Binding interactions and FRET between bovine serum albumin and various phenothiazine-/anthracene-based dyes: A structure-property relationship

Article abstract of DOI:10.1039/d0ra09580j

The present study demonstrates binding interactions and F?rster resonance energy transfer (FRET) between bovine serum albumin (BSA) and a series of structurally and electronically diverse phenothiazine (PTZ) and anthracene (ANT) dyes. Upon selective excitation of tryptophan (Trp) residues of BSA, radiationless energy transfer to a dye takes place, resulting in fluorescence quenching of the former. Fluorescence quenching mechanisms, FRET parameters, possible locations, and binding constants of dyes with the BSA have been examined to deduce a structure-property relationship. The mechanism of quenching is apparently static in nature. PTZ dyes with heteroatoms and a pentyl tail (C5-PTZ) attached to them were found to have a stronger binding affinity with BSA as compared to ANT dyes. Stronger binding affinities of C5-PTZ dyes with BSA result in greater energy transfer efficiencies (ET). A dye with a strong electron-withdrawing group present in it has shown better energy accepting capability. A FRET study with dicyanoaniline (DCA) analogs of PTZ and ANT dyes (C5-PTZDCA and ANTDCA, respectively) revealed that ET depends on electronic and structural factors of molecules. An almost orthogonal geometry between ANT and DCA moieties (～79°) in ANTDCA induces the greater extent of electron transfer from ANT to DCA, showing a higher ET for this dye as compared to C5-PTZDCA in which the torsion angle is only ～38°. Further, the observed facts have been validated by experimentally determined bandgaps (using cyclic voltammetry experiments) for all the dyes. Thus, the hydrophobic character and the presence of interactive substituents along with the electron-accepting abilities majorly control the FRET for such dyes with BSA.

Full text of DOI:10.1039/d0ra09580j

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Binding interactions and FRET between bovine
serum albumin and various phenothiazine-/
anthracene-based dyes: a structure–property
relationship†
Cite this: RSC Adv., 2021, 11, 1679
*
*
Shouvik Bhuin,‡ Sayantan Halder,‡ Subit Kumar Saha
and Manab Chakravarty
The present study demonstrates binding interactions and Forster resonance energy transfer (FRET) between
¨
bovine serum albumin (BSA) and a series of structurally and electronically diverse phenothiazine (PTZ) and
anthracene (ANT) dyes. Upon selective excitation of tryptophan (Trp) residues of BSA, radiationless energy
transfer to a dye takes place, resulting in fluorescence quenching of the former. Fluorescence quenching
mechanisms, FRET parameters, possible locations, and binding constants of dyes with the BSA have been
examined to deduce a structure–property relationship. The mechanism of quenching is apparently static
in nature. PTZ dyes with heteroatoms and a pentyl tail (C5-PTZ) attached to them were found to have
a stronger binding affinity with BSA as compared to ANT dyes. Stronger binding affinities of C5-PTZ dyes
with BSA result in greater energy transfer efficiencies (ET). A dye with a strong electron-withdrawing
group present in it has shown better energy accepting capability. A FRET study with dicyanoaniline (DCA)
analogs of PTZ and ANT dyes (C5-PTZDCA and ANTDCA, respectively) revealed that ET depends on
electronic and structural factors of molecules. An almost orthogonal geometry between ANT and DCA
moieties (ꢀ79ꢁ) in ANTDCA induces the greater extent of electron transfer from ANT to DCA, showing
a higher ET for this dye as compared to C5-PTZDCA in which the torsion angle is only ꢀ38ꢁ. Further, the
observed facts have been validated by experimentally determined bandgaps (using cyclic voltammetry
experiments) for all the dyes. Thus, the hydrophobic character and the presence of interactive
substituents along with the electron-accepting abilities majorly control the FRET for such dyes with BSA.
Received 11th November 2020
Accepted 15th December 2020
DOI: 10.1039/d0ra09580j
rsc.li/rsc-advances
originated selectively from Trp residues of BSA. These Trp
residues act as donors in the energy transfer process with an
1. Introduction
acceptor, and this process can be radiative or non-radiative.
Serum albumin has been identied as the most abundant
protein in plasma. Many researchers remain focused on
studying the interactions of bioactive compounds with plasma
proteins, mainly serum albumin.1 Upon binding with the serum
albumin, the biological activities may be reduced or enhanced
and can provide a transportation path. Bovine Serum Albumin
(BSA) is a well-studied protein due to its structural homology
with human serum albumin and high abundance. BSA has
three structurally distinct domains, I, II, and III, with two sub-
domains (A and B) in each of the areas.2 Out of two Trp residues
in BSA, Trp-134 is located on the surface of domain I, and Trp-
213 is situated in the hydrophobic pocket of domain II. Upon
excitation at ꢀ295 nm, the intrinsic uorescence can be
¨
Forster or uorescence resonance energy transfer (FRET) is
a radiationless process without emission and reabsorption of
photons. Energy exchange takes place between oscillating
dipoles of donor and acceptor of similar resonating frequen-
cies.3 Since the discovery of the FRET, researchers have been
actively utilizing this potential non-radiative tool to unveil
numerous facts related to the eld of supramolecular materials
and biomedical research and development.4,5 There is a rapid
growth in the invention of donor–acceptor combinations for the
FRET applications. However, constructing an appropriate
donor–acceptor pair for the FRET is still a challenge because it
needs to meet primary inuencing criteria such as distance,
spectral overlap, and dipole orientation between donors and
acceptors.6 There are numerous applications where donors and/
or acceptors in FRET are used as uorescence site markers.7,8 In
the present study, a series of phenothiazine and anthracenyl
molecules (Chart 1) are examined as acceptors for the energy
transfer from the donors i.e., Trp residues of BSA. To our
knowledge, both these structural motifs are not tested for FRET
Department of Chemistry, Birla Institute of Technology and Sciences-Pilani,
Hyderabad Campuses, Hyderabad-500078, Telangana, India. E-mail: manab@
hyderabad.bits-pilani.ac.in; sksaha@hyderabad.bits-pilani.ac.in
† Electronic supplementary information (ESI) available. CCDC 2041739 and
2041742. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/d0ra09580j
‡ Shouvik Bhuin and Sayantan Haldar have Contributed equally.
© 2021 The Author(s). Published by the Royal Society of Chemistry
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Chart 1 Phenothiazine and anthracenyl molecules that are studied herein for the FRET with BSA.
1
studies with BSA before. There are a few reports on complicated and H and 13C NMR data (see ESI; pages (3–6) and Fig. (S11–
phenothiazine-linked molecules for metal ion sensing through S16)†). PTZ, ANT, ANTA, and BSA were procured from Alfa-Aesar
FRET.9,10 As benzothiazole type systems are oen found to be and Sigma Aldrich. They were taken into use as received. BSA
suitable for FRET,11,12 the electron-rich phenothiazine core is of protein concentration was kept constant at 5 mM for all
our particular interest.
measurements. HEPES buffer was bought from SRL, India.
Additionally, the questions arise on the requirement of thia H2SO4 and NaOH were procured from SDFCL, India, and SRL,
and aza part in a system for an efficient FRET phenomenon. India, respectively. Quinine sulfate was obtained from Sigma
Therefore, we also intend to explore the FRET on anthracenyl- Aldrich. Spectral grade tetrabutylammonium perchlorate
based uorophores. Notably, anthracene and phenothiazine (TBAP) and MeCN were purchased from TCI, India.
are not analogous due to the fundamental structural differ-
ences; however, we dealt with similar substituents for both the
2.2. Methods
skeletons. Apart from a few known dyes, the dicyanoaniline
A 10 mM solution of HEPES buffer was prepared with Milli-Q
(DCA) analogs [PTZDCA and ANTDCA, Chart 1] with acceptor–
water, and its pH was adjusted to 7.4. H2SO4 and NaOH solu-
tion was used to adjust the pH of the buffer solution. BSA stock
solution was prepared with a freshly prepared buffer solution.
Stock solutions of the different dyes (phenothiazine and
donor–acceptor (A–D–A) architecture bring special attention to
this FRET study.13,14 Furthermore, the FRET analysis using non-
traditional donor–acceptor combinations is very much
appealing.6 Most of these traditional compounds emit at UV-Vis
anthracene series of compounds) were prepared (ꢀ0.5 mM
region but with somewhat commendable Stokes shis
each) in pure methanol for recording the UV-vis absorption and
compared to the previous report.6 Intriguingly, the FRET
uorescence spectra. All stock solutions were carefully degassed
with a pure N2 gas for 15 minutes before use. For the FRET study
between BSA and each individual dye, at a xed concentration
of BSA (5 mM), the concentration of dye was gradually increased
between phenothiazine or anthracene-based molecules and BSA
has enabled them to offer as good acceptors. Such acceptors are
focused on their convincing applications in the FRET. Mecha-
nistic insights are specied to nd quenching of Trp-
in the range of 0 mM ꢂ15 mM. The total volume of each solution
uorescence by each of the dyes. An attempt has been made
was kept at 2 mL. A JASCO (model V-650) UV-vis spectropho-
tometer with a quartz cell of path length 1 cm with a Teon
stopper was used to record the absorption spectra. A FluoroLog-
TM (Horiba Scientic) spectrouorimeter was used for steady-
state uorescence measurements using a quartz cell of path
length 1 cm with a Teon stopper. For uorescence measure-
ments, all the samples were excited at 295 nm. Slit widths for
both excitation and emission were 2.5 nm, that along with the
to nd the possible relation between the structures of dyes and
various properties, such as binding strength/locations and
energy transfer efficiency. The governing effect of substituted
hydrophobic and electron-withdrawing groups, the position of
substitution on the features mentioned above, has also been
elucidated.
scan rates, were maintained constant for all uorescence
measurements. All the uorescence spectra were corrected for
instrument sensitivity and also to avoid any inner lter effect.
2. Experimental section
2.1. Materials
The known phenothiazine and anthracene compounds like C5- Electrochemical assessments were conducted using an Autolab
PTZ, C5-PTZA, and ANTCN were synthesized using reported PGSTAT128N potentiostat. Cyclic Voltammetry (CV) of the
procedures.15,16 C5-PTZCN, C5-PTZDCA, and ANTDCA are the samples were performed by switching the applied voltage in
compounds that were newly synthesized. Their structures were ꢂ3 V to +3 V; using a cleaned glassy carbon (GC) electrode as
veried by FT-IR, LCMS (Liquid Chromatography Mass a working electrode (WE). The Pt-wire and Ag/AgCl were used as
Spectrometry)/HRMS (High-Resolution Mass Spectrometry), a counter electrode (CE) and a reference electrode (RE),
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respectively, and a 0.1 M tetrabutylammonium perchlorate donor–acceptor systems, DCA was installed with the C5PTZ core
(TBAP) in acetonitrile was used as an electrolyte in a typical to afford C5PTZDCA as an acceptor to elucidate the electronic
three-electrode system. A sample solution in acetonitrile was factor in the FRET process. For the comparative study with
mixed with electrolyte to study the electrochemical behavior of a similar tricyclic electron-rich system, anthracene is coupled
samples. The redox reaction that happened on the surface of with the DCA motif to afford ANTDCA. Thus, we investigate the
GC, were recorded in NOVA 2.1.1. soware. HOMO/LUMO scope of changes in FRET with the same trend of functionalities
energy levels were determined from the onset of oxidation/ in these two different scaffolds.
reduction potentials in the fullness of positive/negative scans.
A Jasco-J 1500 CD spectropolarimeter was utilized for recording
the far – UV circular dichroism (CD) spectra of appropriate
systems in the wavelength range of 190–260 nm. A cuvette
having path length as 0.1 cm was used. Each CD spectrum was
recorded at a scan speed of 50 nm minꢂ1 with a spectral
bandwidth of 2.5 nm. The spectra obtained for different BSA
systems were subtracted by buffer spectra for background
correction. All measurements were performed at 298.15 ꢃ 1 K
temperature.
3.1. Synthesis of the compounds
C5-PTZDCA compound was synthesized newly via a one-pot
reaction of aldehyde and malononitrile in the presence of trie-
thylamine (TEA) as depicted in Scheme 1. In the rst step, the
requirement of the base was not mandatory just because of the
highly active methylene group. The dicyano compound, C5-
PTZCN was not isolated separately herein. Instead, the addition
of excess malononitrile and TEA directly produced penta-
substituted benzene, C5-PTZDCA, in 63% yield. Thus, this
strategy is somewhat different than the existing one,18 hitherto
reported.
3. Results and discussions
The compound ANTDCA was synthesized in a manner
An electron-rich and inexpensive phenothiazine core is picked similar to C5-PTZDCA with a 25% yield, possibly due to the
up for the FRET-study with BSA, based on the earlier reports on various reaction scopes with anthracenyl core. A solvent-free
related thiazole as a well-established motif for FRET applica- twisted path19 was also adopted to afford ANTDCA, but the
tions.9,17 However, the presence of pyridyl nitrogen (electron- result was not pleasing.
withdrawing sp2-hydridized) in the thiazole type molecules
All the synthesized compounds are characterized by NMR,
displays the primary difference between phenothiazine and HRMS/LCMS, and FT-IR spectroscopy (see ESI† for details).
benzothiazole cores. To investigate beyond the conventional Mostly, the molecular structure for new compounds C5-
Scheme 1 Synthesis of C5-PTZDCA.
o
ꢀ
Fig. 1 The molecular structures of (a) C5-PTZDCA and (b) ANTDCA. The selected torsion angles ( ) and bond lengths (A) are also given. There are
two molecules in one asymmetric unit for both the systems. Hydrogens are omitted for the C5-PTZDCA for clarity. CCDC Number: ANTDCA
2041742; C5-PTZDCA: 2041739.
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and C5-PTZA, respectively. Still, the emission from C5-PTZA is
unexpected using lex ¼ 295 nm. Albeit, a signicant extent of
overlap between the uorescence band of BSA and absorption
band of C5-PTZA (Fig. 2) along with the uorescence from both
the species indicate a denite possibility of FRET occurring
between them. Among various intrinsic uorophores as part of
the origin of BSA's uorescence, we have focused explicitly on to
excite only Trp residues to avoid the uorescence contribution
from the other amino acid residues of BSA such as phenylala-
nine (Phe) and tyrosine (Tyr). That is why the excitation wave-
length of 295 nm has been selectively preferred.20 Further,
between two Trp residues in BSA, Trp-134 resides in the
hydrophilic environment, whereas Trp-213 chooses a relatively
less polar part of BSA.21 Intriguingly, such FRET behavior was
experienced with all the dyes used herein except molecular
anthracene (ANT).
Fig. 2 Normalized absorption and fluorescence spectra of 5 mM BSA in
the presence of 5 mM C5-PTZA in an aqueous HEPES buffer solution.
lex for fluorescence measurements is 295 nm.
To understand the origin of uorescence quenching for Trp
residues of BSA, the radiative rate constant (kr) of this emission
process is a vital parameter and calculated using Strickler–Berg
equation (eqn (1)) considering no solvent interactions and
PTZDCA and ANTDCA are determined unequivocally with the
help of single-crystal X-ray diffraction studies (Fig. 1). The X-ray
structures help to identify the exact position of cyano, amine,
and methyl groups within the dicyanoaniline (DCA) ring.
Moreover, the torsion angle between the DCA and anthracene or
phenothiazine could also be measured as depicted in Fig. 1.
change in the excited-state geometry:22–24
ð
2
2
kr ¼ 3:13 ꢄ 10ꢂ9y0 3dy x y0 f
(1)
where ꢁy0 represents the wavenumber when the absorption of the
BSA is maximum, 3 is the extinction coefficient, and f denotes
the oscillator strength. The absorption spectra of BSA in the
absence and presence of each molecule (excluding ANT) as
quenchers were recorded to calculate kr and f values in each case
and tabulated in Table 1.
Notably, the change in radiative rate constants of the BSA in
the presence or absence of a dye is not impressive. The changes
in the oscillator strength fall within the range of 0.01–4.61%,
which has an almost negligible contribution to the observed
quenching (29.0–66.9%, Table 1). Therefore, the uorescence
quenching of BSA by the molecules conrms the involvement of
the energy transfer process to a greater extent.
3.2. Systematic absorption and uorescence measurements
to study FRET
In this investigation, the FRET behavior between BSA and all the
organic dyes has been demonstrated by carrying out systematic
absorption and uorescence measurements. The detailed on
FRET are discussed in the ESI.† Before we deal in a sequence, let
us demonstrate the representative plot of FRET with one of the
above-listed compounds C5-PTZA (Fig. 2) to elucidate the
process. The normalized absorption and uorescence spectra of
5 mM BSA solution were recorded in the presence of an extrinsic
uorophore C5-PTZA (5 mM) in an aqueous HEPES buffer
solution. The peak maximum of the more extended wavelength
absorption band of BSA is located at 279 nm, while the
absorption peak maximum of C5-PTZA appeared at 373 nm.
Upon excitation of the sample (a mixture of BSA and C5-
PTZA) with a light of 295 nm, two uorescence bands at 347 nm
and 502 nm were found due to the emissions from both BSA
Next, the uorescence quenching of Trp residues in BSA was
studied at different concentrations for each of the dyes, indi-
vidually except for ANT. This study has enabled us to explain the
mechanism of quenching, binding efficiency with the protein,
and location in the protein. Fluorescence quenching using
Table 1 The radiative rate constant (kr), % change in oscillator strength (f), and % quenching of fluorescence from Trp residues of BSA
%
%
Quencher (dye)
kr ꢄ 10ꢂ10 (without dye) (sꢂ1
)
kr ꢄ 10ꢂ10 (with dye) (sꢂ1
)
change in f
quenching
PTZ
C5-PTZ
1.45
1.45
1.45
1.45
1.45
1.45
1.45
1.45
1.45
1.49
1.51
1.46
1.49
1.42
1.42
1.41
0.01
3.81
4.61
0.11
3.87
2.93
2.26
3.30
31.7
34.9
66.9
49.6
29.0
34.9
30.7
35.8
C5-PTZA
C5-PTZCN
C5-PTZDCA
ANTA
ANTCN
ANTDCA
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quencher is represented by the Stern–Volmer (S–V) equation,35
(eqn (2)):
Fo
¼ 1 þ KSV½Qꢅ ¼ 1 þ kqso½Qꢅ
(2)
F
where Fo and F are the uorescence intensities of Trp residues
of BSA in the absence and presence of a quencher, respectively;
KSV, kq, and so represent S–V quenching constant, bimolecular
quenching rate constant, and the average lifetime of the uo-
rophore in absence of a quencher, respectively. The uores-
cence quenching of Trp residues is analyzed using the S–V plot,
as displayed in Fig. 4a. The observed downward curvature of the
S–V plot indicates the fractional accessibilities of two different
Trp residues of BSA to PTZ.35 The downward curvature in
protein uorescence quenching generally occurs in the pres-
ence of a polar or charged quencher.35 Being polar/charged, the
quencher cannot penetrate the hydrophobic interior of the
protein. Here, the parent compound PTZ thus shows differing
accessibility to the Trp residues. There is no signicant change
in the peak maxima of BSA with an increase in the concentra-
tion of PTZ. The S–V plot has been modied (Fig. 4b) based on
eqn S(1) in Note S1 in ESI† to nd the fraction of Trp residues
accessible to PTZ (fa in eqn S(2)).†35
Fig. 3 Fluorescence spectra of Trp residues of BSA in the presence of
different concentrations of PTZ. [BSA] ¼ 5 mM. lex ¼ 295 nm.
these molecules and analysis of quenching data in each case
has been attempted to decipher the observed facts.
3.2.1 FRET between BSA and phenothiazinyl compounds.
As intended, the series of molecules with phenothiazine (PTZ)
core (Chart 1; Fig. 1a) were initially studied. Upon incremental
addition of PTZ at a xed concentration of BSA (5 mM), there was
a diminution in the uorescence intensity of BSA, which can be
ascribed to the energy transfer from Trp residues to PTZ.
However, no increase in the uorescence intensity of PTZ
could be found, being a non-uorescent dye (Fig. 3). The above-
mentioned calculated values (Table 1) of the radiative rate
constants (kr)/oscillator strength (f) and the analysis of data
have conrmed the energy transfer as a cause for the uores-
cence quenching. The FRET efficiency depends on donor–
acceptor proximity and spectral overlap, not necessarily that the
acceptor should be uorescent.25 There are recent reports on the
use of several non-uorescent acceptors/quenchers, e.g.,
Dabcyl,26–28 the black hole quenchers, BHQ,28–30 QSY-7, and
BHQ2,28 and QSY-9,31 different nucleotides,28 Guanosine,32,33
and Au nanoparticles.27,34 The development of non-uorescent
dye derivatives-based FRET assays is a promising area that
can lead to broad applications in the receptor eld.25 A rela-
tionship between uorescence quenching and concentration of
The correlation coefficient of the linear tting of the modi-
ed S–V plot (Fig. 4b) is found to be 0.9997. The calculated fa
and Ka values from the intercept and the slope of the modied
S–V plot are found to be 0.43 and 1.92 ꢄ 105 Mꢂ1, respectively.
So, 43% of Trp residues in BSA are only accessible to PTZ. The
estimated S–V quenching constant of an accessible fraction (Ka
¼ kqso) is 1.92 ꢄ 105 Mꢂ1. Considering the so for BSA: ꢀ10ꢂ8
s
(ref. 35–37) the bimolecular quenching rate constant, kq appears
as 1.92 ꢄ 1013 Mꢂ1 sꢂ1. However, the maximum value of kq for
a diffusion-controlled quenching process with a biopolymer is
ꢀ2.0 ꢄ 1010 Mꢂ1 sꢂ1.3 It implies that the mechanism of the
quenching of available fraction is static. By assuming the same
and independent binding sites for BSA in case of a static
quenching, the binding constant K0 and the number of binding
sites (n) have been calculated based on eqn S(3) in Note S2.†38
Fig. S1† displays the plot of log[(Fo ꢂ F)/F] versus log(1/([PTZ]
– (Fo ꢂ F)[BSA]/Fo)). The number of binding sites, n, and the
binding constant, K0calculated from the slope and intercept,
Fig. 4 (a) S–V plot for quenching of the Trp fluorescence by PTZ in BSA, and (b) modified S–V plot for quenching the Trp fluorescence by PTZ in
BSA. [BSA] ¼ 5 mM. lex ¼ 295 nm.
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Fig. 5 (a) Fluorescence spectra of Trp residues of BSA with increasing concentration of C5 PTZ, and (b) S–V plot for quenching of the Trp
fluorescence by C5-PTZ. [BSA] ¼ 5 mM. lex ¼ 295 nm.
respectively of the linear tting [R2 ¼ 0.9997] of the data are higher binding constant of C5-PTZ than that for PTZ species
found to be 0.35 and 6.19 ꢄ 103 Mꢂ1, respectively.
the binding of the former at the more hydrophobic site of the
Next, we dealt with the C5-PTZ, possibly more hydrophobic, BSA. Thus, the presence of a hydrocarbon tail enables the dye
as compared to PTZ due to the introduction of the pentyl chain. binding to a comparatively more hydrophobic site.39
Notably, C5-PTZ is also non-uorescent like PTZ. The parame-
The next dye, C5-PTZA, with aldehyde functionality (Chart 1),
ters listed in Table 1 support the energy transfer as a reason for is found to be highly uorescent, unlike PTZ and C5-PTZ. Upon
uorescence quenching of Trp residues in the presence of C5- excitation at 295 nm, the uorescence from both Trp residues of
PTZ as a quencher. Fluorescence spectra of Trp residues of BSA BSA and C5-PTZA have been recorded at different concentra-
at different concentrations of C5-PTZ are shown by Fig. 5a, and tions of the latter, and the corresponding uorescence spectra
Fig. 5b displays the change in uorescence intensity with with an isoemissive point at 445 nm are shown by Fig. 6a.
increasing concentration of C5-PTZ based on eqn (2). The
Fig. 6a illustrates a gradual decrease in the uorescence
observed linear S–V plot (R2 ¼ 0.9995) indicates both the Trp intensity of the Trp residues (donor) of BSA with a concomitant
residues are equally accessible to the dye, C5-PTZ. Unlike PTZ, increase in the uorescence intensity of C5-PTZA (as an
C5-PTZ with a pentyl tail in the molecule can penetrate the acceptor) through the FRET process with increasing concen-
protein and bind to a comparatively interior site, and that tration of C5-PTZA. This trend is well displayed in Fig. 6b, which
facilitates equal accessibility of both the Trp residues.
shows the rise and fall in F/Fo of the acceptor and the donor,
The S–V constant, KSV (¼ kqso) calculated from the linear respectively. Detailed scrutiny revealed that the peak maximum
tting of the quenching data (Fig. 5b) is found to be 3.35 ꢄ 104 of Trp uorescence is blue-shied from 347 nm to 344 nm upon
Mꢂ1. Taking so ¼ 10ꢂ8 s, the kq is calculated to be 3.35 ꢄ 1012 the increasing concentration of C5-PTZA within the range of
Mꢂ1 sꢂ1. As the kq value lies beyond the diffusion-controlled 0 mM to 15 mM. There could be two possible reasons for it. First,
limit, thus it is a static quenching. The binding constant (K0) the shi of peak maximum to a shorter wavelength with
and the number of binding sites, n calculated aer linear tting increasing C5-PTZA concentration could be due to the fact that
[Fig. S2,† R2 ¼ 0.9997] of the quenching data based on eqn S(3),† the Trp uorescence at a longer wavelength is getting quenched
are found to be 2.46 ꢄ 104 Mꢂ1 and 0.73, respectively. The more readily than that at a shorter wavelength.35 Trp-134 being
Fig. 6 (a) Fluorescence spectra of Trp residues of BSA and C5-PTZA at varying concentrations of C5-PTZA, and (b) decrement in fluorescence
intensity of donor (Trp in BSA) with concomitant enhancement in fluorescence intensity of acceptor (C5-PTZA) with increasing concentration of
C5-PTZA in the FRET process. [BSA] ¼ 5 mM. lex ¼ 295 nm.
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Fig. 7 (a) S–V plot for quenching of the Trp fluorescence by C5-PTZA in BSA, and (b) modified S–V plot for quenching of the Trp fluorescence by
C5-PTZA in BSA. [BSA] ¼ 5 mM. lex ¼ 295 nm.
present in a comparatively hydrophilic environment should (Fig. S4,† R2 ¼ 0.9999) of the data based on eqn S(3)† are found
have uorescence at a relatively longer wavelength as compared to be 1.30 ꢄ 105 Mꢂ1 and 1.06, respectively. The higher value of
to Trp-213. Thus as compared to Trp-213, Trp-134 is more binding constant for C5-PTZA as compared to C5-PTZ infers
accessible to C5-PTZA. Secondly, it could also be due to the that –CHO functionality in C5-PTZA induces added binding
changes in secondary structure of BSA in presence of a dye. To interactions with the protein.
verify it, the circular dichroism spectra for BSA in presence of
Subsequently, another dye, C5-PTZCN (Chart 1), has been
different concentrations of C5-PTZA have been recorded and used with much higher electron-accepting capability due to the
given as Fig S3.† The % a-helix of the native BSA is found to be presence of di-cyano functionality linked through a p-bond.
changed from 62.7% to 55.4%.
Fig. 8a shows changes in uorescence spectra of both donor and
The S–V plot, in this case, exhibits an upward curvature, acceptor parts with increasing concentration of C5-PTZCN with
concave towards the y-axis at higher concentrations of the an isoemissive point at ꢀ447 nm. Like the previous dye, C5-
quencher (C5-PTZA) (Fig. 7a). This trend can be well described PTZA, a gradual diminution in the uorescence intensity of the
by using concurrent static and dynamic quenching mecha- Trp residues of BSA with a simultaneous rise in the uorescence
nisms.35 The modied S–V equation for a quenching process intensity of C5-PTZCN are presented in Fig. 8b.
through both static and dynamic quenching can be written as
The blue shi in the uorescence peak maximum of BSA is
eqn S(4) in Note S3.† which can be further modied through due to the same reason, as discussed in the case of C5-PTZA.
eqn S(5) and S(6).†
The S–V plot for this quenching of uorescence presents
Fig. 7b presents the modied S–V plot (R2 ¼ 0.9999) based on a downward curvature (Fig. 9a). The modied S–V plot based on
eqn S(6).† Then, by solving the quadratic equation, we get the eqn S(1)† is shown in Fig. 9b (R2 ¼ 0.9999).
values of the S–V dynamic quenching constant (KD) as 5.96 ꢄ
The fractional accessibility of the dye to the Trp residues of
104 Mꢂ1 and the S–V static quenching constant (KS) as 4.02 ꢄ BSA indicated by the downward curvature in the S–V plot leads
104 Mꢂ1. Assuming that eqn S(3)† is applicable in this case as us to calculate fa and Ka from the intercept and the slope of the
static quenching is in place, the binding constant (K0) and the plot in Fig. 9b based on eqn S(1)†. The fa and Ka values are
number of binding sites (n) calculated from the linear tting estimated to be 0.87 and 2.06 ꢄ 105 Mꢂ1, respectively. So, 87%
Fig. 8 (a) Fluorescence spectra of Trp residues of BSA and C5-PTZCN with increasing concentration of the dye, and (b) decrement in fluo-
rescence intensity of donor (Trp residues in BSA) with concomitant enhancement in fluorescence intensity of acceptor (C5-PTZCN) with
increasing concentration of C5-PTZCN in the FRET process. [BSA] ¼ 5 mM. lex ¼ 295 nm.
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Fig. 9 (a) S–V plot for quenching of Trp fluorescence by C5-PTZCN in BSA, and (b) modified S–V plot for quenching of the Trp fluorescence by
C5-PTZCN in BSA. [BSA] ¼ 5 mM. lex ¼ 295 nm.
of Trp residues of BSA are accessible to the C5-PTZCN. The based on eqn S(1)† was scrutinized (R2 ¼ 0.9965) to nd the
bimolecular quenching constant, kq ¼ 2.06 ꢄ 105 Mꢂ1/10ꢂ8 s ¼ fractional accessibility (fa), as well as the S–V quenching
2.06 ꢄ 1013 Mꢂ1 sꢂ1, which is beyond the diffusion-controlled constant (Ka) those, are found out to be 0.85 and 3.59 ꢄ 104
limit. Thus, the mechanism of quenching appears to be static. Mꢂ1. So, 85% of Trp residues of BSA are accessible to the C5-
The binding constant (K0) and the number of binding sites (n) PTZDCA. The quenching rate constant, kq is found to be 3.59 ꢄ
calculated from the linear tting (Fig. S5,† R2 ¼ 0.9999) of the 1012 Mꢂ1 sꢂ1, reecting the static quenching mechanism. The
quenching data based on eqn S(3)† are found to be 1.21 ꢄ 105 respective values of binding constant (K0) and a number of
Mꢂ1 and 0.63, respectively. Of note, the binding of C5-PTZCN is binding sites (n) calculated from the linear tting of the data in
almost the same as that of C5-PTZA.
Fig. S6† (R2 ¼ 0.9999) based on eqn S(5)† are calculated to be
Besides, the new dye studied in this series is C5-PTZDCA 2.81 ꢄ 105 Mꢂ1 and 0.97.
(Chart 1), dicyanoaniline (DCA) moiety attached to the C5-PTZ
3.2.2 FRET between BSA and anthracenyl compounds.
core. Fig. 10a shows the uorescence spectra of donor–acceptor Similar studies, as discussed above, have also been carried out
pair with increasing concentration of the acceptor, C5-PTZDCA, with an anthracenyl series of molecules given in Chart 1. At the
with an isoemissive point at ꢀ403 nm. The dye, C5-PTZDCA, very beginning, we took a representative example of FRET using
with a strong acceptor–donor–acceptor unit along with a methyl the ANTA molecule. Electron-rich molecular anthracene, ANT,
group, is found to be highly uorescent. The gradual decrease does not act as an acceptor in the FRET process with BSA as
in the uorescence intensity of the Trp residues of BSA with a donor. However, each of its derivatives in the series, such as
a simultaneous rise in the uorescence intensity of C5-PTZDCA ANTA, ANTCN, and ANTDCA with signicant electron-
is represented by Fig. 10b. The blue-shi in the emission peak withdrawing substituents, has shown the FRET phenomenon
maximum of BSA indicating fractional accessibility of Trp as an acceptor. In each case, the energy transfer is responsible
residues and/or changes in secondary structure of BSA is noted for the uorescence quenching of Trp residues of BSA, as sup-
in this case as well.
ported by data given in Table 1.
The S–V plot for this system also displayed a slight down-
Fluorescence quenching of Trp residues by ANTA (a weak
ward curvature (Fig. 11a). The modied S–V plot (Fig. 11b) uorescent molecule) through the FRET process has been
Fig. 10 (a) Fluorescence spectra of Trp residues of BSA and C5-PTZDCA with increasing concentration of the dye, and (b) dropping in fluo-
rescence intensity of donor (Trp in BSA) with concomitant enhancement in fluorescence intensity of acceptor (C5-PTZDCA) with increasing
concentration of C5-PTZDCA in the FRET process. [BSA] ¼ 5 mM. lex ¼ 295 nm.
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Fig. 11 (a) S–V plot for quenching of the Trp fluorescence by C5-PTZDCA in BSA, and (b) modified S–V plot for quenching of the Trp fluo-
rescence by C5-PTZDCA in BSA. [BSA] ¼ 5 mM. lex ¼ 295 nm.
Fig. 12 (a) Fluorescence spectra of Trp residues of BSA and ANTA with increasing concentration of ANTA, and (b) decrement in fluorescence
intensity of donor (Trp residues in BSA) with concomitant enhancement in fluorescence intensity of acceptor (ANTA) with increasing
concentration of ANTA in the FRET process. [BSA] ¼ 5 mM. lex ¼ 295 nm.
shown in Fig. 12a. On the gradual treatment of ANTA, the applicable for concurrent static and dynamic quenching
uorescence intensity for Trp residues of BSA is decreased with mechanism, the quenching data were plotted. Still, we failed to
an increase in uorescence intensity of ANTA, showing an iso- t the data to the said equation. It led us to follow the sphere-of-
emissive point at ꢀ462 nm (Fig. 12a).
action model as an alternate mechanism of the static part of
An upward curvature noted in the S–V plot (Fig. 13a) indi- quenching. The modied S–V equation for the sphere-of-action
cates either a combined dynamic and static mechanism of model is written as eqn S(7) in Note S4.†35 Further modication
quenching or static quenching with the sphere-of-action (Per- done has been shown through eqn S(8) to S(10) and has been
rin) model. Based on a modied S–V equation (eqn (S6)†) discussed in Note S4.†40
Fig. 13 (a) S–V plot for quenching of Trp fluorescence of BSA by ANTA, (b) plot of (1ꢂ (F/Fo))/[ANTA] versus F/Fo, and (c) modified S–V plot for
quenching of the Trp fluorescence of BSA by ANTA. [BSA] ¼ 5 mM. lex ¼ 295 nm.
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Fig. 14 (a) Fluorescence spectra of Trp residues of BSA and ANTCN with increasing concentration of ANTCN, and (b) S–V plot for quenching of
Trp fluorescence of BSA by ANTCN. [BSA] ¼ 5 mM, lex ¼ 295 nm.
Fig. 15 (a) Fluorescence spectra of Trp residues in BSA with increasing concentration of ANTDCA., and (b) lowering in fluorescence intensity of
donor (Trp in BSA) with concomitant enhancement in fluorescence intensity of acceptor (ANTDCA) with increasing concentration of ANTDCA in
the FRET process. [BSA] ¼ 5 mM. lex ¼ 295 nm.
Based on eqn S(10),† the values of KD and V have been quenching data have been plotted based on the actual S–V
calculated from the slope and intercept, respectively by linear equation for the sphere-of-action model (eqn S(7)†) and shown
tting (R2 ¼ 0.9988) of the data in the plot of (1 ꢂ (F/Fo))/[ANTA] by Fig. 13c. The S–V dynamic quenching constant was calcu-
versus F/Fo shown by Fig. 13b. The values of KD and V are found lated from the slope of the linear tting (R2 ¼ 0.9996) of the data
to be 1.79 ꢄ 104 Mꢂ1 and 1.27 ꢄ 10ꢂ17 liters, respectively. The in the plot of Fo/F(eVN [ANTA]/1000) versus [ANTA] based on eqn
AV
radius of the sphere of action is calculated to be 144 nm. Aer S(7).† The KD value is found to be 1.94 ꢄ 104 Mꢂ1, which is
calculating the approximate value of V with an assumption of different from that calculated from the plot in Fig. 13b because
the low value of the term, VNAV[ANTA]/1000 in eqn S(8),† the of the assumption involved in the derivation of corresponding
Fig. 16 (a) S–V plot for quenching of the Trp fluorescence in BSA by ANTDCA, and (b) modified S–V based on eqn S(3).† [BSA] ¼ 5 mM, lex
¼
295 nm.
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eqn S(10).† Assuming that the fraction of molecules presents
within the sphere of action at the time of excitation is non-
uorescent and undergoing static uorescence quenching,
eqn S(3)† has been used to calculate the binding constant (K0)
and the number of binding sites (n) upon linear tting of the
data in Fig. S7† (R2 ¼ 0.9999). The values of K0 and n are found to
be 3.95 ꢄ 104 Mꢂ1 and 1.31.
Next, ANTCN with conjugated dicyano substituents is also
non-uorescent. The uorescence from Trp residues of BSA in
the presence of different concentrations of ANTCN is shown in
Fig. 14a. The radiative rate constant (kr) and oscillator strength
(f) of the donor in the absence and presence of ANTCN and %
quenching data given in Table 1 support the energy transfers as
a cause for the quenching of Trp uorescence. It has been dis-
cussed above that energy transfer is possible even if the acceptor
is non-uorescent. A linear S–V plot (R2 ¼ 0.9995) for the Trp
uorescence quenching is obtained and displayed in Fig. 14b.
The value of S–V quenching constant, KSV calculated from the
slope of the plot is found to be 3.13 ꢄ 104 Mꢂ1, with a corre-
sponding uorescence quenching rate constant (kq) value as
3.13 ꢄ 1012 Mꢂ1 sꢂ1. A kq value beyond the diffusion-controlled
limit denotes the static quenching mechanism. The binding
constant (K0) of ANTCN with BSA and the number of binding
sites (n) have been calculated from the linear tting (R2
¼
0.9999) of the data in Fig. S8 based on eqn S(3)† and the values
are found to be 3.15 ꢄ 104 Mꢂ1 and 1.10, respectively. Thus, the
binding constant of ANTCN is very close to that of ANTA, as
noticed in the case of C5-PTZ analogs.
With our interest in studying a new electron-rich donor–
acceptor–donor (D–A–D) molecular system, ANTDCA molecule
is considered here for the FRET with BSA. There is a consider-
able amount of FRET with an isoemissive point at ꢀ409 nm
(Fig. 15a). The blue-shi in the emission spectrum of Trp resi-
dues of BSA is also observed as discussed above. A trend of
decrease in the uorescence intensity of Trp residues of BSA
and an increase of the uorescence intensity of the dye,
ANTDCA, are represented by Fig. 15b.
Based on the quenching of the BSA–Trp uorescence by
ANTDCA, the S–V plot is identied with slight downward
curvature (Fig. 16a). Therefore, quenching data have plotted
based on modied S–V eqn S(1)† and are shown by Fig. 16b (R2
¼ 0.9997). The values of fractional accessibility (fa) and the S–V
quenching constant (Ka) are calculated to be 0.25 and 5.85 ꢄ 105
Mꢂ1, respectively. So, 25% of Trp residues in BSA are only
accessible to the quencher, ANTDCA. The quenching rate
constant (kq) is found to be 5.85 ꢄ 1013 Mꢂ1 sꢂ1, indicating
a static quenching mechanism. The binding constant (K0) and
a number of binding sites (n) are found to be 2.38 ꢄ 104 Mꢂ1
and 0.63, respectively [calculated from the linear tting
(Fig. S9,† R2 ¼ 0.9998) of the data based on eqn S(3)].†
¨
3.2.3 FRET parameters. The Forster's distance (Ro), the
efficiency of energy transfer (ET) and the distance (r) between an
acceptor quencher dye and donor Trp residues of BSA have been
calculated to create a quantitative analysis of FRET between Trp
residues of BSA and different dyes.36 Details of calculation of
these parameter along with the calculation of the overlap inte-
gral, J(l) have been given in ESI as Note S5.† It is pertinent to
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note that the presence of the two donor moieties needs the
assumption of an average distance between donors and accep-
tors.3 Also, even though the donor is covalently attached to the
protein, but the acceptor dyes are not. However, the high
binding constant of each dye with the BSA reduces the possi-
bility of any high uncertainty allowing us to estimate the average
distance between a donor and an acceptor. It is noteworthy that
all FRET calculations have been done with a ratio of concen-
tration of a dye to BSA of 1 : 1 taking 5 mM concentration of
each. With this concentration of each of the donors and the
acceptors, the intensity ratios, [Intacceptor/Intdonor] are found to
vary from 0.05 to 0.72. Lowest intensity ratio is found in case of
ANTA. However, its relative quantum yield is calculated to be
0.08 which is not very low so that it could contribute a signi-
cant error in calculations. The intensity ratios mentioned above
are increased with increasing concentration of a dye further
reducing the chances of errors in the quenching data.
The overlap between the corrected uorescence spectrum of
Trp residues of BSA as donor and absorption spectrum of C5-
PTZA as acceptor has been shown as a representative one in
Fig. 2. Similar overlapping has also been noted in cases of all
other dyes for which overlap integral values have been calculated.
The calculated FRET parameters are given in a tabular form
in Table 2. The Ro values measured are in the range of 24.2–29.4
ꢀ
ꢀ
A, which corroborates well with the range (20–90 A) for FRET to
occur.3 Thus, the data reect that dyes are binding close to the
Trp residues of BSA. In addition, the band gaps for all dyes have
been calculated based on the data obtained from CV experi-
ments to compare the ET values of the dyes in phenothiazine
and anthracenyl series (see Fig. S10† for details). To further
support the band gaps data, the absorption peak maxima
(lmabasx) of all dyes have been determined. The band gaps and
lmabasx values along with lm ax values are also tabulated in Table 2.
In order to have an overall discussion on the quenching
mechanism, energy transfer processes with different dyes as an
acceptor, and binding affinities of dyes with the BSA, the
quenching data are compiled in Table 3. Few FRET parameters
and band gap data tabulated in Table 2 have been used. It can
be seen that the quenching mechanism is apparently static. In
phenothiazine series, combined static and dynamic quenching
has been noted only in the case of C5-PTZA. Surprisingly,
a combined quenching mechanism has been shown only by
ANTA among all dyes in anthracenyl series following a sphere of
action model of the static part of quenching in this case. Thus,
–CHO functionality has some connection with the combined
quenching mechanism, possibly because of the interaction
between the –CHO group and some active functional group of
amino acids of BSA, such as the –NH functionality of Trp resi-
dues. The binding affinities of dyes in phenothiazine series are
stronger than the corresponding dyes in anthracenyl series
could be due to the presence of pentyl tail as well as hetero
atoms inducing a greater extent of hydrophobic as well as
hydrogen bonding and coulombic attractive interactions in the
former than that in the latter. A higher binding constant value
of C5-PTZ as compared to PTZ is due to the presence of a pentyl
tail that enhances the hydrophobicity in the former. Among all
dyes, including phenothiazine and anthracenyl series, the
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energy transfer efficiency, ET is found to be maximum in the also noticed the expected trend [C5-PTZ < PTZ < C5-PTZDCA <
case of C5-PTZCN (ET ¼ 0.45), proving itself as the most efficient C5-PTZA < C5-PTZCN] in the increase of lmabasx of pure dyes. The
ꢀ
FRET acceptor with a distance of 28.8 A from the donor, which increase in ET with decreasing the band gap except for C5-
is minimum among all other donor–acceptor distances. The PTZDCA can be described with the molecular structural factor,
next dye which shows ET closest to C5-PTZCN is C5-PTZA, with dominating over the electronic factor.
an ET of 0.35. The binding constants for C5-PTZCN and C5-PTZA
As far as anthracenyl series are concerned, their binding
with BSA are found to be very much comparable with the value affinities with BSA are lower than that for dyes in phenothiazine
of 1.21 ꢄ 105 and 1.30 ꢄ 105 Mꢂ1, respectively. Different series as described above. Due to the absence of pentyl tail in
substituents present in these two dyes must be responsible for anthracenyl series of dyes, they probably bind to the compara-
the difference in ET. The presence of more potent electron- tively less hydrophobic/interior sites of the protein. That is why
withdrawing functionalities (two –CN groups) for C5-PTZCN the average distances between the donor and each of dyes,
ꢀ
ꢀ
facilitates the compound to be a more efficient acceptor in ANTA (35.2 A) and ANTCN (36.4 A) are longer than the corre-
ꢀ
comparison to C5-PTZA that carries one electron-withdrawing sponding above-mentioned analogs, C5-PTZA (32.6 A) and C5-
ꢀ
–CHO group. The presence of an electron-withdrawing cyano- PTZCN (28.8 A), with eventually smaller ET values (Table 2).
ester group in a molecule makes it an efficient acceptor, While the respective ET for C5-PTZA and C5-PTZCN are 0.35 and
which has been reported by Vijayakumar et al.41 In phenothia- 0.45, the ET for ANTA and ANTCN are only 0.10 and 0.11,
zine series, the lowest ET is observed for C5-PTZDCA (ET ¼ 0.12) respectively. Now, if we compare between the other two new
though it has got the highest binding constant with BSA (K0 ¼ dyes, C5-PTZDCA and ANTDCA, the fact that the binding
2.81 ꢄ 105 Mꢂ1). A high value of the binding constant of this dye constant of ANTDCA is almost one-tenth of that of C5-PTZDCA,
and high accessibility (85%) of Trp residues to this dye imply the former one binds at the comparatively less hydrophobic
that the dye binds to the interior of the protein, perhaps with region, and as a result, its fractional accessibility to the Trp
a drive of additional hydrophobic interaction due to the pres- residues is lesser than that of the latter one. However, inter-
ence of a benzene ring and hydrogen bonding and coulombic estingly the ET of ANTDCA is higher than that of C5-PTZDCA,
interactions with the BSA as a result of the presence of different and it can be deciphered by the difference in electronic and
functional groups such as –NH2 and –CN. However, the lowest structural factors between these two systems. Anthracene is
ET for this dye can be attributed to the attachment of electron- a at polyaromatic hydrocarbon without any heteroatoms,
donating groups such as amine and methyl that can signi- whereas phenothiazine is a middle ring folded electron-rich
cantly enrich the electron density to the molecule and hamper core with two specic electron donor atoms such as nitrogen
the accepting ability of cyanobenzene.42 Also, the reason due to and sulfur. In our recent research, phenothiazine appears to be
the presence of acceptor moiety, cyano benzene at a distant more electron-rich compared to anthracene.43 From the
position as compared to other dyes cannot be ruled out. This is molecular structure (Fig. 1), the torsion angle between ANT ring
further supported by the longest r (¼35.6 A, Table 2) in this case. and DCA ring is measured to be ꢀ79ꢁ, whereas the torsion angle
ꢀ
It indicates that the phenothiazine ring with pentyl tail is between the C5-PTZ ring and DCA ring is only ꢀ38ꢁ. Thus,
aligned towards the protein's hydrophobic interior, and the a greater extent of charge transfer in ANTDCA might be favor-
substituted benzene ring is protruded towards the outer able for it to be a better acceptor than C5-PTZDCA resulting in
hydrophilic site. Moreover, the molecular structure depicts that a higher ET in FRET.44 Moreover, as the DCA is attached to the
the dicyanoaniline part is tilted with ꢀ38ꢁ torsion angle with C5 carbon of ANTDCA rather than the C2 carbon of C5-PTZDCA,
folded (ꢀ41ꢁ folded middle ring) phenothiazine core (Fig. 1). On the former one remains closer to the donor in BSA (r ¼ 30.9) as
the other hand, C5-PTZCN without any such DCA ring present compared to the latter (r ¼ 35.6). The increasing order of ET of
in the structure shows maximum ET, and almost the same dyes in anthracenyl series is found to be as follows: ANTA <
accessibility (87%) as that is found for C5-PTZDCA that offers ANTCN < ANTDCA (Table 2), which is correlated agreeably with
ꢀ
the shortest average distance (28.8 A) from the Trp residues of the decreasing order of band gaps (Table 2) i.e. ANTA > ANTCN >
max
abs
BSA. The lack of electron-withdrawing substituents for PTZ and ANTDCA while the increasing order of l
of pure dyes is
C5-PTZ compounds can attribute to lower ET as compared to C5- established as (Table 2): ANTA < ANTCN < ANTDCA. In contrast
PTZA and C5-PTZCN. Quenching data also dictate that the to C5-PTZDCA, ANTDCA shows the anticipated energy transfer
binding constants for PTZ and C5-PTZ are comparatively lower efficiency from the order of band gap values in anthracenyl
than the other three dyes in this series. So, the functional series. It depicts that the electronic factor for ANTDCA plays an
groups present in the other three dyes feasibly undergo some important role.
kind of binding interactions with the protein. To check whether
From the results of fractional accessibilities to the four dyes,
the ET has any relation with the band gap of a dye, the band gap PTZ, C5-PTZCN, C5-PTZDCA, and ANTDCA, it can be seen that
values of all dyes have been calculated (Table 2) from CV the % accessibilities are high and almost same for C5-PTZCN
measurements (Fig. S10† displays CV diagrams for pure dyes). and C5-PTZDCA, 87% and 85%, respectively. Thus these two
The increasing ET order for the dyes of phenothiazine series dyes bind to a site from where both the Trp residues are quite
(Table 2) is found with the following trend: C5-PTZDCA < C5- accessible. The binding sites must be in the hydrophobic
PTZ < PTZ < C5-PTZA < C5-PTZCN. However, the decreasing region; otherwise, Trp-213 would not be accessible. For the
order of band gap is as follows (Table 2): C5-PTZ > PTZ > C5- other two dyes, PTZ and ANTDCA fractional accessibilities are
PTZDCA > C5-PTZA > C5-PTZCN. With decreasing band gap, we only 43% and 25%, respectively. Therefore, they bind to
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hydrophilic sites. Thus, hydrophobic substitution on dyes campus, for helping in the CV analysis. CAL lab facilities
makes them more accessible to the Trp residues present in available at BITS-Pilani Hyderabad is acknowledged.
a hydrophobic pocket. Earlier, Karukstis et al.44 also noted the
higher fraction of accessibility of Chlorophyll uorescence by
hydrophobic substitution of naphthoquinones. Linear S–V plot
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the acceptor as supported by the data in Table 1 and accordingly
the FRET parameters have been estimated. However, as
a strategy, the variable mechanisms of quenching in presence of
different dyes as quenchers as noted aer a detailed analysis of
the quenching data have been described.
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8 L. Wu, C. Huang, B. P. Emery, A. C. Sedgwick, S. D. Bull,
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A few easily accessible, structurally and electronically diverse
anthracene and phenothiazine dyes are explored for binding-
studies with BSA. FRET between Trp residues of BSA and the
dyes, mechanism of uorescence quenching, and their possible
locations are examined to deduce a structure–property rela-
tionship. The binding efficiency of phenothiazine dyes with
a pentyl tail is stronger than that of anthracenyl dyes due to the
presence of a hydrocarbon tail and also hetero atoms in the
former inducing hydrophobic as well as hydrogen bonding and
coulombic attractive interactions. Energy transfer between Trp
residues of BSA as a donor and each dye as an acceptor is found
to be responsible for uorescence quenching. Energy transfer
has been noted even in cases of non-uorescent acceptors, PTZ,
C5-PTZ, and ANTCN. The mechanism of quenching is static.
Concurrent static and dynamic mechanisms have been distin-
guished only in cases of C5-PTZA and ANTA with a sphere of
action model of the static part of quenching for the latter dye.
Dyes with a hydrophobic entity (pentyl tail herein) present in
their structures bind with the hydrophobic sites of protein,
showing better energy acceptors. Although the binding constant
is more signicant for C5-PTZDCA than ANTDCA, the favored
energy transfer for ANTDCA could be due to its relatively higher
electron-transfer capabilities within the molecules and lower
acceptor–donor distances. The band gap values of all the dyes
are also very much supportive of the observed behavior. Thus,
the hydrophobic character and the presence of interactive
substituents along with the electron-accepting abilities majorly
control the FRET for such acceptor molecules with BSA.
¨
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Conflicts of interest
The authors declare no conict of interest.
Acknowledgements
˘
14 E. Yalçin, Y. C. Kutlu, V. Korkmaz, E. ¸Sahin and Z. Seferoglu,
SB and MC thank CSIR 02(-0289)/17/EMR-II for the nancial
assistance. The authors thank Susmita Roy, PhD Research
Scholar, Department of Chemistry, BITS Pilani, Hyderabad
2,6-Dicyanoaniline based donor–acceptor compounds: the
facile synthesis of uorescent 3,5-diaryl/hetaryl-2,6-
dicyanoanilines, Arkivoc, 2015, 2015, 202–218.
1692 | RSC Adv., 2021, 11, 1679–1693
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