Chemical Physics Letters 478 (2009) 271–276
Contents lists available at ScienceDirect
Chemical Physics Letters
journal homepage: www.elsevier.com/locate/cplett
Investigations to reveal the nature of interactions between bovine hemoglobin
and semiconductor zinc oxide nanoparticles by using various optical techniques
Gopa Mandal, Sudeshna Bhattacharya, Tapan Ganguly *
Department of Spectroscopy, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India
a r t i c l e
i n f o
Article history:
Received 28 May 2009
In final form 29 July 2009
Available online 3 August 2009
a b s t r a c t
The interaction between bovine hemoglobin (BHb) and zinc oxide (ZnO) nanoparticle is investigated by
UV–vis absorption, fluorescence, synchronous fluorescence, time-resolved fluorescence, FT-IR and circular dichroism techniques under physiological pH 7.4. The static mode of fluorescence quenching of BHb
by ZnO nanoparticle indicates formation of BHb–ZnO complex in the ground state. The process of binding
of ZnO nanoparticles on BHb is a spontaneous molecular interaction procedure in which electrostatic
interaction plays a major role. The CD spectra reveal that a helicity of BHb is reduced by increasing
ZnO nanoparticle concentration although the structure of BHb preserves its a-helical structure.
Ó 2009 Published by Elsevier B.V.
1. Introduction
Interaction of biomolecules with nanomaterials is the area of
fundamental interest due to their immense biological importance.
Nanomaterials which possess novel optical, electronic and chemical properties absent in bulk materials [1], have promising applications in biotechnology and life sciences [2]. Recently, biomolecules
conjugated with ZnO nanoparticles have received much attention
because ZnO nanomaterials are nontoxic, biosafe, and biocompatible [3] and have been used in many applications such as in biosensors [4], biogenerators [5], bioelectrodes [6] etc. The binding study
of drugs with proteins is of great importance in pharmacy, pharmacology and biochemistry. In order to using ZnO nanoparticles as
drug delivery carriers, knowledge regarding binding study of proteins with ZnO nanoparticles is required. In the present investigation the interaction of ZnO nanoparticles with a physiologically
important protein, bovine hemoglobin has been studied as hemoglobin is involved in many clinical diseases such as leukemia, anemia, heart disease, excessive loss of blood, etc. [7]. Hemoglobin is
an iron-containing oxygen-transport metalloprotein in the mammals’ red blood cells and has four oxygen-binding sites. Among
the four globin chains of hemoglobin, two are a-chains and two
are b-chains [8]. Thus, the subunit structure of Hb is a2 b2. The
a-chains contain 141 amino acids whereas b-chains contain 146
amino acids. Each a-chain is in contact with b-chain [9].
In the present investigation, we employed the fluorescence
quenching technique to examine the effect of addition of ZnO
nanoparticles to the solution of bovine hemoglobin. From the plot
* Corresponding author. Fax: +91 33 2473 2805.
E-mail addresses: tapcla@rediffmail.com, sptg@iacs.res.in (T. Ganguly).
0009-2614/$ - see front matter Ó 2009 Published by Elsevier B.V.
doi:10.1016/j.cplett.2009.07.095
of log [(F0 F)/F] vs. log [Q], the binding constants and the numbers of binding sites are computed. Again different thermodynamic
parameters are estimated according to the van’t Hoff equation,
from which the nature of the interacting force among the reactants
has been explored.
2. Experimental section
2.1. Materials
The samples BHb (Fig. 1a) supplied by Fluka, were used as obtained. The procedure of synthesis and characterization of ZnO
nanoparticles (Fig. 1b) is given below.
2.1.1. Synthesis and characterization of ZnO nanomaterial
Nanocrystals of ZnO were prepared by using zinc acetate. In a
typical synthesis a reaction mixture containing 2-propanol, diethanol amine and zinc acetate was prepared. At first diethanol amine
is added to a mixture containing 2-propanol and zinc acetate. The
ZnO nanoparticles are separated out from the solution by centrifugation of the precipitate obtained in basic medium. Finally, ZnO
nanoparticles are washed with water several times and dried at
60 °C. The UV–vis spectroscopy can be used to determine the diameter of semiconductor nanoparticle. The UV–vis spectrum of nanoparticles in Millipore water shows an absorption onset at about
500 nm. Colloidal ZnO solution was prepared by dissolving the desired amount of ZnO particles into Millipore water. The size of the
ZnO nanoparticles was also estimated from high resolution Transmission Electron Micrograph (TEM). The particle diameter varies in
the range of 20–40 nm (Fig. 1b). The average diameter of ZnO
nanoparticle is found to be <40 nm.
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G. Mandal et al. / Chemical Physics Letters 478 (2009) 271–276
of the light path in cm, M is the molecular weight and nr is the number of residues of the protein. FT-IR measurements were carried out
in a Perkin–Elmer Spectrum 100 FT-IR spectrometer using a 0.1 mm
CaF2 cell. The size of the ZnO nanoparticles was estimated from high
resolution Transmission Electron Micrograph (HRTEM) (JEOL, model
JEM-2010).
3. Results and discussion
3.1. Steady state UV–vis absorption studies
2.2. Other chemicals used
The solvent used in these experiments is Tris buffer (pH 7.4).
The buffer Tris was purchased from Merck (Germany), and NaCl,
HCl used for preparing the buffer are of analytical purity. BHb solution (M) was prepared in pH 7.4 Tris–HCl buffer solution (0.05 M
Tris, 0.1 M NaCl).
2.3. Spectroscopic apparatus
Steady state UV–vis and fluorescence emission spectra of dilute
solutions (104–106 M) of the samples were recorded at ambient
temperature (296 K) using 1 cm path length rectangular quartz
cells by means of an UV–vis absorption spectrophotometer (Shimadzu UV–vis 2101PC) and F-4500 fluorescence spectrophotometer (Hitachi), respectively. Fluorescence lifetime measurements
were carried out by the time-correlated single photon counting
(TCSPC) method using HORIBA JOBIN YVON FLUOROCUBE. The
excitation of the sample was carried out by NanoLED-295
(pd < 750 ps), FWHM of which is 690 ps. The quality of fit was assessed over the entire decay, including the rising edge, and tested
with a plot of weighted residuals and other statistical parameters
e.g., the reduced v2 and the Durbin–Watson (DW) parameters.
The time resolution is 68 ps. Circular dichroism (CD) spectra have
been recorded by JASCO, CD Spectrometer; model J-815-150S using
a 0.1 cm path length quartz cell in a wavelength range between
200 and 260 nm. The results were expressed as molar ellipticity
[h] in units of m degree gcm2 dmol1. Molar ellipticity values were
obtained using the relation.
h ¼ ðMhobs Þ=10clnr
where hobs is the observed ellipticity in degrees at a given wavelength, c is the protein concentration in mole/c.c. and l is the length
3.2. Steady state fluorescence studies
BHb contains three Trp residues in each ab dimer, for a total of
six in the tetramer: two a-14 Trp, two b-15 Trp, and b-37 Trp [12].
Of the six Trp residues present in the tetramer, only the b-37 residues are located at the dimer–dimer interface, wherein the structural differences between quaternary states are largest [13]. The
intrinsic fluorescence of BHb primarily originates from b-37 Trp
that plays a key role in the quaternary state change upon ligand
binding [14]. The experimental data by different laboratories indicated that b-37 Trp at the a1b2 interface is the primary contributor
to the fluorescence emission and report on alterations in the R ? T
transition [15]. A valuable feature of intrinsic fluorescence of protein is the high sensitivity of tryptophan to its local environment.
Changes in emission spectra of tryptophan are common in re-
0.4
0
8
0.2
0
Absorbance
Fig. 1b. TEM picture of ZnO nanoparticles.
Absorbance
Fig. 1a. Molecular structure of BHb.
Fig. 2a shows the UV–vis absorption spectra of BHb in buffer of
pH 7.4 and the effect of increasing the ZnO concentration on it.
The molecular weight of BHb is 64 442 Da and the diameter of it
is 280 nm [10]. Hemoglobin has four heme groups which are located in the crevices near the surface of the molecule; there were
two peaks (275 and 407 nm) in its absorption spectrum
(Fig. 2a). The band in the near-UV region with a maximum at
275 nm appears due to phenyl group of tryptophan residues
(Trp) and tyrosines. The sharp peak at 407 nm corresponds to the
characteristic absorption of the porphyrin–Soret band [7]. Along
with the Soret band of BHb a pair of bands of very weak intensity
are also appeared at 540 nm and 576 nm (inset of Fig. 2a) which
are assigned as the Q bands [11]. With gradual addition of ZnO
nanoparticles to BHb solution, the absorbance of the Soret band decreases with the increment of that of the band situated at 275 nm.
So from the significant spectral change in UV–vis absorption spectra of BHb, it is clear that ZnO nanoparticles affect both the Soret
band and the band corresponding to the tryptophan and tyrosine
residues of the BHb.
0.020
0.015
525 550 575
Wavelength / nm
8
0.0
300
400
500
Wavelength / nm
600
Fig. 2a. UV–vis absorption spectra of BHb (conc. 106 M) in presence of ZnO
nanoparticles at ambient temperature in buffer solution. The concentrations of ZnO
in (0) 0, (1) 2 107 M, (2) 6 107 M, (3) 1 106 M, (4) 1.4 106 M, (5)
2 106 M, (6) 2.9 106 M, (7) 3.9 106 M, (8) 4.8 106 M. Inset: Q bands of
BHb.
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G. Mandal et al. / Chemical Physics Letters 478 (2009) 271–276
sponse to protein conformational transitions, subunit association,
substrate binding, or denaturation [16]. Thus, the intrinsic fluorescence of proteins can provide considerable information about their
structure and dynamics, and it is often considered on the study of
protein folding and association reactions.
The effect of ZnO nanoparticles on the fluorescence of tryptophan residues of BHb at temperature 298 K is shown in Fig. 2b. It
was observed that the fluorescence emission band of BHb was
quenched with increasing the concentration of ZnO nanoparticles
when BHb is excited at 275 nm. The same types of fluorescence
quenching are observed at other two temperatures i.e. at 291
and 308 K. It is to be noted that in this experiment the concentration of ZnO nanoparticles kept in such a way that its absorbance at
275 nm is negligible relative to the Trp absorbance (inset of
Fig. 2b).
Fluorescence intensity data at three temperatures are then analyzed according to Stern–Volmer equation:
F 0 =F ¼ 1 þ K½Q ð1Þ
60
Absorbance
Relative intensity
where F0 and F are the fluorescence intensities of BHb in absence
and presence of ZnO, K is the Stern–Volmer constant and [Q] is
the concentration of the quencher. The linear Stern–Volmer plots
observed at the three different temperatures (Fig. 3a) in the case
of BHb and ZnO nanoparticles indicate the nature of the quenching
either of pure dynamic or static mode of quenching [17].
Dynamic and static quenching can be distinguished by their differing dependence on temperature or preferably by lifetime measurements [17]. As a rule, dynamic quenching depends upon
diffusion. Since higher temperatures result in larger diffusion coefficients, the bimolecular quenching constants are expected to in-
0
0.0009
0.0006
0.0003
0.0000
45
300 400 500
Wavelength / nm
8
30
15
300
325
350
375
Wavelength / nm
400
Table 1
Lifetimes of BHb in presence of ZnO nanoparticles at pH 7.4 (kex = 295 nm,
kem = 340 nm).
Conc. of ZnO nanoparticles
(M)
s1
0
1 106
4.8 106
1.4
1.6
1.6
f1
(ns)
s2
f2
(ns)
0.53
0.50
0.50
7.0
7.3
7.0
0.47
0.50
0.50
The biexponential decay: I (k, t) = A1 exp (t/s1) + A2 exp (t/s2), where A1 + A2 = 1.
The fractional contributions, f1 = A1 and f2 = A2.
crease with increasing temperature [17]. The K values decrease
with an increase in temperature for static quenching, but the reverse effect would be observed for dynamic quenching. In the present study, the K value decreases with increase in temperature
which indicates the type of quenching should be of static in nature.
Now to confirm the static nature of quenching mechanism, the
fluorescence lifetime measurements of BHb are made at the different concentrations of ZnO by using 295 nm excitation where only
Trp is excited [18]. It is to be mentioned in this connection that
the similar quenching effect, as observed in the case of 275 nm
excitation, was found with ZnO nanoparticles on the steady state
fluorescence spectra of Trp using the excitation wavelength at
295 nm. The time-resolved fluorescence data (Table 1 and
Fig. 3b) show that the fluorescence decay of BHb is biexponential
with lifetimes 1.4 ns and 7 ns and the magnitudes of the lifetimes
do not change significantly with addition of ZnO nanoparticles. But
along with these two lifetimes a very short lifetime (<68 ps) is also
obtained which is beyond the resolution of our instrument. It is reported that a very short lifetime of 70–90 ps is also obtained in
the case of human hemoglobin along with two lifetimes of ns range
due to three conformers having different tryptophan-heme orientations [18]. So in our case i.e. in the case of BHb, though we can
not assign the exact value of the short lifetime due to the limitation
of the instrument, but we can conclude that the three lifetimes of
BHb arise due to three different tryptophan-heme orientations. In
the present investigation the existence of the third component
(very fast decay of ps order) was apparent from the better statistical values (lower v2 value) obtained by using third exponential decay fittings. The unperturbed values of the lifetimes of BHb even in
presence of ZnO nanoparticles further confirm that the quenching
of fluorescence of BHb in presence of ZnO nanoparticles is of static
in nature.
3.3. Binding constant and number of binding sites
Fig. 2b. Fluorescence emission spectra of BHb (conc. 106 M) in presence of ZnO
nanoparticles at ambient temperature in buffer solution. The concentrations of ZnO
in (0) 0, (1) 2 107 M, (2) 6 107 M, (3) 1 106 M, (4) 1.4 106 M, (5)
2 106 M, (6) 2.9 106 M, (7) 3.9 106 M, (8) 4.8 106 M. Inset: UV–vis
absorption spectra of ZnO nanoparticles (conc. 4.8 106 M).
In order to evaluate the binding constant (Kb) and number of
binding sites (n), the fluorescence intensity data were used and
the following equation [19–23], was applied:
log ½ðF 0 FÞ=F ¼ log K b þ n log ½Q 1.65
308K
298K
291K
F0 /F
1.50
1.35
1.20
1.05
0.0
-6
2.0x10
Q/M
4.0x10 -6
Fig. 3a. S–V plot in case of BHb–ZnO nanomaterial complex at three different
temperatures.
ð2Þ
From the plot of log [(F0 F)/F] vs. log [Q], the values of Kb and n
were obtained from intercept and slope, respectively. The values
of Kb and n were obtained in the case of BHb and ZnO nanoparticle
shown in Table 2. The values of n was found to be approximately
equal to 1 (0.9) at the three temperatures indicating that there
is only one class of binding site of BHb with which ZnO nanoparticles can bind to form the ground state complex at the three temperatures. Again the Kb values are decreased with increasing
temperature indicating that the BHb–ZnO complex would be partly
decomposed with rising the temperature of the system. Though the
above Eq. (2) is theoretically not correct for a general value of n, it is
correct for n = 1 [24]. As we have found that our experimental data
fit Eq. (2) with n nearly equal to 1 therefore our experimental data
can be successfully analyzed on the basis of Eq. (2) with n = 1, which
is theoretically correct. As we get n = 1, therefore Eq. (2) can be re-
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G. Mandal et al. / Chemical Physics Letters 478 (2009) 271–276
Fig. 3b. The fluorescence decay profiles associated with impulse response function (fast decaying curve) of BHb (conc. 106 M) (kex 295 nm, kem 340 nm) in buffer
solution in absence and presence of ZnO nanoparticles (v2 1.12).
Table 2
Determination of binding constants Kb, number of binding sites n and the different thermodynamic parameters of BHb–ZnO complex at different temperatures.
Temperature
(K)
n
log Kb
Kb
(M1)
R2
DG 0
(kcal mol1)
D H0
(kcal mol1) (±0.11)
DS 0
(cal mol1 K1) (±0.24)
291
298
308
0.86
0.88
0.89
4.34
4.25
4.14
2.2 104
1.8 104
1.4 104
0.997
0.999
0.999
5.83
5.79
5.76
4.83
3.24
duced to Eq. (1). This simplification can be possible only in the case
of n = 1.
cates the spontaneity of the binding process of ZnO nanoparticles
with BHb.
3.4. Thermodynamic parameters and the nature of binding forces
3.5. FT-IR studies
To determine the thermodynamic parameters, the binding studies are carried out at three different temperatures 291, 298 and
308 K. The following two thermodynamic equations were used in
this case:
For determining the effect of ZnO nanoparticles on the secondary structure of BHb, FT-IR spectra of BHb and mixture of BHb and
ZnO nanoparticles are measured in aqueous media (Fig. 4a). The
spectra of BHb contain two major bands one at 1650 cm1 and
another at 3405 cm1. The first one arises due to the presence of
C@O stretching vibration of amide group whereas the band at
3405 cm1 corresponds to the stretching vibration of N–H of amide
group. From the Fig. 4a it is apparent that in the presence of ZnO
nanoparticles, the band at 3405 cm1 of BHb becomes broaden
which indicates the possibility of the occurrence of hydrogen
bonding between ZnO nanoparticles and the amide groups present
within the BHb molecule. In hydrogen bonding, there is a significant contribution from electrostatic interaction. May be this is
the reason why we get the binding force between the BHb and
the ZnO nanoparticles is of electrostatic in nature.
ln K b ¼ ðDH0 =RTÞ þ ðDS0 =RÞ
ð3Þ
and
DG0 ¼ DH0 T DS0
ð4Þ
Eqs. (3) and (4) are called van’t Hoff equation and Gibbs–Helmholtz
equation, respectively. DH0 and DS0 were obtained from the slope
and the intercept of the plot of ln Kb vs. 1/T and the values of DG0
at different temperatures were obtained using Eq. (4) as shown in
Table 2. As DH0 is negative and DS0 is positive in this case, it seemingly indicates that the key binding force in this case is electrostatic
interaction [25,26]. The isoelectric point of BHb is 6.8 [27] whereas
that of ZnO nanoparticles is 9.5 [28]. So at physiological pH, BHb is
in negatively charged whereas ZnO is positively charged at this pH.
Therefore, an electrostatic interaction contributes to the binding of
BHb to the ZnO nanoparticles. Again, the negative sign of DG0 indi-
3.6. Investigation on the conformation of BHb
To examine the effect of ZnO nanoparticles on the conformation
of BHb, CD spectra and synchronous fluorescence spectra are
measured.
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G. Mandal et al. / Chemical Physics Letters 478 (2009) 271–276
90
350
0
T%
70
60
1
50
40
1650
0
Relative Intensity
80
300
250
200
7
3405
30
3500
150
2800
2100
-1
Wavenumber (cm )
Fig. 4a. FT-IR spectra of BHb (conc. 1.7 102 M) in absence and presence of ZnO
nanoparticles. Conc. of ZnO nanoparticles in (0) 0, (1) 7.2 103 M.
260
270
280
290
Wavelength / nm
Fig. 5a. The synchronous fluorescence spectra of the BHb (conc. 1 106 M) in
presence of ZnO nanoparticles where Dk 60 nm. The concentration of ZnO
nanoparticles in (0) 0, (1) 2 107 M, (2) 6 107 M, (3) 1 106 M, (4)
2 106 M, (5) 2.9 106 M, (6) 3.9 106 M, (7) 4.8 106 M.
140
210
220
230
240
Wavelength / nm
1.0
0.8
0
-0.5
-1.0
200
0
F / F0
6
0.0
Relative intensity
2
-1
[θ](mdegcm dmol )
0.5
120
0.6
0.0
-6
4.0x10
Q/M
100
80
7
250
6
Fig. 4b. CD spectra of BHb (conc. 1 10 M) in presence of ZnO nanoparticles
(0) 0, (1) 9.9 107 M, (2) 2.9 106 M, (3) 3.9 106 M, (4) 4.8 106 M, (5)
5.7 106 M, (6) 1 105 M.
3.6.1. CD spectra
CD spectroscopy is a sensitive technique for determining structures and monitoring structural changes of biomolecules. The farUV CD spectra give a measure of helical content in the secondary
structure of proteins. The CD spectrum of BHb exhibits intensive
pronounced negative bands at about 208 and 222 nm (Fig. 4b) in
the ultraviolet region, which are characteristics of a-helical content [29]. With the addition of ZnO nanoparticles to BHb, the CD
signals decrease significantly, indicating loss of a-helical content
of the protein. It can be calculated that BHb has a 24% a-helix in
the absence of ZnO while in the presence of maximum conc. of
ZnO (where the molar ratio of ZnO to BHb is 10), it becomes 20%.
However, the CD spectra of BHb in absence and presence of ZnO
nanomaterial are similar in shape, indicating that the structure of
BHb is also predominantly a-helical even in the presence of ZnO
nanoparticles. This is particularly very important in the biomedical
applications as loss of original structure of the protein in drug–
protein interaction may influence its use for such application.
3.6.2. Synchronous fluorescence spectra
Synchronous fluorescence spectroscopy is one of the commonly
used methods to study the conformations of protein which was
introduced by Lloyd [30]. In this experiment, excitation and emission wavelengths are varied simultaneously with a constant offset,
chosen by trial and error, such that the experiment is performed
along the diagonal of the excitation–emission matrix, which passes
through the maximum number of peaks and troughs. The resulting
spectra have a greater number of narrower peaks compared with
that obtained in conventional excitation spectroscopy, allowing
discrimination between fluorophores that have broad and overlap-
60
270
280
290
300
Wavelength / nm
310
Fig. 5b. The synchronous fluorescence spectra of the BHb (conc. 1 106 M) in
presence of ZnO nanoparticles where Dk 15 nm. The concentration of ZnO
nanoparticles in (0) 0, (1) 2 107 M, (2) 6 107 M, (3) 1 106 M, (4)
2 106 M, (5) 2.9 106 M, (6) 3.9 106 M, (7) 4.8 106 M. Inset: the
quenching of synchronous fluorescence of BHb (conc. 1 106 M) in presence
of ZnO nanoparticles. (d) 15 nm, (.) 60 nm.
ping spectra. The synchronous fluorescence spectroscopy provides
information about the molecular environment in a vicinity of the
chromospheres molecules and has several advantages, such as sensitivity, spectral simplification, spectral bandwidth reduction and
avoiding different perturbing effects [31]. Yuan et al. [32] suggested a useful method to study the environment of amino acid
residues by measuring the possible shift in wavelength emission
maximum kmax, the shift in position of emission maximum corresponding to the changes of the polarity around the chromospheres
molecule. When the D-value (Dk) between excitation wavelength
and emission wavelength were stabilized at 15 or 60 nm, the synchronous fluorescence gives the characteristic information of tyrosine residues or tryptophan residues, respectively [33].
It is apparent from Fig. 5a that the emission maxima of tryptophan residue are little red shifted whereas Fig. 5b clearly shows
that the emission maxima of tyrosine residue are not changed in
presence of ZnO nanoparticles. This red shifting may be due to
the fact that the microenvironment of tryptophan residue is changed to a less hydrophobic environment in the presence of ZnO
nanoparticles [34]. From the inset of Fig. 5b it is clear that the slope
is higher when Dk is 60 nm. This indicates that tryptophan residues contribute significantly to the fluorescence of BHb and ZnO
mostly interact with trytophan residues rather than tyrosine
residues.
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G. Mandal et al. / Chemical Physics Letters 478 (2009) 271–276
4. Concluding remarks
The steady state, time-resolved spectroscopic studies on BHb in
presence of ZnO nanomaterials indicate the formation of a ground
state complex via static quenching mechanism. The nature of the
binding force in the formation of the ground state complex is of
electrostatic in nature. Synchronous fluorescence spectra reveal
that the Trp residues environments of BHb are altered by the addition of ZnO nanoparticles whereas the CD spectra indicate that the
content of a-helix of BHb decreases with the addition of ZnO nanoparticles though the structure of BHb remains predominantly ahelical even in the presence of maximum concentration of ZnO
nanoparticles used during the experiment. For biomedical application this is very important as here ZnO cannot change the original
structure of the protein. The present investigation provides important insight into the interaction of the physiologically important
protein BHb with semiconductor ZnO nanoparticles and possesses
potential applications in biotechnology.
Acknowledgments
GM thanks to the Council of Scientific and Industrial Research
(CSIR), New Delhi, India for providing her the NET–CSIR fellowship.
We express our heartiest thanks to Prof. P.K. Das of department of
Biological Chemistry, IACS, for allowing us to measuring the CD
and FT-IR spectra. The authors also thank Mr. Subrata Das of the
department of Spectroscopy for his active help during fluorescence
lifetime measurements.
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