Luminescent study on the binding interaction of bioactive imidazole with bovine serum albumin - A static quenching mechanism

Article abstract of DOI:10.1016/j.saa.2011.09.033

Novel bioactive imidazole derivatives were synthesized and characterized by NMR spectra, mass and CHN analysis. The interaction between the imidazole derivative and bovine serum albumin (BSA) was investigated by fluorescence and UV-vis absorption spectros

Full text of DOI:10.1016/j.saa.2011.09.033

Spectrochimica Acta Part A 84 (2011) 233–237
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Luminescent study on the binding interaction of bioactive imidazole with bovine
serum albumin—A static quenching mechanism
Jayaraman Jayabharathi∗, Venugopal Thanikachalam, Natesan Srinivasan, Marimuthu Venkatesh Perumal
Department of Chemistry, Annamalai University, Annamalainagar 608 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel bioactive imidazole derivatives were synthesized and characterized by NMR spectra, mass and
CHN analysis. The interaction between the imidazole derivative and bovine serum albumin (BSA) was
investigated by fluorescence and UV–vis absorption spectroscopy. The fluorescence quenching of BSA
by the imidazole derivatives may be due to the formation of imidazole–BSA complex. The fluorescence
quenching mechanism of BSA by imidazole was analyzed and the binding constant has been calculated.
The binding distance between imidazole and BSA was obtained based on Forester’s non-radiation energy
transfer (FRET). The effect of some common ions on the binding constant between imidazole and BSA
was also examined.
Received 1 July 2011
Received in revised form
10 September 2011
Accepted 13 September 2011
Keywords:
Bioactive imidazole
FRET
Forester distance
Stern–Volmer plot
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Quenching measurements of albumin fluorescence is an impor-
tant method to study the interactions of compounds with proteins
As a major soluble protein constituent of circulatory system
(∼60% of the total), serum albumin plays an essential role in
binding and transport a wide range of endogenous and exoge-
nous compounds such as fatty acids, heme, bilirubin, metal ions
and drugs, in the bloodstream to their target organs [1]. Its func-
tional and physiological properties have been extensively studied
in recent years [2]. Moreover, these proteins have long been used
as model proteins in both industrial and academic research areas
[3].
In this work, we selected Bovine serum albumin (BSA) as our
protein model, because of its medically important, abundance, low
cost, ease of purification, unusual ligand-binding properties, sta-
bility and all the studies are consistent with the fact that human
and bovine serum albumins are homologous proteins [4]. BSA has a
wide range of physiological functions involving the binding, trans-
port and delivery of fatty acids, porphyrins, steroids, etc. BSA is
constituted by 582 amino acid residues and on the basis of the dis-
tribution of the disulfide bridges and of the amino acid sequence
it seems possible to regard BSA as composed of three homologous
domains linked together. The domains can all be subdivided into
two sub-domains [5].
[6,7]. It can reveal accessibility of quenchers to albumin’s flu-
orophores, help to understand albumin binding mechanisms to
compounds and provide clues to the nature of the binding phe-
nomenon [8]. The study of the interaction features between small
molecules and biological macromolecules such as BSA and discus-
sions regarding the binding force quality, and the mechanism of
the interactions play a critical role in promoting research in pro-
teins. The aim of this work is to study the binding and the effects
of energy transfer between BSA and the imidazole derivative spec-
trofluorimetrically.
2. Experimental
2.1. Materials and methods
Butan-2,3-dione (Sigma–Aldrich Ltd.), 2,4-difluoro benzalde-
hyde (S.D. fine.), 4-methyl aniline, N,N-dimethyl aniline and all
other reagents were used without further purification. Bovine
Serum Albumin was obtained from Sigma-Aldrich Company, Ban-
galore). All BSA solution was prepared in Tris–HCl buffer solution
(0.05 mol L−1 Tris, 0.15 mol L−1 NaCl, pH 7.4) and it was kept
in the dark at 303 K. Tris base (2-amino-2-(hydroxymethyl)-1,3-
propanediol) 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.
∗
Corresponding author. Tel.: +91 9443940735.
E-mail address: jtchalam2005@yahoo.co.in (J. Jayabharathi).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.09.033

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J. Jayabharathi et al. / Spectrochimica Acta Part A 84 (2011) 233–237
Scheme 1. Synthesis of imidazole derivatives 1 and 2.
2.2. Optical measurements and composition analysis
Fig. 1. Absorption spectra of BSA in the presence of imidazole (6) (a–e) and absence
of imidazole (6) (f).
NMR spectra were recorded on a Bruker 400 MHz instrument.
The UV–vis spectra and photoluminescence (PL) spectra were mea-
sured on UV–vis spectrophotometer (PerkinElmer, Lambda 35) and
fluorescence spectrometer (PerkinElmer LS55), corrected for back-
ground due to solvent absorption. MS spectra were recorded on a
Varian Saturn 2200 GCMS spectrometer.
applied to determine KSV by linear regression of a plot of F0/F versus
[Q].
2.5. Calculation of binding parameters
Apparent binding constant kA and binding sites n [19] can be
2.3. General procedure for the synthesis of 2-arylimidazole
derivatives
obtained from
(F0 − F)
log
= log KA + n log[Q]
(2)
F
The 2-arylimidazole derivatives were synthesized from an
unusual four components [9–17] assembling of butan-2,3-dione,
ammonium acetate, substituted anilines and substituted benzalde-
hydes (Scheme 1).
where F0 and F are the fluorescence intensities before and after the
addition of the quencher, [Q] is the total quencher concentration.
By the plot of log (F0 − F)/F versus log [Q], the number of binding
sites n and binding constant KA can be obtained.
2.3.1. 2.3.1.
3. Results and discussion
2-(2,4-Difluorophenyl)-4,5-dimethyl-1-p-tolyl-1H-imidazole (1)
Yield: 55%. 1H NMR (400 MHz, CDCl3): ı 2.05 (s, 3H), 2.34 (d,
6H), 6.64 (bt, 1H), 6.83 (bt, 1H), 6.98 (d, 2H), 7.16 (d, 2H), 7.41
(m, 1H). 13C NMR (100 MHz, CDCl3): ␦ 9.60, 12.74, 21.10, 104.02,
111.38, 116.17, 125.23, 126.98, 129.68, 132.85, 133.87, 134.36,
138.17, 139.83, 144.19, 160.97, 164.06. Anal. calcd. for C18H16F2N2:
C, 72.47; H,5.41; N, 9.39. Found: C, 72.25; H, 5.10; N, 9.09. MS: m/z
298.13, calcd. 298.02.
3.1. Binding interaction of BSA with bioactive imidazole
derivative
The interaction between bioactive imidazole derivative and
bovine serum albumin was investigated using UV–vis and flu-
orescence spectral studies and the fluorescence quenching of
BSA by imidazole derivative was the result of the formation
of BSA–imidazole complex. The binding site number (n) and
apparent binding constant (KA) were measured. The absorption
spectra of BSA in the presence and absence of imidazole were
different (Fig. 1). The absorption band of 210 nm of BSA is the
2.3.2. N,N-Dimethyl-4-(4,5-dimethyl-2-phenyl-1H-imidazol-1-
yl)benzenamine
(2)
Yield: 47%. 1H NMR (400 MHz, CDCl3): ı 2.01 (s, 3H), 2.31 (s, 3H),
3.02 (s, 6H), 6.71 (d, 2H), 7.02 (d, 2H), 7.19 (m, 3H), 7.36 (m, 2H).
13C NMR (100 MHz, CDCl3): ı 9.57, 12.84, 40.40, 112.35, 125.97,
126.53, 127.92, 127.94, 128.39, 131.23, 132.76, 132.99, 145.25,
150.06. Anal. calcd. for C19H21N3: C, 78.32; H, 7.26; N, 14.42. Found:
C, 78.11; H, 7.11; N, 14.20. MS: m/z 291.17, calcd. 291.01.
2.4. Principles of fluorescence quenching
Fluorescence quenching [18] is described by the Stern–Volmer
equation:
F0
= 1 + Kqꢀ0 [Q] = 1 + KSV[Q]
(1)
F
where F0 and F are the fluorescence intensities before and after
the addition of the quencher, respectively. Kq, KSV, ꢀ0 and [Q] are
the quenching rate constant of the bimolecular, the Stern–Volmer
dynamic quenching constant, the average lifetime of the bimolec-
ular without quencher (ꢀ0 = 10−8 s) and the concentration of the
quencher, respectively. Obviously, Kq = KSV, ꢀ0, hence, Eq. (1) was
Fig. 2. Fluorescence quenching spectra of BSA at different concentrations of imida-
zole (2).

J. Jayabharathi et al. / Spectrochimica Acta Part A 84 (2011) 233–237
235
Fig. 3. Fluorescence resonance energy transfer (FRET) mechanism.
Scheme 2. Schematic representation for the formation of the BSA–imidazole com-
plex.
Fig. 4. Stern–Volmer plot of F0/F against [imidazole].
characteristic of ␣-helix structure of BSA. The intensity of
absorbance of BSA was decreased with increasing concentration of
imidazole and the peak was red shifted. The results indicated that
there exists interaction between imidazole and BSA results ground
state complex was formed [20–22].
Fig. 6. Overlapping of Fluorescence spectra of BSA with absorption spectra of imi-
dazole.
Fluorescence quenching spectra of solutions containing BSA of
fixed concentration and different concentrations of imidazole were
depicted in Fig. 2. It can be observed that the fluorescence intensity
of BSA decreases regularly with the increase addition of imida-
zole but there is no significant emission wavelength shift, suggests
that imidazole interact with BSA and quench its intrinsic fluores-
cence. A Forster type fluorescence resonance energy transfer (FRET)
mechanism (Fig. 3) is involved in the quenching of BSA fluores-
cence by imidazole in BSA–imidazole complex. This means that the
possible quenching mechanism of fluorescence of BSA by imidazole
is not initiated by dynamic collision but from the formation of the
BSA-imidazole complex (Scheme 2).
3.2. Fluorescence quenching mechanism
The quenching mechanism of imidazole with BSA was probed
using the Stern–Volmer equation [23]. Eq. (1) was applied to
Fig. 5. Schematic representation of binding interaction of BSA with imidazole.

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J. Jayabharathi et al. / Spectrochimica Acta Part A 84 (2011) 233–237
Fig. 7. Synchronous Fluorescence spectra of BSA in the presence and absence of imidazole (a) at ꢁꢂ = 15 nm (b) at ꢁꢂ = 60 nm.
determine KSV by linear regression from the Stern–Volmer plot
of F0/F against [imidazole] (Fig. 4). Dynamic and static quench-
ing can be distinguished by the quenching constant Kq. According
to the literature [24] for dynamic quenching, the maximum scat-
ter collision quenching constant of various quenchers with the
biopolymer is 2.0 × 101◦ L mol−1 s−1 and the fluorescence lifetime
of the biopolymers is 10−8 s [25]. From Fig. 4, the values of
KSV and Kq (=KSV/ꢀ0) was calculated. The values of Kq are larger
than 2.0 × 1010 L mol−1 s−1, which suggest that the fluorescence
quenching is caused by a specific interaction between BSA and
imidazole (2) and the quenching mechanism mainly arise from
BSA–imidazole complex formation, while dynamic collision could
be negligible in the concentration range studied [26]. The plot
of log [(F0 − F)/F] versus log [imidazole] binding constant (KA) as
2.02 × 104 M−1 and binding site (n) as 1.09 of imidazole with BSA
from the intercept and slope (Fig. 5).
3.4. Synchronous fluorescence spectroscopic studies of BSA
The synchronous fluorescence spectra [28–30] of BSA with
various amounts of imidazole were recorded at ꢁꢂ = 15 nm and
ꢁꢂ = 60 nm (Fig. 7a and b), respectively. The emission wavelength
of the Tyrosine residues is blue-shifted (ꢂmax from 310 to 300 nm
in Fig. 7a) with increasing concentration of imidazole. 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 imidazole with BSA does not affect the con-
formation of tryptophan micro-region. The Tyrosine fluorescence
spectrum may represent that the conformation of BSA is somewhat
changed, leading to the polarity around Tyr residues strengthened
and the hydrophobicity weakened [31]. This is because Tyr con-
tains one aromatic hydroxyl group unlike tryptophan and Tyr can
undergo an excited state ionization, 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 quench-
ing. Hence the aromatic hydroxyl group present in the Tyr residue
is responsible for the interaction of BSA with imidazole.
3.3. Energy transfer from BSA to imidazole derivative
The distance between the BSA and the interacted imidazole was
estimated by Forster’s non-radiative energy transfer (FRET) and the
overlap fluorescence spectra of imidazole and the UV–vis absorp-
tion spectra of BSA were shown in Fig. 6. According to Forster’s
non-radiative energy transfer theory, the energy transfer efficiency
(E) is defined by the following equations,
3.5. Effect of common ions on the binding constants of
imidazole–BSA complex
In the blood system, there exists many metal ions and these
ions can directly influence the binding force of drug with protein.
The effect of metal ions Cu2+, Zn2+, Ca2+,Mg2+, Ni2+, Co2+ and Fe2+
on the binding constants was investigated at 287 K by recording
the fluorescence intensity in the range from 300 to 500 nm upon
excitation at 280 nm and the concentration of BSA and metal ions
were fixed at 2 ␮M (Table 1). The competition between the common
metal ions and imidazole makes the decrease in binding constant
of imidazole–BSA as compared to the binding constant without the
metal ions. Therefore, the common metal ions will shorten the stor-
age time of the drug in blood plasma, which may lead to the need for
R06
(R06/r06)
F
E = 1 −
=
(3)
F0
R06 = 8.8 × 1023[ꢃ2n−4˚DJ(ꢂ)] in Å 6
(4)
(5)
ꢀ
∞
J(ꢂ) =
FD(ꢂ)εA(ꢂ)ꢂ4dꢂ
0
where FD (ꢂ) is the corrected fluorescence intensity of the donor
at wavelength ꢂ to (ꢂ + ꢁꢂ), with the total intensity normalized
to unity and εA(ꢂ) is the molar extinction coefficient of the accep-
tor at wavelength ꢂ. The Forster distance (R0) has been calculated
assuming random orientation of the donor and acceptor molecules.
Table 1
Effects of metal ions on the binding constant of BSA–imidazole (1).
System
Binding constant (104 L mol−1
)
In the present case, ꢃ2 = 2/3, n = 1.334,
˚D = 0.30 and from
BSA + (1)
2.06
2.02
1.96
1.89
1.71
1.51
1.42
1.33
BSA + (1) + Cu2+
BSA + (1) + Zn2+
BSA + (1) + Ca2+
BSA + (1) + Mg2+
BSA + (1) + Ni2+
BSA + (1) + Co2+
BSA + (1) + Fe2+
the available data, it results that J(ꢂ) = 2.37 × 10−15 cm3 L mol−1
,
E = 0.48, R0 = 2.41 nm and r = 2.44 nm were calculated. The donor-
to-acceptor distance is less than the 8 nm, and the long distance
indicates that the quenching mechanism is a dynamic one, which
is strong evidence for static quenching and energy could transfer
from BSA to imidazole [27].

J. Jayabharathi et al. / Spectrochimica Acta Part A 84 (2011) 233–237
237
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