Docking investigation and binding interaction of benzimidazole derivative with bovine serum albumin

Article abstract of DOI:10.1016/j.jphotobiol.2012.08.018

¹H NMR, ¹³C NMR and Mass spectral analysis have been made for 1-(4-fluorobenzyl)-2-(4-fluorophenyl)-1H-benzo[d]imidazole (FBFPB). The mutual interaction of FBFPB with bovine serum albumin (BSA) was investigated using absorption, fluo

Full text of DOI:10.1016/j.jphotobiol.2012.08.018

Journal of Photochemistry and Photobiology B: Biology 117 (2012) 27–32
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Docking investigation and binding interaction of benzimidazole derivative
with bovine serum albumin
⇑
J. Jayabharathi , K. Jayamoorthy, V. Thanikachalam
Department of Chemistry, Annamalai University, Annamalainagar 608 002, Tamilnadu, India
a r t i c l e i n f o
a b s t r a c t
1H NMR, 13C NMR and Mass spectral analysis have been made for 1-(4-fluorobenzyl)-2-(4-fluorophenyl)-
1H-benzo[d]imidazole (FBFPB). The mutual interaction of FBFPB with bovine serum albumin (BSA) was
investigated using absorption, fluorescence and synchronous fluorescence spectral studies. The binding
distance has been determined based on the theory of Forester’s non-radiation energy transfer (FRET).
The calculated quenching constants (Ksv) were 2.84 ꢀ 104, 2.55 ꢀ 104 and 2.37 ꢀ 104 at 301, 310 and
318 K respectively. The Stern–Volmer quenching constant (Ksv), binding site number (n), apparent bind-
Article history:
Received 25 July 2012
Received in revised form 21 August 2012
Accepted 27 August 2012
Available online 5 September 2012
Keywords:
ing constant (KA) and corresponding thermodynamic parameters (
interaction between FBFPB and BSA have discussed by molecular docking technique.
Ó 2012 Elsevier B.V. All rights reserved.
DG, DH and DS) were calculated. The
Benzimidazole
Molecular docking
BSA
Quenching
1. Introduction
2. Experimental
Heterocyclic imidazole derivatives have attracted considerable
attention because of their unique optical properties [1] and used
for preparing functionalized materials [2]. Imidazole nucleus forms
the main structure of human organisms, i.e., the amino acid histi-
dine, Vitamin B12, a component of DNA base structure and also has
significant analytical applications utilizing their fluorescence and
chemiluminescence properties [3,4].
BSA is made up of three homologous domains (I–III), which are
divided into nine loops by 17 disulfide bridges. Each domain is
composed of two sub-domains (A and B). Aromatic and heterocy-
clic ligands were found to bind within two hydrophobic pockets
in sub-domain IIA and IIIA, site I and site II [5,6]. BSA has two try-
ptophans, Trp-134 and Trp-212, embedded in the first sub domain
IB and sub-domain IIA, respectively. There is evidence of conforma-
tion changes of BSA induced by its interaction with low molecular
weight benzimidazole and imidazole ligands [7–10]. It is important
to study the interaction of FBFPB with BSA, and hence become an
important research field in chemistry, life sciences and clinical
medicine. The molecular structure of FBFPB is given in Fig. 1. In
the present research article, we have studied the binding interac-
tion of BSA with 1-(4-fluorobenzyl)-2-(4-fluorophenyl)-1H-ben-
zo[d] imidazole.
2.1. Materials and methods
All BSA solution were prepared in the Tris–HCl buffer solution
(0.05 mol Lꢁ1 Tris, 0.15 mol Lꢁ1 NaCl, pH 7.4) and it was kept in
the dark at 303 K. Tris base (2-amino-2-(hydroxymethyl)-1,3-pro-
panediol) had a purity of not less than 99.5% and NaCl, HCl and
other starting materials were all of analytical purity and doubly
distilled water was used throughout.
2.2. Optical measurements
NMR spectra have been recorded for the FBFPB on a Bruker
400 MHz instrument. The ultraviolet–visible (UV–vis) spectra have
been measured on UV–Vis spectrophotometer (Perkin Elmer,
Lambda 35) and corrected for background due to solvent absorp-
tion. Photoluminescence (PL) spectra have been recorded on a (Per-
kin Elmer LS55) fluorescence spectrometer. Solvents used for
spectral measurements are spectroscopic grade. Mass spectra have
also been recorded on a Varian Saturn 2200 GCMS spectrometer.
2.3. Molecular docking
The rigid molecular docking studies were performed by using
HEX 6.1 software [11], is an interactive molecular graphics pro-
gram to understand the drug-protein interaction. The Structure
of the FBFPB was sketched by CHEMSKETCH (http://www.acdl-
abs.com) and converts it into pdb format from mol format by
OPENBABEL (http://www.vcclab.org/lab/babel/). The crystal
⇑
Corresponding author. Tel.: +91 9443940735.
E-mail address: jtchalam2005@yahoo.co.in (J. Jayabharathi).
1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2012.08.018

28
J. Jayabharathi et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 27–32
NMR (400 MHz, CDCl3): d5.41 (s, 2H), 7.02–7.08 (m, 4H), 7.15–7.18
(t, 3H), 7.22–7.23 (d, 1H) 7.26–7.29 (m, 1H), 7.33–7.36 (m, 1H),
7.65–7.67 (m, 2H), 7.87–7.88 (s, 1H). 13C (100 MHz, CDCl3): d
47.71 (–CH2 carbon), 110.33, 115.93, 116.03, 116.21, 120.07,
122.92, 123.30, 126.16, 126.19, 127.60, 127.63, 127.67, 131.19,
131.25, 131.93, 131.95, 135.89, 143.06, 153.08, 161.31, 162.78,
163.28, 164.77 (Aromatic carbons). MS: m/e 320.1, calcd 321.11
[M + 1].
3. Results and discussion
3.1. Fluorescence spectral studies
The interaction between FBFPB and BSA was investigated by
evaluating fluorescence intensity on the BSA before and after the
addition of the FBFPB [10,12]. Here, the concentrations of BSA
were stabilized at 1.0 ꢀ 10ꢁ5 mol Lꢁ1 and the concentration of
FBFPB varied from 0 to 3.5 ꢀ 10ꢁ5 mol Lꢁ1 at increments of
0.5 ꢀ 10ꢁ5 mol Lꢁ1. The effect of the FBFPB on BSA fluorescence
intensity is shown in Fig. 2. The fluorescence intensity of BSA de-
creases progressively but the emission maximum did not move
to shorter or longer wavelength, due to the interaction of FBFPB
with BSA and quench its intrinsic fluorescence (Trp-212) [13], but
there was no alteration in the local dielectric environment of BSA.
The quenching mechanism of FBFPB with BSA was probed
using the Stern–Volmer equation [14], which can be applied to
determine Ksv by linear regression from the Stern–Volmer plot
of F0/F against [FBFPB] (Fig. 3) at different temperatures. Table 1
summarizes the values of Ksv and KA at different temperatures,
which shows the values of Stern–Volmer quenching constant
Ksv and KA decreases with increase in temperature. According to
the literature [15] for dynamic quenching, the maximum scatter
collision quenching constant of various quenchers with the bio-
polymer is 2.0 ꢀ 1010 L molꢁ1 sꢁ1 and the fluorescence lifetime
of the biopolymer is 10ꢁ8 s. From Fig. 3, the values of Ksv and
Fig. 1. Molecular structure of FBFPB.
structure of HSA (PDB entry 1AO6) is obtained from the Protein
Data Bank (http://www.rcsb.org./pdb). Since the structure of bo-
vine serum albumin (BSA) is unavailable in the PDB, a homology
model was used for the docking studies. All calculations were car-
ried out on an Intel pentium 4, 2.4 GHz based machine running MS
Windows XP SP2 as operating system. Visualization of the docked
pose has been done by using PyMol (http://pymol.sourceforge.net/
) molecular graphic program.
2.4. General procedure for the synthesis of ligands
kq (=Ksv/s0) were calculated. The obtained values of kq were larger
than the limiting diffusion rate constant of the biomolecule
(2.0 ꢀ 1010 L molꢁ1 sꢁ1), which indicate that the fluorescence
quenching is caused by a specific interaction between BSA and
FBFPB. Therefore, the quenching mechanism mainly arises from
the formation of BSA–FBFPB complex rather than dynamic
quenching. So it was implied that the static quenching was dom-
inant in the system. From the plot of log[(F0 ꢁ F)/F] vs log[FBFPB],
binding constants KA and the number of binding sites ‘n’ were cal-
culated from the intercept and slope.
A mixture of 4-fluorobenzaldehyde (2 mmol), o-phenylenedi-
amine (1 mmol) and ammonium acetate (2.5 mmol) has been re-
fluxed at 80 °C in ethanol. The reaction was monitored by TLC
and purified by column chromatography using petroleum ether:
ethyl acetate (9:1) as the eluent.
2.4.1. 1-(4-Fluorobenzyl)-2-(4-fluorophenyl)-1H-benzo[d]imidazole
Yield: 55%. mp = 84 °C, Anal. calcd. for C20H14F2N2: C, 74.98; H,
4.41; F,11.86; N, 8.75. Found: C, 74.49; H, 4.51; F,11.95; N, 9.05. 1H
Fig. 2. Fluorescence quenching spectra of BSA at different concentrations of FBFPB.
Fig. 3. Stern–Volmer plot of F0/F against [FBFPB].

J. Jayabharathi et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 27–32
29
Table 1
D
D
G ¼ ꢁRT ln K
S ¼ ð H ꢁ GÞ=T
ð2Þ
Ksv KA, n and r values of BSA–FBFPB system at 301, 310 and 318 K.
T (K)
Ksv (104 L Molꢁ1
)
KA (104 L Molꢁ1
)
n
r
D
D
ð3Þ
301
310
318
2.84
2.55
2.37
3.76
2.74
2.17
1.2
1.01
0.98
0.99
0.99
0.99
According to the theory of Ross, the positive enthalpy change
H) and entropy change ( S) are associated with hydrophobic
interaction. The negative values of H and S are associated with
hydrogen binding and van der Waals interactions whereas the very
low positive or negative H and positive S values are character-
ized by electrostatic interactions, From the Table 2
H (ꢁ25.77),
S (1.88), it was found that there is electrostatic
(D
D
D
D
D
D
Table 2
D
Thermodynamic parameters of BSA–FBFPB system at 301, 310 and 318 K.
D
G (ꢁ24.55) and
D
T (K)
D
H (kJ molꢁ1
ꢁ25.77
)
D
G (kJ molꢁ1
)
D
S (J molꢁ1Kꢁ1
1.88
)
interaction between BSA and FBFPB [16].
301
310
318
ꢁ24.55
ꢁ25.7
ꢁ24.1
3.3. Energy transfer from BSA to FBFPB
Energy transfer takes place through direct electro-dynamic
interaction between the excited molecule and its neighbours
[17]. The distance between the donor (BSA) and the acceptor
(FBFPB) was estimated by Forster’s non-radiative energy transfer
theory and the overlapping of fluorescence spectra of BSA with
absorption spectra of FBFPB was shown in Fig. 4. According to For-
ster’s non-radiative energy transfer theory, the energy transfer effi-
ciency (E) can be defined as the following equations:
ꢀ
ꢁ
E ¼ 1 ꢁ ðF=F0Þ ¼ R60= R06 þ r06
ð4Þ
ð5Þ
ð6Þ
R60 ¼ 8:8 ꢀ 10ꢁ25½k2nꢁ4UDJðkÞꢂ in Aꢀ
Z
1
JðkÞ ¼
FDðkÞeAðkÞk4 dk
0
where E is the efficiency of transfer between the donor and the
acceptor, R0 is the critical distance when the efficiency of transfer
is 50%. FD(k) is the corrected fluorescence intensity of the donor at
Fig. 4. Overlapping spectra of absorption (i) and emission (ii) of BSA with FBFPB.
wavelength k to (k +
Dk), with the total intensity normalized to
3.2. Determination of thermodynamic parameters
unity and A(k) is the molar extinction coefficient of the acceptor
e
at wavelength k. The Forster distance (R0) has been calculated
assuming random orientation of the donor and acceptor molecules.
Here, k2 = 2/3, n = 1.311, UD = 0.20 and from the available data, it
results that J(k) = 4.03 ꢀ 10ꢁ12 cm3 L molꢁ1, E = 0.34, R0 = 0.73 nm
and r = 0.79 nm. The donor-to-acceptor distance is less than 8 nm
which indicates that the energy could transfer from BSA to FBFPB
[18] with high probability and the distance obtained by FRET with
higher accuracy.
In order to elucidate the interaction between the FBFPB and
BSA, the thermodynamic parameters were calculated from the
van’t Hoff plots. If the enthalpy change (
DH) does not vary signif-
icantly over the temperature range studied then the thermody-
namic parameters
following equations, respectively.
D
H,
D
G and
D
S can be determined from the
ð1Þ
ln K ¼ H=RT þ S=R
D
D
Fig. 5. Synchronous fluorescence spectra of BSA in the presence and absence of FBFPB (a) at
Dk = 15 nm and (b) at Dk = 60 nm.

30
J. Jayabharathi et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 27–32
spectrum may represent that the conformation of BSA is somewhat
changed, due to the blue shift (Fig. 5a), leading to the polarity
around Tyr residues was decreased and the hydrophobicity was in-
creased [22], but the interaction of FBFPB with BSA does not obvi-
ously affect the conformation of tryptophan micro-region [23,24].
The FBFPB could involve the second site (sub-domain IB) with
higher binding affinity and the formation of complex led to the
observation of the blue shift of tyrosine residues fluorescence. This
is because the tyrosine contains one aromatic hydroxyl group un-
like tryptophan and tyrosine can undergo an excited state ioniza-
tion, resulting in the loss of the proton on the aromatic hydroxyl
group. The hydroxyl group can dissociate during the lifetime of
its excited state, leading to quenching. Hence the aromatic hydro-
xyl group present in the tyrosine residues is responsible for the
interaction of BSA with FBFPB.
For reconfirming the structural change of BSA by the addition of
the FBFPB, we have measured the UV–vis absorbance spectra of
BSA with various amounts of the FBFPB. Fig. 6 displays the UV–
vis absorbance spectra of BSA at different concentrations of the
FBFPB. The absorption band of 210 nm of BSA is characteristic of
a-helix structure of BSA. The intensity of absorbance of BSA was
Fig. 6. Absorption spectra of BSA in the presence of FBFPB (a–e) and in the absence
decreased with increasing concentration of the FBFPB and the peak
was red shifted. In addition, the absorption peaks in the UV–vis
spectra at approximately 280 nm rise gradually (from curve (a)
to curve (e)) and blue shifted to about 11 nm with increasing con-
centration of the FBFPB. These results indicating that the interac-
tion between the FBFPB and BSA and the fluorescence quenching
of BSA by FBFPB was the result of the formation of BSA–FBFPB com-
plex [25]. These results confirmed that the quenching was mainly a
static quenching process.
of FBFPB (f).
3.4. Conformation investigation
To exploit the structural change of BSA by addition of the FBFPB,
we have measured synchronous fluorescence spectra of BSA with
various amounts of FBFPB. The synchronous fluorescence spectra
[12,19,20] of BSA with various amount of FBFPB were recorded at
D
k = 15 nm and Dk = 60 nm (Fig. 5a and b). It is apparent from
the figure that the emission wavelength of the tyrosine residues
is blue-shifted (kmax from 345 to 330 nm in Fig. 5a) with increasing
concentration of FBFPB. This blue shift expressed that the confor-
mation of BSA was changed and it suggested a less polar (or more
hydrophobic) environment of tyrosine residue [21]. At the same
time, the tryptophan fluorescence emission is decreased regularly,
but no significant change in wavelength was observed. It suggests
that the interaction of FBFPB with BSA does not affect the confor-
mation of tryptophan micro-region. The tyrosine fluorescence
3.5. Molecular docking
Molecular docking technique is an attractive scaffold to under-
stand the ligand–protein interactions which can substantiate our
experimental results. Descriptions of the 3-D structure of crystal-
line albumin have revealed that BSA is made up of three homolo-
gous domains (I, II, and III): I (residues 1–183), II (184–376), III
(377–583), each containing two subdomains (A and B) that assem-
ble to form heart shaped molecule (Fig. 7), which are divided into
Fig. 7. Modeling of X-ray crystallographic structure of BSA (PDB ID: 1AO6). The domains and subdomains were displayed with different color, the every subdomain and
classical binding site were marked in the corresponding location. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)

J. Jayabharathi et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 27–32
31
Fig. 8. (a) Molecular docked model of FBFPB (sphere representation) located within the hydrophobic pocket of BSA. (b) The hydrogen bond interaction (yellow dashed line)
between FBFPB (stick) and BSA (cartoon). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
nine loops by 17 disulphide bonds, each one formed by six helices,
and its secondary structure is dominated by -helix. It is suggested
(NRB-213/MAT/10-11) for providing funds to this research study.
K. Jayamoorthy is thankful to DST (No. SR/S1/IC-73/2010) for pro-
viding fellowship.
a
that the principal regions of ligand binding to BSA are located in
hydrophobic cavities in subdomains IIA and IIIA, and one trypto-
phan residues (Trp-212) of BSA is in subdomain IIA [26]. There is
a large hydrophobic cavity in subdomain IIA to accommodate the
drug molecule, which play an important role in absorption, metab-
olism, and transportation of BSA. The best energy ranked results
(Fig. 8a) revealed that FBFPB was located within subdomain IIA
hydrophobic cavity in close proximity to positively charge hydro-
phobic residues, such as Asp-118, Asp-129, Leu-138, Phe-126,
Phe-133, Pro-117, Trp-134 and Tyr-137, suggesting the existence
of hydrophobic interaction between them. Hence, this finding pro-
vides a good structural basis to explain the efficient fluorescence
quenching of BSA emission in the presence of the FBFPB. Further-
more, there are also a number of specific electrostatic interactions
and hydrogen bonds, because several ionic and polar residues in
the proximity of the ligand play an important role in stabilizing
the molecule via H–bonds and electrostatic interactions. As shown
in Fig. 8b, there are hydrogen bond interactions between the fluo-
rine atoms of FBFPB and Leu-138 and Asp-118 residues of BSA.
These results suggest that the formation of hydrogen bonds de-
creased the hydrophilicity and increased the hydrophobicity to
keep the FBFPB–BSA system stable. On the other hand, the amino
acid residues with benzene ring can match that of the structure
of FBFPB in space in order to confirm the conformation of the mol-
ecule. On the other hand, the amino acid residues with a benzene
ring can match that of the FBFPB in space in order to firm the con-
formation of the complex stability. Therefore it can be concluded
that the interaction between the FBFPB and BSA was dominated
by hydrophobic forces as well as hydrogen bonds, correlated well
with the binding mode observed by fluorescence quenching mech-
anism of BSA in presence of FBFPB.
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