Biophysical Study on the Interaction between Eperisone

Article pubs.acs.org/molecularpharmaceutics

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 ≈ 104 reflecting 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 © 2017 American Chemical Society

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: Revised: Accepted: Published: 1656

December 14, 2016 March 3, 2017 April 5, 2017 April 5, 2017 DOI: 10.1021/acs.molpharmaceut.6b01124 Mol. Pharmaceutics 2017, 14, 1656−1665

Article

Molecular Pharmaceutics 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 concentrations, 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 halflife 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.

Fcorr = Fobs × e(Aex + Aem)/2

(1)

where Fcorr and Fobs are corrected and observed fluorescence intensities, respectively. The excitation and emission absorbance 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 Fo = KSV[Q ] + 1 = kqτ o[Q ] + 1 F

(2)

where F0 and F are 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 τo is the integral fluorescence lifetime of tryptophan (5.78 × 10−9 s). The modified Stern−Volmer equation was used to determine the quantitative binding constant (Kb) and binding stoichiometry (n) of the HSA−EH complex: ⎡F − F⎤ log⎢ o = log Kb + n log[Q ] ⎣ F ⎥⎦

(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’t Hoff equation: ln K = −

ΔH ◦ ΔS◦ + RT R

(4) −1

−1

where R is the universal gas constant (1.987 cal K mol ) and T is absolute temperature in Kelvin. The Gibbs free energy change (ΔG°) of the process was determined using the relation:

■

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 E1% 280nm = 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

ΔG◦ = ΔH ◦ − T ΔS◦

(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 Naphosphate 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 spectropolarimeter 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−1 and 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 1657

DOI: 10.1021/acs.molpharmaceut.6b01124 Mol. Pharmaceutics 2017, 14, 1656−1665

Article

Molecular Pharmaceutics

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

signals were converted into mean residual ellipticity (MRE) using

MRE =

Θobs 10nCl

(6)

where Θobs is CD in millidegree, n is the number of amino acid residues (n = 585−1), l is the path length of the cell in cm, and C is the molar concentration of HSA (2 μM). Fourier Transform Infrared (FTIR) Spectroscopy. Infrared spectra were recorded on FTIR/FIR (Frontier; PerkinElmer) 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 concentration 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’t Hoff enthalpy (ΔHvH), and melting point temperature (Tm). Esterase-Like Activity of HSA. Changes in the esteraselike 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. ν0 =

Vmax[S] K m + [S]

■

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

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.

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

(7)

where v0 and Vmax are initial and maximum velocities, respectively, [S] is p-NPA concentration, and Km is the Michaelis−Menten constant. Lineweaver−Burk reciprocal plot was constructed by plotting 1/v0 against 1/[S] at varying concentration of p-NPA in the absence or in the presence of EH: Km 1 1 = + ν0 Vmax + [S] Vmax

(8)

In Silico Studies. Molecular docking was performed to gain an insight of EH binding within the active site of HSA. The 1658


Article


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 Experimentsa 25 °C

parameters n (binding stoichiometry, HSA/EH) KSV (Stern−Volmer constant, M−1) Kb (binding constant, M−1) kq (bimolecular quenching rate constant, M−1 s−1) ΔH° (binding enthalpy, kcal mol−1) TΔS° (entropy change, kcal mol−1) ΔG° (Gibbs free energy change, kcal mol−1) a

0.93 9.7 0.49 1.6

± ± ± ±

30 °C

0.11 0.41 × 104 0.11 × 105 0.12 × 1013

17.5 ± 0.24 −6.5 ± 0.13

1.0 7.9 0.99 1.3 11.0 17.8 −6.8

± ± ± ± ± ± ±

0.13 0.34 × 104 0.16 × 105 0.16 × 1013 1.5 0.34 0.18

37 °C 1.0 6.1 1.0 1.0

± ± ± ±

42 °C

0.16 0.29 × 104 0.32 × 105 0.13 × 1013

18.2 ± 0.18 −7.2 ± 0.25

1.1 4.8 1.7 0.83

± ± ± ±

0.12 0.26 × 104 0.23 × 105 0.14 × 1013

18.5 ± 0.38 −7.5 ± 0.31

The data are the means ± standard deviations of three independent trials.

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 CO 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 αhelix 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 macromolecules. 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 estimated33 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/F vs [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 104−105 M−1, which is in accord with those reported for drug/protein interactions (104−105 M) 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 Kb values 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 × 10−9 s (as discussed in UV−Vis Absorption and Fluorescence Spectroscopy). The values of obtained kq were in order of 1.6 ± 0.12 × 1013, 1.3 ± 1659


Article

Molecular Pharmaceutics 0.16 × 1013, 1.0 ± 0.13 × 1013, and 0.83 ± 0.14 × 1013 M−1 s−1 at 25, 30, 37, and 42 °C, respectively (Table 1). The obtained kq values of HSA−EH system are in the range of 1013 M−1 s−1, which is much larger than the upper limit of scattering quenching constant, kq of various quenchers with the biopolymers in aqueous solution (2 × 1010 M−1 s−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) quenching 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 interactions 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’t Hoff plot was generated using previously calculated Kb at four different temperatures (inset of Figure 2B). The thermodynamic parameters (ΔH° and ΔS°) were determined from the slope and intercept of eq 4 (van’t Hoff plot 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−1 at 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) × 104 M, 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

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.

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

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.

transfer (FRET)38 was employed to calculate the binding distance (r) and the energy transfer efficiency (EFRET) using equation: ⎛ R 06 F⎞ E FRET = ⎜1 − ⎟ = F0 ⎠ ⎝ R 06 + r 6 1660

(9) DOI: 10.1021/acs.molpharmaceut.6b01124 Mol. Pharmaceutics 2017, 14, 1656−1665

Article

Molecular Pharmaceutics where F is fluorescence intensity of HSA with EH and F0 is fluorescence intensity of HSA without EH. R0 is critical distance at which the EFRET is 50%. The value of R0 can be calculated from donor emission and acceptor absorption spectra using equation: R 0 6 = 8.79 × 10−25K 2n−4φJ

(10)

2

In eq 10 K is the orientation factor related to the geometry of the donor and acceptor molecule, n is the average refractive index of the buffer, φ denotes the fluorescence quantum yield of HSA (donor), and J is the degree of spectral overlap integral between donor and acceptor. The value of J was calculated by following equation: ∞

J=

∫0 F(λ)ε(λ)λ 4 dλ ∞

∫0 F(λ) dλ

(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 cm3 M−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, R0 was 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 T1m = 57.4 ± 0.03 and a second peak at T2m = 70.2 ± 0.21 °C (Figure 5A). The higher Tm values show the thermogram shifted toward higher temperature; first endothermic peak reflects T1m = 59.4 ± 0.01 and a second endothermic peak reflects T2m = 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 Cp after 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 Cp profiles 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’t Hoff and calorimetric enthalpy we calculated the ΔHvH/ΔHcal ratio, and native HSA gave the highest R value (Table 2). It is known that ΔHvH/ΔHcal ratio shows two things: (i) increase the distance and weak interactions between interacting domains48 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

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 7.4a transition

parameters

1st transition

T1m

2nd transition

ΔH1cal ΔH1vH R1 T2m ΔH2cal ΔH2vH R2

native HSA 57.4 225.9 74.2 0.32 70.2 47.3 67.3 1.42

± 0.033 ± 1.85 ± 0.49 ± 0.21 ± 1.95 ± 2.7

HSA−EH complex 59.4 128.2 104.5 0.81 70.4 62.1 60.6 0.97

± 0.01 ± 0.86 ± 0.47 ± 0.98 ± 0.98 ± 0.99

R or R2 = ΔHvH/ΔHcal. Tm is expressed in °C. ΔH is expressed in kcal mol−1.

a 1

(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 phosphates.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 (Km and 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 Km are 1661


Article


a significantly higher enzymatic activity (28 × 10−2 min−1) than that of HSA (30 × 10−2 min−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 Km values 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,

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 Km and Vmax. (B) Lineweaver−Burk plots: inverse of initial reaction velocity versus inverse of substrate concentration (1/v0 vs 1/[S]) for HSA in absence or presence of different concentrations of EH.

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.

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 Km values of 25 × 10−2, 48 × 10−2, and 63 × 10−2 mM 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 Km and 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 farUV 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

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−1 by fluorescence quenching, respectively). The ΔG values 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

Table 3. Michaelis−Menten Kinetic Parameters of HSA in the Presence of Increasing EH Concentrationsa HSA/drug 1:0 1:5 1:10

RA (%) 100 98 94

Vmax (mM min−1)

Km (mM)

−4

−2

25 × 10 48 × 10−2 63 × 10−2

14.0 × 10 14.6 × 10−4 15.3 × 10−4

kcat (min−1) −2

28 × 10 29 × 10−2 30 × 10−2

kcat/Km (mM−1 min−1) 1.12 0.60 0.47

All measurements were carried out in 20 mM Na-phosphate buffer pH 7.4 at 37 °C. Values of Vmax and Km were 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. a

1662


Article


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

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 Å2 of 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 Å2 after 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

residues Y150 K199 L219 F223 L238 H242 R257 L260 I264 I290 A291

ASA (Å2) in HSA ASA (Å2) in HSA−EH complex 25.01 43.53 12.80 4.33 38.39 11.17 23.30 17.93 13.80 14.33 44.45

9.35 23.65 3.02 0 0.65 0 8.03 5.02 0 0 16.25

ΔASA (Å2) 15.66 19.88 9.78 4.33 37.74 11.17 15.27 12.91 13.8 14.33 28.2

binding site residues before and after complex formation were also calculated (Table 5). Several interacting residues lost more than 10 Å2 of 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. 1663


Article


■

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-3pyrrolidinopropiophenone 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 centrallyacting muscle relaxant, eperisone hydrochloride, on muscle sympathetic 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 myelopathy/ 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.

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: [email protected]. 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 1664


Article

Molecular Pharmaceutics (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 metalloproteinase 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. 2,2,2Trifluroethanol 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. pHInduced 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-fluoro5′-deoxyuridine with human serum albumin under physiological and non-physiological condition: a biophysical investigation. Colloids Surf., B 2014, 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.; KraghHansen, 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.

1665


Biophysical Study on the Interaction between Eperisone

Recommend Documents