Characterization of domain-specific interaction of synthesized dye with serum proteins by spectroscopic and docking approaches along with determination of in vitro cytotoxicity and antiviral activity

Article abstract of DOI:10.1080/07391102.2017.1400468

The interaction between a synthesized dye with proteins, bovine, and human serum albumin (BSA, HSA, respectively) under physiological conditions has been characterized in detail, by means of steady-state and time-resolved fluorescence, UV–vis absorption, and circular dichroism (CD) techniques. An extensive time-resolved fluorescence spectroscopic characterization of the quenching process has been undertaken in conjugation with temperature-dependent fluorescence quenching studies to divulge the actual quenching mechanism. From the thermodynamic observations, it is clear that the binding process is a spontaneous molecular interaction, in which van der Waals and hydrogen bonding interactions play the major roles. The UV–vis absorption and CD results confirm that the dye can induce conformational and micro-environmental changes of both the proteins. In addition, the dye binding provokes the functionality of the native proteins in terms of esterase-like activity. The average binding distance (r) between proteins and dye has been calculated using FRET. Cytotoxicity and antiviral effects of the dye have been found using Vero cell and HSV-1F virus by performing MTT assay. The AutoDock-based docking simulation reveals the probable binding location of dye within the sub-domain IIA of HSA and IB of BSA.

Full text of DOI:10.1080/07391102.2017.1400468

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Characterization of domain specific Interaction
of Synthesized Dye with Serum Proteins by
Spectroscopic and Docking Approaches along with
Determination of Invitro Cytotoxicity and Antiviral
Activity
Suparna Rudra, Somnath Dasmandal, Chiranjit Patra, Biman Kumar Patel,
Suvendu Paul & Ambikesh Mahapatra
To cite this article: Suparna Rudra, Somnath Dasmandal, Chiranjit Patra, Biman Kumar Patel,
Suvendu Paul & Ambikesh Mahapatra (2017): Characterization of domain specific Interaction
of Synthesized Dye with Serum Proteins by Spectroscopic and Docking Approaches along with
Determination of Invitro Cytotoxicity and Antiviral Activity, Journal of Biomolecular Structure and
Dynamics, DOI: 10.1080/07391102.2017.1400468
To link to this article: http://dx.doi.org/10.1080/07391102.2017.1400468
Accepted author version posted online: 01
Nov 2017.
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Date: 02 November 2017, At: 04:15

1
Publisher: Taylor & Francis
Journal: Journal of Biomolecular Structure and Dynamics
DOI: http://doi.org/10.1080/07391102.2017.1400468
Characterization of domain specific Interaction of Synthesized Dye with
Serum Proteins by Spectroscopic and Docking Approaches along with
Determination of Invitro Cytotoxicity and Antiviral Activity
Suparna Rudraa, Somnath Dasmandala, Chiranjit Patraa, Biman Kumar Patela, Suvendu Paulb and
Ambikesh Mahapatraa,*
aDepartment of Chemistry, Jadavpur University, Kolkata 700 032, India
bDepartment of Chemistry, Kalyani University, Kalyani 741235, India
*Corresponding author, Tel.: +91 33 2457 2770 (Office); +91 33 2432 4586 (Residence);
Facsimile: +91 33 2414 6223
e-mail addresses: ambikeshju@gmail.com & amahapatra@chemistry.jdvu,ac.in
Abstract
The interaction between a synthesized dye with proteins, bovine and human serum albumin
(BSA, HSA respectively) under physiological conditions has been characterized in details, by

2
means of steady state and time resolved fluorescence, UV-Vis absorption and circular dichroism
(CD) techniques. An extensive time-resolved fluorescence spectroscopic characterization of the
quenching process has been undertaken in conjugation with temperature-dependent fluorescence
quenching studies to divulge the actual quenching mechanism. From the thermodynamic
observations, it is clear that the binding process is a spontaneous molecular interaction, in which
van der Waals and hydrogen bonding interactions play the major roles. The UV-Vis absorption
and CD results confirm that the dye can induce conformational and micro environmental changes
of both the proteins. In addition, the dye binding provoke the functionality of the native proteins
in terms of esterase-like activity. The average binding distance (r) between proteins and dye has
been calculated using FRET. Cytotoxicity and antiviral effect of the dye has been found using
Vero cell and HSV-1F virus by performing MTT assay. The AutoDock-based docking
simulation reveals the probable binding location of dye within the sub domain IIA of HSA and
IB of BSA.
Keywords: Azo dye, serum albumin, docking, fluorescence
1. Introduction
In modern era there is a phenomenal rise in the growth of different organic dyes owing to their
extensive uses in different industries like food, textile, printing, cosmetics, electronics and

3
pharmaceuticals. Apart from these commercial uses, dyes are also used as probe for
understanding the mechanism of different photo physical processes. (Carter et. al., 1994) Among
different types of dyes, water soluble azo dyes are extensively employed in foodstuff like swiss
rolls, jams, jellies, yoghurts, breadcrumbs; mouthwash and cheese cake mixes etc (Amin et.al.,
2010). However most of these dyes have been found to have carcinogenic effects on human body
that may cause bladder cancer, splenic sarcomas, hepatocarcinomas and nuclear anomalies in
animal models (Medvedev et. al., 1988). The cleavage of azo linkage leads to the development of
a highly toxic carcinogenic molecule benzidine which can adversely affect our urinary organs,
stomach, kidney, brain, and liver (Ratna et. al., 2012). As they are toxic, carcinogenic, mutagenic
and less degradable, the effluent discharged by the textile industries after dyeing process is
concerned as a pollutant (Chowdhury et. al., 1996). The physiological and toxicological actions
of the dyes are related to the binding capacity of the proteins.
Biomacromolecules such as proteins, nucleic acids have very much important role in living
systems. Serum albumins, one of the most plentiful compounds in the blood plasma are the
major constituent of the circulatory system. They expedite the transport process and delivery of
various exogenous and endogenous ligands including long-chain fatty acids, drugs and metal
ions (Xu et. al., 2012). Serum proteins are also responsible for maintaining colloid blood
pressure and are extremely effective for biological functions like membrane preparation,
metabolism and other pharmacokinetic properties (Khan et. al., 2012).
The well-known serum proteins Bovine Serum albumin (BSA) and Human Serum Albumin
(HSA) have formed the center of many-faceted research activities for years. Apart from their
abundance and ease of purification, the intense importance of them in pharmaceutical industries
originating from its functional properties has played a foremost role in modern research area.

4
These most abundant multifunctional proteins in Bovine and Human blood respectively have
almost 76% structural similarity (Peters, 1996). The primary structure of BSA contains 583
amino acids and that of HSA contains 585 amino acid residues. From determined
crystallographic structure of the proteins it is proposed that single Tryptophan residue (Trp-214)
of HSA is located in IIA binding site and in case of BSA two tryptophan moieties viz. Trp-213
and Trp-134 are present in hydrophobic sub domain IIA and less hydrophobic IA respectively
(Sudlow et. al., 1975; Arumugam et.al., 2016) The tertiary structure of them is constituted of
almost 67% helical content and 17 disulphide bridges (Peters, 1985). In pharmaceutical
industries it is accepted that the overall distribution and metabolism of different ligands can be
altered on the basis of their affinity to proteins. In addition, many drug molecules are considered
as ineffective owing to their high affinity towards proteins. So, different types of interaction of
various biologically active compounds with proteins advocate new approach to drug therapy and
design (Roy et. al., 2017; Qin et al., 2016).
Coumarin derivatives are very much important to the modern drug designers owing to that most
of them have antimicrobial, anti-inflammatory, antitumor and anticancer activity. For this vast
range of application they are widely used in pharmaceutical industries. Coumarins are strongly
fluorescent and have high photo stability, so that they are extensively used in cosmetic world.
Normally most of the coumarin compounds are colorless but substitution at different position
make them colorful and used in the application such as laser dyes, textile dyes etc (Chen et. al.,
2013; Bayraktutan et. al., 2017; Akbaya et. al., 2010; Vaarla et.al., 2015; Christie et. al., 2008).
Consequently, in our present investigation we have tried to design a coumarin based azo dye
having antiviral effect as well as protein binding capacity which can be applicable in cosmetics,
textile and pharmaceutical industries. To focus on its protein binding capacity (with two plasma

5
proteins BSA and HSA) various spectroscopic techniques have been asserted. Herein, the dye-
protein binding phenomenon has been observed by steady-state and time-resolved fluorescence
spectroscopy in which the dye induced quenching of the intrinsic fluorescence of the proteins has
been monitored to elucidate the binding strength. For precise evaluation of therapeutic activity of
the synthesized dye on proteins, absorption and circular dichorism studies have been done.
Cytotoxicity and antiviral effect of the dye has been found using Vero cell and HSV-1F virus by
performing MTT assay. We have introduced a computational approach to establish the binding
location of the dye around the macromolecular architecture of the serum proteins in order to
provide theoretical support to the experimental findings.
Scheme 1: Structure of the synthesized azo dye
2. Materials and methods
2.1. Materials
Thiosemicarbazide (puriss. p.a., 98%), carbon disulphide, anhydrous sodium carbonate
(anhydrous, free-flowing, Redi-Dri™, ACS reagent, ≥99.5%), bovine serum albumin (BSA) and

6
human serum albumin (HSA) (lyophilized powder, ≥97% (agarose gel electrophoresis)) have
been purchased from Sigma Aldrich (USA) and used as received. All other chemicals have been
procured from the commercial sources as per requirements and used after purification. The
sample solutions have been prepared in 10 mM tris- HCl/10 mM NaCl buffer (pH = 7.4) using
deionized ultrapure water from Millipore Synergy System (Merck, India). The tris base has been
purchased from Merck, India. Stock solution of BSA and HSA have been prepared in 10 mM
tris- HCl buffer measuring the absorbance at 280 nm with ε value of 43,824 M-1 cm-1 and 42,864
M-1 cm-1respectively (Peters, 1975; Andrade et.al., 2002). All the protein solution have been
stored in the dark at 4 ○C and used within 4 days. Stock solution of dye has been prepared by
methanol-water (1:9) mixture system due to its low solubility in water.
2.2. Synthesis of Azo dye
5-Amino-3H-[1, 3, 4] thiadiazole-2-thione has been prepared according to a literature method
(Popiołek et. al., 2015). To (1.33 g, 0.01 mol) of 5-Amino-3H-[1, 3, 4]thiadiazole-2-thione taken
in a 500-mL RB flask 60 mL of 50% HCl solution has been added and it was stirred at 0 -5 °C.
To this solution an ice-cold solution of sodium nitrite (0.828 g, 0.012 mol) has been added at a
rate such that the temperature should not rise above 5 °C. The cold diazonium chloride solution
thus prepared has been added very slowly with vigorous stirring to cold (0 °C) alkaline 7-
hydroxy-4-methyl-coumarin (1.76 g, 0.01 mol) in a 250-mL RB flask immersed in an ice salt
bath. The reddish brown solid separated was filtered, washed with water, dried, and collected
with 60 %Yield (~ 2.0 g). Microanalytical data : C12H8N4O3S2; Calcd. (Found): C, 44.99 (44.98);
H, 2.52 (2.53); N, 17.49 (17.51) %. 1H-NMR (300 MHz, CDCl3). δ 10.53 (s, H, OHa) , 6.11 (s,
H, Hb), 6.69 (s, H, Hc), 6.78 (d, H, J = 12, Hd), 6.58 (d, H, J = 9, He), 2.34 (s, 3H, Hf). IR:
3492.51 cm-1 (phenolic –O-H), 1667.41 cm-1 (–C=N), 1455.29 cm-1 (azo, -N=N). (NMR and IR

7
spectra have been shown at Figs. S1 and S2, S stands for supporting information).
S
N
anhydrous Na2CO3
absolute ethanol
HN
H2N
NH2
S
C
S
N
NH2
H
S
S
thiosemicarbazide
reflux for 4 hours
5-Amino-3H-[1,3,4]thiadiazole-2-thione
O
OH
O
NaNO2 / HCl
O - 5 degree C
7-hydroxy-4-methyl-coumarin
HN
HO
N
S
N
N
S
O
O
7-Hydroxy-4-methyl-8-(5-thioxo-4,5-dihydro-[1,3,4]thiadiazol-2-ylazo)-chromen-2-one
Scheme 2: Synthesis of (7-Hydroxy-4-methyl-8-(5-thioxo-4, 5-dihydro-[1, 3, 4]thiadiazol-2-
ylazo)-chromen-2-one)
2.3. Protein binding experiment:
2.3.1. Absorption Studies:
UV-Vis absorption spectroscopy has been employed to investigate the interactive phenomena

8
between the synthesized dye and serum albumins using UV-Vis Spectrophotometer (Shimadzu,
UV-1800) equipped with a Peltier temperature controller, TCC-240A in order to sustain a
constant temperature of 298 K (±0.1 K). A quartz cell from Hellma of 1 cm path length has been
used in this study. The dye solution has been added to the fixed amount of buffered protein
solution and during addition of the dye an equal amount of it has been added to both sample and
reference cell in order to cancel out the contribution of the added dye to the absorbance of
protein spectra.
2.3.2. Steady state fluorescence spectroscopy
Steady-state fluorescence measurements have been carried out on a Shimadzu RF-5000
spectrofluorimeter. The intrinsic fluorescence of proteins has been studied upon exciting at 295
nm using excitation and emission band slits of 5 nm each. The effect of proteins on dye
fluorescence has also been studied upon exciting the dye at 320 nm. During fluorometric titration
experiments, the dilution factor of the dye has been corrected.
2.3.3. Time resolved fluorimetric analysis
Fluorescence lifetimes of Trp in albumin proteins have been determined from time-resolved
intensity decays by employing time-correlated single-photon-counting (TCSPC) technique using
Horiba Jobin Yvon at 298 K. A nanosecond diode laser at 300 nm (IBH UK) has been used as an
excitation source with a TBX photon detector and the signals have been recorded at the magic
angle of 54.7°. The number of counts gathered in the channel of maximum intensity has been
fixed at 2500. The data have been analyzed using a nonlinear least-squares iterative method
utilizing the Fluorescence Analysis Software IBH DAS-6. The quality of fits has been judged
from χ2 (0.9 ~ 1.2) data (Lakowicz, 2006). The following equation (1(a)) (Lakowicz, 2006) has

9
been used to investigate the experimental time-resolved fluorescence decays where, F (t) is the
fluorescence intensity at time t and αi is the pre-exponential factor representing the fractional
contribution to the time-resolved decay of the ith component with a lifetime τi. For
multiexponential decays, the average lifetime has been calculated from the following equation
where (τ1, τ2 and τ3) are decay times and (α1, α2 and α3) are the normalized pre-exponential
factors.


t
F(t) 
a exp
(1(a))




i


i


i
       
1 1
2 2
3 3
(1(b))
2.3.4. Circular dichroism Studies:
Circular dichroism spectra of the albumins have also been recorded in presence and absence of
azo dye owing to explain the conformational properties of proteins upon action of dye using a
JASCO J-815 spectropolarimeter attached to a water bath to control the temperature of the
electronic circuit. The concentration of albumins has been kept at 0.4 µM for instrumental
requirement. The CD results have been expressed in terms of mean residue ellipticity (MRE) in
deg cm2 dmol-1 using eq. 2 (Kelly et. al., 2005).
MRE = θobs /10 n l [P]
(2)
Where θobs is the CD in millidegree, n is the number of amino acid residue (583 for BSA, 585 for
HSA), l is the optical path length of the cell, [P] is the molar concentration of the albumin. CD
spectra have been recorded in the wavelength range of 200 - 275 nm with scan speed of 50 nm
min-1. An average of three scans has been done for each spectrum at 298 K. The base line has
been subtracted from each data set.

10
2.3.5. Esterase activity assay
The effect of dye on the esterase activity of serum proteins toward p-nitrophenyl acetate (p-NPA)
has been analyzed on a Shimadzu, UV-1800 UV-Vis spectrophotometer by steady-state kinetics
at 310 K. The concentration of albumins and p-NPA has been kept constant at 25 µM and 50 µM
respectively. The rate of p-NPA hydrolysis has been determined by measuring the appearance of
p-nitrophenol, a yellow product, at 400 nm for 500 s.
2.3.6. Cytotoxic activity assays and antiviral activity
One of the most well-known methods that used for the assessment of cell viability is the 3-[4, 5-
dimethylthiazol-2yl]-2, 5-diphenyltetrazolium bromide (MTT) assay based on the formation and
colorimetric quantification of an enzyme activity (Mosmann, 1983, Mohamadi et. al., 2017).
MTT assay is a colorimetric method based on the rupturing of the yellow water-soluble
tetrazolium salt, MTT, to form water insoluble, dark-blue formazan crystals. MTT splitting takes
place only in living cells by mitochondrial succinate dehydrogenase enzyme. The formazan
crystals are dissolved usually in iso-propanol or DMSO and the absorbance of the out-coming
solution is measured using a spectrophotometer to calculate the concentration of the formazan,
which is proportional to the number of viable cells. In our present contribution African green
monkey kidney cells (Vero cell) morphology has been determined by MTT assay. Vero cells
have been cultured onto 96-well plates at 10 × 106 cells per well and different concentrations of
the dye have been added to each well at a final volume of 100 µL, in triplicate using DMSO
(0.1%) and acyclovir (0–50 µg mL-1 ) as a negative and positive control, respectively. The drug-
treated cells have been incubated at 310 K with 5% CO2 for 2 days and then the MTT reagent
(10 µL) has been added to each well. After 4 h of incubation, the formazan has been solubilized

11
by adding MTT solubilisation solution equal to the original culture media volume and the
absorbance has been recorded at 570 nm with a reference wavelength of 690 nm by an ELISA
reader. Data have been calculated as the percentage of cell viability by the formula.
Cell viability = [(sample absorbance cell free sample blank)/ mean media control absorbance)]/
100%.
In our present contribution the viral strains have been used as HSV-1F (ATCC 734), purchased
from the ATCC (Manassas, VA, USA). Virus stocks have been prepared from infected culture at
a multiplicity of infection (MOI) of 0.5 for 1 h at 310 K. The residual viruses have been then
washed out with phosphate-buffered saline (PBS). We have determined the antiviral effects of
the dye against HSV-1-infected Vero cells (MOI 1) by MTT assay, using acyclovir as positive
and DMSO (0.1%) as negative control.
2.3.7. Theoretical Studies
Molecular simulation is beneficial to understand the interaction of drug molecules with serum
albumin proteins combining to the experimental result. For docking analysis the ground state
geometry of the synthesized azo dye has been optimized introducing density functional theory
(DFT) in connection with B3LYP functional and 6-31G* standard basis set in Gaussian 09
program suit (Koh et. al., 1965). The available crystal structures of the proteins have been
collected from the protein data bank (PDB) with pdb id: 4F5S (BSA) and 4S1Y (HSA). The
required pdb structure of the dye has been collected from the output file of the optimized
structure. The dye has been docked using AutoDock 4.2 into the 3D structure of proteins
(Morris, 1998). AutoDock 4.2 uses the Lamarckian genetic algorithm to investigate the optimum
binding site of small molecules to the protein. To recognize the binding sites in serum albumins,
docking has been done with the grid sizes 126, 126, and 126 along the x-, y-, and z- axes with a

12
0.675 Å grid spacing. The Auto Docking parameters have been used as follows: GA population
size, 150; maximum number of energy evaluations, 250,000. During docking, a maximum of 50
conformers have been considered for each molecule, and the root mean square (rms) cluster
tolerance has been set to 2.0Å. The output from AutoDock has been further analyzed using
PyMOL software (Lano, 2004).
3. Results and Discussion
3.1. Absorption Spectroscopic Study
UV-Vis absorption spectroscopy is an effective method in exploring the structural changes of the
bio-macromolecules and to investigate protein-ligand complex formation. The UV-Vis
absorption spectra of HSA in the absence and presence of dye obtained by utilizing the mixture
of dye and tris buffer at the same concentration as the reference solution are shown in Fig. 1.

13
Fig. 1: UV-Vis spectra of HSA in presence of different concentrations of dye at 298 K and pH
7.4, [HSA] = 5µM, inset shows change of absorption spectra of protein at 278 nm upon action of
dye
Albumins show two absorption peaks, the strong absorption peak at about 212 nm reflects the
framework conformation of the protein, the weak one at about 278 nm appears to be due to the
aromatic amino acids (Trp, Tyr, and Phe) (Zhao et. al., 2010; Lou et. al. 2017). Upon addition of
the synthesized dye to the protein the intensity at 212 nm decreases with a red shift (212 to 220
nm for HSA and 211 to 222 nm for BSA) and the intensity of the peak at 278 nm have minimal
changes (inset of Fig. 1). The results indicate that dye-protein interaction leads to increase the
hydrophobicity of the microenvironment of the HSA (Pan et. al., 2011); similar phenomenon has
been occurred in case of BSA also, which is not shown in the manuscript. Spectral changes in
UV-Vis spectra of the albumins with addition of dye signify the presence of ground state
complexation between them.
3.2. Fluorescence measurements
3.2.1. Steady state fluorescence technique
Fluorescence spectroscopy is a broadly applied technique for investigating protein conformation,
dynamics and intermolecular interactions upon action of different drugs on it.
The intrinsic emission property of the proteins appears mainly due to presence of amino acid
residues like Tryptophan (Trp), Tyrosin (Tyr) and Phenylalanine (Phe). However the

14
contribution of Phe to the total intrinsic fluorescence of Serum proteins is very low due to its low
fluorescence quantum yields. Therefore any change in fluorescence property of Serum Albumin
in presence of dye may be attributed due to the change occurring in the microenvironment of Trp
and Tyr residues (Lakowicz, 2006).
The emission spectra of HSA and BSA have been recorded at λem = 340 and 346 nm respectively
in absence and presence of the dye (Fig. 2) upon excitation at 295 nm to avoid any contribution
of Tyr residue and the resulting emission spectrum is exclusively ascribed to the intrinsic Trp
fuorophore (Efink et. al., 1976).
Fig. 2: Emission spectra of (a) HSA and (b) BSA at various concentration of azo dye. [dye] = 0,
0.75, 1.50, 2.25, 3.00, 3.75, 4.50, 5.25, 6.00, 6.75 µM, [protein] = 5 µM, inset shows change of
protein fluorescence upon action of dye.
Fig. 2 illustrates that upon successive addition of dye, fluorescence intensity of the albumins
decreases accompanying simultaneous enhancement in the fluorescence intensity of the dye at
450 nm. This is because of the fact that upon exciting the protein-dye mixture at 295 nm, some
of the dye molecules get excited and thereby produces emission spectra centered at 450 nm. Such

15
decrease in fluorescence intensity of albumins implies that the dye can alter the structure of
proteins. The dye induced structural changes of the proteins have been exemplified by slight red
shift (340 to 342 nm for HSA and 346 to 348 nm for BSA) in emission spectra. It implies that the
microenvironment across the amino acid residues of the proteins in vicinity of interaction site of
the dye is perturbed, leading to greater extent of exposure of the Trp residue to the more polar
aqueous milieu (Lakowicz, 2006). Such alteration of microenvironment near Trp residue
indicates disruption of native structure of proteins upon action of the dye. Circular dichoric
spectral responses of the proteins at presence of dye affix credence to the observations as
discussed to the following sections.
3.2.2. Influence of Inner Filter Effect (IFE) on emission quenching of HSA/ BSA by the
synthesized dye
IFE refers to the absorption of light at the excitation or emission wavelength by the compounds
present in the experimental solution. In the protein binding studies if ligand molecules possess
strong absorption at the excitation wavelength of the protein, less light reaches to the centre of
the sample and thus the emission intensity of the protein decreases, whereas, a strong absorption
at the emission wavelength of protein would diminish the emitted light that reaches the detector.
In our present investigation, the dye absorbs radiation at excitation wavelength of protein at 295
nm and emission wavelength at 340 nm for HSA and 346 nm for BSA (absorbance spectrum of
the dye has been shown in Fig. S3). So, the IFE of the dye on the emission intensity of both HSA
and BSA has been evaluated. Similar kind of observation has been reported for the binding
interactions of β-conglycinin and glycinin with vitamin B12 (Dan et. al., 2007).
The emission intensities are corrected using the relationship, eq (3) (Lakowicz, 2006)

16
(A  A )
ex
em
2
(3)
F
 F e
obs
corr
Where, Fcorr and Fobs are the corrected and observed emission intensities, respectively. Aex and
Aem are the solution absorbance at the excitation and emission wavelengths respectively.
The plots of (F/F0) of both corrected emission intensities Fcorr and observed emission intensities
Fobs in case of HSA and BSA versus [dye] have been displayed in Fig. 3(a) and (b).
Fig. 3: Effect of inner filter effect (IFE) on the emission spectra of serum proteins for (a) HSA
and (b) BSA at various dye concentration. [dye] = 0, 0.75, 1.50, 2.25, 3.00, 3.75, 4.50, 5.25,
6.00, 6.75µM, [protein] = 5 µM.
The (F/F0) values of corrected and observed emission intensities decreased progressively with
increase of dye concentration. The former is slower than the latter, signifying that the quenching
of emission intensity of proteins is partly caused by IFE of dye. The decrease of corrected
emission intensity of albumins due to presence of dye indicates presence of binding interaction
between them.

17
In the present contribution our synthesized dye exhibits emission spectra at 450 nm upon
excitation at 320 nm. We have studied the effect of proteins on fluorescence of the dye. The
fluorescence intensity of dye decreases upon increment of protein concentration which implies
the presence of drug-protein interaction (Fig. 4).
Fig. 4: Fluorescence emission spectra of azo dye in absence and presence of serum albumins, (a)
HSA and (b) BSA {[dye] = 2 µM, [serum albumin] = 2.5, 5.0, 7.5, 10.0, 12.5 µM}. The arrow
indicates the decrease in fluorescence intensity of the dye upon addition of albumins.
3.2.3. Quenching mechanisms of proteins in presence of dye
In modern science fluorescence technique has vast applications and this can be used to
understand the mechanism of protein-ligand interaction. The quenching of emission spectra
occurs mainly in two processes namely collisional or dynamic quenching through exciplex
formation and static quenching due to ground state complexation (Lakowicz, 2006). As we have
got the evidence of presence of ground state complexation between dye and proteins so the
phenomenon of fluorescence quenching is conventionally described in terms of the well-known
Stern−Volmer equation: (Lakowicz, 2006)

18
F
[Q]
0
1 K
1 k  [Q]
q 0
(4)
sv

F
C
Where, F0 and F are the corrected emission intensities of the proteins in absence and presence of
quencher respectively. kq is the bimolecular quenching constant, τ0 is the average life time of
fluorophore in absence of quencher and [Q] is the concentration of quencher. Ksv is the Stern-
Volmer quenching constant indicating the efficiency of quenching.
Fig. 5: Stern-Volmer plot of dye-serum protein system
According to the Stern-Volmer equation, F0/F versus [dye] (Fig. 5) plot has been drawn for the
quenching of serum proteins by the above mentioned dye. The plots show upward curvature to
the Y axis (Adj. R square value (r) = 0.9948 for HSA and 0.9944 for BSA) within the
investigated concentration of dye. The nonlinearity of the curve implies the presence of both
static and dynamic quenching by the same quencher (Lakowicz, 2006).
Therefore, modified Stern-Volmer equation has been used to analyze the quenching data. The
equation helps in resolving the accessible and inaccessible residues when there is possibility of
differential accessibilities of the proteins to the probe. The emission spectral studies have been
analyzed according to the following modified Stern–Volmer equation (eq. (5)) (Lakowicz, 2006).

19

1 C
1
F0
F


(5)
f K [Q] f
a a
a
Where, F is the difference in corrected emission intensity of HSA/ BSA in the absence and
presence of quencher (dye) Q, (F = F0 - F), fa is the fraction of accessible fluorescence and Ka
is the effective quenching constant for the accessible fluorophores, which is analogous to the
associative binding constant for HSA/ BSA–dye system. From Fig. 6 the plot F0 / F versus 1/
[dye] shows a linear relationship with a slope equal to C° (fa Ka)-1 and intercept equal to 1/ fa.
Fig. 6: Modified Stern-Volmer plot of dye-serum protein system
The value of Ka has been determined from intercept to slope ratio using this equation and found
5.71 × 104 and 7.47 × 104 for HSA and BSA respectively. From the above result it is clear that
the dye forms moderately strong complex with the proteins. The results obtained in our present
study are consistent with the previous literature reports (Sharma et. al., 2014; Tian et. al., 2004).
3.2.4. Determination of stoichiometry of dye-protein complex
Spectral data from the emission titration experiment has been utilized to calculate the
stoichiometry of dye-protein complex using the Benesi–Hildebrand equation [eq. 6] (Benesi et.
al., 1949).

1
1
C


F  F F  F (F  F )K[Q]
0
0
1
0
1

20
(6)
Where, F0 is the emission intensity of HSA/ BSA in the absence of dye, F is the corrected
emission intensity of the serum proteins at intermediate concentration of the dye, F1 is the
emission intensity of HSA/ BSA at infinite concentration of dye, K is the binding constant and Q
is the quencher (dye).
From Fig. 7(a) and (b), it has been seen that the plots of 1/[F0 - F] versus 1/[dye] show a fine
linearity, thereby suggesting one-to-one interaction between dye and HSA/ BSA.
Fig. 7: Benesi–Hildebrand plot of 1/[F0 - F] versus 1/[dye] for binding of dye with (a) HSA and
(b) BSA. Inset shows the modified Benesi–Hildebrand plot of 1/[F0 - F] versus 1/[dye]2 for
HSA/ BSA–dye systems
It is broadly accepted that the linearity in Benesi–Hildebrand plot is indicative of 1:1
complexation (Samanta et. al., 2011). In our present study we have applied the modified Benesi–
Hildebrand equation to validate the existence of 1:1 complexation and to exclude the possibility
of 1:2 complex formations in dye-protein system. From the inset plots of Fig.7, 1/(F0-F) versus

21
1/[dye]2 clearly shows a deviation from linearity indicating the nonexistence of 1:2 complexation
(Samanta et. al., 2011).
Ostensibly, it is very much important at this stage to find the mechanistic interpretation of dye
induced quenching of intrinsic fluorescence of the proteins. The two different quenching
mechanism dynamic (collisional) and static can be distinguished by their differential dependence
on temperature. Higher temperature leads to faster diffusion and greater extent of dynamic
quenching but in case of static process reverse is true. In our present work occurrence of static
quenching is proved from some experimental evidence but due to delve the presence of excited
state complexation or dynamic quenching another important technique, time resolved
fluorimetric analysis has been done.
3.2.5. Time resolved Fluorescence decay studies
Intrinsic Fluorescence life time decay measurement has become a promising tool for determining
the conformational dynamics of the proteins and it is also very much sensitive to identify excited
state interaction (Santhamani et. al., 2013). In our present study to gather clear information about
dye-protein excited state complexation, time resolved fluorometric measurements have been
done. The amendment of Trp life time upon regular addition of azo dye has been investigated
and corresponding decay profiles of it has been displayed at Table 1.
Table 1(a): Fluorescence decay parameter of HSA as a function of added azo dye
Azo dye
τ1(ns)
A1
τ2(ns)
A2
<τ>(ns)
χ2
(µM)
0
4
3.33
3.06
0.3839
0.4256
6.68
6.56
0.6161
0.5744
5.39
5.08
1.05
1.03

22
8
2.95
3.09
2.82
0.4711
0.5042
0.5223
6.51
6.39
6.31
0.5289
0.4958
0.4777
4.83
4.73
4.50
1.02
1.05
1.01
12
16
Table 1(b): Fluorescence decay parameter of BSA as a function of added azo dye
Azo dye
τ1 (ns)
A1
τ1 (ns)
A2
<τ> (ns)
χ2
(µM)
0
3.0204
2.3940
2.0033
1.6289
1.58204
1.5188
0.3069
0.3980
0.5565
0.6213
0.6825
0.7300
6.3696
6.0363
5.8013
5.5318
5.3404
5.0604
0.6931
0.6020
0.4435
0.37875
0.3175
0.2700
5.34
4.58
3.69
3.11
2.77
2.61
1.02
1.06
1.02
1.04
0.99
1.05
4
8
12
16
20

23
Fig. 8: Lifetime decay curves of serum proteins in the presence of various concentrations of dye
From the Fig. 8 it has been seen that the added dye decreases protein lifetime indicating excited
state interaction between them. The Trp emission has been fitted to a bi-exponential decay, and
the respective data have been summarized in Table 1(a) for HSA and Table 1(b) for BSA. As it
is a non trivial task to assign the explicit mechanistic interpretation for the individual decay
components within multi-exponential fluorescence decays in complex biological environments,
we have taken an attempt to consider variation of the relative amplitudes of the two components
of albumins upon gradual addition of dye (Berezin et. al., 2010; De et. al., 2013).
The decay profiles of both the proteins help us to conclude the presence of dynamic quenching
upon action of quencher. The extent of dynamic fashion in the quenching mechanism of
aforementioned dye-protein systems has been evaluated using the equation (Lakowicz, 2006)


[Q]
0
(7)
1 K
D

C
Where τ0 and τ are the fluorescence lifetimes of the proteins in the absence and presence of the
dye, respectively, and KD is the dynamic quenching constant. The observation based on life time
data and the plot τ0/τ versus [dye] (Fig. S4) of dye-protein system, the KD has been determined as
1.18 × 104 and 5.56 × 104 for HSA and BSA respectively. From the steady state and time
resolved spectroscopy it is concluded that in our present contribution the dye quenches the
emission intensity of the serum albumins by both static and dynamic fashion. In the mechanistic
assay of the quenching process the contribution of static fashion has been determined using the
following equation (Lakowicz, 2006)
F  F



0
C


F
[Q]


 (K  K )  K K
S D
(8)
s
D

[Q]
C

24
Using the slope of the plot [(F0 − F)/F]/[Q] versus [Q] (Fig. S5), Ks have been calculated as 2.15
× 105 and 8.54 × 104 for HSA and BSA respectively. The results illustrate the greater amount of
static quenching constant than that of dynamic quenching constant for both the proteins which
indicates that the quenching mechanism is predominantly static rather than dynamic.
3.2.6. Binding constants and number of binding sites:
A quantitative assessment for the dye-protein binding interaction can be derived in terms of
estimation of the binding constant (Kb) and the free energy change (ΔG) for the interaction
process. The binding constant plays a vital role because of the binding ability and subsequent
influence of drugs on protein stability will govern the therapeutic efficacy of the drug to a large
extent. For static quenching process when a small molecule binds independently to a set of
equivalent sites on macromolecule, then the equilibrium between the free molecule and bound
molecule is given by double-logarithmic equation (Lakowicz, 2006)






F  F
[Q]






0
log
 log(K )  nlog
b
(9)

F
C
.Where Kb is the binding constant and n is the number of binding sites. According to the equation
the values of Kb and n can be obtained from the intercept and slope of the above equation. The
results for the present investigation at various temperatures have been summarized in Table 2
and the representative plots of log [(F0 − F)/F] versus log ([dye]/C°) have been displayed in Fig.
9.

25
Fig. 9: Plots of log [(F0-F)/F] versus log([dye]/ C°) at different temperatures representing effect
of temperature on binding of dye with (a) HSA and (b) BSA, pH 7.4
Table 2: Summary of the Binding Parameters for the dye−protein interaction from Fluorescence
Quenching Data for HSA and BSA respectively
HSA
10-6 × Kb
1.28
0.61
0.26
BSA
Temperature (K)
n
10-6 × Kb
2.82
n
293
298
303
1.25
1.21
1.15
1.26
1.19
1.05
0.98
0.15
From the data collected in Table 2 we see that our results evidently demonstrate that the
dye−protein binding strength becomes weak with increasing temperature which appears to
reinforce the postulate of a predominant static quenching mechanism rather than dynamic
process in the presently studied system in confirmation to the aforementioned discussion.
3.2.7. Thermodynamic Parameters and Nature of the Binding Forces
The knowledge of the actual binding forces originating from the drug−protein interaction process
is of immense importance in protein chemistry, and deciphering of the thermodynamics of the

26
interaction process provides a profound acumen to the mechanistic aspects involved. Usually, the
interaction forces between drugs and biomolecules may be assorted including electrostatic,
hydrogen bonding, van der Waals interaction, hydrophobic, and steric contacts within the
binding site, and so forth (Paul et. al., 2011; Klotz et. al., 1973; Ross et. al., 1981). The sign and
magnitudes of thermodynamic parameters are very much important to insight the principle
binding forces (Klotz et. al., 1973; Ross et. al., 1981).
If we consider that there is no significant variation of the enthalpy change (ΔH) within the range
of temperature studied, both the enthalpy change (ΔH) and the entropy change (ΔS) can be
evaluated from van’t Hoff’s equation: (Klotz et. al., 1973)
H
R
1
S
R
ln K  
 
(10)
T
Here, R is the universal gas constant, and T is the temperature in kelvin. The free energy change
(ΔG) of the process is then evaluated from the following relationship:
G  H TS
(11)
The thermodynamic parameters have been calculated from the van’t Hoff plot (lnK vs. 1/T)
which is displayed in (Fig. S6). In protein chemistry the various individual kinds of interaction
are characterized with the thermodynamic parameters such as: (Paul et. al., 2014)
(i) ΔH > 0, ΔS > 0 correspond to hydrophobic forces;
(ii) ΔH < 0, ΔS < 0 correspond to van der Waals interaction, hydrogen-bond formation;
(iii) ΔH < 0, ΔS > 0 correspond to electrostatic/ionic interactions.
The derived thermodynamic parameters suggest that dye-protein interaction is enthalpically
favorable (-117.6 and -213.2 kJ mol-1 for HSA and BSA respectively and both are < 0) but

27
entropically not favorable (-284.2 and -603.8 J mol-1 K-1 for HSA and BSA respectively and both
are < 0). With this we also get that the overall negative Gibbs free energy change (-22.9 and -
33.5 kJ mol-1 for HSA and BSA respectively) implies the spontaneity of the binding interaction.
The negative entropy and enthalpy change signifies presence of van der Waals interaction or
hydrogen bond formation between dye and serum proteins.
3.3. Effect of ionic strength on dye-protein interaction
From the thermodynamic parameters it is clear that the binding interaction of azo dye with serum
proteins does not involve electrostatic interaction in our present study but to ensure this, a strong
electrolyte NaCl has been added to the dye-protein system. In order to study the effect of ionic
strength on the binding interaction, the NaCl concentration has been varied from 0 to 50 mM. No
considerable change in binding constant has been observed in the presence of high concentration
of NaCl (figure not shown). The incident clearly indicates that electrostatic forces have no
significant contribution in the drug−protein interaction process. Thus, van der Waals interaction
or H-bond formation is major contributing binding force for dye-protein system.
3.4. Fluorescence Resonance Energy Transfer from protein to azo dye
Fluorescence Resonance Energy Transfer (FRET) is a distance dependent interaction between
the different electronic excited states of molecules in which excitation energy is transferred from
donor molecule to acceptor molecule without emission of a photon from the former molecular
system (Förster, 1959). When the emission spectrum of a fluorophore (protein) overlaps with the
absorption spectrum of acceptor (ligand) then FRET occurs. The spectral overlap between the
UV absorption spectrum of dye and the fluorescence emission spectrum of HSA has been
displayed in the Fig. 10. Similar spectral overlap has been also observed in case of BSA also

28
(spectra not shown).
Fig. 10: Spectral overlap of UV–Vis absorption spectrum of synthesized dye with the
fluorescence emission spectrum of HSA, [HSA] = [dye] = 5.0 µM, T = 298 K.
The degree of energy transfer depends on the distance between donor and acceptor and extent of
overlap. According to Förster’s nonradiative energy transfer theory, the energy transfer
efficiency (E) is defined by the following equation:
6
R
F
0
E 1

(12)
6
6
F
R  r
0
0
Where F and F0 are fluorescence intensities of serum proteins in presence and absence of dye, r
is the distance between acceptor and donor, and R0 is the critical energy transfer distance when
transfer efficiency is 50%. The value of R0 has been evaluated using the equation below:
25 2 4
(13)
R  8.810  N J
0

29
Where κ2 is spatial orientation factor of the dipole, N is refractive index of the medium, Φ is the
fluorescence quantum yield of the donor, and J is the overlap integral of the fluorescence
emission spectrum of donor and absorption. Now J is given by the following equation:
4
F     
   
J  
(14)
F  
 

Where F(λ) is the fluorescence intensity of the fluorescent donor of wavelength λ, ε(λ) is the
molar absorption coefficient of the acceptor at wavelength λ (Tian et.al., 2010). In our present
contribution κ2 = 2/3, N = 1.336 and 1.36, Φ = 0.118 and 0.15 for HSA and BSA respectively
(Cui et. al., 2003; Li et. al., 2007).
Utilizing above equations the value of overlap integral (J), R0, E, and r have been calculated and
found to be 3.07 × 10−14 cm3 L mol−1, 2.96 nm, 0.17 and 3.85 nm for HSA and 2.84 × 10−14 cm3
L mol−1, 4.42 nm, 0.36 and 4.84 nm for BSA respectively. For energy transfer process an
essential criterion is that the distance between donor and acceptor must be within 2−8 nm
(Förster, 1959). This criterion is satisfied in the present study and hence quenching of protein
(HSA and BSA) fluorescence in the presence of the dye is attributed to energy transfer.
3.5. Conformation Investigation: Circular Dichroism (CD) Spectral Study
Circular dichroism (CD) spectroscopy has long been recognized as a powerful technique to
investigate the conformational alteration of a variety of bimolecular systems including proteins
upon action of different drug molecules (Sreerama et. al., 2000). Fig. 11 reveals far UV circular
dichorism spectra of proteins in absence and presence of different dye concentrations.