Insights into the binding of thiosemicarbazone derivatives with human serum albumin: Spectroscopy and molecular modelling studies

Article abstract of DOI:10.1080/07391102.2015.1075905

4-[(1Z)-1-(2-carbamothioylhydrazinylidene)ethyl]phenyl acetate [Ace semi],4-[(1Z)-1-(2-carbamothioylhydrazinylidene) ethyl]phenyl propanoate [Pro semi] from the family of thiosemicarbazones derivative has been newly synthesized. It has good anticancer activity as well as antibacterial and it is also less toxic in nature, its binding characteristics are therefore of huge interest for understanding pharmacokinetic mechanism of the drug. The binding of thiosemicarbazone derivative to human serum albumin (HSA) has been investigated by studying its quenching mechanism, binding kinetics and the molecular distance (r) between donor (HSA) and acceptor (thiosemicarbazone derivative) was estimated according to Forster’s theory of non-radiative energy transfer using fluorescence spectroscopy. The binding dynamics has been elaborated using synchronous fluorescence spectroscopy, and the feature of thiosemicarbazone derivative induced structural changes of HSA has been studied by circular dichorism, Fourier transform infrared spectroscopy. Molecular modelling simulations explore the hydrophobic interaction and hydrogen bonding which stabilizes the interaction.

Full text of DOI:10.1080/07391102.2015.1075905

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Insights into the Binding of thiosemicarbazone
derivatives with Human Serum Albumin:
Spectroscopy and Molecular Modelling studies
Subramani Karthikeyan, Ganesan Bharanidharan, Manish Kesherwani,
Karthik Ananth Mani, Narasimhan Srinivasan, Devadasan Velmurugan,
Prakasarao Aruna & Singaravelu Ganesan
To cite this article: Subramani Karthikeyan, Ganesan Bharanidharan, Manish Kesherwani,
Karthik Ananth Mani, Narasimhan Srinivasan, Devadasan Velmurugan, Prakasarao Aruna
& Singaravelu Ganesan (2015): Insights into the Binding of thiosemicarbazone derivatives
with Human Serum Albumin: Spectroscopy and Molecular Modelling studies, Journal of
Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2015.1075905
To link to this article: http://dx.doi.org/10.1080/07391102.2015.1075905
Accepted online: 14 Sep 2015.
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Date: 20 September 2015, At: 08:32

Publisher: Taylor & Francis
Journal: Journal of Biomolecular Structure and Dynamics
DOI: http://dx.doi.org/10.1080/07391102.2015.1075905
Insights into the Binding of thiosemicarbazone derivatives with Human Serum
Albumin: Spectroscopy and Molecular Modelling studies
Subramani Karthikeyana, Ganesan Bharanidharana* Manish Kesherwanib, Karthik Ananth
Manid, Narasimhan Srinivasand, Devadasan Velmuruganb,c,,Prakasarao Arunaa, Singaravelu
Ganesana*
aDepartment of Medical Physics, Anna University, Chennai-600 025, India.
bCentre of Advanced Study in Crystallography and Biophysics, University of Madras,
Chennai-600 025, India.
cBioinformatics Infrastructure Facility, University of Madras, Chennai-600 025, India.
dDepartment of Chemistry, Asthagiri Herbal Research Foundation, Perungudi Industrial
Estate, Perungudi, Chennai600 096, India
*Corresponding authors
Dr.S.Ganesan
Department of Medical Physics
Anna University, Chennai – 600 025
Phone: +91-44-22358685
Email: sganesan@annauniv.edu , ganesansingaravelu@gmail.com

Dr.G.Bharanidharan
Department of Medical Physics
Anna University, Chennai – 600 025
Phone: +91-044-2235 8702
Email: ganesanbharani@annuniv.edu, ganesanbharani@yahoo.co.in
Abstract
4-[(1Z)-1-(2-carbamothioylhydrazinylidene)ethyl]phenyl acetate [Ace semi],4-[(1Z)-
1-(2-carbamothioylhydrazinylidene)ethyl]phenyl propanoate [Pro semi] from the family of
thiosemicarbazones derivative has been newly synthesised. It has good anticancer activity as
well as antibacterial and less toxic in nature, its binding characteristics are therefore of huge
interest for understanding pharmacokinetic mechanism of the drug. The binding of
thiosemicarbazone derivative to Human Serum Albumin has been investigated by studying its
quenching mechanism ,binding kinetics and the molecular distance (r) between donor
(HSA) and acceptor (thiosemicarbazone derivative) was estimated according to Forster’s
theory of non-radiative energy transfer using fluorescence spectroscopy. The binding
dynamics has been elaborated using synchronous fluorescence spectroscopy, and the
feature of thiosemicarbazone derivative induced structural changes of human serum albumin
has been studied by circular dichorism , Fourier transform infrared spectroscopy
(FTIR).Molecular modelling simulations explore the hydrophobic interaction and
hydrogen bonding which stabilizes the interaction.
Keywords: Anticancer, Drug binding pocket, Florescence resonance energy transfer,
Molecular modeling.
Introduction
Cancer is the uncontrolled cell growth and it can affect any part of the body. The
growths may often invade surrounding tissue and can metastasize to different sites. Nearly
90-95% of cancer is caused by factors such as tobacco, infections, radiations and 5-10% due
to heredity (Anand & Kunnmakkara 2008). World Health Organization (WHO) reported that
the death rate due to cancer may increase to 12 million by 2030. Generally, a different type of
cancers has been treated using various techniques like, radiotherapy, surgery and
chemotherapy. In chemotherapy it is treated using anti-cancer drugs available in the market;
in this regard recently there is a requirement for new anti-cancer agents worldwide
(http://www.who.int/cancer/country/en/).

Thiosemicarbazones (TSC) belongs to a class of compounds that occupy a wide range
of biological activities and have been studied for their activity against tuberculosis (Klayman,
Scovill, Barosevich & Mason 1979). Structure- Activity Relationship (SAR) studies show
that a large number of TSCs of an N-heterocyclic compound have low π density at the side
chain part and ring N atom should be reasonably a good electron pair donor to form
coordination compounds (Mylonas & Mamalis 2005).The N N S ligand system (Fig. 1b and
c) was a common feature for all compounds (Dilovic, Ruccic, Vrdoljak, Karaljevic& Cindric
1970), and also there is a strong correlation between tumor growth rate and the enzyme
Ribonuclieoside Diphospate Reductase (RDR)( Elford, Frees, Passamani & Morris 1970).
A considerable number of Thiosemicarbazones (TSC) derivatives have been reported
as antibacterial, antiviral and anti proliferative compounds. In particular, certain TSCs
showed a selective inhibition of herpes simplex virus (HSV) infection in vitro, based on their
inactivation of the ribonucleotide reductase enzyme (Blumenkopf et al. 1992; Prichard et al.
1995) and also TSCs were active inhibitors of HSV genital infection in guinea pigs and mice
in vivo(Sidwell, Huffman, Schafer & Shipman 1990). The effect of TSCs against human
immunodeficiency virus (HIV) was also reported to target the structural protein synthesis of
HIV(Teitz, Ronen, Vansover, Stematsky & Riggs 1994). In contrast, thiadiazolines,
compounds obtained by heterocyclization of TSCs, were reported only for their antibacterial
activity(Ali & Alam 1994).
Some synthetic analogues of thiosemicarbazone already exist in market like, Trapine,
and Marboran (shown in Fig. 1b). Trapine is potent ribonucleotidereductase inhibitor and
used in cancer treatment. 3-aminopyridine-2carboxaldehyde thiosemicarbazone (Trapine) is a
tridentate chelator that ligatse Fe via sulphur and two nitrogen atoms (Yu et al 2006;
Kalinowski & Richardson 2005). A recent study reported that the triapine-Fe (II) complex
was significantly more active at inhibiting ribo nucleotide reductase than free triapine(Shao,
Zhou & Di Billo 2006). 1-N-methylisatin-β-thiosemicarbazone (Marboran) is a good antiviral
agent and also a activity against pox viruses, Maloney leukaemia viruses and recently against
HIV(Sebastian, Desai, Shampur, Perumal, Sriram& Vasanthapuram 2008).
Studies on molecular interactions of drugs with biological molecules have
significantly contributed to the understanding of the structures and functions of
biomacromolecules and some biophysical processes. By binding to Human serum albumin
(HSA), most drugs circulate in plasma and reach the target tissues and their distribution is
mainly controlled by HSA (Peters 1995). Therefore, drug binding to proteins has become an
important determinant of pharmacokinetics. HSA is the most abundant protein in human

blood plasma and has high affinity to many endogenous and exogenous compounds, also
transporter for drugs and other organic molecules to their targets (Carter et al. 1989). For this
purpose, HSA is commonly used as a model protein for biophysical and physicochemical
studies(Sudlow et al. 1976; Curry et al 1998; ; Curry et al 1999).The globular protein consists
of a single polypeptide chain of 585 amino acid residues and is composed of three
structurally similar domains (I, II, and III) (Fig. 1a).
The changes in the photophysical characteristics interaction can be used to probe the
nature of the binding due to drugs with bio macromolecules .The results of binding studies
may be very useful in designing new and promising drugs for chemotherapy. In this context,
thiosemicarbzone derivatives (Fig. 1c) binding properties with serum albumin was carried out
using various optical spectroscopic techniques. Computational study, also describes the
binding region and affinity of various substrates with a serum albumin protein (Ding et al.
2012; Sugio et al. 1999).
Materials and Methods
Materials
Human serum albumin was purchased from Hi Media Laboratory Pvt. Limited,
Mumbai, India and it was used without any further purification. Millipore water was used for
preparing solution throughout the experiments. HSA solution was prepared in phosphate
buffer solution of pH 7.4. HSA solution was kept in the dark at 4ºC.4-[(1Z)-1-(2-
carbamothioylhydrazinylidene)ethyl]phenyl acetate (Ace semi),4-[(1Z)-1-(2-
carbamothioylhydrazinylidene)ethyl]phenyl propanoate(Pro semi) were newly synthesised
thiosemicarbazone derivatives, and the stock solution of these derivative was also prepared
using the same buffer.
Synthesis of 4-[(1-(2-carbamothioylhydrazinylidene) ethyl] phenyl acetate (Ace
Semi)
Step I: Acetic acid 4-acetyl-phenyl ester
Clean and dry 250ml two neck RB flask was taken and fitted with condenser
and addition funnel. 0.5mol of 4- hydroxyl aceto phenone was taken and 200ml of
chloroform was added to it with stirring. The reaction mixture was cooled at 5-10oC and
0.5mol of acetyl chloride was added drop wise to the reaction mixture. Stirring was continued
for another 15mins and 0.5mol of potassium carbonate was slowly added. Reaction was
continued for another 4h and monitored using TLC. The reaction mass was transferred into 1l
beaker and washed twice with water (2x250ml). The chloroform layer was separated and
washed with 10% NaOH solution (2x250ml). The chloroform layer formed was separated

and dried with anhydrous sodium sulphate. The chloroform layer was filtered and
concentrated under reduced presser using rotary vacuum, cooled and hexane was added to it.
Solid was precipitated which is filtered and the product was air dried (Fig. 2a).
Step-II : 4-[(1-(2-carbamothioylhydrazinylidene)ethyl]phenyl acetate
Acetic acid 4-acetyl-phenyl ester (0.1mole) was dissolved in 50 ml of ethanol and
thiosemicarbazide (0.1mole) was added to it. The reaction mixture was refluxed for 10-20
mins until a white solid was formed, the solid product was collected by filtration, washed
with Hexane and the product was air dried (Fig. 2b).
Synthesis of4-[(1-(2-carbamothioylhydrazinylidene) ethyl] phenylpropanoate (Pro
Semi)
Step I: Propionic acid 4-acetyl-phenyl ester
Clean and dry 250ml two neck RB flask was taken and fitted with condenser
and addition funnel. 0.5mol of 4- hydroxyacetophenone was taken and 200ml of chloroform
was added to it with stirring. The reaction mixture was cooled at 5-10oC. 0.5mol of propinoyl
chloride was added drop wise to the reaction mixture. Stirring was continued for another 15
mins and 0.5 mol of potassium carbonate was slowly added. Reaction was continued for
another 4h and monitored using TLC. The reaction mass was transferred into 1l beaker and
washed twice with water (2x250ml). The chloroform layer was separated and washed with
10% NaOH solution (2x250ml). The chloroform layer formed was separated and dried with
anhydrous sodium sulphate. The chloroform layer was filtered and concentrated under
reduced presser using rotary vacuum, cooled and hexane was added to it. Solid was
precipitated which is filtered and the product was air dried (Fig. 2c).
Step-II : 4-[(1-(2-carbamothioylhydrazinylidene)ethyl ]phenyl propanoate
Propionic acid 4-acetyl-phenyl ester (0.1mole) was dissolved in 50 ml of ethanol and
thiosemicarbazide (0.1mole) was added to it. The reaction mixture was refluxed for 10-20
mins until a white solid was formed, the solid product was collected by filtration, washed
with Hexane and the product was air dried (Fig. 2d).
Methods
Absorption spectra were recorded in the wavelength range of 190-400 nm using UV-
Visible absorption spectrophotometer (Perkin-Elmer Lamda35, Waltham, MA). The steady
state fluorescence emission measurements were obtained using a commercially available
spectrofluorometer (Fluoromax-2, ISA; Jobin-Yuvon-Spex, Edison, NJ) and the slit width

was maintained as 5 nm in both excitation and emission monochromators. The emission
spectrum was recorded in the wavelength region 300-540 nm at 280 nm excitation.
Synchronous fluorescence spectra were recorded by simultaneously scanning the excitation
(λex) and emission (λem) monochromators with two different constant wavelength intervals
(Δλ) such as 15 nm and 60 nm with the help of Fluoromax-2 equipped with a excitation
source (150W ozone free xenon arc lamp) coupled to the monochromators which delivers
light to the sample spot at a desired wavelength and the fluorescence emission from the
sample is collected by a photomultiplier tube (R928; Hamamatsu, Shizuoka-Ken, Japan). In
addition, using the absorption spectrum of thiosemicarbazone derivative, and fluorescence
spectrum of HSA, (Concentration ratio of drug and protein is 1:1 at pH 7.4) the fluorescence
resonance energy transfer and energy transfer efficacy of HSA with various concentration of
drug is also evaluated.
Fourier transforms infrared spectra (FTIR) of the protein solutions were carried out at
room temperature using FTIR spectrometer (Interspec 2020, Estonia) equipped with a
attenuated total reflection (ATR) accessory with resolution 4cm-1 and 32 scans. Spectra of the
sample solution and the buffer solution were collected at the same condition. Circular
Dichroism (CD) measurements of HSA in the presence and absence of thiosemicarbazone
derivative were made in the UV-region (200-260nm) on a spectropolorimeter (Jasco J715,
MD, USA) using a quartz cell of 0.1cm. The CD profiles were obtained by employing a scan
speed 20 nm/min. Appropriate baseline corrections in the CD spectra were made. All the
spectra were recorded at room temperature.
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The X-ray crystal structure of Human Serum albumin (PDB ID: 4L8U)( Wang et al.
2013) and thiosemicarbazone derivatives was taken for molecular docking studies. Structures
were energetically minimized using OPLS2005 force field in impact module and subsequent
minimized structure was used for flexible docking approach using Induced fit module (Glide)

of Schrodinger 2014. Binding mode of thisemicarbazone derivative with HSA was analysed
in terms of binding energy (Glide Energy).
Docked complex of thiosemicarbazone derivatives with HSA and as well as free drug
and free HSA was used for molecular simulation study for comparison of stability and
calculation of binding free energy using AMBER 12 (Case et al., 2012). To prepare the initial
system for simulation, the free HSA and their thiosemicarbazone derivatives complex was
protonated and solvated using TIP3P water box with a dimension of 10Å and counter ions
were added appropriately to make the total charge zero. The initial coordinates and
corresponding topology files were created using molecular mechanics (ff99SB) force field for
protein and using antechamber for thiosemicarbazone derivatives. The free HSA and their
thiosemicarbazone derivatives complex system were relaxed with the help of two phase of
energy minimization. The first phase of energy minimization was carried with 1000 cycle of
steepest descent and 1500 cycle of conjugate gradient with solute atom restrained by a
harmonic potential with force constant by 10Kcal/molÅ2. The second phase of energy
minimization was carried out using conjugate gradient method for 2500 cycle for full system
minimization without any restraint.
Further, the two phase of equilibration, first with positional restraint on solute atom
(protein and thiosemicarbazone derivatives) and other one without restraint were run for
bringing the temperature and pressure of each system with 300K and 1 atm respectively for
making isothermal-isobaric ensemble system. The temperature and pressure of each system
was stabilized around 300K using Barendsen temperature coupling (Thermostat) and
langevinbaro stat respectively. The equilibration was done for 1ns with 2fs integration time
step, all atoms belonging to solute atoms were restrained by harmonic potential with force
constant 10kcal/molÅ2.SHAKE algorithm was used to constrained hydrogen linked bonds.
The equilibrated system with periodic boundary condition was used to perform constant
temperature and pressure simulation for 15 ns with 1 fs integration time step using leag-frog
integrator. Simulation trajectory was saved on 1ps of simulation time and further used for
structural analysis.
Result and Discussion
UV–Vis absorption studies
Among various optical methods, UV–Vis spectroscopy is considered to be a simple
method to study the drug binding interaction(Yan-qing, Bo-ping, Hong-mei, Qiu-hua &Gen-
cheng 2009).Human Serum Albumin has the absorption sort band at 280 nm(Wang, Zhang &

Zhou 2009). Fig. 3a and 3b show the UV–Vis absorption spectra of HSA–Ace semi and
HSA-Pro semi system in the presence of thiosemicarbazone derivatives (Ace semi and Pro
semi). The absorbance of HSA is formed to increase with increase of the concentration of
Ace semi and Pro semi. Further it is observed that there is a blue shift in the maximum peak
position for both systems. Also a new peak appeared and increased at longer wavelength
between 300 and 400 nm, indicating that there is a strong interaction of thiosemicarbazone
derivatives (Ace semi, Pro semi) and HSA complex may form, the observed changes in the
absorbance of HSA–Ace semi and HSA–Pro semi complexes have indicated that an
interaction occurred between these drugs and HSA; and the considerable blue shift in
maximum peak positions may be accounted for the change in polarity around the tryptophan
residue and hence changes in hydrophobicity in both HSA-drug complexes(Katrahalli,
Jaldappagari, & Kalanur 2010).
Firstly, the UV–Vis absorption spectrum of HSA shows a strong band with a
maximum at 220 nm and a weak band with a maximum at 280 nm which arise from the
transition of n-π* of the characteristic polypeptide backbone structure C=O and the transition
of π-π* of the phenyl groups in tryptophan, tyrosine and phenylalanine in HSA, respectively.
The degree of complex formation between HSA and thiosemicarbazone derivative
was evaluated using equation 1
ꢀ
ꢁ
ꢂ
HSA + thiosemicarbazonederivative ⇔ HSA : thiosemicarbazonederivatie
[1]
[2]
K is the association constant and it is given as
K = [HSA:thiosemicarbazone derivative]/[[HSA][thiosemicarbazonederivative]]
The binding constant of complex formation were computed from the change in the
intensity to the absorption peak at 280nm, according to Benesi-Hildebrand relation,
1/ (Aobs-A0) = 1/ (Ac-A0) + 1/Kapp [Ac-A0] .1/ [thiosemicarbazone derivative]
[3]
Where A0 and A is the absorption of HSA solution at 1µM and HSA with different
concentrations of thiosemicarbazone derivative respectively. Ac is the final absorption of the
ligated HSA. The plot of 1/ (Aobs-A0) against 1/ [thiosemicarbazone derivative] is found to be
linear (Fig. 3c and d) and the value of the binding constant (K) for HSA-thiosemicarbazone
derivative complex is found to be 1.19×104M-1,0.70×104M-1 Complex formed between HSA

and thiosemicarbazone derivative (Ace semi, Pro semi) was evident from the data of UV-vis
absorption.
Fluorescence studies
Fluorescence spectroscopy is fundamentally a probe method for monitoring the local
environmental changes of the fluorophore, understanding the binding mechanisms of protein
to compounds and providing evidences to the nature of binding phenomenon(Ding, Huang,
Lin, Li, Liu, Jiang & Sun 2009) .HSA has only one tryptophan residue (Trp-214) located in
IIA sub domain and 20 tyrosine residues localized mostly in sub domain IB (Tyr 140, 148,
150, 156, 157) and IIIA (Tyr 401, 411, 452 and 497)( Domotor et al. 2014; Równicka-Zubik
et al 2009). Protein fluorescence is usually obtained by exciting at its absorption maxima
around 280 nm. The absorption of proteins at 280 nm is because of both tyrosyl and
tryptophanyl residues, whereas at longer wavelength at 295 nm, is mainly due to Trp
(Garland et al 2003). Fig. 4a and 4b shows the fluorescence emission spectra of HSA with
various molar ratios of Ace semi and Pro semi systems. HSA presented a strong fluorescence
emission with a peak at 351nm at 280 nm excitation, while the drugs have no intrinsic
fluorescence at this wavelength of excitation. The fluorescence emission intensity of HSA
decreased regularly with increase of the concentrations of Ace semi and Pro semi into the
HSA system (Kuznetsova IM et al 2012). The presence of Ace semi and Pro semi caused the
quenching of fluorescence of HSA. This indicated that the Ace semi and Pro semi interacted
with HSA. Further, it is observed that there is no notable shift at the maximum emission
wavelength, this suggests a decline in polarity, and that drug presence may be due to the
increase of hydrophobicity around tryptophan and folding of protein (Rinco et al 2009;
Zhang, Chen, Zhou & Wang 2009).
Determination of quenching mechanism
The quenching of fluorescence emission can be induced by different mechanisms,
which are in general classified into dynamic, static and it may be due to the combined
quenching mechanisms (Tang, Luan & Chen 2006).Dynamic quenching refers to collision
between fluorophore and quencher during the lifetime of the excited state. Static quenching
refers to the formation of the non-fluorescent complex of fluorophore–quencher(Zhang, Que
& Pan 2008).To examine the nature of fluorescence quenching mechanism induced by the
drugs, the well-known Stern–Volmer equation was used:
F0/F=1+kqτ0 [Q] =1+KSV [Q]
[4]

where F0 and F are the fluorescence emission intensity in the absence and presence of the
quencher (Ace semi, Pro semi), respectively, kq is the bimolecular quenching constant
(kq = KSV × τ0−1), τ0 is the lifetime of the fluorophore without the quencher (10−8 s), KSV is the
Stern–Volmer quenching constant, and [Q] denotes the concentration of the quencher (Qian
et al 2010). The Stern–Volmer curves (F0/F against [Q]) for the fluorescence quenching at
the excitation wavelength of 280 nm of HSA-Ace semi, HSA-Pro semi systems are indicated
in Fig. 4c and 4d. The results have displayed a good linear relationship (Hediyeh Iranfar et al
2012). As it can be seen the Stern–Volmer plot does not demonstrate any deviation from
linearity toward y-axis when the concentration of the drug is higher which can be the
evidence for the incidence of the static quenching(Wu, Ji, Wu & Wan 2006; Wei, Li, Dong,
Shuang, Liu & Huie 2006). The Stern–Volmer quenching constants, Kq at two different
temperatures (293K, 303K) were calculated for HSA-Ace semi complex and HSA- Pro semi
complex (Table 2). It is known that the maximum scattering collision quenching constant Kq,
of various quenchers with macromolecules is about 2.0 × 1010 l mol−1 s−1. As mentioned
above, the Kq values for all interacting systems could be observed that the quenching constant
was much higher than 2.0 × 1010 l mol−1 s−1.This clearly indicates that the nature of
quenching is not the result of dynamic collision quenching mechanism, rather its state is due
to the complex formation between HSA and both drugs (Cui, Wang, Cui & Li 2006).
Determination of number of binding sites
The relationship between the fluorescence peak area and total concentration of drug can be
determined using the following equation.
F0/F=K [Dt] F0/ (F0-F)-nK [Pt]
[5]
Where [Pt] and [Dt] are the total concentration of protein and drug respectively. Keeping
fixed protein concentration and varying the drug concentration, the binding constant and
number of binding site was obtained using least-squares algorithm. As depicted in Fig. 4e and
4f, the double-logarithmic plot yielded a straight line with a slope value of approximately
equal to 1 and the corresponding results are summarized in Table 2.
Thermodynamic analysis
Thermodynamic parameters are important for understanding the mechanism of a
biomolecule–ligand reaction. To evaluate the binding force between thiosemicarbazone
derivative and HSA, thermodynamic parameters, including the enthalpy change (ΔH°),

entropy change (ΔS°) and free energy change (ΔG°) of the interaction were determined using
the following equations
∆ꢈ°
∆ꢎ°
ꢃꢄꢅ ꢆꢇ = − ꢉ.ꢊꢋꢊꢌꢍ
+
[6]
[7]
ꢉ.ꢊꢋꢊꢌ
ΔG° = ΔH° - T ΔS° = -RT ln Ka
Where R is the gas constant. Two different temperatures of 298K and 303K were used.
In general, the interaction forces between small molecules and macromolecules may be
manifold including electrostatic, hydrogen bonding, van der Waals interaction, hydrophobic
and steric contacts within the binding sites. The signs and magnitudes of thermodynamic
parameters for the protein–ligand binding reaction have been invoked to provide a simple
way to account for the principal binding forces. Under the assumption of no significant
variation of the enthalpy change (ΔH°) within the range of temperature studied (JamshidKhan
Chamani et al 2011), both the enthalpy change (ΔH°) and the entropy change (ΔS°) for the
thiosemicarbazone derivative–protein interaction process can be evaluated by applying the
van’t Hoff equation [6]. The calculated thermodynamic parameters are summarized in Table
2. A negative value for ΔH° indicates the exothermicity of the binding reaction while a
negative free energy change (ΔG°) represents a spontaneous occurrence of the reaction. Ross
and Subramanian have previously demonstrated the characterization of various individual
kinds of interaction with thermodynamic parameters that may take place in the protein
association process.
The model of interaction can be summarized as follows on the basis of the thermodynamics
data:
(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 interaction.
The derived thermodynamic parameters presented in Table 2 suggest that the HSA–
thiosemicarbazone binding interaction is enthalpically as well as entropically favored (ΔH°<
0 and ΔS° > 0), with an overall negative Gibbs free energy change implying the spontaneity
for the occurrence of the interaction. Further, the negative enthalpy change along with a
positive entropy change indicates the predominance of electrostatic interaction for the HSA–
thiosemicarbazone derivative (Ace semi, Pro semi) interaction process.
Time resolved fluorescence measurement
The fluorescence lifetime measurement is the most definitive method for
differentiating static and dynamic quenching processes (Lakowicz et al. 2006). Time resolved

fluorescence lifetime measurement was carried out to substantiate the nature of quenching
mechanism due to the interaction of thiosemicarbazone derivatives with HSA system. The
time resolved fluorescence decay curve of HSA in the absence and presence of
thiosemicarbazone derivatives is depicted in Fig. 5a and 5b. (HSA-Ace complex, HSA-Pro
complex), the fluorescence decay curve of HSA is triple exponential with a lifetime value of
6.90 ns. Upon the addition of drug, the excited state lifetime of HSA-thiosemicarbazone
derivative showed considerable change (6.50 ns). It is well established that the static
quenching mechanism is generally associated with stable fluorescence lifetime values (Table
1)( Paramaguru, Kathiravan, Selvaraj, Venuvanalingam & Renganathan 2010).
Synchronous fluorescence spectral studies
Synchronous fluorescence spectroscopy method was introduced by Lloyd and Evett in
1971(Ding, Huang, Lin, Li, Liu, Jiang & Sun 2009).It provides information about the
molecular environment surrounding the chromospheres molecules (Ge, Chen, Liu, Han,
Xiong & Zhao 2010). In the synchronous spectra, the sensitivity of fluorescence is
maintained while presenting several advantages, such as spectral bandwidth reduction,
simplification of spectra and avoiding various perturbing effect(Yue, Zhang, Zhou, Qin &
Chen 2008).When the wavelength interval (Δλ) between excitation and emission wavelengths
is 15 and 60 nm, the synchronous fluorescence spectra provide the characteristics of tyrosine
(Tyr) and the tryptophan (Trp) residues of HSA, respectively (Xiang & Wu 2010). By
investigating the synchronous spectra of Tyr residues and Trp residues, the conformational
changes of HSA could be studied. The effect of the drugs on the synchronous fluorescence
spectra is displayed in Fig. 6. It can be seen from Fig. 6a and 6b that the addition of the drugs
led to decrease in the synchronous fluorescence intensity of Tyr in the systems of [HSA–Ace
semi complex] and [HSA–Pro semi complex] at Δλ = 15 nm. The maximum emission
wavelength of HSA – thiosemicarbazone derivative complexes had a weak blue shift (only
3 nm), which indicated that the polarity surrounding the tyrosine residues was decreased and
the hydrophobicity was increased. On the other hand Fig. 6c and 6d shows Trp systems of
[HSA-Ace semi complex] and [HSA- Pro semi complex] at Δλ = 60 nm and these results
show that no shift has occurred in the tryptophan region. Therefore, the conformation of HSA
may be altered upon the addition of the drugs in both HSA-Ace complex and HSA-Pro
complex during the binding process (Zhang, Li, Ge, Liu Jiang & Liu 2010).
Energy transfer between thiosemicarbazone derivative and HSA

The transfer efficiency can be used for the quantitative estimation of the distance
between the relevant protein residue (donor) and the bound ligand (acceptor). The energy
transfer efficiency between thiosemicarbazone derivative (Ace semi, Pro semi) and HSA was
studied according to the Forester’s energy transfer theory (Förster 1996). The Forester theory
shows that energy transfer is affected not only by the distance between the acceptor and the
donor, but also by the critical distance of energy transfer (R0),(Sattar Z et al 2012) which can
be calculated by using the following equation (7):
ꢑ
ꢌ
ꢐ
E=1-(F/F0); ꢏ = ꢒꢌ ꢓꢔ ꢕ
[7]
ꢑ
ꢑ
ꢐ
where E is the energy transfer efficiency, F and F0 the fluorescence intensities of bio
molecule in the presence and absence of quencher (Sareh Sarzehi et al 2010), r the donor–
acceptor distance and R0 is the critical distance where the transfer efficiency is 50%:
Equation (8)
ꢉ
ꢗꢛ
ꢗꢉꢘꢁ
ꢂ
ꢖꢋ = 8.79 × 10
ꢙ ꢚ ꢜꢝꢞ
[8]
2
Where K is the space factor of orientation, N the refractive index of the medium, F the
fluorescence quantum yield of donor, and J is the effect of the spectral overlap between the
emission spectrum of the donor and the absorption spectrum of the acceptor, which could be
calculated as:
Equation (9)
J λ = ∞ Fꢢ λ εꢣ λ λ dλ
[9]
ꢛ
ꢟ ꢠ
ꢟ ꢠ ꢟ ꢠ
ꢡ
ꢋ
Where FD (λ) is the fluorescence intensity of the donor at wavelength λ to λ +Δλ, with the
total intensity normalized to unity and εA (λ) is the molar extinction coefficient of the acceptor
at wavelength (λ).
Figure 7a and 7b show the overlap of the UV–vis absorption spectrum of thiosemicarbazone
derivatives, Ace semi and Pro semi respectively with the fluorescence emission spectrum of
2
HSA. In the present case, K = 2/3, N = 1.336 and φ = 0.188 for HSA (Cyril et al. 1961).
Eqs. (7), (8) and (9) gave the following values for [Ace-HSA complex, Pro-HSA
complex]: J = 1.703 × 10−21 cm3 L mol−1,6.721 × 10−21 cm3 L mol−1, R0 = 0.1782 nm,0.2238
nm, E = 0.0553,0.0233 and r = 0.28 nm, 0.41nm. Since, the average distance r < 7 nm
(Valeur et al. 1999) and 0.5R0 < r < 1.5 R0, the energy transfer from HSA to
thiosemicarbazone derivative occurred with high probability. The average distance is shorter

than 7 nm upon interaction of thiosemicarbazone derivative with HSA, which is well within
the conditions required for applying the Förster’s non-radiative energy transfer theory, again
supporting the static quenching interaction between thiosemicarbazone derivative and HSA.
Three-dimensional fluorescence spectroscopy
Three-dimensional fluorescence spectroscopy is another powerful method for
providing conformational and structural information of protein (Zhang, Que, Pan & Guo
2008). The three-dimensional fluorescence spectra of HSA – thiosemicarbazone
derivative(Ace semi, Pro semi) systems were shown in Fig .8(a,b,c) and the main peak
reveals that the spectral feature of Tyr and Trp residues. Because when HSA is exited at 276
nm, it primarily display the intrinsic fluorescence of Tyr and Trp residues, and the
phenylalaline residue fluorescence can be negligible. In the absence of thiosemicarbazone
derivative the relative fluorescence intensity of HSA (λem = 356 nm) (Fig 8a), and the
presence of thiosemicarbazone derivative(Ace semi, Pro semi) in HSA fluorescence intensity
were (λem = 355 nm, λem = 354 nm)(Fig 8b and 8c) respectively. After the addition of
thiosemicarbazone derivative in HSA the relative fluorescence intensity is almost changed in
both HSA-Ace semi,Pro semi complex systems, which indicates that the interaction between
HSA and thiosemicarbazone derivative (Ace semi, Pro semi) induced microenvironment and
confirmation change in HSA and this result was consistent with that of synchronous
fluorescence spectra (Ding et al. 2009; Guo et al. 2014).
FT-IR Studies
The FT-IR spectrum was recorded to verify the conformational changes in HSA due
to the binding of thiosemicarbazone derivative (Ace semi, Pro semi). Generally the amide I
peak of HSA lies between the regions of 1600-1700 cm-1 (C=O stretch) and amide II band in
the regions nearly 1500-1600 cm-1 (C-N stretch couple with N-H bending mode). It is noted
that these bands are related with secondary structure of protein. Among amide I and II, amide
I is more sensitive than amide II band (Chinnathambi et al 2014). From the FT-IR spectra
(Fig 9) it is observed that there is a slight decrease in the absorption of amide I of the protein
which may be due to the interaction of thiosemicarbazone derivative (Ace semi, Pro semi)
indicating that C=O stretching vibration of amide group is disturbed. From the Fig 9
interaction of thiosemicarbazone derivative with HSA resulted in the decrease of the
absorbance intensity without any shift which clearly indicates that the α-helix structure of
protein is not disturbed.

CD Spectral Analysis
The far UV CD shows a possible stabilization of the secondary structure of HSA on
interaction with thiosemicarbazone derivative (Ace semi, Pro semi). The CD spectra recorded
from 190nm to 260nm show the characteristic bands for HSA at 208 and 222 nm (Fig 10). On
addition of thiosemicarbazone derivative both the bands shows gain in negative intensity,
hinting at a more stable secondary structure. The α-helical content decreases from 54.31% for
pure HSA to 49.62% (HSA-Ace semi complex) and 48.43% (HSA-Pro semi complex) in the
presence of thiosemicarbazone derivatives. This is consistent with all of the experimental
results discussed so far. An interaction of protein with the thiosemicarbazone derivative
denatures the protein, which would in fact disturb the helicity and rearrange the secondary
structure of protein.
Molecular Docking and Simulation
Results from above fluorescence quenching and spectroscopic techniques suggest that
thiosemicarbazone derivative (Ace semi, Pro semi) have binding affinity against HSA with a
binding constant of KAce-semi =1.19×104 M-1, KPro-semi = 0.70×104 M-1 from UV- Vis
absorbtion studies. It suggest that Pro complex with HSA has stronger binding affinity
compared to Ace complex of HSA. To understand the mechanism of the binding
computational analysis were carried out (Karthikeyan et al., 2015). Molecular modelling is
very fruitful for understanding the binding interaction of thiosemicarbazone derivative with
HSA at atomistic level. Thus we carried out computational studies to analyse the binding
mode of Ace semi, Pro semi with HSA using molecular docking (Glide) and dynamic
simulation, which helps us in understanding stability of protein-ligand complex in terms of
thermodynamics parameter. Flexible docking (Induced Fit) result of thiosemicarbazones
with HSA suggest that both drugs favourably bind to lower region of proximal site I and II to
interact (Fig 11) with alpha helices H7, H8 and H9; similarly to cocrystal oncogenic drug
camptothecin with HSA (Pdbid: 4L8U) and Pro complex binds more favourable in terms of
glide energy and have hydrogen bond and hydrophobic interactions with active site such as
Arg117, Tyr138,Ile142,Phe149,Tyr161 and Gly189 (Fig 11)( Wallace, Laskowski, &
Thornton, 1995).
Simulation results of docked complex of thiosemicarbazone drug with HSA and as
well as free HSA were analysed in terms of potential energy, structural deviation of overall
fold of HSA and residual fluctuation. Potential energy graph of these drug complexes suggest
that complex are stable in their native solvent (water) environment (Fig 13). Root mean

square deviation (RMSD) and radius of gyration (RGYR) were calculated for each
conformation of thisemicarbazon with HSA and compared with free HSA with respect to
simulation time (Fig12a and b) which suggests that more structural restriction occurred in
Pro- HSA complex due to binding of drug in sub domain IB compared to free HSA and Ace-
HSA complex, and drug binding doesn’t disturbed the overall folding of HSA as represented
by radius of gyration and stable during simulation time. Residual fluctuations and b-factor in
backbone atoms (N, Cα and C) of drug complex with HSA suggest smaller conformation
fluctuation in active site residues (Arg 117,Tyr138,Phe149,Tyr161,Leu185 and Arg186)
occurred compared to that of Ace complex and drug free HSA (Figure 12 c and d). Hydrogen
bonding interaction with Pro-complex was more stable in terms of count of hydrogen bond
between drug and HSA during simulation time (Figure 14). Calculated binding free energy
using MMGBSA approach from simulated conformations Pro-HSA complex showed better
compared to Ace because of less standard deviation in free energy (Table 4) ( Garg et al.,
2013; Yeggoni et al., 2014). Besides, the Gibbs free energy (ΔG°) change from the
MMGBSA calculation (Table 4) has been almost same order of the thermodynamic
calculation (Table 2).
Conclusion
In summary, the interaction of thiosemicarbazone derivative with HSA has been
investigated using different optical spectroscopic techniques viz., UV-Vis absorption,
Fluorescence spectroscopy (Fluorescence emission, Synchronous, Excitation Emission
Matrix). From UV-Vis spectral characterization of HSA, it is observed that there is an
enhancement of absorption of HSA in the presence of thiosemicarbazone derivative (Ace
semi, Pro semi). This elevated absorption may be attributed to formation of ground state
complex due to intermolecular interaction. The observed fluorescence of HSA decreases in
the presence of thiosemicarbazone derivatives is due to the static quenching for the both
drugs. This is further confirmed from the fluorescence decay kinetics. From the results of
synchronous fluorescence spectroscopy, it is confirmed that the binding of thiosemicarbazone
derivatives may also induces conformational changes in HSA. The distance between Ace
semi-HSA and Pro semi-HSA (r =0.285 nm, 0.417 nm) was also calculated using
fluorescence resonance energy transfer. Besides, the Gibbs free energy (ΔG°) change from
the MMGBSA free energy calculation (Table 4) has been almost same order of the
thermodynamic calculation (Table 2). Computational analysis in addition to spectrometric
experiment has revealed significantly good binding affinity of HSA with thiosemicarbazone
derivatives.

Acknowledgments
The authors are thankful to Centre for Research, Anna University, Chennai for offering Anna
Centenary Fellowship (Lr. No. CRF/ACRF/ Jan. 2015/36) to Subramani Karthikeyan,
Department of Medical Physics, Anna University, Chennai-25. This study was supported
by the Board of Research in Nuclear Sciences, Department of Atomic Energy,
Government of India, Project no.2009/34/38/BRNS/3206.We also thankful for
Mr.karuppusamy shanmugam, for helping FTIR studies, Department of Physics, Anna
University, Chennai-25 , and also thankful to Dr. N. Nishad Fathima for allowing to utilize
CD spectroscopy, CLRI, Adyar, Chennai-600 020.
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Fig.1. (a).Human serum albumin (PDB ID : 4L8U) , chemical structure of
(b)triapine Marbone (c) thiosemicarbazone derivative.
Fig.2. Synthesis of 4-[(1-(2-carbamothioylhydrazinylidene) ethyl] phenyl
acetate (Ace Semi) Step I (a) Step II (b). Synthesis of4-[(1-(2-
carbamothioylhydrazinylidene) ethyl] phenylpropanoate (Pro Semi) Step
I (c) Step II (d).
Fig.3. The UV absorption spectra of HSA varying with the different
concentration of thiosemicarbazon derivative in the distilled water
solution at room temperature. Concentrations: HSA (0.1×10-5M), semi
derivative [Ace semi(a),Pro semi(b)] (0.5-7.5 µM). Benesi-Hildebrand
plot of HSA (0.1×10-5 M) in the presence of semi derivative [Ace
semi(c), Pro semi (d)] (0.5-7.5 µM).
Fig.4. Fluorescence emission spectra of HSA-semi derivative [Ace semi(a),Pro
semi(b)] (pH = 7.4). HSA (0.1×10-5M) in the presence of 0 to 7.5×10-5
M respectively. Stern Volmer plot at two different temperature(293K,
303K).[Ace semi(c), Pro semi(d)]. The plot of log (F0-F)/F versus Log
[Q] [Ace semi (e), Pro semi (f)].
Fig.5. The time resolved fluorescence decay profiles of HSA in absence and
presence of [Ace semi (a),Pro semi(b)] in Tris–HCl buffer of pH 7.4, 35
µM concentration for both drugs at 280nm excitation.
Fig.6. Synchronous fluorescence and its spectra, at ∆λ=15nm [Ace semi(a),Pro
semi(b)] and ∆λ=60nm [Ace semi(c),Pro semi(d)]) of HSA (0.1×10-5
M) in the presence of (0- 3.5) µM thiosemicarbazone derivative
respectively.
Fig.7. The overlap of the fluorescence emission spectrum of HSA and the
absorption coefficient spectrum of thiosemicarbazonederivative[Ace
semi(a),Pro semi(b)]). ([HSA]: [thiosemicarbazone derivative] =1:1), pH
=7.4.
Fig.8. The fluorescence contour maps of (a) HSA, (b) Ace semi-HSA and (c)
Pro semi-HSA. Concentration of HSA is 10 µM and both drugs is 30
µM.

Fig.9 FT-IR Spectra of HSA in the absence and presence of thiosemicarbazone derivative in
Phosphate buffer solution (pH=7.40) in the region of 1400-1800 cm-1.HSA = 10×10-
6, thiosemicarbazone derivative (Ace semi, Pro semi) = 10×10-6.
Fig.10 CD spectra of HSA(10µM) in the absence and presence of thiosemicarbazone
derivative(Ace semi(10µM), Pro semi(10µM)).
Fig.11. Shows the pymol and ligplot view of hydrogen bonding and
hydrophobic interactions of both Ace semi and Pro semi with HSA of
Native site [Ace semi (a) (b), Pro semi(c) (d), cocrystal(e) (f) ] with
HSA complex.
Fig.12. Shows the Molecular simulation of (a) Root Mean Square Deviation
(RMSD) (b) Radius of gyration(Rgyr) (c) Root Mean Square
Fluctuation(RMSF). Inset shows active site residues region (d) Root
Mean Square Fluctuation b-factor.
Fig.13. Potential energy plot for HSA with Ace semi, Pro semi complex.
Fig.14. Intermolecular interaction plot for HSA with Ace semi, Pro semi
complex.

Fig 1
(a)
(b)
(c)

Fig 2

Fig 3
Fig 4

Fig 5

Fig 6
Fig 7