA were studied on JASCO-715 CD
spectropolarimeter. For CD experiments, the protein concentration
and path lengths used were 10 × 10−6 mol dm−3 and 0.1 cm, respec-
tively. The spectropolarimeter was sufficiently purged with 99.9%
dry nitrogen before measurement. The spectra were collected at a
scan rate speed of 50 nm min−1, and a response time of 1 s. Each
spectrum was baseline corrected, and the final plot was taken as an
average of three accumulated plots in the range of 200–250 nm. The
molar ellipticity [Â] was calculated from the observed ellipticity Â
as
[Â] = 100 ×
Â
c × l
(8)
where c is the concentration of the protein (10−6 mol dm−3) and l is
the path length of the cell (cm). These experiments were performed
for the [FA]/[BSA] molar ratios as 1:1, 1:2, 1:3, 1:4 and 1:5. Effect
of temperature on the native and protein–ligand complex was car-
ried out in the same spectropolarimeter equipped with a Peltier
accessory to maintain a heating rate of 274 K/min for providing
an adequate time for equilibration. Changes in [Â] of each protein
sample were measured in the temperature range of 293–358 K.
Thermal denaturation of 10 × 10−6 mol dm−3 BSA was measured
in the absence and presence of 10 × 10−6 mol dm−3 FA.
2.6. Isothermal titration calorimetry measurements
The energetics of the binding of FA to BSA was determined using
an isothermal titration calorimeter (VP-ITC, Microcal, Northamp-
ton, MA). ITC measurements on the interaction of BSA with FA
were performed at 298 K and pH 7.4. Before loading, the solu-
tions were thoroughly degassed by using Thermo Vac degassing
unit supplied by Microcal, USA. The reference cell was filled with
the respective degassed phosphate buffer. The protein solution
(45 × 10−6 mol dm−3) was kept in the sample cell and working solu-
tion FA (412 × 10−6 mol dm−3) was filled in the syringe of volume
300 ␮L.
The FA solution was added sequentially in 8 ␮L aliquots (for a
total of 35 injections, 20 s duration each) at 4 min intervals with
stirring at 264 rpm. Injections were started after baseline stability
had been achieved.
The values of heats of dilution of FA and BSA in phosphate buffer
were subtracted from the integrated ITC profile of BSA titrating
with FA. The resulting data were fitted with a two-site model using
MicroCal ORIGIN software supplied with the instrument; the bind-
ing constant (K), the binding stoichiometry (N), change in enthalpy
( H), change in entropy ( S) and change in free energy ( G) were

Fig. 2. Fluorescence spectra of BSA in the presence of FA at pH 7.4 in
10 × 10−3
mol dm−3
phosphate buffer containing 0.15 mol dm−3
NaCl. BSA concen-
tration was kept fixed at 10 × 10−6
mol dm−3
and FA concentration was varied from
(a) 0 to (l) 52 × 10−6
mol dm−3
. The measurement conditions were: excitation wave-
length at 295 nm, slit width 4.0 nm, dwell time 0.2 s and wavelength increment
0.5 nm.
thus obtained. The two-site binding model defines the equilibrium
binding constants as
K1 =
1
(1 − 1)L
(9)
K2 =
2
(1 − 2)L
(10)
where is the fractional ligand occupancy
Lt = L + M1(N1 1 + N2 2) (11)
Lt is the total ligand concentration, and K is binding constant
Lt = L +
N1MtLK1
1 + LMt
+
N2MtLK2
1 + LK2
(12)
The heat content Q after any ith injection is then expressed as
Qi = MtV0(N1 1 H1 + N2 2 H2) (13)
Binding models were fitted to the data by a non-linear least square
Marquardt algorithm until 2 values were achieved.
3. Results and discussion
3.1. Fluorescence spectral measurements
Fluorescence quenching during intermolecular interaction is a
common phenomenon that occurs due to various processes, such as
excited-state reactions, molecular rearrangements, energy trans-
fer, ground-state complex formation and collision quenching [24].
The fluorescence spectra of BSA–FA have a strong emission band
( em) at 336 nm due to the tryptophan residues of BSA (Fig. 2).
Since FA is also fluorescent in nature; FA fluorescence was inves-
tigated in the spectral region of interest (310–450 nm) spectrum
at ex = 295 nm. Fig. 3 shows the fluorescence emission spectrum
of FA (52 × 10−6 mol dm−3) with the spectral maximum at 420 nm
which is away from the emission maximum of BSA (336 nm) and
FA has almost negligible emission intensity at 336 nm.
The maximum intensity of BSA in absence of FA is observed
at 336 nm and further increase in FA concentration causes a con-
centration dependent quenching of intrinsic fluorescence of BSA
accompanied with a red shift in the maxima from 336 nm to
343 nm. Similar spectral shift was reported by Li et al. [20]. The
red shift suggested an increase in polarity of the microenvironment
around the tryptophan residues after binding of FA with BSA. It was
Fig. 3. The fluorescence emission spectrum of FA (52 × 10−6
mol dm−3
) in
10 × 10−3
mol dm−3
phosphate buffer with 0.15 mol dm−3
NaCl, pH 7.4.
probably due to the loss of the compact structure of hydropho-
bic subdomain IIA where tryptophan-214 is placed [25]. Similarly
Seetharamappa and Kmat [26] and Tian et al. [27] have reported
the quenching of BSA fluorescence intensity accompanied with red
shift in the maxima upon binding with molecules vinblastin sulfate
and scutellarian, respectively. Therefore it can be inferred that bind-
ing interaction between FA and BSA resulted in enhancement of
hydrophilicity around the tryptophan residues of BSA. Further, the
observation of a well defined isobestic point at 413 nm wavelength
in emission spectra of BSA (Fig. 2) has suggested the existence of
equilibrium between bound and free form of FA and provided a
direct evidence of FA binding to BSA.
The data were further analyzed with Stern–Volmer equation to
investigate quenching mechanism for FA–BSA binding. The upward
curvature of plot (Fig. 4a) indicated involvement of both dynamic
quenching and static quenching [28]. Modified Stern–Volmer plot
based on Eq. (2) provides better description of the quenching
data when both dynamic quenching and static quenching coex-
ist [26]. The value of dynamic quenching constant (Kq) was
obtained from the slope of (F0/FeV[FA]) − 1 versus [FA] (Fig. 4b)
while assuming the value of static quenching constant (V). The val-
ues of V and Kq at 298 K are found to be 3.5 × 104 mol−1 dm3 and
7.46 × 103 mol−1 dm3, respectively.
The dynamic quenching basically depends on diffusion of the
quencher molecule and subsequently collision with the fluo-
rophore, while static quenching is initiated by a complex formation
between the fluorophore and the quencher and this complex is
non-fluorescent in nature. The value of bimolecular quenching rate
constant (kq) was obtained according to Eq. (14).
F0
F
= 1 + Ksv[FA] = 1 + kq 0[FA] (14)
where kq is bimolecular quenching rate constant which is different
from Kq as given in Eq. (2) and 0 is the lifetime of BSA in the absence
of quencher and Ksv is the Stern–Volmer constant.
The 0 value for BSA is reported in many studies and generally
0 value was found to be 10−9 s.
The result showed that the value of kq (1013 mol−1 dm3 s−1)
was much greater than the maximum scattering collision quench-
ing constant (2 × 1010 mol−1 dm3 s−1) [26] which indicated that the
probable quenching mechanism is not a dynamic quenching but the
complex formation. However Li et al. [20] reported that quenching
of BSA upon binding with FA might be due to dynamic quench-
ing that is limited by diffusion but our result suggested that the
quenching owed to complex formation between FA and BSA.
3.2. Fluorescence anisotropy measurement: motional
information of the ligand
Fluorescence anisotropy provides useful information on the
change in orientation of a small molecule upon binding with

Fig. 4. (a) Stern–Volmer plot F0/F versus [FA] for the binding of BSA with FA and (b) modified Stern–Volmer plot [F0/F(eV[FA]
)] − 1 versus [FA] for the binding of BSA with FA.
Table 2
Fluorescence anisotropy and lifetime decay of 15 × 10−6
mol dm−3
FA in phosphate buffer of pH 7.4 at different concentrations of BSA at 298 K.
BSA (10−6
mol dm−3
) Anisotropy Lifetime (ns) Amplitude (%) Average lifetime (ns) a 2
1 2 A1 A2
0 0.16 0.15 6.75 97 3 0.34 1.15
15 0.28 0.07 7.74 89 11 0.91 1.04
a
Chi square is statistical fitting parameter.
macromolecules. Table 2 shows that the anisotropy value (r) of
FA was increased from 0.16 (in the absence of BSA) to 0.28 upon
binding with BSA. Anisotropy value (r) of a small molecule may
vary from 0 (randomly oriented molecule) to 0.4 when there is no
rotation or restricted motion of molecule. Rossi and Taylor [29]
have suggested that for a small molecule which is more likely to
reorient, the binding of a small fluorescent molecule to a large pro-
tein increases the r value. Thus, increase in the anisotropy values
suggests the binding of FA with BSA.
3.3. Fluorescence lifetime measurement
Fluorescence excited state lifetimes are very sensitive to the
structure and dynamics of a fluorophore. Fluorescence decay pro-
files of FA were obtained both in the absence and presence of BSA
(Fig. 5). Double exponentially decaying constants with short ( 1)
and long ( 2) decay components were observed (Table 2). Although
in FA lifetime decay profile, two decay constants were observed, but
the dominant component was due to the fast decay constant 1. In
case of free FA, the numerical values of 1 and 2 were 0.15 ns and
6.75 ns, respectively. The relative contributions from these compo-
nents were 97% and 3% for 1 and 2, respectively and the average
lifetime ( 0) was calculated as 0.34 ns. In the presence of BSA the
numerical values of 1 and 2 have changed to 0.07 ns and 7.74 ns,
respectively. The relative contribution of 1 has decreased to 89%
from 97% and the contribution of 2 has increased from 3% to 19%
(Table 2). The average lifetime value has increased from 0.34 ns
to 0.91 ns in the presence of BSA. This significant change in the
numerical values of each individual decay components as well as
that of average lifetime decay and relative percentage contributions
strongly suggested change in the microenvironment of FA upon
binding with BSA [23]. Li et al. [20] have reported that there was
no change in lifetime distribution of BSA upon binding with FA
due to static quenching mechanism. Similar lifetime distribution
pattern of BSA was obtained in the present study; however, the
Fig. 5. Fluorescence decay profile of FA (15 × 10−6
mol dm−3
) in the absence and
presence of BSA (15 × 10−6
mol dm−3
) in 10 × 10−3
mol dm−3
phosphate buffer
(0.15 mol dm−3
NaCl) of pH 7.4, measurement conditions: ex = 310 nm and
em = 420 nm. (a) Instrument response profile with Ludox, (b) FA concentration was
fixed at 15 × 10−6
mol dm−3
and (c) BSA–FA complex, the concentration of both FA
and BSA was kept at 15 × 10−6
mol dm−3
.
fluorescence lifetime of FA showed definite change upon interac-
tion with BSA (Table 2) suggested binding of FA with BSA.
3.4. Analysis of binding equilibria
The binding parameters, K and n, as obtained from the plot of
log(F0 − F)/F versus log[FA] are 40.15 ± 0.02 × 104 mol−1 dm3 and
1.22 ± 0.05 at 298 K, respectively (Fig. 6). The values of binding
parameters at different temperature and thermodynamical param-
eters are summarized in Table 3. Each value in the table is an
average of two or three independent experiments. The numerical
value of n suggested there is one independent class of binding site
on BSA for FA. Based on the similar studies and reported data, FA
appears to bind in the hydrophobic pocket located in sub-domain

Table 3
Binding and thermodynamical parameters of BSA–FA system based on fluorescence measurement.
Temperature (K) K × 104
(mol−1
dm3
) n H (kJ mol−1
) S (J K−1
mol−1
) G (kJ mol−1
)
295 97.87 ± 0.03 1.31 ± 0.07 −128.06 ± 7.78 −320.85 ± 14.64 −32.45 ± 3.42
298 40.15 ± 0.02 1.22 ± 0.05
305 24.21 ± 0.03 1.17 ± 0.05
310 10.23 ± 0.04 1.06 ± 0.09
Fig. 6. The plot of log(F0 − F)/F0 against log[FA] to determine the value of binding
constant and number of binding site.
IIA containing the tryptophan-214 [30]. The value of K (104) sug-
gested major role of non-covalent interactions in the formation of
BSA–FA complex [31].
The binding parameters reported elsewhere have been summa-
rized in Table 1, suggesting that the binding constant (K) varies
from 4.1 ± 0.07 × 103 mol−1 dm3 to 5.6 × 104 mol−1 dm3 at 298 K
and physiological pH [17–20] with single class of binding sites.
The observed binding constant value is 10–100 times higher
than the binding constant values reported elsewhere [17–20,32].
Rawel et al. reported K value 4.1 ± 0.07 × 103 mol−1 dm3 based
on HD method which is 100 times lower than binding constant
obtained in the present study since HD is a solid/solution technique
and the use of column may interfere with binding processes which
is also highlighted by He et al. [32]. However the trend of binding
affinity of FA with BSA was found to decrease with temperature in
contrast to the trend of binding affinity reported by Li et al. [20]. He
et al. [32] investigated the binding affinity of FA with BSA through
affinity capillary electrophoresis (ACE) at five different tempera-
tures and fluorescence quenching method at 298 K. The binding
constants were found to decrease in magnitude with temperature
and same trend is obtained in the current study. Rawel et al. [17]
have also reported for quercetin binding to BSA so that the thermal
denaturation of BSA causes polymerization of BSA which may lead
to decrease in the binding affinity.
3.5. Nature of interaction force between FA and BSA
The interaction forces between small molecule and
biomolecules usually comprise of hydrophobic force, electro-
static interactions, Vander Waals interactions and hydrogen bonds
[33]. Due to a possible dependence of binding constant on temper-
ature, a thermodynamic process was considered to be responsible
for the formation of the complex.
H and S are obtained from the plot of log K versus 1/T (Fig. 7)
by fitting the binding constants values as obtained at four temper-
atures, 295, 298, 305 and 310 K into Eq. (4). The derived values of
thermodynamical parameters are given in Table 3.
Fig. 7. Van’t Hoff plot for the interaction of BSA and FA in phosphate buffer at
pH = 7.4.
Ross and Subramanian have investigated in detail and character-
ized the sign and magnitude of these thermodynamic parameters
associated with various individual kinds of the interaction [34].
Table 3 shows that H and S both have negative values viz.,
−128.06 ± 7.78 kJ mol−1 and −320.85 ± 14.64 J mol−1 K−1, respec-
tively. The negative value of G (−32.45 ± 3.42 kJ mol−1) is
suggestive of spontaneous nature of the binding process. How-
ever, He et al. [32] reported negative change in enthalpy change
( H) and positive change for entropy change ( S) i.e. values
−14.76 ± 1.76 kJ mol−1 and 40.71 ± 6.23 J mol−1 K−1, respectively,
for FA binding with BSA with possible dominance of hydropho-
bic forces and electrostatic forces with significant role played by
hydrogen bonding. In the present study the negative contribution
from both the enthalpy change and entropy change has suggested
that binding process is enthalpy driven and the hydrogen bond-
ing and electrostatic forces play the dominant role [35]. Secondly
there is difference in values of thermodynamic parameters from
reported values. In order to understand the thermodynamic of this
binding mechanism in more detail, the ITC study was conducted
for titration of BSA with FA at 298 K.
3.6. Circular dichroism spectral measurements
CD spectra of BSA in the absence and presence of different con-
centrations of FA were studied in the far-UV region to obtain an
insight into the structure of BSA (Fig. 8a). The CD spectra BSA exhib-
ited two negative absorption bands with minima at 208 nm and
222 nm, which is typical of the ␣-helical secondary structure of
this class of proteins. The CD spectrum of BSA–FA upon addition of
10 × 10−6 mol dm−3 and 20 × 10−6 mol dm−3 FA does not differ sig-
nificantly from that of the native BSA. However, a significant change
was observed from 30 × 10−6 mol dm−3 to 50 × 10−6 mol dm−3 of
FA in the form of decrease in band intensity at all wavelengths.
The changes in the secondary structure in terms of ␣-helical
content were measured by determining the [Â]2 2 2 as a function of

Fig. 8. (a) CD spectra of BSA–FA system obtained in 10 × 10−3
mol dm−3
phosphate
buffer with 0.15 mol dm−3
NaCl of pH 7.4 at room temperature. BSA concen-
tration was kept fixed at 10 ␮M. FA concentration was (a) 0 × 10−6
mol dm−3
;
(b) 10 × 10−6
mol dm−3
; (c) 20 × 10−6
mol dm−3
; (d) 30 × 10−6
mol dm−3
; (e)
40 × 10−6
mol dm−3
and (f) 50 × 10−6
mol dm−3
. (b) Thermal denaturation of
10 × 10−6
mol dm−3
BSA in the absence and presence of 10 × 10−6
mol dm−3
FA at
pH 7.4.
the ligand concentration with the help of an equation as given by
Chen et al. [36]. The native protein was found to have 72.4% ␣-helix
which was decreased finally to 55.1% ␣-helical content of BSA with
the addition of 50 × 10−6 mol dm−3 of FA. It is thus evident from
CD spectra that FA causes conformational change in the protein
structure upon binding to BSA. However, FA does not completely
alter the secondary structure of BSA upon binding. Rawel et al. [17]
suggested that the non-covalent binding interactions of phenolic
compounds have no effect on the secondary structure of the pro-
teins. But the present study suggested that non-covalent force does
not cause large change in secondary structure but the structural
changes are significant. Furthermore, these findings are in agree-
ment with our fluorescence binding studies. This experiment was
done to understand if the FA provides any thermal stability to the
protein as a result of binding. Fig. 8b shows that there occurs an
increase in melting temperature (Tm) from 341 K for native BSA to
348 K in the presence of 10 × 10−6 mol dm−3 FA. It is well known
that protein thermal stability is modified by ligand binding due to
coupling between two mutual processes under equilibrium. The
increase in melting temperature (Tm) of BSA indicated that the
binding of FA increases the thermal stability of BSA. The significant
increase in thermal stability of BSA in the presence of FA suggested
that FA binds strongly with BSA [23].
3.7. Isothermal titration calorimetric (ITC) measurement
ITC provides a direct method to determine thermodynamic
characterization of non-covalent, equilibrium interactions involv-
ing small molecules with macromolecule (protein) [37]. A
representative calorimetric titration profile of titration of FA
Fig. 9. (a) Raw data for the titration of 412 × 10−6
mol dm−3
FA with
45 × 10−6
mol dm−3
BSA at pH 7.4 and 298 K, showing the calorimetric response as
successive injections of the ligand are added to the sample cell. (b) Integrated heat
profile of the calorimetric titration is shown in panel a. The solid line represents the
best non-linear-squares fit to a two-site binding model.
(412 × 10−6 mol dm−3) with 45 × 10−6 mol dm−3 BSA at pH 7.4 and
298 K is shown in Fig. 9. Each peak in the binding isotherm (see
Fig. 9, panel a) represents a single injection of the FA into the BSA
solution. Panel b of the figure shows the plot of the amount of heat
liberated per injection as a function of the molar ratio of the FA to
BSA. A standard nonlinear least squares regression binding model
involving two binding sites fitted well to the data. The solid line
shown in Fig. 9 (panel b) is the best fit to the experimental. The
thermodynamic data accompanying the binding of FA with BSA
are summarized in Table 4. Each value in this table is an average
of two to three independent experiments. The observed enthalpy
does not have significant contribution from the buffer ionization
because phosphate has a small value for enthalpy of ionization
( Hioniz = 3.6 kJ mol−1) [38]. As seen in Fig. 9, the ITC titrations
of 412 × 10−6 mol dm−3 FA with 45 × 10−6 mol dm−3 BSA yielded
negative heat deflection, which indicated that the binding is an
exothermic process with a high affinity constant at 298 K. Total
enthalpy change, total entropy change and total free energy change,
H = −455.79 kJ mol−1, S = −1237.28 J mol−1 K−1 suggested that
binding is entropically opposed but enthalpically favored.
Site I pocket of BSA which is known to be flexible and may bind
to more than one FA molecule is the site where FA binds with bind-
ing constant (1.87 ± 0.63) × 106 mol−1 dm3 and N2 = 2.200 ± 0.143,
while site II pocket of BSA seems to be smaller than site I where the
binding is stereoselective in nature and is the ideal site for FA to bind
with BSA with the binding constant (9.70 ± 2.64) × 108 mol−1 dm3
and relatively small of N1 = 0.470 ± 0.003.

Table 4
Binding parameters accompanying the titration of 412 × 10−6
mol dm−3
FA with 45 × 10−6
mol dm−3
BSA at pH 7.4 in the presence of 0.15 mol dm−3
NaCl at 298 K using ITC
study.
K1 (mol−1
dm3
) N1 H1 (kJ mol−1
) S1 (J mol−1
K−1
) K2 (mol−1
dm3
) N2 H2 (kJ mol−1
dm3
) S2 (J mol−1
dm3
K−1
)
(9.7 ± 2.64) × 108
0.470 ± 0.003 −244.74 ± 0.47 −647.9 (1.87 ± 0.63) × 106
2.200 ± 0.143 −211.05 ± 0.70 −589.38
Since FA is a negatively charged molecule at pH 7.4 (pK = 4.58),
it is expected that the sites comprise the positively charged amino
acids [39,40].
The strong binding constants for binding of FA with BSA at two
binding sites also support the melting temperature CD study result
where the noticeable increase in the thermal stability of BSA upon
binding with FA indicated the involvement of strong binding inter-
actions.
Contrarily, the fluorescence data showed single binding sites for
FA binding to BSA, but the ITC data suggested two binding sites. One
of the binding sites showed high affinity with low stoichiometry
while the second one was with low affinity and high stoichiometry.
Similar observation was reported by Zhang et al. [41] for binding
of vitamin C with HSA. The cause of deviation was the limitation
of fluorescence measurement (based on tryptophan emission) that
it may not give information about the protein regions which were
far away from tryptophan residue. The authors have reported on
the basis of ITC data that vitamin C binds to protein at other bind-
ing sites far from tryptophan residues. Sun et al. [42] reported that
with the binding of matrine with BSA, the ITC data showed that
BSA has two binding sites for matrine. On comparing the calori-
metric and fluorescence data the authors concluded that the second
class of binding site as showed by ITC data was the main cause
of fluorescence quenching. Hence, reported studies suggested that
fluorescence technique may have limitation to detect the binding
sites which are detected by more sensitive techniques like ITC. The
advantage of ITC is that it measures directly the heat of reactions
for titration of protein with ligand and generates primary data to
determine the thermodynamical and binding parameters simulta-
neously.
The strong exothermicity observed in the binding suggests the
involvement of electrostatic forces. The phenolic hydroxyl group of
FA can interact with C, O, and N H of the main polypeptide chain
of the protein by forming strong hydrogen bonding, resulting in
the rearrangement of polypeptide carbonyl hydrogen bonding net-
work [34]. He et al. [32] have demonstrated that the role of hydroxyl
groups of phenolic compounds plays a significant factor in bind-
ing the interaction between phenolic compounds with BSA. Thus
the present study suggested that both the electrostatic forces and
hydrogen bonding are involved in such interactions at physiological
pH.
The difference between the Van’t Hoff enthalpy (derived from
fluorescence data) and calorimetric enthalpy is due the fact that
Van’t Hoff enthalpy is calculated from indirect methods based on
derived parameters, and calorimetric enthalpy is directly measured
through ITC measurements. The inconsistency between the calori-
metric enthalpy and Van’t Hoff enthalpy may also indicate that
there is an appreciable conformation change in BSA upon ligand
binding [43]. This is in agreement to red shift in maxima as observed
emission spectra of BSA in presence of FA (Fig. 2).
4. Conclusions
The binding interaction between FA and BSA results sug-
gested that BSA fluorescence was quenched by FA through static
quenching mechanism. Fluorescence anisotropy and lifetime study
indicated significant change in micro-environment of FA thereby
suggesting binding interaction. Thermodynamic study indicated
that binding interaction is spontaneous in nature and electrostatic
forces and hydrogen bonding are the dominant binding forces. In
contrast to spectroscopy techniques, ITC measurement suggested
that there are two binding sites for FA. The CD results revealed a
change in the secondary structure of BSA upon interaction with FA
besides the fact that the thermal stability of BSA increases upon
binding with FA.
The biological significance of this work is the binding interac-
tion of FA with albumin protein, which will influence the delicate
equilibrium between the bound and the free active forms of FA
and thus FA will have greater bioavailability and bio-distribution.
The results of this study are expected to serve useful purpose for
research groups to engage in establishing the drug potential of FA.
Acknowledgments
Authors are thankful to DRDO, India for funding this study. One
of the authors (Krishnanand Mishra) is thankful to University Grant
Commission (New Delhi) for senior research fellowship.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tca.2012.08.016.
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