Displacement reaction using ibuprofen in a mixture of bioactive imidazole derivative and bovine serum albumin - A fluorescence quenching study

Article abstract of DOI:10.1007/s10895-011-0878-3

The mutual interaction of imidazole derivative (PIPP) with bovine serum albumin (BSA) was investigated using photoluminescent studies. The fluorescence quenching mechanism of BSA by PIPP was analyzed and the binding constant was calculated. The binding di

Full text of DOI:10.1007/s10895-011-0878-3

J Fluoresc (2011) 21:1825–1830
DOI 10.1007/s10895-011-0878-3
ORIGINAL PAPER
Displacement Reaction Using Ibuprofen in a Mixture
of Bioactive Imidazole Derivative and Bovine Serum
Albumin—a Fluorescence Quenching Study
Jayaraman Jayabharathi & Venugopal Thanikachalam &
Marimuthu Venkatesh Perumal &
Kanagarathinam Saravanan
Received: 6 January 2011 /Accepted: 23 February 2011 /Published online: 18 March 2011
#
Springer Science+Business Media, LLC 2011
Abstract The mutual interaction of imidazole derivative
PIPP) with bovine serum albumin (BSA) was investigated
Investigations related to proteins are hot research subject
in life sciences, pharmacology, chemistry and so on [4–8].
The most abundant protein in blood plasma is serum
albumin. One of the significant way of transport of drugs in
the blood is binding with serum albumin. The binding
ability of drug to the serum albumin in blood may have a
considerable impact on distribution, free concentration and
metabolism of drugs. Therefore, drug–protein interaction
has a considerable significance in pharmacology and the
binding parameters are helpful in the study of pharmaco-
kinetics [9]. BSA is highly stable and comparatively cheap
and its structure is similar to human serum albumin in 76%
[10]. So it is often used to replace human serum albumin in
drug–protein studies [4–6].
(
using photoluminescent studies. The fluorescence quenching
mechanism of BSA by PIPP was analyzed and the binding
constant was calculated. The binding distance between PIPP
and BSAwas obtained based on the theory of Forester’s non-
radiation energy transfer. Displacement experiments were
performed by using ibuprofen to identify PIPP binding site in
BSA. The effect of some common ions on the binding
constant between PIPP and BSA was also examined.
.
.
.
Keywords Bioactive imidazole FRET Forester distance
.
Stern-Volmer Plot Ibuprofen
BSA is composed of a single chain of 582 amino acid
residues and can be divided into three homologous
domains. Each domain can be subdivided into two sub-
domains (A and B). The binding sites of BSA for
endogenous and exogenous ligands may be in these
domains and the principal region of drug binding sites of
albumin are often located in hydrophobic cavities in sub-
domains IIA and IIIA. So called sites I and II are located in
subdomain IIA and IIIA of albumin, respectively. Many
ligands bind specifically to serum albumin, for example,
ketoprofen in site I [11], flufenamic acid and ibuprofen in
site II [12]. After having thorough literature survey, we
have interested to identify the binding interaction behaviour
of a newly designed imidazole derivative namely, 2-(1-
phenyl-1 H-imidazo[4,5-f][1, 10]phenanthrolin-2-yl)phenol
(PIPP) with BSA. Due to the presence of phenanthroline
function with two diiminic nitrogen atoms in PIPP, this
imidazole derivative behaves as a highly active Lewis base.
We have also exploited the fluorescence resonance energy
transfer (FRET) from PIPP to the BSA. The binding
characteristic between PIPP and BSA was investigated in
Introduction
Recently, heterocyclic imidazole derivatives have attracted
considerable attention because of their unique optical proper-
ties [1]. These compounds play very important role in
chemistry as mediators for synthetic reactions, primarily for
preparing functionalized materials [2]. Imidazole nucleus
forms the main structure of some well-known components of
human organisms, i.e., the amino acid histidine, Vitamin
B , a component of DNA base structure, purines,
1
2
histamine and biotin and present in structure of many
natural or synthetic drug molecules, i.e., azomycin,
cimetidine and metronidazole and also have significant
analytical applications utilizing their fluorescene and
chemiluminescence properties [3].
:
:
:
J. Jayabharathi (*) V. Thanikachalam M. V. Perumal
K. Saravanan
Department of Chemistry, Annamalai University,
Annamalainagar, Tamilnadu 608002, India
e-mail: jtchalam2005@yahoo.co.in

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J Fluoresc (2011) 21:1825–1830
detail by using fluorescence and UV–Vis absorption. The
aim of this work is to explore the interaction mechanism,
the specific binding site and the effect of PIPP on the
conformational changes of BSA.
CDCl ): δ 115.86, 119.42, 121.50, 121.85, 122.40, 122.91,
3
127.53, 127.61, 128.30, 129.05, 129.72, 130.67, 135.20,
136.40, 137.40, 149.4, 150.00, 154.1, 156.02. MS: m/z
388.13,calcd 388.42.
Experimental
Results and Discussion
Materials and Methods
Analysis of Fluorescence Quenching of BSA by PIPP
1
,10-phenanthroline-5,6-dione (Sigma-Aldrich Ltd.), sali-
The fluorescence intensity can be corrected for absorption
of exciting light and re-absorption of emitted light using the
following relationship [23]:
cylaldehyde (S.D. fine.), aniline (S.D. fine) and all the other
reagents used without further purification. Bovine Serum
Albumin (BSA) was obtained from Sigma-Aldrich Company,
Bangalore. All BSA solution was prepared in the Tris–HCl
Fcor ¼ Fobs ꢀ eðAex=Aem=2Þ
ð1Þ
−1
−1
buffer solution (0.05 mol L Tris, 0.15 mol L NaCl, pH
.4) and was kept in the dark at 303 K. Tris base (2-amino-2-
hydroxymethyl)-1,3-propanediol) had a purity of no less
7
(
where Fcor and Fobs are the fluorescence intensity corrected
and observed, respectively and Aex and Aem are the
absorption and emission wavelength, respectively. There
exists obvious absorption for PIPP in the range of 270–
400 nm. So the intensity of fluorescence used is corrected
in this study. There are three types of fluorophores in BSA,
namely tryptophan residues (Trp), tyrosine residues (Tyr)
and phenylalanine (PA) residues. Although both Trp and
Tyr residues can be excited at 280 nm, because of energy
transfer effect between amino acid residues, the recorded
intrinsic fluorescence of BSA excited at 280 nm mostly
comes from Trp residues [24]. In order to determine
whether both Trp and Tyr residues are involved in the
interaction with PIPP, the fluorescence of BSA excited at
280 and 295 nm, respectively and compared in the presence
of PIPP. When 280 nm is used as excitation wavelength,
fluorescence of the albumin comes from both Trp and Tyr
residues, whereas 295 nm wavelength only excites Trp
than 99.5% and NaCl, HCl and other starting materials were
all of analytical purity and doubly distilled water was used
throughout.
Optical Measurements and Composition Analysis
NMR spectra were recorded for PIPP on a Bruker
5
00 MHz The ultraviolet–visible (UV–Vis) spectra were
measured on UV–vis spectrophotometer (Perkin Elmer,
Lambda 35) and corrected for background due to solvent
absorption. Photoluminescence (PL) spectra were
recorded on a (Perkin Elmer LS55) fluorescence spec-
trometer. An MS spectrum was recorded on a Varian
Saturn 2200 GCMS spectrometer.
General Procedure for the Synthesis of 2-(1-phenyl-1
H-imidazo[4,5-f][1,10]phenanthro- lin-2-yl)phenol (PIPP)
residues [12]. The plot of F/F against [PIPP]/[BSA] is
0
shown in Fig. 1, which indicates that the fluorescence of
BSA excited at 280 nm obviously differs from BSA
exciting at 295 nm in the presence of PIPP. This significant
difference between quenching of serum albumin fluores-
cence shows that both Tyr and Trp residues participated in
the molecular interaction between PIPP and BSA. At the
excitation wavelength of 280 nm, the fluorescence quench-
ing spectra of BSA with growing amounts of PIPP are
shown in Fig. 2. The intensity of BSA fluorescence
regularly decreased but the maximum emission wavelength
of BSA did not apparently shift with the increase of PIPP
concentration. Fluorescence quenching is the decrease of
the quantum yield of fluorescence from a fluorophore
induced by a variety of molecular interactions with
quencher molecules. Under the conditions of fixed pH,
temperature and ionic strength, fluorescence quenching
may result from ground complex formation, energy transfer
and dynamic quenching processes [25]. Dynamic quench-
The experimental procedure was used as the same as
described in our recent papers [13–22]. The imidazole
derivative, PIPP was synthesized from an unusual four
components assembling of 1,10-phenanthroline-5,6-
dione, ammonium acetate, aniline and salicylaldehyde
(
Scheme 1).
2
-(1-Phenyl-1 H-imidazo[4,5-f][1,10]phenanthro- Lin-2-yl)
phenol (PIPP)
Yield: 55%. mp=289°C, Anal. calcd. for C H N O: C,
2
5 16 4
7
1
7.3; H, 4.15; N, 14.42. Found: C, 76.65; H, 4.08; N,
1
3.98. H NMR (500 MHz, CDCl ): δ 4.90 (s, 1 H), 6.63
3
(
[
d, 1 H), 6.81 (m, 2 H), 7.01–7.16 (m, 1 H), 7.29 (d, 2 H)
o-hydroxy phenyl ring], 7.59–7.75 (Aromatic protons of
aniline ring), 9.04 (d, 2 H, H-aryl, J=4.5 Hz), 9.10 (d, 2 H,
1
3
J=8.0 Hz), 9.20 (d, 2 H, J=5.6 Hz). C (100 MHz,

J Fluoresc (2011) 21:1825–1830
1827
Scheme 1
ing refers to a process that the fluorophore and the quencher
come into contact during the lifetime of the excited state,
whereas static quenching refers to fluorophore- quencher
complex formation. To shed light on the fluorescence
quenching mechanism, the fluorescence quenching data
are analyzed by the Stern–Volmer equation [26]:
by PIPP is not initiated by dynamic collision but from the
formation of a complex.
Binding Constants and Binding Points
The apparent binding constant, K , and the number of
a
binding sites, n, can be calculated using the following
equation [29]:
ꢀ
F0 F ¼ 1 þ Kqt0½Qꢁ ¼ 1 þ KSV½Qꢁ
ð2Þ
F0 ꢂ F
where F0 and F are the fluorescence intensities in the
log
¼ n log Ka
F
absence and presence of quencher, respectively. K is the
q
ꢃ
ꢁ
ꢂꢄ
bimolecular quenching rate constant, C0 is the average
lifetime of the biomolecule in the absence of quencher, [Q]
is the concentration of quencher, and K =K /C is the
F0 ꢂ F
þ n log ½Qꢁ ꢂ ½Pꢁ
ð3Þ
t
t
F0
sv
q 0
Stern–Volmer quenching constant. The fluorescence life-
where F and F are the fluorescence intensities before and
−
8
0
time of biopolymer is about 10 s [27]. The Stern–Volmer
plot is shown in Fig. 3, based on the measured fluorescence
data determined at room temperature and the corresponding
after the addition of the quencher, [Q] and [P] are the total
t
t
quencher concentration and the total protein concentration,
respectively. The main advantage of this equation is that it
uses the total concentration of quencher instead of the free
3
KSV value was calculated as 3.816 x 10 (r=0.997). The
1
2
13
value of K was also obtained in the range of 10 to 10
q
concentration of quencher. K and n values were calculated
−
1 −1
a
M s . Obviously, the rate constant of protein quenching
procedure initiated by PIPP is much greater than the
maximum scatter collision quenching constant of the
4
as 1.224×10 and 1.13. The correlation coefficient is larger
than 0.999 indicating that the interaction between PIPP and
BSA agrees well with the site-binding model underlined in
the above equation. The value of n (1.13) indicates the
existence of one association site.
1
0
−1 −1
biomolecule (K =2.0×10 M s ) [28]. This means that
q
the possible quenching mechanism of fluorescence of BSA
Fig. 2 Fluorescence quenching spectra of BSA with increasing
Fig. 1 Plot of F/F
0
versus [PIPP]/[BSA]
concentration of PIPP

1
828
J Fluoresc (2011) 21:1825–1830
0
Fig. 3 Stern-Volmer plot of F /F versus [PIPP]
Forester Energy Transfer Efficiency from BSA to PIPP
Fig. 5 Plot of F2/F1×100% versus [Ibuprofen]
The overlap of the UV–Vis absorption spectrum of PIPP
with the fluorescence emission spectrum of BSA is shown
in Fig. 4. The importance of the energy transfer in
biochemistry is that the efficiency of transfer can be used
to evaluate the distance between the PIPP and the
tryptophan residues in BSA. According to Forster non-
radiative energy transfer theory [30], the efficiency of
energy transfer mainly depends on the fluorescence of
donor, overlap emission spectrum of donor and absorption
spectrum of acceptor and the distance between the donor
and the acceptor (usually shorter than 8 nm). Based on the
theory of Forster energy transfer, the distance between PIPP
and BSA can be calculated by the equation:
at which the efficiency of transfer is 50% and R
calculated as follows:
can be
0
ꢇ
ꢈ
6
R ¼ 8 : 8 ꢀ 10 k2nꢂ46DJðlÞ in + ;
6
23
ð5Þ
0
2
where .
is the relative orientation of the donor and
acceptor molecule, n is the refractive index of the medium,
ΦD is the quantum yield of the donor in absence of the
acceptor and J(l) is the overlap integral of the fluorescence
emission spectrum of the donor and the absorption
spectrum of the acceptor. The overlap integral J(l) for a
donor-acceptor pair is defined as
F
ꢀꢅ
ꢆ
6
0
6
0
6
0
Z 1
E ¼ 1 ꢂ
¼ R R þ r
ð4Þ
F0
4
JðlÞ ¼
FDðlÞ"AðlÞl dl
ð6Þ
0
where r is the distance between the donor and acceptor, R
0
0
is the Forster’s distance or critical energy transfer distance,
where FD (l) is the corrected fluorescence intensity of the
donor at wavelength l to (l+Δl), with the total intensity
normalized to unity and ε (l) is the molar extinction
A
coefficient of the acceptor at wavelength l. The Forster
distance (R ) has been calculated assuming random orientation
0
2
of the donor and acceptor molecules. In the present case, . =
Table 1 Effects of metal ions on the binding constant of BSA–PIPP
System
Binding constants
Error (±)
4
−1
(−10 L mol−1)
5
(−10 L mol
)
BSA+PIPP
1.224
1.200
1.194
1.163
1.146
0.20
0.26
0.25
0.23
0.18
2
+
+
+
BSA+PIPP+Cu
2
2
BSA+PIPP+Zn
BSA+PIPP+Ca
2
+
BSA+PIPP+Mg
Fig. 4 Overlap of absorption spectrum of PIPP (i) with the emission
spectrum of BSA (ii)
Number of measurements: 4 times; Temperature: 303 K

J Fluoresc (2011) 21:1825–1830
1829
2
/3, n=1.422, Φ =0.62 and from the available data, it results
The Displacement Experiment Using Ibuprofen
D
−16
that J(l)=4.315×10 , E=0.18, R =3.8 nm and r=1.8 nm
0
were calculated. The binding distance r=1.8 nm is less
BSA has a large hydrophobic cavity that can accommodate
two or more ligands. When two ligands (denoted with
Ligand1 and Ligand2) bind to BSA simultaneously, two
types of interaction can occur [32]:
than 8 nm and 0.5 R <r<1.5 R which indicates that the
0
0
energy transfer from BSA to PIPP occurs with high
probability [31].
(
a) Competitive binding
+
BSA
+ Ligand 2
ð7Þ
Ligand 1
Ligand 2-BSA + Ligand 1
Ligand 1-BSA
(
b) Non-Competitive binding
+
BSA
+ Ligand 2
ð8Þ
Ligand 1
Ligand 2 + BSA-Ligand 1
Ligand 1-BSA
In order to identify the location of the PIPP binding site
on BSA, the displacement experiments were carried out
using ibuprofen. PIPP to BSA ratio was kept at 5:1 in order
to keep non-specific binding of ibuprofen to a minimum.
The percentage of fluorescence probe displaced by the drug
was determined by measuring the changes in fluorescence
intensity according to the method proposed by Sudlow et
al. [33]:
at 287 K by recording the fluorescence intensity in the
range from 300 to 500 nm upon excitation at 280 nm and
the concentrations of BSA and metal ions were fixed at
2 μM (Table 1). The competition between the common
metal ions and PIPP makes the binding constant of PIPP–
BSA decrease as compared to the binding constant without
the metal ions. Therefore, the common metal ions will
shorten the storage time of the drug in blood plasma,
which may lead to need for more doses of PIPP to achieve
the desired therapeutic effect [5].
F =F ꢀ 100%
ð9Þ
2
1
where F and F denote the fluorescence of the PIPP plus
Conclusions
1
2
BSA without the ibuprofen and with the ibuprofen,
respectively. The fluorescence spectra of the BSA+PIPP
mixture solution upon addition of ibuprofen was recorded
in the range from 300 to 500 nm upon excitation at 280 nm.
The plot of F /F against site ibuprofen concentration
In this paper, the interaction of PIPP with BSA has been
investigated by fluorescenc spectrum and UV–vis spectrum
and the experimental results indicated the energy transfer
from BSA to PIPP occurred. The fluorescence quenching
results show that both of Trp and Tyr residues participated
in the interaction between PIPP and BSA and the probable
quenching mechanism of fluorescence of BSA by PIPP is a
static quenching process. The results also reveal the
presence of a single binding site and a complex formation
is in 1:1 mole ratio; hydrophobic interaction played a major
role in stabilizing the complex. The distance (r) between
BSA and PIPP was calculated to be 1.8 nm which indicated
the energy transfer with high probability. All the experi-
mental results clarified that PIPP could bind with BSA
effectively which could be a useful guideline for imidazole
drug design.
2
1
(
Fig. 5) reveals that the fluorescence was remarkably
affected by adding ibuprofen to the same solution. This
result indicates that ibuprofen displaced PIPP from the
binding site.
Effect of Common Ions on the Binding Constants
of PIPP–BSA Complex
There exists many metal ions in the blood system and
these ions can directly influence the binding force of
2
+
2+
drug with protein. The effect of metal ions Cu , Zn ,
2
+
2+
Ca and Mg on the binding constants was investigated

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J Fluoresc (2011) 21:1825–1830
Acknowledgment One of the authors Dr. J. Jayabharathi, Associate
professor, Department of Chemistry, Annamalai University is thankful
to Department of Science and Technology [No. SR/S1/IC-07/2007]
and University Grants commission (F. No. 36-21/2008 (SR)) for
providing fund to this research work.
16. Gayathri P, Thiruvalluvar A, Saravanan K, Jayabharathi J, Butcher
RJ (2010) 4,5-Dimethyl-1,2-diphenyl-1H-imidazole monohydrate.
Acta Crystallogr E 66:o2219
17. Gayathri P, Jayabharathi J, Srinivasan N, Thiruvalluvar A, Butcher
RJ (2010) 2-(4-Fluorophenyl)-1,4,5-triphenyl-1H-imidazole. Acta
Crystallogr E 66:o2519
1
8. Jayabharathi J, Thanikachalam V, Srinivasan N, Saravanan K
2010) Synthesis, structure, luminescent and intramolecular proton
(
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