Antioxidant benzimidazole bind bovine serum albumin

Article abstract of DOI:10.1016/j.jphotobiol.2012.06.014

1-(4-Methoxybenzyl)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole (MBMPB) was synthesized and characterized by ¹H NMR, ¹³C NMR, Mass and IR spectral analysis. The mutual interaction of MBMPB with bovine serum albumin (BSA) was investigate

Full text of DOI:10.1016/j.jphotobiol.2012.06.014

Journal of Photochemistry and Photobiology B: Biology 115 (2012) 85–92
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Journal of Photochemistry and Photobiology B: Biology
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Antioxidant benzimidazole bind bovine serum albumin
⇑
J. Jayabharathi , V. Thanikachalam, K. Jayamoorthy
Department of Chemistry, Annamalai University, Annamalainagar, Tamilnadu 608 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 June 2012
Received in revised form 26 June 2012
Accepted 30 June 2012
Available online 11 July 2012
1-(4-Methoxybenzyl)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole (MBMPB) was synthesized and char-
acterized by 1H NMR, 13C NMR, Mass and IR spectral analysis. The mutual interaction of MBMPB with
bovine serum albumin (BSA) was investigated using solution spectral studies. The binding distance has
been calculated based on the theory of Forester’s non-radiation energy transfer (FRET). The Stern–Volmer
quenching constant (Ksv) were calculated at different temperature. The binding site (n), apparent binding
constant (KA) and corresponding thermodynamic parameters (DG,DH and DS) were calculated. Antioxi-
Keywords:
Antioxidant
Benzimidazole
BSA
Quenching
FRET
dant analyses such as DPPH radical scavenging analysis, Superoxide anion scavenging analysis and
Hydroxyl radical scavenging analysis have been carried out for MBMPB and it shows potential antioxi-
dant property due to the presence of electron releasing methoxy group.
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
ligands were found to bind within two hydrophobic pockets in
sub-domain IIA and IIIA, site I and site II [9,10]. BSA has two trypto-
Fused imidazole derivatives such as benzoimidazoles and phen-
anthroimidazoles [1–3] have been used in the fabrication of light-
emitting devices, employing them as electron-transporting layer
and as sensitizers in dye-sensitized solar cells [4,5] due to their
wide optical absorption, bright luminescence and bipolar transport
characteristics. Imidazole nucleus forms an important structure of
human organisms, i.e., the amino acid histidine, vitamin B12, a
component of DNA base structure and also has significant analyti-
cal applications utilizing their fluorescence and chemilumines-
cence properties [6].
phans, Trp-134 and Trp-212, embedded in the first subdomain IB
and sub-domain IIA, respectively. These changes appear to affect
the secondary and tertiary structure of albumin. So, it is important
to study the interaction of MBMPB with BSA, and hence become an
important research field in chemistry, life sciences and clinical
medicine. The molecular structure of MBMPB was given in Fig. 1.
In the present research article, we have analyses the antioxidant
property of MBMPB and the binding interaction of BSA with
MBMPB.
Free radicals, the partially reduced metabolites of oxygen and
nitrogen, are highly toxic and reactive. Free radicals are linked with
the majority of diseases like aging, atherosclerosis, cancer, diabe-
tes, liver cirrhosis, cardiovascular disorders, etc., [7]. The most
common reactive oxygen species are superoxide anion ðOÅ2ꢀÞ,
hydrogen peroxide (H2O2), peroxyl radical (ROOÅ) and highly reac-
tive hydroxyl radical (OHÅ). The nitrogen derived free radicals are
nitric oxide (NO) and peroxy nitrite anion (ONOOÅ). Oxidation pro-
cess is one of the most important routes for producing free radicals
in food, drugs and living systems. Antioxidants are the substances
that when present in low concentration significantly delay or re-
duce the oxidation of the substrate [8].
2. Experimental
2.1. Materials and methods
All BSA solution were prepared in the Tris–HCl buffer solution
(0.05 mol Lꢀ1 Tris, 0.15 mol Lꢀ1 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.
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 heterocyclic
2.2. In vitro antioxidant activity
2.2.1. DPPH radical scavenging activity
The stable free radical DPPH method is an easy, rapid and sen-
sitive way to survey the antioxidant activity of a specific com-
pound. The DPPH radical scavenging activity of MBMPB were
determined by the reported method [11].
⇑
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.2012.06.014

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J. Jayabharathi et al. / Journal of Photochemistry and Photobiology B: Biology 115 (2012) 85–92
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.
2.4. Synthesis of 1-(4-methoxybenzyl)-2-(4-methoxyphenyl)-1H-
benzo[d]imidazole
A mixture of 4-methoxybenzaldehyde (2 mmol), o-phenylene-
diamine (1 mmol) and ammonium acetate (2.5 mmol) has been re-
fluxed at 80 °C in ethanol for appropriate time. The reaction was
monitored by TLC and purified by column chromatography using
petroleum ether: ethyl acetate (9:1) as the eluent. The 1H NMR
spectrum of MBMPB was given in Fig. 2 and 13C NMR spectrum
was given in Fig. 3. Yield: 60%. mp = 162 °C, Anal. calcd. for
C22H20N2O2: C, 76.72; H, 5.85; N, 8.14; O, 9.29. Found: C, 77.02;
H, 5.29; N, 8.33; O, 9.36. 1H NMR (400 MHz,CDCl3): d 3.78 (s,3H),
3.85 (s,3H), 5.38 (s,2H), 6.84–6.86 (d,2H), 6.96–6.98 (d,2H),
7.02–7.04 (d,2H), 7.21–7.22 (d,2H), 7.24–7.31 (m,2H), 7.62–7.64
(d,2H), 7.83–7.85 (s,1H). 13C (100 MHz,CDCl3): d 47.91 (ꢀCH2 car-
bon), 55.32, 55.40 (methoxy carbons), 110.43, 114.20, 114.44,
119.73, 122.47, 122.54, 122.75, 127.23, 128.51, 130.73, 136.11,
143.19, 154.15, 159.12, 160.91 (Aromatic carbons). MS: m/e
344.2, calcd 345.15[M + 1].
Fig. 1. Molecular structure of MBMPB.
2.2.2. Superoxide radical scavenging activity
Superoxide anion radical scavenging activity of MBMPB were
determined by the reported method [12].
3. Results and discussion
2.2.3. Hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity of synthetic imidazole
derivatives were determined by the reported method [13].
3.1. FT-IR characteristics of MBMPB–BSA
Fourier transform infrared (FT-IR) technique was provided
information about the nature of interaction between the MBMPB
and BSA. Fig. 4a shows the FT-IR spectrum of MBMPB. Fig. 4b
shows MBMPB bound to the BSA. The spectrum of pure MBMPB
shows the >C@N stretching vibration at 1595 cmꢀ1. This band is
2.3. Optical measurements
NMR spectra have been recorded for the MBMPB on a Bruker
400 MHz instrument. The ultraviolet–visible (UV–vis) spectra have
Fig. 2. 1H NMR spectrum of MBMPB.

J. Jayabharathi et al. / Journal of Photochemistry and Photobiology B: Biology 115 (2012) 85–92
87
Fig. 3. 13C NMR spectrum of MBMPB.
shifted from 1595 cmꢀ1 to 1619 cmꢀ1 for MBMPB bound to the
BSA.
the biopolymer is 10ꢀ8 s. From Fig. 7, the values of KSV and Kq
(=KSV 0) were calculated. The obtained values of Kq were larger
/s
than the limiting diffusion rate constant of the biomolecule
(2.0 ꢁ 1010 L molꢀ1 sꢀ1), which indicate that the fluorescence
quenching is caused by a specific interaction between BSA and
MBMPB. Therefore, the quenching mechanism mainly arises from
the formation of BSA–MBMPB complex rather than dynamic
quenching. So it was implied that the static quenching was domi-
nant in the system. From the plot of log [(F0 ꢀ F)/F] vs log [MBMPB],
binding constants KA and the number of binding sites ‘n’ were cal-
culated from the intercept and slope.
3.2. Fluorescence spectral studies
The interaction between MBMPB and BSA was investigated by
evaluating fluorescence intensity on the BSA before and after the
addition of the MBMPB. Here, the concentrations of BSA were stabi-
lized at 1.0 ꢁ 10ꢀ5 mol Lꢀ1 and the concentration of MBMPB varied
from 0 to 3.5 ꢁ 10ꢀ5 mol Lꢀ1 at increments of 0.5 ꢁ 10ꢀ5 mol Lꢀ1
.
The effect of the MBMPB on BSA fluorescence intensity is shown
in Fig. 5. The fluorescence intensity of BSA decreases progressively
but the emission maximum did not move to shorter or longer wave-
length, due to the interaction of MBMPB with BSA and quench its
intrinsic fluorescence (Trp-212) [14], but there was no alteration
in the local dielectric environment of BSA. Forster type fluorescence
resonance energy transfer (FRET) mechanism (Fig. 6) is involved in
the quenching of fluorescence by MBMPB in BSA–MBMPB complex.
Therefore the possible quenching mechanism of fluorescence of
BSA by MBMPB is not initiated by dynamic collision but from the
formation of the BSA–MBMPB complex.
3.3. Determination of thermodynamic parameters
In order to elucidate the interaction between the MBMPB 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
D
H,
D
G and
D
S can be determined from the
following equations respectively.
ln K ¼
D
H=RT þ
G ¼ ꢀRT ln K
S ¼ ð H ꢀ GÞ=T
Table 2 shows the values of
site from the van’t Hoff plot. The negative sign for free energy (
shows that the binding process is spontaneous. The negative en-
thalpy ( H) and positive entropy ( S) values of the interaction
D
S=R
ð1Þ
ð2Þ
ð3Þ
The quenching mechanism of MBMPB with BSA was probed
using the Stern–Volmer equation [15], which can be applied to
determine KSV by linear regression from the Stern–Volmer plot of
F0/F against [MBMPB] (Fig. 7) 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 [16] for dynamic quenching, the maximum scatter
collision quenching constant of various quenchers with the bio-
polymer is 2.0 ꢁ 1010 L molꢀ1 sꢀ1 and the fluorescence lifetime of
D
D
D
D
D
H and
D
S obtained for the binding
G)
D
D
D
of the MBMPB and BSA indicate that the specific electrostatic inter-
actions played major role in the reaction [17].

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J. Jayabharathi et al. / Journal of Photochemistry and Photobiology B: Biology 115 (2012) 85–92
Fig. 4. (a) FT-IR spectrum of MBMPB (b) FT-IR spectrum of MBMPB bound with BSA.
.ꢀ
ꢁ
E ¼ 1 ꢀ ðF=F0Þ ¼ R6 R06 þ r06
ð4Þ
ð5Þ
ð6Þ
3.4. Energy transfer from BSA to MBMPB
0
R60 ¼ 8:8 ꢁ 10ꢀ25
½j
2nꢀ4UDJðkÞꢂ in Å
Energy transfer takes place through direct electro-dynamic
interaction between the excited molecule and its neighbors [18].
The distance between the donor (BSA) and the acceptor (MBMPB)
was estimated by Forster’s non-radiative energy transfer theory
and the overlapping of fluorescence spectra of BSA with absorption
spectra of MBMPB was shown in Fig. 8. According to Forster’s non-
radiative energy transfer theory, the energy transfer efficiency (E)
can be defined as the following equations:
Z
1
JðkÞ ¼
FDðkÞeAðkÞk4dk
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 wavelength k to (k +
Dk), with the total intensity normalized to

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89
Table 2
Thermodynamic parameters of BSA–MBMPB system at 301, 310 and 318 K.
T (K)
D
H (kJ molꢀ1
ꢀ27.67
)
D
G (kJ molꢀ1
)
D
S (J molꢀ1 Kꢀ1
6.48
)
301
310
318
ꢀ23.55
ꢀ24.70
ꢀ23.10
Fig. 5. Fluorescence quenching spectra of BSA at different concentrations of
MBMPB.
Fig. 8. Overlapping fluorescence spectra of BSA (i) with absorption spectra of
MBMPB (ii).
assuming random orientation of the donor and acceptor molecules.
Here,
j
2 = 2/3, n = 1.311, UD = 0.20 and from the available data, it
results that J(k) = 4.07 ꢁ 10ꢀ12 cm3 L molꢀ1, E = 0.30, R0 = 0.70 nm
and r = 0.89 nm. The donor-to-acceptor distance is less than 8 nm
which indicates that the energy could transfer from BSA to MBMPB
[19] with high probability and the distance obtained by FRET with
higher accuracy.
Fig. 6. Fluorescence resonance energy transfer (FRET) mechanism.
3.5. Conformation investigation
To exploit the structural change of BSA by addition of the
MBMPB, we have measured synchronous fluorescence spectra of
BSA with various amounts of MBMPB. The synchronous fluores-
cence spectra [20] of BSA with various amount of MBMPB were re-
corded at
Dk = 15 nm and Dk = 60 nm (Fig. 9a and b). It is apparent
from the figure that the emission wavelength of the tyrosine resi-
dues is blue-shifted (kmax from 353 to 340 nm in Fig. 9a) with
increasing concentration of MBMPB. This blue shift expressed that
the conformation 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 reg-
ularly, but no significant change in wavelength was observed. It
suggests that the interaction of MBMPB with BSA does not affect
the conformation of tryptophan micro-region. The tyrosine fluores-
cence spectrum may represent that the conformation of BSA is
somewhat changed, due to the blue shift (Fig. 9a), leading to the
polarity around Tyr residues was decreased and the hydrophobicity
was increased [22], but the interaction of MBMPB with BSA does
not obviously affect the conformation of tryptophan micro-region
[23]. The MBMPB 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
unlike tryptophan and tyrosine can undergo an excited state ioni-
zation, 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 hydroxyl
Fig. 7. Stern–Volmer plot of F0/F against [MBMPB].
Table 1
Ksv KA, n and r values of BSA–MBMPB system at 301, 310 and 318 K.
T (K)
Ksv (104 L molꢀ1
)
KA (104 L molꢀ1
)
n
r
301
310
318
2.9
2.61
2.42
3.71
2.69
2.12
1.23
1.04
1.00
0.99
0.99
0.99
unity and eA(k) is the molar extinction coefficient of the acceptor
at wavelength k. The Forster distance (R0) has been calculated

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J. Jayabharathi et al. / Journal of Photochemistry and Photobiology B: Biology 115 (2012) 85–92
Fig. 9. Synchronous fluorescence spectra of BSA in the presence and absence of MBMPB (a) at
Dk = 15 nm and (b) at Dk = 60 nm.
% of DPPHÅ scavenging ¼ ½A0 ꢀ A1=A0ꢂ ꢁ 100
ð7Þ
group present in the tyrosine residues is responsible for the inter-
action of BSA with MBMPB.
where A0 was the absorbance of the control and A1 was the absor-
bance in the presence of the sample of MBMPB. MBMPB shows
DPPH radical quenching activity in a concentration dependent
manner.
For reconfirming the structural change of BSA by the addition of
the MBMPB, we have measured the UV–vis absorbance spectra of
BSA with various amounts of the MBMPB. Fig. 10 displays the
UV–vis absorbance spectra of BSA at different concentrations of
the MBMPB. The absorption band of 210 nm of BSA is characteristic
of a–helix structure of BSA. The intensity of absorbance of BSA was
3.6.2. Superoxide anion scavenging assay
decreased with increasing concentration of the MBMPB and the
peak was red shifted. In addition, the absorption peaks in the
UV–vis spectra at approximately 290 nm rise gradually (from
curve (a) to curve (e)) and blue shifted to about 11 nm with
increasing concentration of the MBMPB. These results indicating
that the interaction between the MBMPB and BSA and the fluores-
cence quenching of BSA by MBMPB was the result of the formation
of BSA–MBMPB complex [24]. These results confirmed that the
quenching was mainly a static quenching process.
It is well known that superoxide anions damage biomacromol-
ecules directly or indirectly by forming H2O2, OHÅ, peroxylnitrite, or
singlet oxygen during pathophysiologic events such as ischemic-
reperfusion injury. PMSred (phenazine methosulphate) convert oxi-
dized nitro blue tetrazolium (NBToxi) to the reduced form (NBTred),
which formed a violet colored complex. The color formation indi-
cates the generation of superoxide anion, which was measured
spectrophotometrically at 560 nm. A decrease in the formation of
color after addition of the antioxidant was a measure of its super-
oxide scavenging activity.
3.6. In vitro antioxidant activity
The superoxide radical scavenging activities of MBMPB were
evaluated based on their ability to quench the superoxide radical
3.6.1. DPPH radical scavenging activity
MBMPB, at various concentrations ranging from 10, 20, 40, 60,
80 and 100 lM, were mixed in 1 mL of freshly prepared 0.5 mM
DPPH ethanolic solution and 2 mL of 0.1 M acetate buffer at pH
5.5. The resulting solutions were then incubated at 37 °C for
30 min and measured at 517 nm in a Shimadzu UV-1601 spectro-
photometer. DPPHÅ scavenging activities of the MBMPB were calcu-
lated from the decrease in absorbance from 517 nm in comparison
with the negative control (Fig. 11).
generated from the PMS/NADH reaction. 1 mL of NBT (100
of NBT in 100 mM phosphate buffer, pH 7.4), 1 mL of NADH
(468 mol in 100 mM phosphate buffer, pH 7.4) solution and vary-
ing volume of imidazoles (10,20,40,60,80 and 100 M) were
mixed well. The reaction was started by the addition of 100 l of
PMS (60 mol/100 mM phosphate buffer, pH 7.4). The reaction
mixture was incubated at 30 °C for 15 min. The absorbance was
measured at 560 nm in a spectrophotometer. Incubation without
the compound was used as blank. Decreased absorbance of the
reaction mixture indicates increased superoxide anion scavenging
activity (Fig. 12). The percentage scavenging was calculated as
follows:
lmol
l
l
l
l
Fig. 11. Radical scavenging potential of MBMPB by DPPH method at different
Fig. 10. Absorption spectra of BSA in the presence of MBMPB (a–e) and in the
concentrations (lg/mL).
absence of MBMPB (f).

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91
4. Conclusion
In this article, we investigated the interaction of 1-(4-Methoxy-
benzyl)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole (MBMPB)
with bovine serum albumin (BSA) by solution spectral studies.
The experimental results of fluorescence showed that the quench-
ing of BSA by MBMPB is a result of the formation of BSA–MBMPB
complex; static quenching and non-radiative energy transferring
were confirmed to result in the fluorescence quenching. Binding
reaction is play major role in the reaction is showed by thermody-
namic parameters. Synchronous fluorescence spectrum shows the
interaction of the MBMPB with BSA affects the conformation of
tyrosine residues micro-region. MBMPB shows potential antioxi-
dant property due to the couple of electron releasing methoxy
group.
Fig. 12. Scavenging potential of MBMPB at different concentrations (
superoxide radicals generated by the PMS/NADH system.
lg/mL) on
% of scavenging ½OÅ2ꢀꢂ ¼ ½A0 ꢀ A1=A0ꢂ ꢁ 100
ð8Þ
Acknowledgments
where A0 was the absorbance of the control and A1 was the absor-
bance in the presence of the sample of MBMPB. MBMPB shows
Superoxide anion quenching activity in a concentration dependent
manner.
One of the authors Prof. J. Jayabharathi is thankful to Depart-
ment of Science and Technology [No. SR/S1/IC-73/2010], University
Grants commission (F. No. 36-21/2008 (SR)) and Defence Research
and Development Organisation (DRDO) (NRB-213/MAT/10-11) for
providing funds to this research study.
3.6.3. Hydroxyl radical scavenging
In this assay, hydroxyl radical was produced by reduction of
H2O2 by the transition metal (iron) in the presence of ascorbic acid.
The generation of OHÅ is detected by its ability to degrade deoxyri-
bose to form products, which on heating with thiobarbituric acid
(TBA) form a pink color chromogen. Addition of MBMPB with
deoxyribose for OHÅ and diminishes the color formation. The incu-
bation mixture in a total volume of 1 mL contained 0.1 mL of buf-
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