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Biophysical Study on the Interaction between Eperisone
Hydrochloride and Human Serum Albumin Using Spectroscopic,
Calorimetric, and Molecular Docking Analyses
Gulam Rabbani,
†
Mohammad Hassan Baig,
†
Eun Ju Lee,
†
Won-Kyung Cho,
‡
Jin Yeul Ma,
‡
and Inho Choi*
,†
†
Department of Medical Biotechnology, YeungNam University, 280 Daehak-ro, Gyeongsan, Gyeongbuk-38541, Republic of Korea
‡
Korean Medicine (KM) Application Center, Korea Institute of Oriental Medicine (KIOM), Donggu, Daegu-41062, Republic of
Korea
ABSTRACT: Eperisone hydrochloride (EH) is widely used as
a muscle relaxant for patients with muscular contracture, low
back pain, or spasticity. Human serum albumin (HSA) is a
highly soluble negatively charged, endogenous and abundant
plasma protein ascribed with the ligand binding and transport
properties. The current study was undertaken to explore the
interaction between EH and the serum transport protein, HSA.
Study of the interaction between HSA and EH was carried by
UV−vis, fluorescence quenching, circular dichroism (CD),
Fourier transform infrared (FTIR) spectroscopy, Förster’s
resonance energy transfer, isothermal titration calorimetry and
differential scanning calorimetry. Tryptophan fluorescence
intensity of HSA was strongly quenched by EH. The binding constants (Kb) were obtained by fluorescence quenching, and
results show that the HSA−EH interaction revealed a static mode of quenching with binding constant Kb≈104reflecting high
affinity of EH for HSA. The negative ΔG°value for binding indicated that HSA−EH interaction was a spontaneous process.
Thermodynamic analysis shows HSA−EH complex formation occurs primarily due to hydrophobic interactions, and hydrogen
bonds were facilitated at the binding of EH. EH binding induces α-helix of HSA as obtained by far-UV CD and FTIR
spectroscopy. In addition, the distance between EH (acceptor) and Trp residue of HSA (donor) was calculated 2.18 nm using
Förster’s resonance energy transfer theory. Furthermore, molecular docking results revealed EH binds with HSA, and binding site
was positioned in Sudlow Site I of HSA (subdomain IIA). This work provides a useful experimental strategy for studying the
interaction of myorelaxant with HSA, helping to understand the activity and mechanism of drug binding.
KEYWORDS: circular dichroism, differential scanning calorimetry, eperisone hydrochloride, esterase-like activity,
human serum albumin, isothermal titration calorimetry, molecular docking, muscle relaxant
■INTRODUCTION
Eperisone hydrochloride (EH) is an antispastic agent that was
formulated by Japanese and is now commercialized in Japan,
Korea, India, and Far East under the trademark name Myonal.
In fact, Kim et al. recently reported that ∼25% of EH
prescribed in Korea is used in combination with aceclofenac
and is frequently prescribed for the treatment of muscular
pain.
1
EH reduces alpha and gamma afferent motor neuron
activities and inhibits spinal cord activities as demonstrated by
its action on the spinal cord and supraspinal structures.
1
EH is a
centrally acting, hydrophobic drug and acts as a muscle relaxant
and calcium antagonist with vasodilatory and antispastic effects
used to reduce spasticity.
2,3
Its treatment shows improvement
in conditions of myotonic situation caused by scapulohumeral
periarthritis, low back pain (LBP), neck−shoulder−arm
syndrome, and in paralysis.
4
Skeletal muscle is composed of tubular cells (myocytes or
myofibers), formed in a process known as myogenesis.
5
Skeletal
muscles are endowed with contractibility, extensibility,
elasticity, and excitability and constitute 40% of body mass.
6
Periphery and centrally acting myorelaxants are used to treat
muscle spasticity of neurological origin. EH is utilized for the
treatment of problems, such as acute LBP, and their medication
reduces the adverse effects on the central nervous system
(CNS).
7
EH was initially introduced for the treatment of
painful conditions caused by muscle contracture and is now
considered to be an antispastic agent with an improved safety
profile.
8−10
The effects of EH are believed to be due to the
blockade of Na+-channels.
11
EH related compounds such as
silperisone hydrochloride and tolperisone hydrochloride shows
significant inhibitory effects on the voltage-gated Na+/Ca+-
Received: December 14, 2016
Revised: March 3, 2017
Accepted: April 5, 2017
Published: April 5, 2017
Article
pubs.acs.org/molecularpharmaceutics
© 2017 American Chemical Society 1656 DOI: 10.1021/acs.molpharmaceut.6b01124
Mol. Pharmaceutics 2017, 14, 1656−1665
channels.
12
EH and its derivatives exert inhibitory effects on
spinal reflex through inhibition in the presynaptic release of
transmitter from primary afferent nerve fibers.
13
Their receptors
are localized on axonal terminals of afferent fibers.
14
As
reported in clinical trials of patients infected with myelopathy,
15
bladder dysfunction,
16
or muscle cramps in liver disease,
17
EH
did not show any sedative effect on the CNS.
A majority of drugs and small bioactive molecules reversibly
binds with HSA, which functions as a carrier molecule to them.
HSA often solubilizes long chain fatty acid in plasma, increases
their stability, and modulates their deliveries to cellular
receptors.
18
Most of the drugs reversibly bind to serum
albumin (SA) for their transport in the blood.
19,20
It has been
well documented that the interaction of drugs with SA in the
circulation determines their distributions, bound/free concen-
trations, metabolisms, and elimination.
21
SA serves as a channel
to carry the plasma insoluble lipophilic drugs to their target
sites.
19
Furthermore, drug−protein interactions increase half-
life of drug by preventing drug metabolism and elimination and
thus provide slow drug release.
19
However, the binding of drug
to their target sites is known to trigger structural changes, and
thus, it could potentially affect the biological functions of drug
bound proteins.
22
In the current study, we explored the binding between HSA
and EH by Trp fluorescence quenching, far-UV circular
dichroism (CD), Fourier transform infrared (FTIR) and
Förster’s resonance energy transfer (FRET). A comprehensive
thermodynamic characterization of HSA−EH complex has
been subsequently obtained by isothermal titration calorimetry
(ITC) and differential scanning calorimetry (DSC) study. The
EH induced functionality (esterase-like activity) of HSA was
assayed by measuring the hydrolysis of p-nitrophenyl acetate.
The binding location of EH within the HSA explored from
AutoDock-based molecular docking simulation. The findings of
this study will provide understanding of pharmacological and
structural changes underlying the binding effects of EH with
HSA.
■MATERIALS AND METHODS
Chemicals and Reagents. HSA (A1887; fatty acid and
globulin free) and p-nitrophenyl acetate (N8130) were
purchased from Sigma-Aldrich. Eperisone hydrochloride
(>99% purity index, Mw = 295.85) was from Santa Cruz
Biotechnology (Dallas, USA). HSA and EH solutions were
prepared by dissolving in Na-phosphate buffer (20 mM, pH
7.4). Before sample preparation HSA stock was dialyzed in 20
mM Na-phosphate buffer at 4 °C. The concentration of HSA in
buffer was determined from absorbance measurement using
molar absorption coefficient E280nm
1% = 5.3. The aqueous solution
of drug was prepared on the weight/volume (w/v) basis.
UV−Vis Absorption and Fluorescence Spectroscopy.
UV−vis absorption spectra of EH, HSA, and HSA−EH
complex systems were obtained using PerkinElmer Lambda
45 double beam UV−vis spectrophotometer, and temperature
of cell holder was controlled by a Peltier temperature
programmer (PTP-1).
The fluorescence quenching experiments were carried out on
a Varian Cary Eclipse fluorimeter and connected to a circulating
water bath. The deviation of fluorescence quenching was
experimentally recorded, and correction of inner filter effects
for fluorescence intensity in solution absorbance was calculated
by eq 1:
23
=×
+
FFe
AA
corr obs ()/2
ex em
(1)
where Fcorr and Fobs are corrected and observed fluorescence
intensities, respectively. The excitation and emission absorb-
ance wavelengths of HSA are shown by Aex and Aem,
respectively. In all emission spectral scanning both λex and
λem slit widths were set to 3.0 nm, and λex was set to 295 nm.
The HSA concentration was maintained at 5 μM in absorption
and 2 μM in Trp quenching measurements. However, the
quenching of Trp residues were analyzed by using Stern−
Volmer equation.
24
τ=+= +
F
FKQ k Q[] 1 [] 1
oSV q o (2)
where F0and Fare the fluorescence intensities in absence and
presence of the quencher. [Q] is the molar concentration of
quencher (EH), Stern−Volmer quenching constant is denoted
by KSV, the bimolecular rate constant of the quenching reaction
is denoted by kq, and τois the integral fluorescence lifetime of
tryptophan (5.78 ×10−9s). The modified Stern−Volmer
equation was used to determine the quantitative binding
constant (Kb) and binding stoichiometry (n) of the HSA−EH
complex:
−=+
⎡
⎣
⎢⎤
⎦
⎥
FF
FKnQ
l
og log log[
]
ob
(3)
The thermodynamics of HSA−EH interaction was calculated
directly from the binding constant data performed at various
temperatures. Within the studied temperature range, the
enthalpy (ΔH°) and entropy (TΔS°) changes were calculated
from the slope and intercept of the van’tHoffequation:
=−
Δ+Δ
◦◦
KH
RT
S
R
l
n
(4)
where Ris the universal gas constant (1.987 cal K−1mol−1) and
Tis absolute temperature in Kelvin.
The Gibbs free energy change (ΔG°) of the process was
determined using the relation:
Δ
=Δ − Δ
◦◦
◦
GHTS
(5)
ITC Measurements. Binding thermodynamics of EH to
HSA was performed at 25 °C on a titration microcalorimeter
(VP-ITC). The degassed HSA solution (15 μM) and Na-
phosphate buffer (20 mM, pH 7.4) were loaded in sample and
reference cells of the calorimeter, respectively. Degassed EH
solution (1.5 mM) was sequentially injected (10 μL in each
injection) into the sample cell containing HSA. The rotating
speed of injector was set at 307 rpm, and reference power of
ITC was set at 16 μcal s−1. The time duration of each injection
was 20 s, and delay time between next injections was 180 s. To
reduce the involvement of thermal effects due to EH, the
control experiments were carried by injecting EH solution into
the buffer solution in an identical manner, and resulting thermal
effects were subtracted from integrated data before curve fitting.
Far-UV CD Spectropolarimetry. A Jasco J-815 spectro-
polarimeter was used for CD measurements at 25 °C. The final
CD spectra are an average of four repeated scans performed
with an interval of 1 nm and scan speed of 50 nm min−1and
corrected with baseline. Solutions for baseline correction
(blank, 20 mM Na-phosphate buffer pH 7.4) were prepared
in the identical manner except that HSA was omitted. The CD
spectra of solutions (HSA + EH) were acquired, and changes in
far-UV CD spectral results were analyzed. The obtained CD
Molecular Pharmaceutics Article
DOI: 10.1021/acs.molpharmaceut.6b01124
Mol. Pharmaceutics 2017, 14, 1656−1665
1657
signals were converted into mean residual ellipticity (MRE)
using
=Θ
nCl
M
RE 10
obs
(6)
where Θobs is CD in millidegree, nis the number of amino acid
residues (n= 585−1), lis the path length of the cell in cm, and
Cis the molar concentration of HSA (2 μM).
Fourier Transform Infrared (FTIR) Spectroscopy. Infra-
red spectra were recorded on FTIR/FIR (Frontier; PerkinElm-
er) spectrophotometer using an attenuated total reflection
(ATR) sampling accessory. Solutions of HSA and HSA−EH
complex were loaded in the well and data measurement
acquired at 25 °C in the range of 1700−1600 cm−1. Typically,
40 scans were collected and averaged for a single spectrum with
resolution of 4 cm−1. The background was corrected before
scanning the samples. The FITR spectra of HSA were collected
in the absence and presence of EH and then absorbance of
buffer were subtracted from the spectra of HSA and HSA−EH
complex. The molar ratio of HSA/EH was 1:5 and the used
concentration of HSA was 75 μM.
Calorimetric Measurements for Thermostability. The
thermal unfolding was investigated by differential scanning
calorimetry (DSC) in 20 mM Na-phosphate buffer (pH 7.4)
between 20 to 90 °C at a scan rate of 1.0 °C min−1. The
thermal unfolding experiments were carried out using 15 μM
HSA, and the molar ratio of HSA/EH was 1:5. Individual buffer
scan (baseline) was determined before each sample scan under
the same experimental conditions. The collected excess heat
capacity curves were subtracted from buffer scans and
normalized the subtracted curves by the used HSA concen-
tration before curve fitting. The normalized excess heat capacity
curves were analyzed according to a non-two-state model using
Origin 7.0 software to calculate the calorimetric enthalpy
(ΔHcal), van’tHoffenthalpy (ΔHvH), and melting point
temperature (Tm).
Esterase-Like Activity of HSA. Changes in the esterase-
like activity can be assessed with spectrophotometric method.
The 50 mM p-nitrophenyl acetate (p-NPA) stock solution was
prepared in acetonitrile. 5 μM HSA was incubated at various
HSA/EH molar ratios (1:0, 1:5, and 1:10) for 12 h. The
concentration of the substrate (p-NPA) ranged from 0.1 to 0.7
mM. The absorbance of colored reaction product of p-NPA
with HSA was recorded at 405 nm by measuring the emergence
of p-nitrophenol measured over 2 min. The enzyme activities
are reported as observed absorbance differences between final
and initial. Initial reaction velocities (v0) were determined from
the linear portion of graph between 0 and 2 min.
ν
=+
VS
KS
[]
[]
0max
m(7)
where v0and Vmax are initial and maximum velocities,
respectively, [S]isp-NPA concentration, and Kmis the
Michaelis−Menten constant. Lineweaver−Burk reciprocal plot
was constructed by plotting 1/v0against 1/[S] at varying
concentration of p-NPA in the absence or in the presence of
EH:
ν=++
K
VSV
1
[]
1
0
m
max max (8)
In Silico Studies. Molecular docking was performed to gain
an insight of EH binding within the active site of HSA. The
crystal structure of HSA was retrieved from the Protein Data
Bank (PDB code: 2BXB). The structure was purified by
removing all the heteroatoms and solvent molecules. This clean
structure of HSA was further subjected to energy minimization
using the Charmm force field. The structure of EH (pubchem
id: 123698) was downloaded from the Pubchem compound
database. EH was docked within the active site of HSA using
Autodock 4.0 and the Lamarkian genetic algorithm. The total
number of runs was set at 15. Optimal docking was selected
based on considerations of binding free energy.
Accessible Surface Area Calculations. Differences
between the accessible surface areas (ASAs) of HSA in native
and in complex with EH were calculated using NACCESS
version 2.1.1.
25
■RESULTS AND DISCUSSION
Absorption Spectroscopic Studies for Physical
Changes upon EH Interaction. The absorption spectral
changes can be used to investigate the structural alteration, after
drug−protein complexation. The UV−vis absorption spectra of
HSA and HSA−EH complex are shown in Figure 1A. Native
HSA exhibited a strong absorption peak at 278 nm, which was
mostly recognized as the absorptions of Trp and Tyr.
26
The
maximum absorption wavelength increases with blue shift after
addition of increasing concentration of EH.
27
The results
indicate that observed changes in the absorbance of HSA−EH
complexes clearly assigned the strong interaction between EH
and HSA. The significant blue shift in maximum peak positions
was possibly caused by a change in the polarity, and hence
hydrophobicity increases, around the tryptophan residue.
27
Reformation of Secondary Structure of HSA Studied
by CD and FTIR Spectroscopy. CD spectroscopic studies
were carried out to explore the secondary structural change in
HSA after binding of EH. In this work, molar ratios ([HSA]/
[EH]) of 1:0, 1:5, and 1:10 were used. The far-UV CD
spectrum of native HSA gives two negative peaks at 208 and
222 nm, a characteristic feature of α-helix structure of HSA
(Figure 1B). A logical elucidation of two negative bands 222
nm (contributed to n−π*transfer of α-helical structure of
protein) and ∼208 nm (contributed to π−π*transfer of α-helix
of protein) shows an interrogating secondary structure such as
α-helix.
28,29
The outcome was in agreement with the previous
finding, advocating the dominance of α-helix conformation
(65−67%) in native HSA.
28
As shown in Figure 1B, the rise in
the molar ratio of EH (from 1:0 to 1:10) induces the α-helical
Figure 1. (A) UV−visible absorption spectra of HSA in the absence
and presence of eperisone hydrochloride at different concentrations.
(B) Circular dichroic spectral profiles of HSA with increasing EH
concentration.
Molecular Pharmaceutics Article
DOI: 10.1021/acs.molpharmaceut.6b01124
Mol. Pharmaceutics 2017, 14, 1656−1665
1658
percentage of HSA.
29
Our finding showed that free HSA
contained 48% α-helix, and after complexation with 10 and 20
μM of EH, the proportion of α-helix of HSA increases from
48% to 54% and 59%, respectively. An increase in the α-helix
structure was also reported for a flavonoid diosmetin/HSA
interaction.
30
From the above results, it was evident that EH
caused secondary structure changes in the HSA and increased
the α-helix stability.
Amide I and II bands are two major frequencies in the IR
region; the frequencies of amide I band (1700−1600 cm−1) are
more responsive to small vibrations in molecular geometry in
the secondary structure of proteins, giving rise to CO
stretching frequency.
31
The FTIR of free HSA and HSA−EH
complex at 1:5 molar ratio showed presence of 52% α-helix,
18% β-sheet, 14% turn, 12% disordered, and 4% β-antiparallel
in free HSA. The composition of α-helix was increased to 55%
in HSA−EH complex (figure not shown), which agrees with
the previously reported data.
32
Thus, the percentage of α-helix
tends to increase as a result of binding of EH with HSA. This
finding confirms an agreement with the result obtained by CD
experiments. The retention of the secondary structure of α-
helix of HSA is the primary requirement for biomedical
applications.
Steady-State Fluorescence Quenching of HSA in the
Presence of EH. Fluorescence quenching provides useful
information on the specific ligand binding sites in macro-
molecules. Intrinsic fluorescence probes are provided by
aromatic amino acids, allowing to access the conformation of
protein as well as protein dynamics and their intermolecular
interactions. HSA excitation wavelength of 295 nm was used to
selectively excite the Trp residue and emissions at 340 nm were
examined. A concentration-dependent regular decrease in the
emission signal of HSA suggests Trp residue is situated at or
nearby the drug binding site. In addition to the accessibility of a
fluorophore by a ligand, protein conformation changes can also
be estimated
33
or inferred by reduced Trp emission induced by
a nearby quencher residue, such as His or Arg.
34
Due to the
presence of only one Trp residue (W214) in the Sudlow site I
of HSA, this residue is often used as an intrinsic probe to
investigate the interaction of ligands with HSA.
35,36
Binding Affinity of EH with HSA. The plot of F0/Fvs [Q]
is presented in Figure 2A, and its slope obtained by linear
regression yielded the Stern−Volmer constant (Figure 2A), as
previously described.
37
The plots were linear and the rise in
temperature causes decrease in the slopes, indicating that
quenching phenomena was static rather than dynamic. Stern−
Volmer constants were found to fall in the range 104−105M−1,
which is in accord with those reported for drug/protein
interactions (104−105M) in vivo.
38
Furthermore, from eq 3,
intercepts and slopes provide the binding constant (Kb) and
binding stoichiometry (n) of EH with HSA (Figure 2B). The
Kbvalues as shown in Table 1 were of 4 orders of magnitude,
indicating a strong binding between HSA and EH.
Type of HSA Quenching by EH. Usually, the fluorescence
quenching of protein by small molecules can be either dynamic
or static. In dynamic quenching, rise in temperature causes
faster diffusion and more quantity of collisions, which increases
quenching constants, while in static quenching, rise in
temperature weakens the complex stability maintained by
intermolecular forces (H bonding and van der Waals
interactions) and henceforth reducing the quenching constants,
providing strength to hydrophobic effect.
39
The value of the
bimolecular rate constant (kq) was calculated by considering
the fluorescence lifetime of Trp 5.78 ×10−9s (as discussed in
UV−Vis Absorption and Fluorescence Spectroscopy). The
values of obtained kqwere in order of 1.6 ±0.12 ×1013, 1.3 ±
Figure 2. (A) Stern−Volmer plots and (B) double-logarithm plot for the quenching of HSA by EH at four different temperatures.
Table 1. Binding and Thermodynamic Parameters of HSA and EH at 25, 30, 37, and 42 °C Obtained from Fluorescence
Quenching Experiments
a
parameters 25 °C30°C37°C42°C
n(binding stoichiometry, HSA/EH) 0.93 ±0.11 1.0 ±0.13 1.0 ±0.16 1.1 ±0.12
KSV (Stern−Volmer constant, M−1) 9.7 ±0.41 ×1047.9 ±0.34 ×1046.1 ±0.29 ×1044.8 ±0.26 ×104
Kb(binding constant, M−1) 0.49 ±0.11 ×1050.99 ±0.16 ×1051.0 ±0.32 ×1051.7 ±0.23 ×105
kq(bimolecular quenching rate constant, M−1s−1) 1.6 ±0.12 ×1013 1.3 ±0.16 ×1013 1.0 ±0.13 ×1013 0.83 ±0.14 ×1013
ΔH°(binding enthalpy, kcal mol−1) 11.0 ±1.5
TΔS°(entropy change, kcal mol−1) 17.5 ±0.24 17.8 ±0.34 18.2 ±0.18 18.5 ±0.38
ΔG°(Gibbs free energy change, kcal mol−1)−6.5 ±0.13 −6.8 ±0.18 −7.2 ±0.25 −7.5 ±0.31
a
The data are the means ±standard deviations of three independent trials.
Molecular Pharmaceutics Article
DOI: 10.1021/acs.molpharmaceut.6b01124
Mol. Pharmaceutics 2017, 14, 1656−1665
1659
0.16 ×1013, 1.0 ±0.13 ×1013, and 0.83 ±0.14 ×1013 M−1s−1
at 25, 30, 37, and 42 °C, respectively (Table 1). The obtained
kqvalues of HSA−EH system are in the range of 1013 M−1s−1,
which is much larger than the upper limit of scattering
quenching constant, kqof various quenchers with the
biopolymers in aqueous solution (2 ×1010 M−1s−1).
40
These
findings confirm the interaction between HSA and EH appear
to occur through static quenching. The static quenching refers
to formation of nonfluorescence complex between the
fluorophore and quencher. The collisional (dynamic) quench-
ing is differentiated from static one because type of quenching
can be determined in response to temperature and viscosity
changes.
41
Thermodynamic of Complexation between HSA and
EH. The thermodynamic parameters of protein−drug inter-
actions at different temperatures can be exploited to identify the
major forces that contributes to protein−drug complex
formation.
42
To identify the main driving forces involved in
HSA−EH complex formation, a van’tHoffplot was generated
using previously calculated Kbat four different temperatures
(inset of Figure 2B). The thermodynamic parameters (ΔH°
and ΔS°) were determined from the slope and intercept of eq 4
(van’tHoffplot inset of Figure 2B). It was observed that the
formation of HSA−EH complex was an exothermic process
with large amount of positive enthalpy and entropy, suggesting
the dominance of hydrophobic interactions in the formation of
complex. Here, positive value of TΔS°signals a strong
indication of expulsion of water molecules from the binding
site. However, hydrophobic interactions are involved because of
the internalization of W214 after addition of EH.
43
The
changes in thermodynamic parameters are summarized in
Table 1 and reveal that the binding process between HSA and
EH is entropy driven (ΔH°> 0 and ΔS°> 0). Negative Gibbs
free energy change (ΔG°) in the present study reveals that the
binding process was spontaneous (−6.5 ±0.13, −6.8 ±0.18,
−7.2 ±0.25, and −7.5 ±0.31 kcal mol−1at 25, 30, 37, and 42
°C, respectively). The positive values of TΔS°and ΔH°suggest
hydrophobic interaction plays a major role in the binding of
EH.
44
Isothermal Titration Calorimetric Measurements. EH
interaction with HSA was studied by ITC at 25 °C(Figure 3).
The obtained titration data allows the calculation of binding
affinity (Ka), thermodynamics of binding, i.e., entropy changes
(ΔS°), enthalpy changes (ΔH°), and binding stoichiometry
(n). After correction of heat of EH dilution effect the ITC data
was analyzed with the single site binding model. In Figure 3
each of the heat burst peaks corresponds to a single injection of
EH into the HSA solution in the calorimeter cell (upper panel).
The lower panel corresponds to the corrected heats per mole of
injection, plotted against molar ratio of HSA/EH. The
calorimetric titration profile of EH with HSA resulted in the
negative heat deflection, indicating binding was an exothermic
reaction (Figure 3). The value of the binding constant (Ka) was
in order of (2.73 ±0.14) ×104M, and the binding
stoichiometry (n) was 0.970 ±0.10. A large negative value of
enthalpy change (ΔH°=−31.2 ±1.1 kcal mol−1) indicates
dominance of electrostatic interaction and hydrogen bonding
between lone pair electron of the benzene ring of EH molecule
and amino acid residues of HSA.
37
Similarly, negative value of
entropy change (TΔS°=−24.5 ±0.61 kcal mol−1) suggests
favorable electrostatic, van der Waals forces, and redistribution
of the hydrogen bonding network between EH and HSA.
42
The
calculated value of ΔG°is negative (−6.7 ±0.2 kcal mol−1)
indicating the interaction process was spontaneous.
Förster Energy Transfer between HSA and EH. The
overlap spectra of fluorescence of HSA and absorption of EH
indicates the transfer of energy from HSA (donor i.e Trp) to
EH molecule (acceptor) (Figure 4). Förster’s resonance energy
transfer (FRET)
38
was employed to calculate the binding
distance (r) and the energy transfer efficiency (EFRET) using
equation:
=− = +
⎛
⎝
⎜⎞
⎠
⎟
EF
F
R
Rr
1
FRET
0
06
066 (9)
Figure 3. ITC profiles for the binding of EH to HSA. The top panels
represent raw data for the sequential injection of EH solution into
HSA solution. The bottom panels show integrated heat data after
correcting of heat of EH dilution. The data points (●)reflect
experimental injection heats, while the solid lines represent the
calculated fit of the data.
Figure 4. Overlap of the fluorescence emission spectrum of HSA with
the absorption spectrum of EH. The molar ratio of protein and EH
was 1:1. The protein concentration was 2 μM.
Molecular Pharmaceutics Article
DOI: 10.1021/acs.molpharmaceut.6b01124
Mol. Pharmaceutics 2017, 14, 1656−1665
1660
where Fis fluorescence intensity of HSA with EH and F0is
fluorescence intensity of HSA without EH. R0is critical distance
at which the EFRET is 50%. The value of R0can be calculated
from donor emission and acceptor absorption spectra using
equation:
φ=×
−−
R
Kn
J
8.79 10
062524 (10)
In eq 10 K2is the orientation factor related to the geometry of
the donor and acceptor molecule, nis the average refractive
index of the buffer, φdenotes the fluorescence quantum yield
of HSA (donor), and Jis the degree of spectral overlap integral
between donor and acceptor. The value of Jwas calculated by
following equation:
∫
∫
λελλ λ
λλ
=
∞
∞
J
F
F
()() d
()d
0
4
0(11)
For the ligand−HSA interaction, K2= 2/3, n= 1.336, and φ=
0.15.
38
Using the eqs 9−11, the values obtained to be J= 2.23
×10−15 cm3M−1,R0= 1.98 nm, EFRET = 0.35, and r= 2.18 nm.
Values of F0= 450 and F= 290 at 25 °C are taken to calculate
the EFRET. Thus, R0was 1.98 nm and r= 2.18 nm for HSA−EH
system is within the scale of 2−8 nm. The relation 0.5R0<r<
1.5R0, validates that energy transfer occurred between HSA to
EH, and findings are in good correlation with high possibility of
complex formation between HSA and EH indicative of static
quenching.
Differential Scanning Calorimetry of HSA and HSA−
EH Complex for Thermostability. The effect of EH on the
thermal stability of HSA was measured by DSC and heat
capacity (Cp) vs temperature curve is shown in Figure 5. The
measured transition temperature and the enthalpies of thermal
unfolding of HSA are summarized in Table 2. In the absence of
EH, HSA has a first peak at Tm
1= 57.4 ±0.03 and a second
peak at Tm
2= 70.2 ±0.21 °C(Figure 5A). The higher Tmvalues
show the thermogram shifted toward higher temperature; first
endothermic peak reflects Tm
1= 59.4 ±0.01 and a second
endothermic peak reflects Tm
2= 70.4 ±0.98 °C in comparison
to native HSA peaks, which confirms the binding of EH
affected thermal stability of HSA. As shown in Figure 5B, a
downward shift in Cpafter addition of EH indicates reduction
in the hydrophobic surface area of HSA, which agreed well with
our fluorescence results. Corresponding results for the HSA−
EH complex revealed experimental Cpprofiles can be
deconvoluted using two components up to 70 °C, which
suggest stabilization of the IA−IB−IIA domain induced by
ligand binding impacts of the other energetic domain. After
denaturation, the protein started to aggregate and the thermal
transition became irreversible, which agrees with previous
studies.
45,46
The ratio of ΔHvH/ΔHcal is an index to measure
the transition process to the unfolding states during thermal
denaturation.
47
According to obtained van’tHoffand
calorimetric enthalpy we calculated the ΔHvH/ΔHcal ratio,
and native HSA gave the highest Rvalue (Table 2). It is known
that ΔHvH/ΔHcal ratio shows two things: (i) increase the
distance and weak interactions between interacting domains
48
and (ii) the weak internal forces that governs the stability of
proteins.
49
The calculated enthalpy ratios (ΔHvH/ΔHcal),
defined as if obtained ratios, are greater than unity mean
greater distances and smaller intramolecular forces for native
HSA domains as compared to HSA−EH complexes. The
differences in ΔHcal of the uncomplexed and complexed HSA
(225.9 ±1.85 and 128.2 ±0.86 kcal mol−1) suggest the extent
of exposure of hydrophobic region caused by thermal
unfolding.
Effect of EH Binding on Esterase-Like Activity of HSA.
HSA exhibits esterase-like activity for the hydrolysis of various
compounds, such as p-NPA, esters, amides, and phos-
phates.
19,50
A variety of drugs inhibiting the prominent
esterase-like activities of HSA are very closely related to the
binding sites of drugs with the substrate (p-NPA). The kinetic
parameters (Kmand Vmax) for hydrolysis were estimated by
fitting initial velocity versus (p-NPA) concentration to the
Michaelis−Menten equation using eq 7, shown in Figure 6A. In
addition, the reciprocal of the substrate (p-NPA) concentration
and initial velocity was plotted in the form of a Lineweaver−
Burk plot (Figure 6B). The calculated values of kcat and Kmare
Figure 5. (A) Calorimetric melting profile of HSA (black line) at pH
7.4, and the best fit of the curves to the non-two-state transition model
(thin red line). (B) Thermal unfolding profile of HSA and eperisone
hydrochloride (black line) in the presence of 15 μM HSA and 75 μM
eperisone hydrochloride (1:5 molar ratio).
Table 2. Thermodynamic Parameters for the Thermal
Unfolding of HSA and the HSA−EH Complex Obtained by
Differential Scanning Calorimetry at pH 7.4
a
transition parameters native HSA HSA−EH complex
1st transition Tm
157.4 ±0.033 59.4 ±0.01
ΔHcal
1225.9 ±1.85 128.2 ±0.86
ΔHvH
174.2 ±0.49 104.5 ±0.47
R10.32 0.81
2nd transition Tm
270.2 ±0.21 70.4 ±0.98
ΔHcal
247.3 ±1.95 62.1 ±0.98
ΔHvH
267.3 ±2.7 60.6 ±0.99
R21.42 0.97
a
R1or R2=ΔHvH/ΔHcal.Tmis expressed in °C. ΔHis expressed in
kcal mol−1.
Molecular Pharmaceutics Article
DOI: 10.1021/acs.molpharmaceut.6b01124
Mol. Pharmaceutics 2017, 14, 1656−1665
1661
listed in Table 3. The R410 and Y411, critical amino acid
residues of HSA located in the center of site II (subdomain
IIIA), are participating in its esterase-like activity.
51
The
catalytic function of HSA toward p-NPA was investigated to
determine the involvement of R410, Y411, and K199 residues
in the binding of EH to HSA. Y411 is the first amino acid
residue of HSA to be acetylated rapidly by p-NPA.
52
Kinetic
constants for hydrolysis of p-NPA by HSA gives Kmvalues of
25 ×10−2,48×10−2, and 63 ×10−2mM at a molar ratio of
1:0, 1:5, and 1:10, respectively (Table 3). Vmax remained the
same at all studied molar ratios suggesting that EH interacts
with the same catalytic part of HSA. The increase in Kmand no
change in Vmax indicate the inhibitor (EH) binds with active site
of HSA where substrate usually occupies and exhibits
competitive type of inhibition. The large enhancement in Km
after the binding of EH indicates that conformational changes
of structural element (secondary and tertiary structure) in HSA
are occurring, as results obtained by the fluorescence and far-
UV CD spectral measurements. The EH-induced structural
changes detected in HSA caused partial denaturation of the
HSA. The rate-determining step, which limits the Vamx of the
enzymatic reaction (expressed as kcat) and the propensity of the
substrate turn over to product (Km), is calculated by ratio of
kcat/Km. The HSA used in this study was defatted and displayed
a significantly higher enzymatic activity (28 ×10−2min−1) than
that of HSA (30 ×10−2min−1), as determined by Watanabe et
al.
51
The reduction in catalytic efficiency (kcat/Km) indicates
that EH binds in active site of HSA and induces the
conformation that is more favorable for catalysis associated
with high Kmvalues
Molecular Docking of Eperisone Hydrochloride and
HSA. Molecular docking study was performed to examine the
intermolecular interactions between the amino acid residues in
the subdomain IIA cavity of HSA and EH (Figure 7A). EH,
being a fairly bulky molecule, was found to get accommodated
in the cavity near W214 residue and make a favorable
interaction profile within this cavity. EH was found to interact
with a binding free energy of −6.8 kcal mol−1, which is
confirmed with experimental results (−6.7 ±0.2 kcal mol−1
obtained by ITC and −6.5 ±0.13 kcal mol−1by fluorescence
quenching, respectively). The ΔGvalues obtained from three
different methods (molecular docking, fluorescence quenching,
and calorimetry) are literally the same. This indicates that
obtained docking energy is verified with fluorescence
quenching and calorimetric results. Within the interaction
cavity, EH interacts via hydrogen bonds with R257 (zoom of
Figure 7B). EH was surrounded by the hydrophobic cavity of
subdomain IIA lined with the following amino acid residues;
Y150, K199, L219, F223, L238, H242, R257, L260, I264, I290,
and A291 (Table 4). Docking studies revealed R257 form
hydrogen bonding with the oxygen atom of EH at distance of
2.88 Å. While several binding site residues of HSA were
showing hydrophobic interaction with EH in the range of of
3.28−3.89 Å. The carboxyl groups of the cyclic anhydride form
hydrogen bonds with the K199 residues, with a distance of 3.75
and 3.54 Å. These findings support the ITC results, showing
Figure 6. (A) Relationship between initial velocity v0(mM min−1) and
substrate (p-NPA) molar concentration [S]. Values were fitted in
Michaelis−Menten plot to determine Kmand Vmax.(B)Line-
weaver−Burk plots: inverse of initial reaction velocity versus inverse
of substrate concentration (1/v0vs 1/[S]) for HSA in absence or
presence of different concentrations of EH.
Table 3. Michaelis−Menten Kinetic Parameters of HSA in the Presence of Increasing EH Concentrations
a
HSA/drug RA (%) Vmax (mM min−1)Km(mM) kcat (min−1)kcat/Km(mM−1min−1)
1:0 100 14.0 ×10−425 ×10−228 ×10−21.12
1:5 98 14.6 ×10−448 ×10−229 ×10−20.60
1:10 94 15.3 ×10−463 ×10−230 ×10−20.47
a
All measurements were carried out in 20 mM Na-phosphate buffer pH 7.4 at 37 °C. Values of Vmax and Kmwere derived from Lineweaver−Burk, eq
8. RA, relative activity. kcat/Km, catalytic efficiency. kcat, catalytic constant (Vmax =kcat ×enzyme concentration). The concentration of HSA was 5 μM.
Figure 7. (A) Molecular docking of EH with HSA: HSA is represented
as a cartoon. (B) The ligand structure is represented by green color.
The important interacting residues of HSA are shown in ligplot.
Molecular Pharmaceutics Article
DOI: 10.1021/acs.molpharmaceut.6b01124
Mol. Pharmaceutics 2017, 14, 1656−1665
1662
involvement of hydrogen bonds between EH and binding site
residues of HSA.
Molecular docking analysis revealed that the major ligand
binding region of HSA is located in the hydrophobic cavity of
Sudlow Site I.
53,54
Table 4 lists the binding score of EH and
HSA. The ligand binding site in HSA was found to be located
in a hydrophobic cavity surrounded by 11 amino acids. EH
binding is dominated largely by the hydrophobic interactions.
Change in the Accessible Surface Area of HSA. The
accessible surface areas of the residues were calculated for HSA
and HSA/EH complex. Comparisons of the ASA changes
caused by binding provide insights of the goodness of packing
of amino acid residues in a protein structure and their
importance with respect to ligand binding.
55
Residues that lose
more than 10 Å2of accessible surface area upon switching from
the uncomplexed to complexed state are considered to be
actively involved in the interaction.
56,57
Several studies have
reported the use of this approach to reveal important binding
residues.
25
Table 5 presents the total change in the accessible surface
area (ΔASA) of HSA upon moving from the uncomplexed to
complexed state with EH. The uncomplexed HSA had a total
ASA of 27126.46 Å2, which was reduced to 26938.87 Å2after
binding with EH. This large reduction in accessible surface area
provides solid support for the effectiveness of EH binding to
HSA. This finding shows that EH binds effectively and tightly
to the active site of HSA. The ΔASA values for important
binding site residues before and after complex formation were
also calculated (Table 5). Several interacting residues lost more
than 10 Å2of accessible surface area after complex formation.
For example, R257 showed an ASA reduction from 23.30 to
8.03 Å2, and other interacting residues, i.e., Y150, K199, L219,
F223, L238, H242, L260, I264, I290, and A291 also showed
large reductions in ASA, which suggests a large change in the
microenvironment of HSA. Therefore, this finding is identical
to well explained fluorescence quenching of HSA in the
presence of EH.
Table 4. Binding Efficacy of Eperisone Hydrochloride against HSA and the Amino Acid Residues Involved in Their Complex
Formation with EH and the Distance between Specific Atoms of EH with Specific Residue of HSA
residues involved
protein compound binding free energy (kcal mol−1) hydrogen bond hydrophobic interaction
HSA eperisone hydrochloride −6.8 R257: NH2···O1 2.88 Å Y150: CE2···C17 3.66 Å
Y150: CE2···C14 3.47 Å
Y150:CZ···C14 3.79 Å
K199:CE···C18 3.54 Å
K199:CE···C19 3.75 Å
L219: CD1···C7 3.81 Å
L219: CD2···C7 3.54 Å
F223: CE2···C6 3.88 Å
L238:CG···C19 3.78 Å
L238: CD1···C3 3.78 Å
L238: CD1···C13 3.52 Å
L238: CD1···C16 3.42 Å
L238: CD2···C5 3.8 Å
L238: CD2···C7 3.61 Å
L238: CD2···C16 3.80 Å
L238: CD2···C19 3.82 Å
H242: CD2···C19 3.56 Å
H242: CE1···C17 3.74 Å
R257:CG···C10 3.89 Å
L260:CG···C3 3.64 Å
L260: CD2···C3 3.28 Å
L260: CD2···C5 3.53 Å
I264: CD1···C3 3.49 Å
I264: CD1···C5 3.31 Å
I264: CD1···C6 3.64 Å
I264: CD1···C4 3.80 Å
I290:CB···C8 3.73 Å
I290: CG2···C8 3.42 Å
I290: CG2···C4 3.32 Å
A291:CB···C12 3.85 Å
Table 5. Changes in the ASA (Å2) Values of the Interacting
Residues of HSA before and after Binding of Eperisone
Hydrochloride
residues ASA (Å2) in HSA ASA (Å2) in HSA−EH complex ΔASA (Å2)
Y150 25.01 9.35 15.66
K199 43.53 23.65 19.88
L219 12.80 3.02 9.78
F223 4.33 0 4.33
L238 38.39 0.65 37.74
H242 11.17 0 11.17
R257 23.30 8.03 15.27
L260 17.93 5.02 12.91
I264 13.80 0 13.8
I290 14.33 0 14.33
A291 44.45 16.25 28.2
Molecular Pharmaceutics Article
DOI: 10.1021/acs.molpharmaceut.6b01124
Mol. Pharmaceutics 2017, 14, 1656−1665
1663
■CONCLUSIONS
The present study examined the complex formation of EH with
human serum albumin. HSA was found to be an ideal small
molecule for examining the interactions between the drug,
chemicals, fatty acids, etc. The CD and FTIR spectroscopy
show the interaction of EH leads to increase in the secondary
structures of HSA. The distance (r) 2.18 nm obtained through
FRET indicates that donor (HSA) and acceptor (EH) are close
to each other. The obtained results from molecular docking
showed that the EH molecule enters the hydrophobic cleft of
subdomain IIA (Sudlow’s site I) near W214 and form specific
hydrogen bonds with R257 and K199, thereby causing static
fluorescence quenching of W214. The thermodynamic and
molecular docking study suggests interaction between HSA and
EH molecule is governed by hydrogen bonding and the
hydrophobic interactions. These interactions are believed to
make the local microenvironment of HSA (in complex with
EH) more hydrophobic than its native state. This study
provides an effective approach to inspect the drug-induced
microenvironmental changes in the protein, which can be
further utilized toward the development of medicines and
improving drug delivery. This study not only provides
important insights into the binding of HSA with EH but it
also supports the medicinal background of EH.
■AUTHOR INFORMATION
Corresponding Author
*E-mail: inhochoi@ynu.ac.kr.
ORCID
Inho Choi: 0000-0002-5293-8231
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
This study was supported by Research Grant of Yeungnam
University, Republic of Korea (2017). This work was supported
by the grant K16281 awarded to the Korea Institute of Oriental
Medicine (KIOM) from Ministry of Education, Science and
Technology (MEST), Republic of Korea.
■ABBREVIATIONS
ASA, accessible surface area; CD, circular dichroism; DSC,
differential scanning calorimetry; EH, eperisone hydrochloride;
ITC, isothermal titration calorimetry; ΔH, enthalpy; HSA,
human serum albumin; Km, Michaelis−Menten constant; λmax,
wavelength maxima; MRE, mean residue ellipticity; Tm,
midpoint temperature; Vmax, maximum velocity
■REFERENCES
(1) Kim, M. J.; Lim, H. S.; Noh, Y. H.; Kim, Y. H.; Choi, H. Y.; Park,
K. M.; Kim, S. E.; Bae, K. S. Pharmacokinetic interactions between
eperisone hydrochloride and aceclofenac: a randomized, open-label,
crossover study of healthy Korean men. Clin. Ther. 2013,35, 1528−
1535.
(2) Sakai, Y.; Matsuyama, Y.; Nakamura, H.; Katayama, Y.; Imagama,
S.; Ito, Z.; Okamoto, A.; Ishiguro, N. The effect of muscle relaxant on
the paraspinal muscle blood flow: a randomized controlled trial in
patients with chronic low back pain. Spine (Philadelphia) 2008,33,
581−587.
(3) Melilli, B.; Piazza, C.; Vitale, D. C.; Marano, M. R.; Pecori, A.;
Mattana, P.; Volsi, V. L.; Iuculano, C.; Cardi, F.; Drago, F. Human
pharmacokinetics of the muscle relaxant, eperisone hydrochloride by
liquid chromatography-electrospray tandem mass spectrometry. Eur. J.
Drug Metab. Pharmacokinet. 2011,36,71−78.
(4) Sartini, S.; Guerra, L. Open experience with a new myorelaxant
agent for low back pain. Adv. Ther. 2008,25, 1010−1018.
(5) Lee, E. J.; Jan, A. T.; Baig, M. H.; Ashraf, J. M.; Nahm, S. S.; Kim,
Y. W.; Park, S. Y.; Choi, I. Fibromodulin: a master regulator of
myostatin controlling progression of satellite cells through a myogenic
program. FASEB J. 2016,30, 2708−2719.
(6) Jan, A. T.; Lee, E. J.; Choi, I. Fibromodulin: A regulatory
molecule maintaining cellular architecture for normal cellular function.
Int. J. Biochem. Cell Biol. 2016,80,66−70.
(7) Elenbaas, J. K. Centrally acting oral skeletal muscle relaxants. Am.
J. Hosp. Pharm. 1980,37, 1313−1323.
(8) Morikawa, K.; Oshita, M.; Yamazaki, M.; Ohara, N.; Mizutani, F.;
Kato, H.; Ito, Y.; Kontani, H.; Koshiura, R. Pharmacological studies of
the new centrally acting muscle relaxant 4′-ethyl-2-methyl-3-
pyrrolidinopropiophenone hydrochloride. Arzneimittelforschung 1987,
37, 331−336.
(9) Matsunaga, M.; Uemura, Y.; Yonemoto, Y.; Kanai, K.; Etoh, H.;
Tanaka, S.; Atsuta, Y.; Nishizawa, Y.; Yamanishi, Y. Long-lasting
muscle relaxant activity of eperisone hydrochloride after percutaneous
administration in rats. Jpn. J. Pharmacol. 1997,73, 215−220.
(10) Iwase, S.; Mano, T.; Saito, M.; Ishida, G. Effect of a centrally-
acting muscle relaxant, eperisone hydrochloride, on muscle sym-
pathetic nerve activity in humans. Funct. Neurol. 1992,7, 459−470.
(11) Sakaue, A.; Honda, M.; Tanabe, M.; Ono, H. Antinociceptive
effects of sodium channel-blocking agents on acute pain in mice. J.
Pharmacol. Sci. 2004,95, 181−188.
(12) Farkas, S. Silperisone: a centrally acting muscle relaxant. CNS
Drug Rev. 2006,12, 218−235.
(13) Kocsis, P.; Farkas, S.; Fodor, L.; Bielik, N.; Than, M.; Kolok, S.;
Gere, A.; Csejtei, M.; Tarnawa, I. Tolperisone-type drugs inhibit spinal
reflexes via blockade of voltage-gated sodium and calcium channels. J.
Pharmacol. Exp. Ther. 2005,315, 1237−1246.
(14) Tallent, M. K. Presynaptic inhibition of glutamate release by
neuropeptides: use-dependent synaptic modification. Results Probl. Cell
Differ. 2008,44, 177−200.
(15) Nakagawa, M.; Nakahara, K.; Maruyama, Y.; Kawabata, M.;
Higuchi, I.; Kubota, H.; Izumo, S.; Arimura, K.; Osame, M.
Therapeutic trials in 200 patients with HTLV-I-associated myelop-
athy/ tropical spastic paraparesis. J. NeuroVirol. 1996,2, 345−355.
(16) Murayama, K.; Katsumi, T.; Tajika, E.; Nakamura, T. Clinical
application of eperisone hydrochloride to neurogenic bladder.
Hinyokika Kiyo 1984,30, 403−408.
(17) Kobayashi, Y.; Kawasaki, T.; Yoshimi, T.; Nakajima, T.; Kanai,
K. Muscle cramps in chronic liver diseases and treatment with
antispastic agent (eperisone hydrochloride). Dig. Dis. Sci. 1992,37,
1145−1146.
(18) Larsen, M. T.; Kuhlmann, M.; Hvam, M. L.; Howard, K. A.
Albumin-based drug delivery: harnessing nature to cure disease. Mol.
Cell Ther. 2016,4,3.
(19) Theodore Peters, J. All about Albumin: Biochemistry, Genetics and
Medical Applications; Academic Press: San Diego, 1996.
(20) Yue, Y.; Dong, Q.; Zhang, Y.; Li, X.; Yan, X.; Sun, Y.; Liu, J.
Synthesis of imidazole derivatives and the spectral characterization of
the binding properties towards human serum albumin. Spectrochim.
Acta, Part A 2016,153, 688−703.
(21) Yue, Y.; Liu, J.; Liu, R.; Sun, Y.; Li, X.; Fan, J. The binding
affinity of phthalate plasticizers-protein revealed by spectroscopic
techniques and molecular modeling. Food Chem. Toxicol. 2014,71,
244−253.
(22) Yue, Y.; Liu, R.; Liu, J.; Dong, Q.; Fan, J. Experimental and
theoretical investigation on the interaction between cyclovirobuxine D
and human serum albumin. Spectrochim. Acta, Part A 2014,128, 552−
558.
(23) Sur, S. S.; Rabbani, L. D.; Libman, L.; Breslow, E. Fluorescence
studies of native and modified neurophysins. Effects of peptides and
pH. Biochemistry 1979,18, 1026−1036.
Molecular Pharmaceutics Article
DOI: 10.1021/acs.molpharmaceut.6b01124
Mol. Pharmaceutics 2017, 14, 1656−1665
1664
(24) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.;
Springer, 2006.
(25) Baig, M. H.; Ahmad, K.; Hasan, Q.; Khan, M. K.; Rao, N. S.;
Kamal, M. A.; Choi, I. Interaction of glucagon g-protein coupled
receptor with known natural antidiabetic compounds: multiscoring in
silico approach. Evid. Based Complement Alternat Med. 2015,2015,
497253.
(26) Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. How to
measure and predict the molar absorption coefficient of a protein.
Protein Sci. 1995,4, 2411−2423.
(27) Dangkoob, F.; Housaindokht, M. R.; Asoodeh, A.; Rajabi, O.;
Rouhbakhsh Zaeri, Z. Verdian Doghaei, A. Spectroscopic and
molecular modeling study on the separate and simultaneous bindings
of alprazolam and fluoxetine hydrochloride to human serum albumin
(HSA): with the aim of the drug interactions probing. Spectrochim.
Acta, Part A 2015,137, 1106−1119.
(28) Woody, R. W. Theory of circular dichroism of proteins. In
Circular Dichroism and the Conformational Analysis of Biomolecules;
Fasman, G. D., Ed.; Plenum Press: New York, 1996; pp 25−67.
(29) Ahmad, E.; Rabbani, G.; Zaidi, N.; Ahmad, B.; Khan, R. H.
Pollutant-induced modulation in conformation and beta-lactamase
activity of human serum albumin. PLoS One 2012,7, e38372.
(30) Zhang, G.; Wang, L.; Pan, J. Probing the binding of the
flavonoid diosmetin to human serum albumin by multispectroscopic
techniques. J. Agric. Food Chem. 2012,60, 2721−2729.
(31) Yang, H.; Yang, S.; Kong, J.; Dong, A.; Yu, S. Obtaining
information about protein secondary structures in aqueous solution
using Fourier transform IR spectroscopy. Nat. Protoc. 2015,10, 382−
396.
(32) Froehlich, E.; Mandeville, J. S.; Jennings, C. J.; Sedaghat-Herati,
R.; Tajmir-Riahi, H. A. Dendrimers bind human serum albumin. J.
Phys. Chem. B 2009,113, 6986−6993.
(33) Yue, Y.; Sun, Y.; Yan, X.; Liu, J.; Zhao, S.; Zhang, J. Evaluation
of the binding of perfluorinated compound to pepsin: Spectroscopic
analysis and molecular docking. Chemosphere 2016,161, 475−481.
(34) Ariga, G. G.; Naik, P. N.; Nandibewoor, S. T.; Chimatadar, S. A.
Study of fluorescence interaction and conformational changes of
bovine serum albumin with histamine H(1) -receptor-drug epinastine
hydrochloride by spectroscopic and time-resolved fluorescence
methods. Biopolymers 2015,103, 646−657.
(35) Liu, J.; Yue, Y.; Wang, J.; Yan, X.; Liu, R.; Sun, Y.; Li, X. Study of
interaction between human serum albumin and three phenanthridine
derivatives: fluorescence spectroscopy and computational approach.
Spectrochim. Acta, Part A 2015,145, 473−481.
(36) Yue, Y.; Sun, Y.; Dong, Q.; Liu, R.; Yan, X.; Zhang, Y.; Liu, J.
Interaction of human serum albumin with novel imidazole derivatives
studied by spectroscopy and molecular docking. Luminescence 2016,
31, 671−681.
(37) Rabbani, G.; Khan, M. J.; Ahmad, A.; Maskat, M. Y.; Khan, R. H.
Effect of copper oxide nanoparticles on the conformation and activity
of beta-galactosidase. Colloids Surf., B 2014,123,96−105.
(38) Ahmad, E.; Rabbani, G.; Zaidi, N.; Singh, S.; Rehan, M.; Khan,
M. M.; Rahman, S. K.; Quadri, Z.; Shadab, M.; Ashraf, M. T.;
Subbarao, N.; Bhat, R.; Khan, R. H. Stereo-selectivity of human serum
albumin to enantiomeric and isoelectronic pollutants dissected by
spectroscopy, calorimetry and bioinformatics. PLoS One 2011,6,
e26186.
(39) Bijari, N.; Shokoohinia, Y.; Ashrafi-Kooshk, M. R.; Ranjbar, S.;
Parvaneha, S.; Moieni-Aryac, M.; Khodarahmi, R. Spectroscopic study
of interaction between osthole and human serum albumin:
Identification of possible binding site of the compound. J. Lumin.
2013,143, 328−336.
(40) Rabbani, G.; Ahmad, E.; Zaidi, N.; Khan, R. H. pH-dependent
conformational transitions in conalbumin (ovotransferrin), a metal-
loproteinase from hen egg white. Cell Biochem. Biophys. 2011,61,
551−560.
(41) Yue, Y.; Liu, J.; Liu, R.; Dong, Q.; Fan, J. Binding of helicid to
human serum albumin: a hybrid spectroscopic approach and
conformational study. Spectrochim. Acta, Part A 2014,124,46−51.
(42) Ross, P. D.; Subramanian, S. Thermodynamics of protein
association reactions: forces contributing to stability. Biochemistry
1981,20, 3096−3102.
(43)Sen,P.;Ahmad,B.;Rabbani,G.;Khan,R.H.2,2,2-
Trifluroethanol induces simultaneous increase in alpha-helicity and
aggregation in alkaline unfolded state of bovine serum albumin. Int. J.
Biol. Macromol. 2010,46, 250−254.
(44) Afrin, S.; Riyazuddeen; Rabbani, G.; Khan, R. H. Spectroscopic
and calorimetric studies of interaction of methimazole with human
serum albumin. J. Lumin. 2014,151, 219−223.
(45) Flora, K.; Brennan, J. D.; Baker, G. A.; Doody, M. A.; Bright, F.
V. Unfolding of acrylodan-labeled human serum albumin probed by
steady-state and time-resolved fluorescence methods. Biophys. J. 1998,
75, 1084−1096.
(46) Juarez, J.; Lopez, S. G.; Cambon, A.; Taboada, P.; Mosquera, V.
Influence of electrostatic interactions on the fibrillation process of
human serum albumin. J. Phys. Chem. B 2009,113, 10521−10529.
(47) Rabbani, G.; Ahmad, E.; Zaidi, N.; Fatima, S.; Khan, R. H. pH-
Induced molten globule state of Rhizopus niveus lipase is more resistant
against thermal and chemical denaturation than its native state. Cell
Biochem. Biophys. 2012,62, 487−499.
(48) Zhu, Y.; Zhang, R.; Wang, Y.; Ma, J.; Li, K.; Li, Z. Biophysical
study on the interaction of an anesthetic, vecuronium bromide with
human serum albumin using spectroscopic and calorimetric methods.
J. Photochem. Photobiol., B 2014,140, 381−389.
(49) Ishtikhar, M.; Rabbani, G.; Khan, R. H. Interaction of 5-fluoro-
5′-deoxyuridine with human serum albumin under physiological and
non-physiological condition: a biophysical investigation. Colloids Surf.,
B2014,123, 469−477.
(50) Kragh-Hansen, U. Molecular and practical aspects of the
enzymatic properties of human serum albumin and of albumin-ligand
complexes. Biochim. Biophys. Acta, Gen. Subj. 2013,1830, 5535−5544.
(51) Watanabe, H.; Tanase, S.; Nakajou, K.; Maruyama, T.; Kragh-
Hansen, U.; Otagiri, M. Role of arg-410 and tyr-411 in human serum
albumin for ligand binding and esterase-like activity. Biochem. J. 2000,
349 (Pt3), 813−819.
(52) Lockridge, O.; Xue, W.; Gaydess, A.; Grigoryan, H.; Ding, S. J.;
Schopfer, L. M.; Hinrichs, S. H.; Masson, P. Pseudo-esterase activity of
human albumin: slow turnover on tyrosine 411 and stable acetylation
of 82 residues including 59 lysines. J. Biol. Chem. 2008,283, 22582−
22590.
(53) Sudlow, G.; Birkett, D. J.; Wade, D. N. The characterization of
two specific drug binding sites on human serum albumin. Mol.
Pharmacol. 1975,11, 824−832.
(54) Sudlow, G.; Birkett, D. J.; Wade, D. N. Further characterization
of specific drug binding sites on human serum albumin. Mol.
Pharmacol. 1976,12, 1052−1061.
(55) Samanta, U.; Bahadur, R. P.; Chakrabarti, P. Quantifying the
accessible surface area of protein residues in their local environment.
Protein Eng., Des. Sel. 2002,15, 659−667.
(56) Sahoo, B. K.; Ghosh, K. S.; Dasgupta, S. Molecular interactions
of isoxazolcurcumin with human serum albumin: spectroscopic and
molecular modeling studies. Biopolymers 2009,91, 108−119.
(57) Baig, M. H.; Rizvi, S. M.; Shakil, S.; Kamal, M. A.; Khan, S. A
neuroinformatics study describing molecular interaction of Cisplatin
with acetylcholinesterase: a plausible cause for anticancer drug induced
neurotoxicity. CNS Neurol. Disord.: Drug Targets 2014,13, 265−270.
Molecular Pharmaceutics Article
DOI: 10.1021/acs.molpharmaceut.6b01124
Mol. Pharmaceutics 2017, 14, 1656−1665
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