Journal of Luminescence 175 (2016) 135–140

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Journal of Luminescence
journal homepage: www.elsevier.com/locate/jlumin

Full length article

Fluorescence spectral studies on interaction of fluorescent probes

with Bovine Serum Albumin (BSA)
Kaushik Ghosh n, Sweety Rathi, Deepshikha Arora
Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India

art ic l e i nf o a b s t r a c t

Article history: Interaction of 2-(1-(naphthale-1-ylimino)ethyl)phenol (1), 2-methoxy-4-(((4-methoxyphenyl)imino)

Received 18 March 2015 methyl)phenol (2) and 2-methoxy-4-((naphthalene-1-ylimino)methyl)phenol (3) with Bovine Serum
Received in revised form Albumin (BSA) was examined. Fluorescence spectral data were obtained from the probes by varying the
24 December 2015
concentration of BSA as well as from BSA by varying the concentration of probes. Synchronous fluor-
Accepted 20 January 2016
Available online 23 February 2016
escence measurements were performed and binding constants of the probes were calculated. To
understand mode of quenching, Stern–Volmer plot, absorption spectral studies and life time measure-
Keywords: ments were performed. Förster resonance energy transfer (FRET) was also scrutinized.
Fluorescent probes & 2016 Elsevier B.V. All rights reserved.
BSA
Stern–Volmer quenching
Binding constant
Tryptophan
FRET

1. Introduction distributor for several endogenous and exogenous ligands [12,13].

Fluorescence spectral studies on proteins are significantly
important for their biochemical applications in the cells [1,2].
Intrinsic fluorescence of protein is due to three amino acids
namely phenylalanine, tyrosine and tryptophan. Among these
amino acids tryptophan is more dominant as well as more sensi-
tive to alteration in local environment [3]. Serum albumin protein
is one of the most abundant proteins and is the main transport
protein in blood plasma and it also contributes to osmotic pressure
[4]. Although it is well known that there are many different
transport proteins that are present in blood plasma, however the
unique property of albumin is to bind reversibly and transport a
variety of ligands [5] During intravenous administration serum
proteins and DNA are main target molecules for drug or bioactive
molecules. [6] Albumins have the ability to bind with drugs and
other bioactive molecules [7–8]. This prompted significant interest
in the study of interaction of these molecules with albumins.
Bovine Serum Albumin (BSA) is well characterized, abundant and
cost effective serum albumin and is homologous to Human Serum
Albumin (HSA) [9–11]. The above mentioned properties made BSA
a potent tool for protein–drug interaction studies and their specific
and firm binding with small molecules, drugs and dyes can be
used for drug delivery. BSA is also found to be a carrier as well as a
n
Corresponding author. Fax: þ 91 1332 273560.
E-mail address: ghoshfcy@iitr.ac.in (K. Ghosh).
Therefore these types of interactions have been widely studied
[14]. Schiff's base derived compounds have dragged much atten-
tion of the researchers due to excellent chelating tendency
towards metal ions [15–17]. These Schiff's base as well as their
complexes impart very crucial role in antitumor activity [18],
antioxidative activities [19,20] and photophysical properties
[19,21] and utilized as a vital component of drug [22–24]. More-
over, Schiff’s base having phenol in their ligand frame are also
medicinally important. Phenolic compounds also exhibit anti-
oxidative [25], antimutagenic and anticarcinogenic effect [26].
Introduction of hydroxyl group to the molecule increases its
solubility in water along with the interaction with biomolecules
[27]. Metal coordinated phenols are effective in generating phe-
noxyl radical and exhibit importance in enzymes like galactose
oxidase and glyoxal oxidase [28]. Investigation of literature clearly
shows that there are Schiff’s bases as well as compounds having
phenolato function interacting with serum proteins [26,27,29–32].
However, to the best of our knowledge, there are very few reports
of Schiff's base having phenolato function and BSA interaction
studies [33,34]. This prompted us to design a Schiff's base ligand
having phenolato function and to study the interaction of BSA with
such molecules. Recently, we have reported the biological appli-
cation of metal complexes derived from the ligand having phenolic
as well as imine moiety [35]. In vivo binding of serum albumin
with the small organic molecule and most of the drugs is well
known. The drug protein interaction affects the biological activity

http://dx.doi.org/10.1016/j.jlumin.2016.01.029
0022-2313/& 2016 Elsevier B.V. All rights reserved.
136 K. Ghosh et al. / Journal of Luminescence 175 (2016) 135–140
Fig. 1. Structural representation of fluorescent probes 1, 2 and 3 respectively used
in present study.
of the drug. Hence it is important to study the interaction and
behavior of these molecules towards BSA as these molecules could
be utilized in pharmacological application. These studies could
provide the valuable information on the structural feature that
determines the efficiency of drugs. Utilization of sensitive and easy
to handle fluorometric techniques for the interaction of fluor-
escent molecules with BSA is an interesting area of research.
Recently, we have reported Fe(III) sensing via turn-on fluorescence
by 2-(1-(naphthale-1-ylimino)ethyl)phenol (1) and 2-methoxy-4-
((naphthalene-1-ylimino)methyl)phenol (3) [36]. The structural
representation of fluorescent probes is shown in Fig. 1. These
fluorescent probes exhibited remarkable selectivity and specificity
among the served metal ions. These results prompted us to exploit
these fluorescent probes for the interaction studies with BSA. In
this present study we have investigated the interaction of 1, 2 and
3 with BSA using absorption and fluorescence spectral studies.
2. Experimental section
2.1. Materials
Fluorescent probes 2-(1-(naphthale-1-ylimino)ethyl)phenol (1),
2-methoxy-4-(((4-methoxyphenyl)imino)methyl)phenol (2) [36]
and 2-methoxy-4-((naphthalene-1-ylimino)methyl)phenol (3) [37]
were synthesized and purified as reported earlier. All the char-
acterizations were performed to establish the proper synthesis and
characterization of molecules. The purity of probes was justified
using TLC plate. Bovine serum albumin (BSA) was purchased from
Sigma Aldrich, Steinheim, Germany and used without further pur-
ification. Tris buffer was prepared in deionised water. All the sol-
vents used were HPLC grade and purified by standard procedure
before use.
2.2. Instrumentation
The absorption and emission spectra were recorded on a
Schimadzu UV–vis UV-2450 spectrophotometer and Schimadzu
RF-5301PC spectrofluorometer respectively. The synchronous
fluorescence experiments were performed on Horiba Scientific
FluoroMax-4. During study, the sample concentration was main-
tained in micromolar range in order to avoid aggregation and
reabsorption effects [38]. All experiments were performed using
freshly prepared solutions at 298 K.
2.3. Procedures
Stock solutions of fluorescent probes were prepared in DMF
due to insolubility in buffer (100 mM phosphate buffer, pH 7.4).
The electronic absorption spectra of fluorescent probes in phos-
phate buffer (pH 7.4) have been given in Fig. S1. All the studies
were performed using freshly prepared solutions of BSA as well as
fluorescent probes at ambient temperature. During the study total
concentration of DMF was taken up to 5%. All the experiments
were performed in triplicates.
2.3.1. Titration of fluorescent probes against BSA
Fluorescence studies were performed at fixed concentration of
fluorescent probes 1, 2 (50 μM) and 3 (25 μM) in 100 mM phos-
phate buffer, pH 7.4. Concentration of BSA was varied as follows: 1
(0–1 μM), 2 (0–4 μM) and 3 (0–17 μM). The freshly prepared
stocks were used for the studies and the solutions were incubated
for 1 h. The excitation wavelength was used 285 nm.
2.3.2. Titration of BSA against fluorescent probes
To understand the interactions between fluorescent probes and
BSA, titrations were carried out at fixed concentrations of BSA.
During these studies the concentration of BSA (17 μM) was kept
constant for all the three fluorescent probes while the con-
centration of fluorescent probes was varied from 0–0.104 mM (1),
0–0.004 mM (2) and 0–0.012 mM (3). Freshly prepared stocks
were used for studies and the solutions were incubated for 1 h.
Spectrum was obtained at an excitation wavelength of 280 nm.
2.3.3. Synchronous fluorescence measurements
During synchronous fluorescence studies a fixed concentration
of BSA (17 μM) was taken with the variation of concentration of
fluorescent probes. The fluorescence spectra were collected at
Δλ ¼15 nm and Δλ ¼60 nm.
2.3.4. Absorption studies
Electronic spectral studies were also carried out at a fixed
concentration of BSA (17 μM) and the concentrations of fluor-
escent probes were changed from 0-8 μM at 298 K. The scans were
recorded in the range of 200–800 nm.
2.3.5. Time-resolved measurements
Time-resolved fluorescence studies were carried out in absence
as well as the presence of the probes in BSA. A laser excitation
source of 295 nm was used, and the decay kinetics response was
observed at 340 nm. Three exponential decay curves were
observed for all the probes with and without BSA. Samples were
prepared in mixed solvent media (5% DMF in 100 mM phosphate
buffer, pH 7.4).
3. Results and discussion
3.1. Emission spectral studies
3.1.1. Titration of fluorescent probes against BSA
The effect of interaction with BSA on the fluorescence emission
spectra of probes can be evaluated using increasing addition of
BSA. During the present study, a ratiometric fluorescence detec-
tion of BSA was observed with the increasing addition of BSA to a
fixed concentration of probe. With increase in concentration of
BSA to probe 1 and 2 a similar behavior was observed. (Fig. 2
(probe 1), Figs. S2–S3 (probe 2–3)). Enhancement in fluorescence
emission intensity was observed with increase in the concentra-
tion of BSA along with the generation of new band at lower
wavelength (λmax  340 nm). Solvent water acts as a quencher in
the charge transfer (CT) state due to the interactions such as
intermolecular hydrogen bonding [39–42]. In absence of BSA,
fluorescent probes 1 and 2 are more exposed to polar aqueous
media which is responsible for quenching as stated above. With
the increased concentration of BSA, probe molecules are less
exposed to the polar environment of water when present inside
the hydrophobic cavity of BSA and thus resulting in deactivation of
non-radiative decay [43–46]. The blue-shift in the present study
can be attributed to the confinement of the probe in the hydro-
phobic interior of the protein backbone compared to that in aqu-
eous buffer medium [41] (Fig. S4). BSA contains two tryptophan
 K. Ghosh et al. / Journal of Luminescence 175 (2016) 135–140
Fig. 2. Changes observed in emission spectra of probe 1 with increasing concentration
of BSA in phosphate buffer pH 7.4 (λexc ¼ 285 nm).
residue Trp-213 and Trp-134. Among these two tryptophan resi-
due, Trp-213 is present in the cleft or in a hydrophobic fold [47–51]
while Trp-134 is present on surface [49,51,52]. The experimental
finding suggested that probes are mainly interacting with the
tryptophan residue present in hydrophobic cavity (Trp-213) of the
protein.
Further Benesi–Hildebrand Eq. (1) is utilized for better under-
standing of binding stoichiometry of fluorescent probes with BSA.
1 1 1 were performed using fixed concentration of BSA with the addition
¼ þ ð1Þ
ðI I 0 Þ ðI 1  I 0 Þ ðI 1 I 0 ÞK ½BSA
where I0, I and I1 are the emission intensities in absence, at
intermediate and infinite concentration respectively. A plot of
1/[I  I0] vs. 1/[BSA] gives a straight line indicating the formation of
a 1:1 complex between probe and BSA as shown in Figs. S5–S7.
Contrary to this, addition of BSA to the fluorescent probe 3, a
decrease in the emission intensity was observed around 450 nm
[53] while a new band appeared at 340 nm as shown in Fig. S3 and
Benesi–Hildebrand plot is deposited in Fig. S7.
3.1.2. Titration of BSA against fluorescent probes
Upon increasing the concentration of fluorescent probe the
effect on emission intensity of BSA at 340 nm was observed. With
the addition of probe the decrease in emission intensity as well as
a hypsochromic shift was observed in all the probes as evident by
the spectra in Fig. 3 (probe 1), Figs. S8–S9 (probe 2–3). In probe 1
and 3 along with the decrease in emission intensity a new band
generation was observed. The decrease in emission intensity in all
the probes is probably due to the interaction of probes with
tryptophan residue or unfolding of protein in the region of sub-
domain IIA where tryptophan is placed (Figs. S10–S12) [54,55]. The
appearance of a clear isoemissive point at higher wavelength is
possibly due to the complex formation between the probe and BSA
[56]. On successive addition of probe a concentration is reached
where uncomplexed form in solution becomes dominant. The
band generated is having maximum emission wavelength as that
of free fluorescent probes (Fig. S13). These findings suggest that no
structural modification of probes is seen during the titration
against probe.
3.1.3. Synchronous fluorescence
Synchronous fluorescence spectroscopy is used to distinguish
overlapped excitation peaks of aromatic residue in typical fluores-
cence spectra. To observe the change in the conformation of BSA in
presence of fluorescent probe we have performed synchronous
fluorescence experiments. In synchronous fluorescence experiments
137
Fig. 3. Changes observed in emission spectra of BSA with increasing concentration
of probe 1 in phosphate buffer (pH 7.4, λexc ¼ 280 nm).
change in polarity around chromophore microenvironment induces
decrease and/or shifts in emission intensity. A blue-shift in maximum
emission intensity indicates that amino acid residues are in hydro-
phobic environment [3]. On the other hand a red-shift is due to
exposure of amino acid residue into polar solvent environment. As
Δλ ¼15 nm, synchronous fluorescence offers characteristics of tyr-
osine residues, while when Δλ ¼60 nm; it provides the characteristic
information of tryptophan residues. The synchronous experiments
of concentration of fluorescent probes 1 (Fig. 4A and B), 2 (Fig. S14)
and 3 (Fig. S15). During studies it was found that decrease in emis-
sion intensity was more in Δλ ¼ 60 nm rather than in Δλ ¼ 15 nm
(Figs. S16–S21) [57]. These results designate that probes binds mainly
to the tryptophan site which causes the changes in its micro-
environment around tryptophan residue.
3.1.4. Quenching mechanism
Intrinsic fluorescence of BSA is quenched with the addition of
fluorescent probes. Quenching process may be classified into two
processes as static quenching (ground state complex formation) and
dynamic quenching (collisional) [3]. During dynamic quenching
process the quencher diffuses to the fluorophore in excited state
and returns to the ground state without emission of a photon.
In static quenching a nonfluorescent complex formation occurs
between fluorophore and quencher [3]. Mode of quenching can be
determined using Stern–Volmer equation, absorption spectral stu-
dies and time-resolved measurements. The Stern–Volmer Eq. (2)
used to investigate extent of quenching is as follows
I0
¼ 1 þ K q τ0 ½Q  ¼ 1 þ K SV ½Q  ð2Þ
I
K SV
Kq ¼ ð3Þ
τ0
where I and I0 are the steady state fluorescence intensities in pre-
sence and absence of quencher respectively. Ksv is Stern–Volmer
quenching constant and [Q] is concentration of quencher. τ0 is the
average life time of the protein without the quencher. At high
concentration of probes the Plot I0/I vs [probe] was not found to be
linear. This indicates a more complex quenching process. Hence
Stern–Volmer quenching constant was calculated by the slope of
regression curves in the range of linearity. Value of KSV and Kq for 1,
2 and 3 were calculated using Eqs. (2)–(3) and taking average life
time of molecular fluorescence τ0 10  8 s [56,57]. The results have
been deposited in Table 1. Fig. 5 (Probe 1), Figs. S22–S23 (Probe
2–3) depicts Stern–Volmer plots of fluorescent probe 1, 2 and 3
138 K. Ghosh et al. / Journal of Luminescence 175 (2016) 135–140

Fig. 4. Effect on synchronous fluorescence spectra of BSA with the addition of fluorescent probe 1 (A) Δλ ¼60 nm and (B) Δλ ¼15 nm.

Table 1
The Stern–Volmer constant for the fluorescent probe-BSA.

Fluorescent probe Ksv Kq

1
1 4
5.78  10 M 5.78  1012 M  1 s  1
2 1.02  102 M  1 1.02  1010 M  1 s  1
3 5.44  105 M  1 5.44  1013 M  1 s  1

Fig. 6. Fluorescence decay profile of BSA in absence and presence of 1, 2 and 3 in

mixed-solvent media (5% DMF in 100 mM phosphate buffer, pH 7.4).

Table 2
Effect of addition of probes on fluorescence lifetimes (ns) of BSA (λexc ¼ 295 nm,
λem ¼ 340 nm).

Probe τ1(ns) τ2 (ns) τ3 (ns) α1 α2 α3 τavg (ns) χ2

BSA 2.36 0.25 6.54 16.19 2.65 81.16 5.7 1.18

1 2.53 1.08 6.17 32.95 19.23 47.82 3.9 1.13
2 2.83 1.08 6.32 23.36 10.59 66.05 4.9 1.10
Fig. 5. Stern–Volmer plot of fluorescent probe 1 at λexc ¼ 280 nm.
3 2.25 0.52 6.24 22.77 3.97 73.27 5.1 1.13

respectively. Collisional quenching constant of various kinds of

quenchers with biopolymer is 2.0  1010 L mol  1 s  1 [58,59]. The
results clearly indicate that the bimolecular quenching constants
are larger than the limiting diffusion rate constant of the biomole-
cule in case of probe 1, 2 and 3. This ruled out the possibility of
process as purely dynamic quenching.
For further confirmation of static quenching of fluorescent
probes, absorption spectral studies were performed. The increase
in the concentration of quencher in presence of a fixed con-
centration of BSA was optimized with the help of electronic
spectral studies as shown in Fig. S24 (A–C). When there is an
increase in the concentration of quencher changes were observed
in the absorption band of BSA along with the generation of a new
band. The changes observed during variation of quencher also
support a ground state complex formation during absorption
spectral studies. This also indicated that the static quenching is
involved. Collisional quenching affects only the excited state of
molecule hence no change in the absorption spectra. Time-
resolved measurements were also performed to further confirm
the static nature of quenching (Fig. 6). Small change in life-time on
addition of probe to BSA supports the static nature of quenching.
The data has been deposited in Table 2. The Stern–Volmer plot,
changes in absorption spectra and time-resolved measurements
clearly indicate the probably predominantly static nature of
quenching with mixed quenching in nature [60].
3.1.5. Binding constants and the number of binding sites
During static quenching behavior relation between the fluor-
escence emission intensity and concentration of quenching can be
described as in Eq. (4) [61]


log ðF 0  F Þ=F ¼ logK b þ nlog½Q  ð4Þ
where Kb denotes degree of interaction of protein with probes and
n is the number of binding sites. The value of Kb and number of
 K. Ghosh et al. / Journal of Luminescence 175 (2016) 135–140
binding sites n have been described in the Table 3. The data has
been shown in Fig. 7(A–C).
Values of K and binding sites (n) have been derived from the
plots of log (F0 F)/F vs. log [Q]. The high value of binding constant
shows a strong interaction between BSA and fluorescent probe and
value of n depicts that there is a binding site available on BSA for
the binding of probe.
3.1.6. FRET studies
Förster resonance energy transfer (FRET) is used as a powerful
tool for measuring the distance between donor fluorophore and
acceptor fluorophore in vitro and in vivo [38]. FRET is an electro-
dynamic interaction between the electronic excited states of donor
and acceptor molecules in which excitation is transferred from a
donor molecule to an acceptor molecule. The FRET efficiency is
dependent on following parameters [3]: (i) the distance between
donor and acceptor must be in the range of 2–8 nm; (ii) A sig-
nificant overlap between donor fluorescence and acceptor
Table 3
The binding parameters for the fluorescent probe-BSA.
Fluorescent probe Kb
1 5
1.0  10 M 1
2 7.9  1010 M  1
3 1.3  108 M  1
Fig. 7. Double-log plots of fluorescent probe (A) 1 (B) 2 and (C) 3 on BSA in phosphate buffer (pH 7.4).
139
absorption bands; (iii) there must be a proper orientation of the
transition dipole of the donor and acceptor. Implying Förster’s
theory a distance between the donor and acceptor fluorophore can
be calculated [62]. The critical energy transfer distance (R0) cal-
culated by using the following Eq. (5):
h  i 
R60 ¼ 0:2108  K 2 η  4 ΦD J λ Å ð5Þ
K2 is the spatial orientation factor and value is 2/3 [63]; η ¼1.33
is the refractive index of the medium, ΦD ¼0.118 is the fluores-
cence quantum yield of the donor and J is degree of spectral
overlap between the donor emission and the acceptor absorption.
Value of J has been calculated using following Eq. (6):
P  
F λ ɛðλÞλ Δλ
4
JðλÞ ¼ P ð6Þ
FðλÞΔλ
where F(λ) is the fluorescence intensity of the fluorescent donor at
wavelength λ and ε(λ) is the molar absorption coefficient of the
acceptor at wavelength λ. Spectral overlap between the fluores-
cence emission spectrum of BSA and the UV–vis absorbance
spectrum of probes are shown in Fig. S25(A–C) and data have been
deposited in Table S1. These data suggest the effective energy
n transfer between the probe and BSA. FRET studies indicate that
1.0
donor and acceptor are in the range of critical distance and energy
1.3 will transfer from BSA (donor) to fluorescent probe (acceptor) [64].
1.4 We have also compared our data with the other reported
molecules, drugs and dyes interacting with the BSA as shown in
140 K. Ghosh et al. / Journal of Luminescence 175 (2016) 135–140

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