Fluorescence quenching of bovine serum albumin by NNMB

J. Jayabharathi

, K. Jayamoorthy, V. Thanikachalam, R. Sathishkumar

Department of Chemistry, Annamalai University, Annamalainagar, Tamilnadu 608 002, India
h i g h l i g h t s
" The interactions between NNMB and
BSA were investigated.
" Fluorescence quenching arises from
the formation of BSANNMB
complex.
" Fluorescence spectra to exploit the
structural change of BSA.
" Thermodynamic parameters were
determined.
g r a p h i c a l a b s t r a c t
a r t i c l e i n f o
Article history:
Received 20 October 2012
Received in revised form 26 January 2013
Accepted 28 January 2013
Available online 15 February 2013
Keywords:
Fluorophore
BSA
Fluorescence quenching
FRET
a b s t r a c t
A new type of uorophore 2-(naphthalen-1-yl)-1-((naphthalen-1-yl)methyl)-1H-benzimidazole (NNMB)
has been prepared and characterized by
1
H NMR,
13
C NMR, mass and IR spectral analysis. Absorption,
uorescence and synchronous uorescence spectral studies have been made for the mutual interaction
of NNMB with bovine serum albumin (BSA). Absorption spectroscopy proved the formation of a ground
state BSA. . .NNMB complex. Fluorescence spectrum of BSA in the presence of NNMB clearly shows that
NNMB acts as a quencher. Based on the theory of Foresters non-radiation energy transfer (FRET) binding
distance has been deduced. The SternVolmer quenching constant (K
SV
), binding site number (n),
apparent binding constant (K
A
) and corresponding thermodynamic parameters (DG, DH and DS) were
determined.
2013 Elsevier B.V. All rights reserved.
Introduction
BSA is made up of three homologous domains (IIII), which are
divided into nine loops by 17 disulde 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 [1,2]. BSA has two
tryptophans, Trp-134 and Trp-212, embedded in the rst sub
domain IB and sub-domain IIA, respectively. There is evidence of
conformation changes of BSA induced by its interaction with low
molecular weight benzimidazole ligands. These changes appear
to affect the secondary and tertiary structure of albumin. So, it is
important to study the interaction of benzimidazole ligand with
BSA, and hence become an important research eld in chemistry,
life sciences and clinical medicine. Bovine serum albumin (BSA)
has been selected as our protein model due to its water-soluble
nature which is important for interaction studies [3,4]. It contains
582 amino-acid residues with a molecular weight of 69,000, and
two tryptophan moieties at positions 134 and 212 as well as tyro-
sine and phenylalanine [5]. The intrinsic uorescence of BSA is due
to aromatic amino-acid residues. Organic ligands act as biosensors
in the chemical and biochemical elds, and their applications are
becoming more extensive. These probes have been applied to
ultrasensitive detection of proteins, DNA sequencing, clinical diag-
nostics etc. Recently we reported the interaction of imidazoles
with bovine serum albumin using uorescence spectroscopy [69].
1386-1425/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.01.092

Corresponding author. Tel.: +91 9443940735.

E-mail address: jtchalam2005@yahoo.co.in (J. Jayabharathi).
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 108 (2013) 146150
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
j our nal homepage: www. el sevi er . com/ l ocat e/ saa
Study of photophysical properties of heterocyclic organic mole-
cules has achieved a considerable importance recently because of
many of these molecules form an integral part of intermediates
[10], ne product for drugs [11] and pesticides [12], color
industries [13], redox systems for solar energy [14], organized
assemblies [15], laser dyes [16] and complex forming agents
[17]. Imidazole derivatives have attracted considerable attention
because of their unique optical properties [18]. These compounds
play very important role in chemistry as mediators for synthetic
reactions, primarily for preparing functionalized materials [19].
Imidazole nucleus forms well-known components of human
organisms and used as laser, polymer stabilizer; Raman lters
environmental probes in bio-molecules [20], etc. by utilizing their
uorescence and chemiluminescence properties. In this article, A
new type of uorophore 2-(naphthalen-1-yl)-1-((naphthalen-
1-yl)methyl)-1H-benzimidazole (NNMB) has been prepared and
characterized by
1
H NMR,
13
C NMR, mass and IR spectral analysis
and also we discussed the interaction of BSA and napthyl substi-
tuted imidazole as the rst attempt.
Experimental
Materials and methods
All BSA solution were prepared in the TrisHCl 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-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.
Optical measurements
NMR spectra have been recorded for the NNMB on a Bruker
400 MHz instrument. The ultravioletvisible (UVvis) spectra have
been measured on UVVis 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) uorescence spectrometer. Solvents used for
spectral measurements are spectroscopic grade. Mass spectra of
the sample have been recorded on Thermo Fischer LC-Mass spec-
trometer in FAB mode.
General procedure for the synthesis of ligand
A mixture of 1-napthaldehyde (2 mmol), o-phenylenediamine
(1 mmol) and ammonium acetate (2.5 mmol) has been reuxed
at 80 C in ethanol. The reaction was monitored by TLC and puried
by column chromatography using petroleum ether: ethyl acetate
(9:1) as the eluent.
2-(naphthalen-1-yl)-1-((naphthalen-1-yl)methyl)-
1H-benzo[d]imidazole
Yield: 60%. mp = 130 C, Anal. calcd. for C
28
H
20
N
2
: C, 87.47; H,
5.24; N, 7.29. Found: C, 87.67; H, 5.10; N, 7.23.
1
H NMR
(400 MHz, CDCl
3
): d5.75 (s, 2H), 7.247.32 (m, 4H), 7.377.41 (t,
3H), 7.447.47 (m, 1H) 7.507.57 (m, 4H), 7.757.76 (t, 2H),
7.887.94 (m, 4H), 7.988.01 (t, 2H).
13
C NMR (100 MHz, CDCl
3
):
d46.12 (CH
2
carbon), 110.78, 120.25, 122.06, 122.68, 123.15,
123.61, 124.85, 124.91, 125.43, 125.58, 126.07, 126.33, 126.42,
126.50, 127.42, 127.78, 128.12, 128.22, 128.36, 128.97, 130.18,
130.28, 131.21, 133.59, 133.67, 135.42, 143.38, 151.36, 153.17
(aromatic carbons). MS: m/e 384.1, calcd. 385.38 [M + 1].
Results and discussion
FT-IR characteristics of NNMBBSA
The nature of interaction between the NNMB and BSA has been
studied by Fourier transform infrared (FT-IR) technique. FT-IR
spectrum of NNMB (solid line) and NNMB bound to the BSA (bro-
ken line) visualised in Fig. 1. The spectrum of pure NNMB shows
the >C@N stretching vibration at 1596 cm
1
. This band is shifted
from 1596 cm
1
to 1621 cm
1
for NNMB bound to the BSA. This
conrms the complex formation between NNMB and BSA.
Fluorescence spectral studies
Fluorescence spectroscopy is the powerful technique to study
the interaction between NNMB and BSA. Here, the concentrations
of BSA were stabilized at 1.0 10
5
mol L
1
and the concentration
of NNMB varied from 0 to 3.5 10
5
mol L
1
at increments of
0.5 10
5
mol L
1
. Fig. 2 shows the effect of the NNMB on BSA
uorescence intensity. The uorescence intensity of BSA decreases
progressively but the emission maximum remains constant. This is
due to the interaction of NNMB with BSA and quenches its intrinsic
uorescence (Trp-212) [21], but there was no alteration in the local
dielectric environment of BSA. A Forster type uorescence reso-
nance energy transfer (FRET) mechanism is involved in the
quenching of uorescence by benzimidazole in BSANNMB com-
plex. Therefore the possible quenching mechanism of uorescence
of BSA by NNMB is not initiated by dynamic collision but from the
Fig. 1. FT-IR spectra of NNMB (solid line) and NNMB bound with BSA (broken line).
Fig. 2. Fluorescence quenching spectra of BSA at different concentrations of NNMB.
J. Jayabharathi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 108 (2013) 146150 147
formation of the BSANNMB complex. The quenching mechanism
of NNMB with BSA was probed using the SternVolmer equation
[22], which can be applied to determine K
SV
by linear regression
from the SternVolmer plot of F
0
/F against [NNMB] Fig. 3 at
different temperatures. The values of K
SV
and K
A
at different
temperatures are tabulated in Table 1. It shows the values of
SternVolmer quenching constant K
SV
and K
A
decreases with in-
crease in temperature. According to the literature [23] for dynamic
quenching, the maximum scatter collision quenching constant of
various quenchers with the biopolymer is 2.0 10
10
L mol
1
s
1
and the uorescence lifetime of the biopolymer is 10
8
s [24]. From
Fig. 3, the values of K
SV
and k
q
(=K
SV
/s
0
) were calculated. The
obtained values of k
q
were larger than the limiting diffusion rate
constant of the biomolecule (2.0 10
10
L mol
1
s
1
), which
indicate that the uorescence quenching is caused by a specic
interaction between BSA and NNMB. Therefore, the quenching
mechanism mainly arises from the formation of BSANNMB com-
plex rather than dynamic quenching. So it was implied that the sta-
tic quenching was dominant in the system. From the plot of log
[(F
0
F)/F] vs log [NNMB], binding constants K
A
and the number
of binding sites n were determined from the intercept and slope.
Determination of thermodynamic parameters
In order to calculate the interaction between the NNMB and
BSA, thermodynamic parameters were calculated from the vant
Hoff plots. If the enthalpy change (DH) does not vary signicantly
over the temperature range studied then the thermodynamic
parameters DH, DG and DS can be determined from the following
equations:
lnK DH=RT 1
DG RTlnK 2
DS DH DG=T 3
The values of DH and DS obtained for the binding site from the vant
Hoff plot are visualised in Table 2. The negative sign for free energy
(DG) shows that the binding process is spontaneous. The negative
enthalpy (DH) and positive entropy (DS) values of the interaction
of the NNMB and BSA indicate that the specic electrostatic interac-
tions played major role in the reaction [25].
Energy transfer from BSA to NNMB
Energy transfer takes place through direct electro-dynamic
interaction between the excited molecule and its neighbours
[26]. The distance between the donor (BSA) and the acceptor
(NNMB) was estimated by Forsters non-radiative energy transfer
theory and the overlapping of uorescence spectra of BSA with
absorption spectra of NNMB was shown in Fig. 4. According to
Forsters non-radiative energy transfer theory, the energy transfer
efciency (E) can be dened as the following equations:
E 1 F=F
0
R
6
0
=R
6
0
r
6
0
4
R
6
0
8:8 10
25
j
2
n
4
U
D
JkinA

5
Jk
Z
1
0
F
D
ke
A
kk
4
dk=Fkdk 6
where E is the efciency of transfer between the donor and the
acceptor, R
0
is the critical distance when the efciency of transfer
is 50%. F
D
(k) is the corrected uorescence intensity of the donor
at wavelength k to (k + Dk), with the total intensity normalized to
unity and e
A
(k) is the molar extinction coefcient of the acceptor
at wavelength k. The Forster distance (R
0
) has been calculated
assuming random orientation of the donor and acceptor molecules.
Here, j
2
= 2/3, n = 1.311, U
D
= 0.21 and from the available data, it
results that J(k) = 4.68 10
12
cm
3
L mol
1
, E = 0.32, R
0
= 0.80 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 NNMB
[27] with high probability and the distance obtained by FRET with
higher accuracy.
Conformation investigation
To exploit the structural change of BSA by addition of the
NNMB, we have measured synchronous uorescence spectra of
Fig. 3. SternVolmer plot of F
0
/F against [NNMB].
Table 1
K
SV
K
A
, n and r values of BSANNMB system at 301, 310 and 318 K.
T (K) K
SV
(10
4
L Mol
1
) K
A
(10
4
L Mol
1
) n r
301 2.90 3.75 1.19 0.99
310 2.61 2.70 1.05 0.99
318 2.42 2.11 1.01 0.99
Table 2
Thermodynamic parameters of BSANNMB system at 301, 310 and 318 K.
T (K) DH (kJ mol
1
) DG (kJ mol
1
) DS (J mol
1
K
1
)
301 26.11 23.72 6.52
310 24.63
318 24.59
Fig. 4. Overlapping of (i) absorption spectra of NNMB with (ii) uorescence spectra
of BSA.
148 J. Jayabharathi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 108 (2013) 146150
BSA with various amounts of NNMB. The synchronous uorescence
spectra [2830] of BSA with various amount of NNMB were
recorded at Dk = 15 nm and Dk = 60 nm (Fig. 5a and b). It is appar-
ent from the gure that the emission wavelength of the tyrosine
residues is blue-shifted to 15 nm with increasing concentration
of NNMB. This blue shift expressed that the conformation of BSA
was changed and it suggested a less polar environment of tyrosine
residue [31]. At the same time, the tryptophan uorescence emis-
sion is decreased regularly, but no signicant change in wave-
length was observed. It suggests that the interaction of NNMB
with BSA does not affect the conformation of tryptophan micro-
region. The tyrosine uorescence spectrum may represent that
the conformation of BSA is somewhat changed, due to the blue
shift, leading to the polarity around Tyr residues was decreased
and the hydrophobicity was increased [32], but the interaction of
NNMB with BSA does not obviously affect the conformation of
tryptophan micro-region [33,34]. The NNMB could involve the sec-
ond site (sub-domain IB) with higher binding afnity and the for-
mation of complex led to the observation of the blue shift of
tyrosine residues uorescence. This is because the tyrosine con-
tains one aromatic hydroxyl group unlike tryptophan and tyrosine
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
quenching. Hence the aromatic hydroxyl group present in the tyro-
sine residues is responsible for the interaction of BSA with NNMB.
For reconrming the structural change of BSA by the addition of
the NNMB, we have measured the UVvis absorbance spectra of
BSA with various amounts of the NNMB. Fig. 6 displays the
UVvis absorbance spectra of BSA at different concentrations of
the NNMB. The absorption band of 210 nm of BSA is characteristic
of a-helix structure of BSA. The intensity of absorbance of BSA was
decreased with increasing concentration of the NNMB and the peak
was red shifted. In addition, the absorption peaks in the UVvis
spectra at approximately 280 nm rise gradually (from curve (a)
to curve (e)) and blue shifted to about 15 nm with increasing con-
centration of the NNMB. These results indicating that the interac-
tion between the NNMB and BSA and the uorescence quenching
of BSA by NNMB was the result of the formation of BSANNMB
complex [35]. These results conrmed that the quenching was
mainly a static quenching process.
Conclusion
In this paper, we investigated the interaction of 2-(naphthalen-
1-yl)-1-((naphthalen-1-yl)methyl)-1H-benzo[d]imidazole (NNMB)
with bovine serum albumin (BSA) by absorption and emission
spectroscopy. Fluorescence experimental results showed that the
quenching of BSA by NNMB is a result of the formation of BSA
NNMB complex; static quenching and non-radiative energy trans-
ferring were conrmed to result in the uorescence quenching.
Binding reaction is play major role in the reaction is showed by
thermodynamic parameters. A synchronous uorescence spectrum
shows the interaction of the NNMB with BSA affects the conforma-
tion of tyrosine residues micro-region.
Acknowledgments
One of the authors Prof. J. Jayabharathi is thankful to DST [No.
SR/S1/IC-73/2010], UGC (F. No. 36-21/2008 (SR)) and DRDO
(NRB-213/MAT/10-11) for providing funds to this research study.
Mr. K. Jayamoorthy is thankful to DST [No. SR/S1/IC-73/2010] for
providing fellowship.
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