Binding of Tolperisone Hydrochloride with Human Serum Albumin

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Binding of tolperisone hydrochloride with human serum albumin: effects on the conformation, thermodynamics, and activity of HSA Gulam Rabbani, Eun Ju Lee, Khurshid Ahmad, Mohammad Hassan Baig, and Inho Choi Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00976 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018

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Molecular Pharmaceutics

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Binding of tolperisone hydrochloride with human serum albumin: effects on the conformation, thermodynamics, and activity of HSA

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Gulam Rabbani*, Eun Ju Lee, Khurshid Ahmad, Mohammad Hassan Baig and Inho

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Choi*

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Department of Medical Biotechnology, Yeungnam University, 280 Daehak-ro, Gyeongsan,

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Gyeongbuk-38541, Republic of Korea

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Fax: +82 53 810 4769; E-mail: [email protected] [email protected]

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Running Title: Binding between tolperisone hydrochloride and HSA

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Abbreviations: CD: Circular dichroism; DMEM: Dulbecco’s Modified Eagle’s Medium; DSC: differential scanning calorimetry; FBS: Fetal bovine serum; ITC: Isothermal titration calorimetry; ∆H: Enthalpy; HSA: Human serum albumin; Km: Michaelis-Menten constant; λmax: Wavelength maxima; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TH: tolperisone hydrochloride; MRE: mean residue ellipticity; p-NPA: p-nitrophenyl acetate; Tm: midpoint temperature, Vmax: maximum velocity

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Abstract

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Tolperisone hydrochloride (TH) has muscle relaxant activity, and has been widely used for several

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years in clinical practice to treat pathologically high skeletal muscle tone (spasticity) and related

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pains. The current study was designed to explore the binding efficacy of TH with human serum

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albumin (HSA) using a multispectroscopic approach, FRET, esterase-like activity, and a molecular

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docking method. A reduction in fluorescence emission at 340 nm of HSA was attributed to

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florescence quenching by TH via a static quenching type. Binding constants (Kb) were evaluated at

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different temperatures, obtained Kb value were ~104 M-1, which demonstrated high affinity of TH

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for HSA. A calculated negative ∆Gº value indicated spontaneous binding of TH to HSA. Far-UV

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CD spectroscopy revealed that the α-helix content was increased after TH binding. The binding

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distance between donor and acceptor was calculated to be 2.11 nm based on Förster’s resonance

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energy transfer theory. ITC results revealed TH interacted with HSA via hydrophobic interactions

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and hydrogen bonding. The thermal stability of HSA was studied using DSC and results showed

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that in the presence of TH the structure of HSA was significantly more thermostable. The esterase-

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like activity of HSA showed fixed Vmax and increased Km suggesting that TH binds with HSA in a

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competitive manner. Furthermore, molecular docking results revealed TH binds in the cavity of

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has, that is, subdomain IIA (Sudlow site I), and that it hydrogen bonds with K199 and H242 of

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HSA. Binding studies of drugs with HSA are potentially useful for elucidating chemico-biological

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interactions that can be utilized in the drug design, pharmaceutical, pharmacology and

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biochemistry fields. This extensive study provides additional insight of ligand binding and

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structural changes induced in HSA relevant to the biological activity of HSA in vivo.

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Keywords: Muscle relaxant, Circular dichroism, Tolperisone hydrochloride, Esterase-like activity,

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Human serum albumin, Molecular docking

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Introduction

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Skeletal muscle occupies 30-40% of body weight and functions to move the body via the

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contractions and relaxations of sarcomeres in the muscular fibers 1, 2. Skeletal muscle relaxants are

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a diverse group of medicines that have the ability to relax muscle. Tolperisone hydrochloride (TH)

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is a piperidine derivative widely used in the treatment of different pathological conditions like

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multiocular sclerosis, myelopathy, and painful muscle spasms in orthopedic and rheumatologic

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diseases. TH is a centrally acting muscle relaxant that provides safe and effective treatment of

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elevated muscle tone and tension without any side effects 3. The chemical structure of TH (2-

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methyl-1-(4-methylphenyl)-3-(piperidinyl)-1-propanone hydrochloride) is shown in figure 1. TH

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is extremely soluble in water and more stable in acidic medium (pH < 4.5). TH inhibits segmental

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spinal reflexes by blocking voltage gated Ca+2 and Na+ channels 4.

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Human serum albumin (HSA) is the most abundant multifunctional plasma protein in mammals 5.

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It is synthesized in the liver and continuously secreted into the intravascular space at a rate of 9-12

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g/day 6. HSA is a model protein widely used in biophysical and biochemical studies and is of great

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interest to the pharmaceutical industry. HSA, mostly globular α-helical protein made up of 585

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amino acid residues divided into three functional domains (I, II and II). These three domains have

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similar 3D structures and are composed of residues 1-195 (domain I), residues 196-383 (domain

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II), and residues 384-585 (domain III), which are each stabilized by 17 disulfide bridges 7. These

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three domains are further sub-divided into pair of sub-domains (A and B) that possess common

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structural motifs 8. HSA is capable of binding and transport of different endogenous substances

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(hormones vitamins, unesterified fatty acid) and exogenous substances like drug molecules 9.

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Under natural conditions, the overall HSA structure is flexible during ligand binding. X-ray

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crystallographic studies have confirmed that HSA has only one Trp (W214) located in the 3 ACS Paragon Plus Environment


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hydrophobic cavity of subdomain IIA (Sudlow site I) and this s used as a probe in

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spectrophotometric studies10. In addition, HSA has many special active residues, such as, K199,

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R410, and Y411, which are essential for esterase-like activity

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interact with endothelial and tubular epithelial cells and to activate NF-κB

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and proliferate better in the presence of serum albumins because they transport biological

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molecules, such as, growth factors, hormones, and fatty acids 14. Furthermore, HSA is an essential

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protein that initiates muscle growth and repair by activating satellite cells in adult muscle 15.

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In a previous study, we reported on the binding between the myorelaxant, eperisone hydrochloride

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(EH) and HSA

16

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, and was recently reported to 12, 13

. Many cells live

. In this study, we explored the binding mechanism between HSA and the TH

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analogue of eperisone hydrochloride (Fig. 1). TH (50-400 µM) is a more effective potential

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inhibitor

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hydrochloride (200-800 µM)

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and TH with HSA using our previous data and that reported by Rabbani et al 16.

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The aim of this study was to explore binding between TH and HSA using a combined

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experimental/computational approach. We used UV-vis, circular dichroism and fluorescence

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spectroscopy collectively to confirm probable structural alterations in HSA caused by TH binding.

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ITC was used to characterize the binding thermodynamics of HSA-TH systems. Calculated,

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binding constants, binding stoichiometries, and thermodynamic parameters showed binding occurs

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between TH and HSA. Binding site, interacting residues and the position orientation of TH in

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HSA were identified by molecular docking simulation. The findings of this study advance

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understanding of this unique transporter protein and offer new biophysical insights of

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pharmacodynamic and pharmacokinetic prediction methods.

than its analogues, that is, eperisone, lanperisone, silperisone and inaperisone 17

. Our aim was to identify differences between the bindings of EH

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2. Materials and Methods

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2.1 Chemicals and reagents

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Albumin from human serum (A1887; globulin and fatty acid free) lyophilized powder and p-

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nitrophenyl acetate (N8130) were purchased from Sigma Chemical Co. (St. Louis, Mo, USA).

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Tolperisone hydrochloride (>99% purity index) was obtained from Tokyo Chemical Industry Co.

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Ltd (TCI Tokyo, Japan). The other reagents used in this study were of analytical purity.

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2.2 Solution preparation

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HSA stock solution (75 μM) was prepared in 20 mM sodium phosphate buffer of pH 7.4 and

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dialyzed in same buffer at 4 °C in dark. The concentration of HSA was verified by

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1% spectrophotometrically using E 280 nm = 5.3. A hydrophilic stock solution of TH was prepared by

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dissolving (5 mg ml-1) in 20 mM sodium phosphate buffer at pH 7.4. Dilutions of HSA and TH

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stock were made using the same buffer for further experiments.

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2.3 Cell Culture

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Mouse myoblast C2C12 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). The

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C2C12 cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium; HyClone

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Laboratories, UT, USA) and supplemented with 10% FBS (fetal bovine serum, HyClone, CA,

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USA) containing 1% penicillin/streptomycin (P/S, Invitrogen, CA, USA). C2C12 cells were

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cultured at 37°C in a humidified 5% CO2 atmosphere and the medium was changed every two days.

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2.4 Toxicity Testing

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Toxicity testing was performed using a MTT assay. Briefly, cells were cultured in DMEM+10%

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FBS+1% PS containing TH (0, 1, 2.5 or 5 µM) for 1 day. For the MTT assay, media was removed

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and cells were washed with phosphate-buffered saline (PBS), MMT solution was added (5 µg ml-1,

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Sigma Aldrich, MO, USA), and cells were then incubated at 37°C for 3 h. After incubation the 5 ACS Paragon Plus Environment


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MTT medium was removed and dimethyl sulfoxide (DMSO, 1 ml per 12 wells) was added, and

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cells were then incubated in the dark at 37°C for 10 min with gentle shaking. Cell viability was

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determined by measuring absorbance at 540 nm (Tecan Group Ltd., Männedorf, Switzerland).

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2.5 Ultraviolet-visible absorption spectra measurements

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UV-vis absorptions were measured using a Cary 100 (Varian) spectrophotometer and a 1.0 cm

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quartz cuvette. UV-vis spectra of TH, HSA with or without different TH concentrations were

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recorded between 240 and 340 nm. HSA concentration was maintained at 5 µM for all absorption

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spectroscopy measurements. All measurements were performed at room temperature and

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background spectrum was collected before measurements for minimize baseline absorption.

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2.6 Circular dichroism (CD) spectropolarimetry

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Circular dichroism (CD) measurements of HSA with or without TH were carried out on a Jasco (J-

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815) spectropolarimeter at 25±0.2°C in a cylindrical cuvette of path-length 0.1 cm (Hellma, USA).

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The spectra presented were baseline corrected and four repetitions were used to obtain average CD

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spectra, which were obtained from 200 to 250 nm at a scan rate of 50 nm min-1. Buffer solution

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was used for baseline correction and was prepared in the same manner but without has. Scanning

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was performed using the same conditions and baseline was automatically subtracted from HSA

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spectra. To further understand the TH/HSA interaction, far-UV CD spectral data was use to access

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the secondary structure of HSA. Mean residue ellipticities (MRE) of native HSA and HSA-TH

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complex were calculated using:

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MRE =

Θobs (mo )

10 × n × C × l

(6)

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In equation (6), Θobs is the observed ellipticity in mdeg, C is the molar concentration of HSA (2

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µM), n is the number of peptide bonds (n = 585-1) in the protein chain and l is the path length of


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cuvette in cm. The α-helical contents of HSA were calculated from MRE values at 222 nm using

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the equation derived by Chen et al. 18.  MRE 222 nm − 2,340  % α - helix =   ×100 30,300  

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(7)

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2.7 Fluorescence quenching and data analysis

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Steady-state fluorescence (SSF) spectra were recorded on a Jasco spectrofluorometer (FP-8300)

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equipped with an attached water circulator. For HSA/TH fluorescence quenching experiments,

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HSA stock solution was diluted with buffer solution to 2 μM and titrated by repeatedly adding TH

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until the HSA binding site was saturate. For SSF spectra, an excitation wavelength of 295 nm was

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chosen to avoid Tyr excitation, and emission intensities were recorded from 300 to 400 nm using

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excitation and emission slit widths of 5 and 2.5 nm, respectively. Inner filter effects of HSA and

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TH absorbances were measured at excitation and emission wavelengths (295 and 340 nm) and

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corrected using:

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Fcorr = f obs

( Aex + Aem ) 2 ×e

(1)

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where Fcorr and Fobs are the corrected and observed fluorescence intensities. Aex and Aem are sample

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absorptions at excitation and emission wavelengths of 295 and 340 nm, respectively. The

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quenching effects of Trp residues were accessed using the Stern-Volmer equation 19:

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Fo = Ksv [Q ] + 1 = kqτ o[Q ] + 1 F

(2)

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where Fo and F are the emission fluorescence intensities of HSA in the absence and presence of

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quencher (TH), respectively, [Q] is the molar concentration of quencher (TH), KSV is the Stern-

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Volmer quenching constant, and kq is the bimolecular quenching rate constant. τo is the average

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excited state lifetime of HSA without quencher, that is, 5.78×10-9 s. 7 ACS Paragon Plus Environment


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2.8 Determination of binding constant and binding stoichiometry

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The values of equilibrium binding constant (Kb) and number of binding stoichiometries (n) of the

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HSA-TH interaction at different temperature were determined by intercept and slope of modified

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Stern-Volmer equation:

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F − F  log  o  = log K b + n log[Q ]  F 

(3)

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2.9 Thermodynamic analysis of the binding process

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In order to explain the nature of interacting forces between HSA and TH, were calculated from Kb

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data obtained at different temperatures. Assuming no significant enthalpy change (∆Hº) variation

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within the temperature range studied. The thermodynamic parameters of binding, entropy change

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(T∆Sº) and enthalpy change (∆Hº) can be obtained from van’t Hoff equation:

ln K = −

∆H o ∆S o + RT R

(4)

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here, K is the binding constant at a given temperature, R is the ideal gas constant (1.987 cal K-1

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mol-1), and T is absolute temperature in Kelvin. The free energy change (∆Gº) of binding

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processes can be (is?) determined using:

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∆ G o = ∆H o − T ∆ S o

(5)

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2.10 Isothermal titration calorimetry (ITC) measurements

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A VP-ITC microcalorimeter (MicroCal, Inc., Northampton, MA, USA) was used to measure

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enthalpy changes associated with ligand-protein interactions. Solutions of HSA and TH for ITC

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experiments were prepared using 20 mM sodium phosphate buffer (pH 7.4). To prevent air bubble

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formation, HSA (18 µM) and TH (0.129 mM) solutions were degassed in a Thermovac unit before

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loading solutions into the calorimeter cell. Total 28 consecutive injections of 10 µl aliquots of TH

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solution were injected into the protein solution using a rotating syringe (307 rpm) for continuous 8 ACS Paragon Plus Environment

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mixing of TH to HSA. The temperature and reference power of the ITC cells were set at 37 °C and

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16 µcal s-1 respectively. Injections were made over 20 s and then 180 s delay was allowed to

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achieve complete equilibration before the next injection. The enthalpy of TH dilution was run in a

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new set of experiments by injecting TH solution into buffer solution. The obtained HSA-TH

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isotherm was corrected by subtracting heat of dilution (buffer and TH). The isothermogram was

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analyzed by non-linear least squares fit with the one set of binding sites model in Microcal Origin

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7.0 software package to obtain the number of binding stoichiometry (N), association constant (Ka),

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binding enthalpy (∆Hº), binding entropy (T∆Sº), and binding free energy (∆Gº).

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2.11 Calorimetric measurements

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A VP-DSC microcalorimeter (MicroCal, Northampton, MA) was used for thermal unfolding

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experiments. Protein samples for DSC were prepared in 20 mM sodium phosphate buffer (pH 7.4)

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and stored at 4°C for 24 h to allow complex formation between HSA and TH at different molar

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ratios. Samples were scanned at 1.0°C min-1 from 20 to 90 °C. Thermal unfolding of 15 µM HSA

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and of HAS-TH complex (1:5 molar ratio excess of TH) were performed. Before HSA loading 5

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stable baselines were obtained by loading buffer and in both sample and reference cells, and after

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completing baseline acquisition, buffer was removed from sample cell and HSA was loaded.

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Baseline acquisition was performed for HSA-TH complex by loading buffer in both cells

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containing 75 µM TH. The phosphate buffer baseline was subtracted from the thermogram of

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protein samples and obtained calorimetric data were normalized with respect to protein

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concentration. These corrected thermograms were used for further analysis. The obtained heat

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capacity curve data were applied into



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a non-linear fitting algorithm in Origin 7.0 software to calculate thermodynamic parameters:

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calorimetric enthalpy (∆Hcal), van’t Hoff enthalpy (∆HvH) and midpoint temperature (Tm). The

3

van’t Hoff enthalpy was calculated with the help of a commonly used equation 20

∆HvH =

4

4RTm2 Cexess p ∆Hcal

(8)

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excess In equation (8) R is the ideal gas constant, Tm is the melting temperature, Cp is constant-

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pressure excess heat capacity and ∆Hcal is the calorimetry enthalpy at Tm that is area under

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excess endotherm of Cp .

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2.12 Enzymatic assay of the esterase-like activity of HSA

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The effect of TH binding on the functionality of HSA was evaluated by measuring the esterase-

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like activity of HSA. 50 mM fresh stock solution of p-nitrophenyl acetate (p-NPA) was prepared

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by dissolving 18.11 mg of p-NPA in 2.0 ml in pure acetonitrile. Reaction mixtures containing 5

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µM HSA, which enabled complex formation at different molar ratios of HSA:TH (1:0, 1:5, and

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1:10), and samples were incubated for 12 h at 37 °C. Changes in enzyme activities were

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determined using different substrate (p-NPA) concentrations in the range 0.1 to 0.8 mM. After

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substrate addition final reaction mixture volume was 1.0 ml. The appearance of p-nitrophenol was

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determined by measuring absorbance at 405 nm in 1.0 cm path-length cuvette and monitoring it

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for up to 2 min until the absorbance started to decrease. The retained activity of HSA (in the

18

absence of TH and increasing molar ratio of HSA:TH) was expressed in terms of initial velocity

19

(

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2.13 Calculation of Kinetic parameters

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To determine the mode of substrate inhibition by TH, kinetic parameters were calculated by

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increasing TH concentrations. Enzyme inhibition kinetics were determined using a nonlinear

vo), which was obtained from the plot p-nitrophenol absorbance from 0 to 2 min.


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vo) were from the slopes of

1

regression method in Graph-Pad Prism version 5.0. Initial velocities (

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plots of absorbance vs. time (from 0 to 2 min) obtained using the Michaelis-Menten equation at

3

different p-NPA concentrations:

Vo=

4

Vmax [S] K m + [S]

(9)

vo is initial velocity, Vmax is maximal velocity respectively, [S] is substrate concentration,

5

where

6

and Michaelis-Menten constant (Km) is the substrate concentration at which the initial velocity

7

half-maximal velocity (

8

reciprocal plot) were obtained by plotting 1/

vo) is

half maximum velocity (Vmax). Lineweaver-Burk plots (double

vo versus 1/[S] in the absence or presence of TH:

K 1 = m V o Vmax

9

 1  1   +  [S]  Vmax

(10)

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2.14 Molecular docking

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We used AutoDock 4.2 for predicting the mode and affinity of TH binding to HSA. The three

12

dimensional crystal structure of HSA (PDB entry code: 1AO6) was obtained from the RCSB

13

Protein Data Bank,

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structure of TH (pubchem id: 92965). Before performing the docking, all water molecules and

15

ions bound to HSA were removed and then Gasteiger charges were computed. The docking

16

simulations were performed using the Lamarckian genetic algorithm (LGA) by applying default

17

parameters. For docking analysis, the grid size was set at 33.175, 30.604, and 34.136 along X, Y

18

and Z axes, respectively, to define domain I and subdomain IIA. The grid spacing was set to 0.375

19

Å. After generating the grid map docking simulations were performed using the following

20

parameters: population size 150, maximum number of energy evaluations 2.5 ×106, and maximum

21

number of iterations 27,000; remaining parameters were set at default values. The best

22

conformation of HSA-TH complex with least energy was identified among 15 different

7

and the PubChem Database was used to retrieve the three dimensional



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conformers and used for docking analysis. The PyMOL software package was used to visualize

2

docked conformations 21.

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2.15 Accessible surface area calculation

4

Differences between accessible surface areas (ASAs) of HSA before and after complex formation

5

were calculated using NACCESS version 2.1.1, 22 which calculates the exposed surface areas of all

6

atoms in a molecule. After complex formation changes in ASA for a specific residue ‘n’ were

7

calculated using ∆ASA = ASAfree HSA - ASAHSA-TH complex. If this formula showed a residue lost >10

8

Å2 ASA after complex formation, the residue was considered to be involved in the interaction.

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3. Results and Discussion

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3. 1 Toxicity analysis of TH in myoblasts

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Cell toxicity was analyzed using different TH concentrations (0, 1, 2.5 and 5 µM) in C2C12 cells,

12

cell morphology and growth was not changed by TH treatment (Fig. 2). Cell proliferation assay

13

showed that dose dependent TH treatment improves significantly myoblast proliferation rate

14

compared to control cells (Fig. 2B).

15

3.2 UV-visible spectrophotometry

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UV-visible absorption measurements of HSA solutions were performed in the absence and

17

presence of increasing TH concentrations (Fig. 3A). The inset of figure 3A, shows two peaks in

18

absorption spectrum of HSA and these peaks are consistence after TH addition. The absorption

19

peak near 218 nm is related to the backbone structure of HSA, while second at 278 nm is

20

associated with the absorption of aromatic amino acids (i.e., W, Y and P) assigned to the π-π*

21

transition of benzene 23. These findings agree with those of other studies 24-26. The peak intensity at

22

218 nm exhibited a red shift of ~2 nm, after addition of TH indicates alteration in the

23

microenvironment around the amide bonds of HSA. The peak intensity at 278 nm moved upward 12 ACS Paragon Plus Environment

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after increasing of TH concentrations; however, the absorption peak at 278 nm did not shifted

2

either left or right. This observation indicates that the microenvironment around the aromatic

3

amino acid residues was altered by HSA-TH complex formation.

4

3.3 Reformation of the secondary structure of HSA

5

Circular dichroism spectroscopy is used to characterize structural changes in proteins and

6

polypeptides resulting from interaction with small molecules. Elements of secondary structures,

7

such as, α-helices, β-sheets, β-turns, and random coil structures have specific peaks with unique

8

shapes and magnitudes in the far ultraviolet region

9

HSA-TH complex are shown in Fig. 3B, and exhibited two negative absorption bands at 208 and

27

. The far-UV CD spectra of free HSA and

28 29

10

222 nm characteristic of the α-helical structure of HSA

11

observed in CD-spectral behavior when the concentration of TH was increased in HSA solution.

12

Free HSA contains 51% α-helix, after the gradual additions of TH at 10 and 20 µM, the α-helical

13

content increased from 51% to 53% and then to 55%. Moreover, the negative bands of HSA at 208

14

nm and 222 nm increased steadily, suggesting complex formation due to increased α-helical

15

content. These obtained results showed that the biding of TH induces conformational change in

16

HSA, which is consistent with previous reports 16, 30, 31.

17

3.4 Analysis of fluorescence quenching of HSA in presence of TH

18

SSF spectrometry is commonly used to explore micro-environmental information of Trp present in

19

proteins after ligand binding. The aromatic fluorophores of proteins (W and Y) moieties, are

20

mostly responsible for intrinsic fluorescence, and fluorophores may be affected by ligand binding

21

32

. Fig. 3B shows the marginal changes

. In HSA, W214 lies in the subdomain IIA (Sudlow's binding site I) and contributes to intrinsic

22

fluorescence. It is well-known the greater quantum yield and higher fluorescence of W214 than

23

other aromatic moieties (Y and P) is largely accountable for the intrinsic fluorescence of HSA.



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Fluorescence spectrum of HSA give maximum emission at 340 nm, due to the presence of W214

2

residue of HSA 16. The fluorescence emission at 340 nm of HSA decreased regularly after adding

3

TH concentration indicates that the TH/HSA interaction quenched HSA fluorescence. This finding

4

suggests that the W214 residue of HSA is located at or near the TH binding site.

5

3.5 Type of quenching and its mechanism

6

Fluorescence quenching phenomenon is typically classified as dynamic or static quenching.

7

Dynamic quenching occurs due to collisions between a ligand and a protein, quencher. Dynamic

8

and static quenching can be differentiated by changing the temperature or viscosity of a solution 33.

9

For static quenching, Ksv decreases with an increase in temperature and the complex formed is

10

weakened, whereas for dynamic quenching, increasing temperature in diffusion and collision rates,

11

which increase Ksv 33.

12

To check the essence of the fluorescence quenching data well known Stern-Volmer plot of Fo/F vs

13

[Q] for Ksv is presented in Fig 4A. As can be concluded from equation 2, Ksv was obtained by its

14

slope determined by linear regression. The corresponding outcomes yielded the Stern-Volmer

15

constant

16

different temperatures. The magnitudes of Ksv values in the steady state fell by ~104 M-1 (Table 1),

17

which agreed with Stern-Volmer constants reported for interactions between drugs and proteins in

18

the 104-106 M-1 range in vivo

19

constant (kq) were 8.4±0.19×1012, 6.9±0.12×1012 and 6.0±0.13×1012, and 5.1±0.23×1012 M-1 s-1 at

20

25, 30, 37 and 42 °C respectively. The kq value at all studied temperatures for the HSA-TH

21

complex were in the order of 1012, was significantly much larger than the reported value of

22

dynamic quenching constant (2×1012 M-1 s-1) 32 Additionally, the values of Ksv and kq reduced with

34

. Table 1 shows the calculated Stern-Volmer constant (Ksv) values for TH binding at

5, 35

. Furthermore, the calculated values of bimolecular quenching


Page 15 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

raising temperature continuous which further confirmed the ground state complex formation

2

between HSA and TH.

3

3.6 Evaluation of the TH binding constant and binding stoichiometry

4

Binding constants (Kb) are crucial for understanding the distribution of a drug in plasma since

5

weak binding can lead to poor drug distribution. The values of Kb and binding stoichiometries (n)

6

for HSA-TH interaction at different temperatures were calculated from the intercept and slope of

7

modified Stern-Volmer plot between log (Fo/F-1) vs log [TH], as shown in Fig. 4B. The values of

8

Kb at 25, 30, 37 and 42 °C (determined using intercepts of equation 3) were as 2.3±0.13×104,

9

1.8±0.11×104, 1.5±0.16×104 and 1.4±0.26×104 M-1, respectively. The magnitudes of obtained Kb

10

values were of the order of 104, indicating a moderately strong interaction between HSA and TH,

11

which agreed with literature values (Table 1)36-38. In the present study, plots were linear and the

12

slopes obtained yielded binding stoichiometries (n) that suggested TH interacted with HSA at a

13

molar ratio of one-to-one at all temperatures studied, indicating strong binding between TH and

14

HSA39. Moreover, an inverse correlation between Kb and temperature, indicate that TH reversibly

15

bound with HSA, indicating an increase in temperature is likely to decrease complex stability.

16 17 18

3.7 Evaluations of thermodynamic parameters and the natures of binding forces

19

hydrogen bond formation, hydrophobic interactions, van der Waals interactions and

20

contacts

21

such as, ∆G°, ∆H° and T∆S° can be utilized to identify the main drivers of protein-drug complex

22

formation 40. Using the binding constants (Kb) obtained at 25, 30 37 and 42 °C, thermodynamics of

23

TH and HSA interactions were calculated by van’t Hoff equation 4 (ln K versus 1/T, Fig. 4C) and

24

values are presented in Table 1. Negative values of free energy (∆G°) revealed the binding of TH

The interaction forces between drugs and proteins are mainly includes electrostatic interactions,

36

steric

. The signs and magnitudes of thermodynamic parameters of protein-drug interactions,



Page 16 of 44

1

with HSA is spontaneous process. According to Ross and Subramanian40, a negative ∆H° value of

2

-4.9±0.11 kcal mol-1 and positive T∆S° values for the interaction between TH and HSA indicate the

3

presence of electrostatic interaction between the amino acid residues of HSA and TH 41.

4

3.8 Isothermal titration calorimetry (ITC)

5

In order to obtain more reliable quantitative approach to measure the binding and thermodynamic

6

parameters, such as, binding stoichiometry (N), binding affinity (Ka), dissociation constant (Kd),

7

enthalpy (∆H°) and entropy change (∆S°) was obtained by isothermal titration calorimetry (ITC)

8

from single experiment. During the experiment it was observed that size of produced peaks

9

decreases continuously after the injection of TH into the ITC cell was due to the binding between

10

TH with HSA and saturation was achieved (Fig. 5). Using ITC experiments, the binding

11

stoichiometry of TH was determined based on measuring the reaction which was not dependent on

12

the location of W214. The upper panel in Figure 5 shows the pulse signals produced (Dp in µcal s-

13

1

) on titrating TH against HSA at 37 °C. Each downward (negative) peak in upper panel of Fig. 5

14

shows an injection of TH into HSA solution and plotted against time. The differential isotherm

15

was obtained by integrating the obtained pulse signal and then subtracting the dilution heats of

16

buffer-TH and HSA-TH. The obtained data was fitted using a standard nonlinear least square

17

regression binding model, involving independent single set of binding sites and corrected data

18

plotted against molar ratio of HSA to TH as shown in Figure 5 (lower panel). The binding constant

19

(Ka) of TH to HSA was determined to be 2.15±0.12×105 M-1 with a binding stoichiometry (n) of

20

0.96 at 37 °C, which is similar to the previously reported values for EH-HSA complexes

21

obtained Ka between HSA and TH is moderate compared to those of other strong protein ligand

22

complexes, which have binding constants ranging from 107-108 M-1. The binding of TH is an

23

exothermic process with a reaction enthalpy (∆Hº) of -18.72±0.80 kcal mol-1. The negative value

16

. The


Page 17 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

of ∆Hº is mainly due to electrostatic interactions and hydrogen bonding between substituent’s on

2

the benzene ring of TH and the amino acid residues of HSA. The negative T∆Sº of -11.6 kcal mol-

3

1

contributes to TH/HSA complex formation. The negative values of ∆Hº and T∆Sº suggest the

4

involvement of hydrogen bond formation during HSA-TH complex formation. These results show

5

that the binding reaction between HSA and TH is entropy driven. The negative value of binding

6

free energy (∆Gº = -7.12 kcal mol-1) suggests that the binding of TH with HSA was spontaneous

7

process.

8

3.9 Energy transfer efficiency and HSA to TH binding distance

9

Förster resonance energy transfer (FRET) has been extensively used to measure the distance

10

between the donor molecule excited at specific excitation wavelength (due to W214 of HSA) and

11

an acceptor molecule (TH) in the ground state42. FRET depends on the relative orientations of

12

donor and acceptor dipoles and distances (r) between donor and acceptor molecules. The overlap

13

between the fluorescence spectrum of HSA and absorbance spectrum of TH is shown in Fig. 6.

14

According to FRET, the efficiency of energy transfer between HSA (W214) and TH was

15

calculated using equation 11:  R6 F   = 6 o 6 E FRET = 1 − Fo  R o + r 

16

(11)

17

where EFRET is energy transfer efficiency, The value of Ro was calculated using the equation: 18

R o6 = 8.79 × 10 −25 K 2 n −4ϕJ (12)

19

where K2 is the absorption spectrum. In the present case, we used these values, it has been

20

previously reported for HSA that K2 = 2/3, n = 1.336, φ = 0.15, and J, as was determined using

21

following equation:



∫ J=

∞

o

1

Page 18 of 44

F (λ)ε (λ)λ 4 dλ

∫

∞

o

F (λ)dλ

(13)

2 3 4

where F(λ) is emission intensity of donor at wavelength λ and ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ. According to the Eqs. (11-13) obtained values are as following, J = 2.45 × 10-15 cm3 M-1, Ro = 2.01 nm, E = 0.42 and r = 2.11 nm at 25 ̊C (Table 2).

5 6 7 8

For energy transfer donor-to-acceptor distance r must be within 2- 8 nm and the obligatory condition 0.5 Ro < r < 1.5 Ro must be fulfilled. It was found that, an ‘r’ value larger than Ro indicates efficient fluorescence quenching of HSA by TH via static mode. Furthermore, this confirmed the high probability of energy transfer from HSA to TH 16.

9

3.10 Assessment of thermal stability of HSA by differential scanning calorimetry

10

Temperature dependent thermal stability of proteins can be investigated by differential scanning

11

calorimetry (DSC)

12

biopharmaceuticals 44. The effects of TH on the thermal stability of HSA were investigated in the

13

absence and presence of TH. Fig. 7A shows a thermogram of HSA at pH 7.4 that includes two

14

transitions, that is, a first peak ( Tm1 ) at 57.41±0.03 and a second ( Tm2 ) at 70.28±0.21 °C. As shown

15

in Fig. 7B, the heat capacity function of HSA-TH complexes was lower than native HSA,

16

suggesting they underwent structural changes during unfolding. HSA:TH at a molar ratio of 1:5

17

exhibited higher Tm values than HSA with a Tm1 of 59±0.02 and a Tm2 of 68.23±0.13 °C, showing

18

that TH binding with HSA affects the thermal stability of HSA. As shown in Fig 7B, the addition

19

of TH caused a downward shift in heat capacity in obtained thermogram, suggesting a decrease in

20

the hydrophobic surface area of HSA, which is discussed in fluorescence quenching results. The

21

enthalpy ratio [van’t Hoff enthalpy (∆HvH)/ calorimetric enthalpy (∆Hcal)] is a parameter for

43

, and has proved to be useful during the development and formulation of


Page 19 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

cooperativity measurements (Table 3). ∆HvH/∆Hcal ratio suggests the existence of perturbation

2

during unfolding, which is coupled to the equilibrium between the folded and unfolded protein

3

species

4

unfolding process. Deviations from unity indicate reduced cooperativity and a non-two state

5

unfolding process

6

irreversible unfolding as observed in 1st transition of native HSA (Table 3). The enthalpy ratio of

7

∆HvH/∆Hcal was, calculated from the thermogram data of native HSA had the highest R value than

8

the HSA-TH complex (Table 3). Enthalpy (∆HvH/∆Hcal) ratios show two things, (i) larger distances

9

between domains

45

. If the ∆HvH/∆Hcal ratio is unity complete cooperativity is indicated and a two state

46

. If ratio of ∆HvH/∆Hcal smaller than unity is clearly indicates a kind of

47

and (ii) reduced participation of internal forces. Furthermore, a low

10

∆HvH/∆Hcal ratio was observed for TH modiﬁed HSA, indicating it had lower stability than native

11

HSA.

12

3.11 TH induced modulation in the functionality of HSA: esterase-like activity

13

The well known esterase-like activity of HSA was determined, as previously described

14

In addition, the esterase-like activity of HSA can be used for pharmaceutical purposes, as it

15

involves the conversion of prodrugs into drugs, such as, aspirin, nicotinic acid and ketoprofen

16

glucoronide

17

salicylate and absorbed in the gut, while the remainder is hydrolyzed by circulating serum albumin

18

52

51

11, 16, 48-50

.

. It has been shown that 30% of orally administered aspirin is hydrolyzed to

. From the site directed mutagenesis studies it has been reported that two reactive residues R410 16, 50, 53, 54

19

and Y411 located in subdomains IIIA are pivotal for the esterase-like activity of HSA

20

Active residues K199, H242, and R257 are responsible for esterase activity and constitute the most

21

catalytically efficient active sites

22

determined by measuring the formation of p-nitrophenol from p-NPA. Changes in the esterase-like

23

activity of HSA in the presence of increasing concentrations of TH are shown in Fig 8A. It was

55

.

. In the present study, the esterase-like activity of HSA was



Page 20 of 44

1

observed that retained activity reduced on increasing TH concentration (Table 4). To identify the

2

inhibition mode of TH, kinetic constants (Km and Vmax) were calculated using the Lineweaver-

3

Burk equation (10) at increasing TH molar ratios (1:0, 1:5 and 1:10). As shown in Fig. 8B the

4

Lineweaver-Burk plot (1/vo versus 1/[S]) was linear with an intercept of 1/Vmax and a slope of

5

Km/Vmax. The Km, Vmax and kcat values obtained are presented in Table 4. The Km values increased

6

from 26.7×10-2 to 53.0×10-2 and 91.2×10-2 mM and Vmax remained same at (HSA:TH molar ratios,

7

1:0, 1:5 and 1:10) all studied molar ratios. The increased Km value and no change in Vmax can be

8

concluded that the substrate (p-NPA) and the inhibitors (TH) compete for same binding to the

9

same active site. These findings are accord with those reported for the myorelaxant, eperisone 16

10

hydrochloride, which binds to HSA in a competitive manner

. Turn over number or catalytic

11

constant (kcat = Vmax/[E]) calculated from Vmax value, and the results shows that in the absence of

12

TH, the kcat value was 30.0×10-2 min-1. While at 1:5 and 1:10 molar ratios of TH to HSA, the value

13

of kcat increased to 32.8×10-2 and 34.6×10-2 min-1 respectively. The catalytic efficiency (kcat/Km) of

14

the hydrolysis of p-NPA by HSA fell significantly at higher TH to HSA molar ratios. The decrease

15

in the value of kcat/Km was attributed to the TH binds with HSA and induces the conformational

16

flexibility along with it provides adaptation towards drug binding.

17

3.12 Molecular docking

18

In order to provide the binding mode of a ligand with binding site in a macromolecule can be

19

predicted by Molecular docking analysis 56. HSA has a heart-like shape with a central channel that

20

allows small molecules to enter and bind with its subdomains

21

following similar domains (I, II and III), each comprised of two subdomains (A and B) and

22

composed of α-helices. Site I is known binding pocket inside the core of subdomain IIA (Fig. 9A),

23

which consists of 6 helices and loop-helix feature (amino acid residues 148-154) contributed by

57

. HSA is composed of three


Page 21 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

sudomain IB. The interior of this pocket is hydrophobic and is predominantly composed of W214,

2

L219, F223, L238, H242, L260, I264, S287, I290, A291 and Y150 (an inner cluster residue).

3

Molecular docking enabled the interaction between HSA and TH to be visualized in Fig. 9B.

4

Docking showed TH enters the cavity of subdomain IIA and interacts by hydrogen bonding with

5

K199 (2.94 Å) and H242 (2.64 Å) (Fig. 9C), and that TH can interact with the residues of HSA

6

located in the hydrophobic cleft lined by the amino acid residues Y150, E153, K199, L238, H242,

7

R257, A261, I264, S287, I290 and A291. Atoms involved in H-bonding and their bond distances

8

and interacting hydrophobic residues are shown in Table 5. The binding site of HSA is mainly

9

surrounded by hydrophobic amino acids that contribute to the stability of HSA-TH complex by

10

participating in hydrophobic interactions, as illustrated in the LigPlot in Figure 9C. For HSA-TH

11

binding the free energy was -7.0 kcal mol-1, which is close to the experimental values -5.96±0.16

12

and -7.12 kcal mol-1 obtained from fluorescence quenching and ITC data at 37 °C. Thus, molecular

13

docking studies provided structural evidence for quenching by TH located within site I of HSA.

14

3.13 Changes in the Accessible surface areas (ASAs) of HSA and HSA-TH complex

15

Calculation of accessible surface area (ASA) analysis enables the most likely binding mode of a

16

ligand within the binding site of a macromolecule. ∆ASA is defined the lost accessible area after

17

complex formation. In previous studies reported that ligand binding affinity depends on loss of

18

∆ASA of proteins, i.e. burning of more surface area had high-affinity complexes with ligands 58, 59.

19

Amino acid residues that lose ASA more than 10 Å2, after complex formation, are considered that

20

strongly take part in the interaction. HSA alone has a total ASA of 56019.61 Å2, but after TH

21

binding, this was reduced to 28394.078 Å2, which is indicative of strong TH to HSA binding.

22

Changes in ASA values of specific residues after TH to HSA binding are provided in Table 6.

23

Maximum reduction of ASA (from 36.98 to 3.66 Å2) was shown by A291, suggesting its



Page 22 of 44

1

involvement in TH to HSA binding. The interaction between TH and K199 corresponded to

2

hydrogen bond formation and caused a total ∆ASA reduction of 17.19 Å2. Amino acids Y150,

3

L238, R257 and I290 also showed ∆ASA losses of >10 Å2 and belong to the large hydrophobic

4

pocket of site I. ∆ASA calculations also demonstrated hydrogen bonds and hydrophobic

5

interactions play major roles in the binding of TH with HSA and concurred with determined

6

thermodynamic parameters from fluorescence quenching and DSC results obtained in this study.

7

4. Conclusion

8

Binding mechanism of TH with HSA was explored by analyzing multi-spectroscopic, calorimetric

9

and in silico approach. In addition, our results compared with previous work16 were also disused.

10

TH was found to have a hyper-chromic effect on the HSA absorption peak centered at 278 nm.

11

The emission spectra studies showed that the quenching of HSA by TH is a result of static

12

quenching mechanism. Analysis of CD spectra suggested binding of TH causes an increase in the

13

α-helical content of HSA. ITC data revealed that complex formation between TH and HSA which

14

detects high binding affinity and molecular docking results suggested the hydrogen boding (with

15

K199 and H242) and hydrophobic interactions contributed to the interaction. The molecular

16

docking results indicated that the preferred binding site of TH was located in IIA subdomain

17

(Sudlow site I). In accordance with FRET theory the binding between donor (W214 of HSA) and

18

acceptor (TH) was evaluated as 2.11 nm which indicated energy transfer between donor and

19

acceptor molecule. In our previous study on quenching of HSA by EH, the ∆Gº of binding was

20

found to be -6.5 kcal mol-1

21

was -5.92 kcal mol-1. The obtained results in the present study were complement those of our

22

previous study16. However, the conformations and the α-helix contents of EH/HSA and TH/HSA

23

differed slightly. Computational analysis revealed that both TH and EH bound at site I; the two

16

, while in the present study the free energy of TH binding by HSA


Page 23 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

only differed in terms of hydrogen bonding, that is, whereas TH formed hydrogen bonds with

2

K199 and H242, its analogue EH formed a hydrogen bond with R257. The molecular docking

3

results also revealed that the involvement of two hydrogen bonds between TH and HSA and

4

higher binding affinity between TH and HSA than between EH and HSA. In summary, current

5

study provides potential insights into the mechanism responsible for the binding of TH by HSA,

6

and improves understanding of the myorelaxant effect of TH on skeletal muscle during its

7

transport and distribution in the blood. This study provides an accurate and comprehensive

8

valuable insight to the pharmacological response of myorelaxant and design of dosage necessary to

9

achieve the desired effect but also support the toxicity background of TH.

10

Acknowledgements

11

Gulam Rabbani gratefully acknowledge to Research Grant of Yeungnam University, Republic of

12

Korea (2017) for supporting this work. We are thankful to Prof. Seok Gyu Kim, department of

13

chemistry, Yeungnam University, for providing experimental facilities CD and fluorescence

14

measurements.

15

References

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

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23. Shen, G. F., Liu, T. T., Wang, Q., Jiang, M., Shi, J. H. Spectroscopic and molecular docking studies of binding interaction of gefitinib, lapatinib and sunitinib with bovine serum albumin (BSA). J. Photochem. Photobiol. B 2015, 153, 380-390. 24. Liu, X. H., Xi, P. X., Chen, F. J., Xu, Z. H., Zeng, Z. Z. Spectroscopic studies on binding of 1phenyl-3-(coumarin-6-yl)sulfonylurea to bovine serum albumin. J. Photochem. Photobiol. B 2008, 92, 98-102. 25. Wang, Y. Q., Tang, B. P., Zhang, H. M., Zhou, Q. H., Zhang, G. C. Studies on the interaction between imidacloprid and human serum albumin: spectroscopic approach. J. Photochem. Photobiol. B 2009, 94, 183-190. 26. Zhang, H., Wang, Y., Zhu, H., Fei, Z., Cao, J. Binding mechanism of triclocarban with human serum albumin: Effect on the conformation and activity of the model transport protein. J. Mol. Liq. 2017, 247, 281-288. 27. Whitmore, L., Wallace, B. A. Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 2008, 89, 392-400. 28. Matei, I., Hillebrand, M. Interaction of kaempferol with human serum albumin: a fluorescence and circular dichroism study. J. Pharm. Biomed. Anal. 2010, 51, 768-773. 29. Sreerama, N., Woody, R. W. Computation and analysis of protein circular dichroism spectra. Methods Enzymol. 2004, 383, 318-351. 30. Rabbani, G., Baig, M. H., Jan, A. T., Ju Lee, E., Khan, M. V., Zaman, M., Farouk, A. E., Khan, R. H., Choi, I. Binding of erucic acid with human serum albumin using a spectroscopic and molecular docking study. Int. J. Biol. Macromol. 2017, 105, 1572-1580. 31. 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. 32. 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. 33. Lakowicz, J. R. Principles of fluorescence spectroscopy, Kluwer Academic Publishers/Plenum Press, New York,(1999). 34. 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 Biointerfaces 2014, 123, 96-105. 35. Kratochwil, N. A., Huber, W., Muller, F., Kansy, M., Gerber, P. R. Predicting plasma protein binding of drugs: a new approach. Biochem. Pharmacol 2002, 64, 1355-1374. 36. Hu, Y. J., Liu, Y., Xiao, X. H. Investigation of the interaction between berberine and human serum albumin. Biomacromolecules 2009, 10, 517-521. 37. Chakrabarty, A., Mallick, A., Haldar, B., Das, P., Chattopadhyay, N. Binding interaction of a biological photosensitizer with serum albumins: a biophysical study, Biomacromolecules 2007, 8, 920-927. 38. Chi, Z., Liu, R. Phenotypic characterization of the binding of tetracycline to human serum albumin. Biomacromolecules 2011, 12, 203-209. 25 ACS Paragon Plus Environment


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39. Kragh-Hansen, U. Molecular aspects of ligand binding to serum albumin. Pharmacol. Rev. 1981, 33, 17-53. 40. Ross, P. D., Subramanian, S. Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry 1981, 20, 3096-3102. 41. Olsson, T. S., Williams, M. A., Pitt, W. R., Ladbury, J. E. The thermodynamics of proteinligand interaction and solvation: insights for ligand design. J. Mol. Biol. 2008, 384, 1002-1017. 42. Förster, T. 10th Spiers Memorial Lecture. Transfer mechanisms of electronic excitation. Discuss. Faraday Soc. 1959, 7-17. 43. Pico, G. A. Thermodynamic features of the thermal unfolding of human serum albumin. Int. J. Biol. Macromol. 1997, 20, 63-73. 44. Lundblad, R. L. Introduction to biopharmaceutical conformational analysis, in approaches to the conformational analysis of biopharmaceuticals, Chapman and Hall/CRC C1-Issues and Methods. 2009, pp 1-18. 45. Horn, J. R., Russell, D., Lewis, E. A., Murphy, K. P. van't Hoff and calorimetric enthalpies from isothermal titration calorimetry: are there significant discrepancies?. Biochemistry 2001, 40, 1774-1778. 46. 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. 47. Novokhatny, V., Ingham, K. Thermodynamics of maltose binding protein unfolding. Protein Sci. 1997, 6, 141-146. 48. 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. 49. Sakurai, Y., Ma, S. F., Watanabe, H., Yamaotsu, N., Hirono, S., Kurono, Y., Kragh-Hansen, U., Otagiri, M. Esterase-like activity of serum albumin: characterization of its structural chemistry using p-nitrophenyl esters as substrates. Pharm. Res. 2004, 21, 285-292. 50. 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, 813-819. 51. Dubois-Presle, N., Lapicque, F., Maurice, M. H., Fournel-Gigleux, S., Magdalou, J., Abiteboul, M., Siest, G., Netter, P. Stereoselective esterase activity of human serum albumin toward ketoprofen glucuronide. Mol. Pharmacol. 1995, 47, 647-653. 52. Seymour, R. A., Williams, F. M., Ward, A., Rawlins, M. D. Aspirin metabolism and efficacy in postoperative dental pain. Br. J. Clin. Pharmacol. 1984, 17, 697-701. 53. Kragh-Hansen, U. Molecular and practical aspects of the enzymatic properties of human serum albumin and of albumin-ligand complexes. Biochim. Biophys. Acta 2013, 1830, 55355544. 54. Walker, J. E. Lysine residue 199 of human serum albumin is modified by acetylsalicyclic acid. FEBS Lett. 1976, 66, 173-175. 26 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 11 12 13


55. Phuangsawai, O., Hannongbua, S., Gleeson, M. P. Elucidating the origin of the esterase activity of human serum albumin using QM/MM calculations. J. Phys. Chem. B 2014, 118, 11886-11894. 56. Sousa, S. F., Fernandes, P. A., Ramos, M. J. Protein-ligand docking: current status and future challenges. Proteins 2006, 65, 15-26. 57. Kragh-Hansen, U. Structure and ligand binding properties of human serum albumin. Dan. Med. Bull. 1990, 37, 57-84. 58. Kastritis, P. L., Bonvin, A. M. Are scoring functions in protein-protein docking ready to predict interactomes? Clues from a novel binding affinity benchmark. J. Proteome Res. 2010, 9, 2216-2225. 59. Chen, J., Sawyer, N., Regan, L. Protein-protein interactions: general trends in the relationship between binding affinity and interfacial buried surface area. Protein Sci. 2013, 22, 510-515.

14 15 16 17 18 19 20 21 22 23 24 25 26



Page 28 of 44

1

Figure Legends

2

Figure 1: (A) Molecular structure of tolperisone hydrochloride (B) eperisone hydrochloride.

3

Figure 2: Different TH concentration was added for 1 day in C2C12 cells. (A) Cells in the absence

4

and presence of TH. (B) Cell proliferation assay. 0 µM TH indicate controls and results presented

5

as the means±SDs of three independent experiments.

6

Figure 3: (A) UV-visible absorption spectra of HSA in the absence and presence of

7

tolperisone hydrochloride at different concentrations. (B) Circular dichroism of the free HSA and

8

HSA-TH complexes. The free HSA and HSA-TH complexes in aqueous solution after addition of

9

TH (1:0, 1:5 and 1:10 molar ratio).

10

Figure 4: (A) Stern-Volmer plots and (B) double-logarithm plot for the quenching of HSA by TH

11

at four different temperatures (C) The van’t Hoff plot for calculation of thermodynamic

12

parameters.

13

Figure 5: Isothermal titration calorimetry profile of TH. Upper panel shows the raw heat data

14

obtained from the consecutive injections of TH. Bottom panel is the integrated binding isotherms

15

as a function of HSA-TH complex at 37 °C. Solid line represents the fitted data obtained from

16

single set of binding site model.

17

Figure 6: The spectral overlaps of the fluorescence emission spectrum of HSA with the UV-vis

18

absorption spectrum of TH. The molar ratio of protein and TH was 1:1. The protein concentration

19

was 2 µM.

20

Figure 7: (A) Calorimetric melting profile of HSA (black line) at pH 7.4 and the best ﬁt of the

21

curves to the non-two-state transition model (thin red line) (B) Thermal unfolding profile of HSA

22

and TH (black line) in the presence of 15 µM HSA and 75 µM TH (1:5 molar ratio).

23

Figure 8: (A) Effect of TH on the reaction rate for the hydrolysis of p-NPA by HSA. Kinetic data

24

obtained by fitting of

25 26 27

Burk plot for the steady state kinetics of p-NPA hydrolysis by HSA in the absence and presence of TH. Figure 9: (A) HSA (PDB: 1AO6) docked with the TH molecules. The protein backbone is shown

28

schematically as a ribbon. (B) The binding site was magnified to show the interactions of TH with

29

HSA. The HSA is represented by ribbon structure whereas TH by stick model. (C) Two-

30

dimensional schematic representation of TH at site I of HSA with its hydrogen bond interactions

31

shown by LigPlot.

vo and [S], according to the Michaelis-Menten equation 9 (B) Lineweaver-


Page 29 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2 3 4 5 6 7 8 9

10 11 12 13 14

Fig .1

15 16 17 18 19 20 21 22 29 ACS Paragon Plus Environment


Page 30 of 44

1 2 3 4 5 6 7

A

B

8 9 10 11 12

Fig. 2

13 14 15 16 17


Page 31 of 44

1

0.24

3

3

Absorbance

A

2

Absorbance

0.18

4 5

1.2 0.6

250

300

350

Wavelength (nm)

Native 1:2 1:4 1:6 1:8 1:10

7

0 240

9

1.8

200

0.12

0.06

8

2.4

0

6

260

280

300

320

340

Wavelength (nm)

10

10

11

B

5

12 13

CD (m deg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


14 15

0 -5 -10 -15

1:0 1:5 1:10

16

-20 17

-25 18 19

200 Fig. 3

210

220

230

240

250

Wavelength (nm)

20 21 22 23



Page 32 of 44

1.9

1 1.6

Fo/F

2 3

1.3

4

25 °C 30 °C 37 °C 42 °C

5 6

1 0.0E+00 5.0E-06

7 8

1.0E-05

1.5E-05

2.0E-05

[TH]

0.5

B 0

9 log(Fo/F-1)

10 11

-0.5

-1

12

25 °C 30 °C 37 °C 42 °C

-1.5

13 14

-2 -6.5

15

-6

-5.5

-5

-4.5

log [TH]

10.1

16

C

17

10

18

9.9

19

lnK

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

9.8

20 9.7

21 9.6

22 23

y = 2466.4x + 1.7246

Figure 4:

9.5 3.15E-03 3.20E-03 3.25E-03 3.30E-03 3.35E-03 3.40E-03

1/T (K-1)


Page 33 of 44

1 2 3 4 5

Time (min)

6

0 10 20 30 40 50 60 70 80 90

7

0.00

µcal sec

-1

8 -0.20

9 10

-0.40

11 0.0

12 -3.0

13 -6.0

14

-1

kcal mol of injectant

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-9.0

15

-12.0

16 0.0

0.5

1.0

1.5

17

Molar Ratio 18 19

Figure 5:

20 21 22 23 24 25



1 2 3 4 5 6

Normalized fluorescence intensity

7 8 9 10 11 12 13 14

1.0

Fluorescence Absorbance

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

0.0

Normalized absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 44

300 320 340 360 380 400 420 440

15 16

Wavelength (nm) Figure 6:

17 18 19 20 21 22 23 24 25


Page 35 of 44

1 2 3

20

A

Cp (kcal mole-1 oC-1)

4 5 6 7 8 9

15 10 5 0

10

20 30 40 50 60 70 80 90 100

11

o

Temperature ( C)

12 13

20

14 15

Cp (kcal mole-1 oC-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


16 17 18 19 20

15 10 5 0

21

20 30 40 50 60 70 80 90 100 o

22 23

B

Temperature ( C) Figure 7:

24 25



1 2 3

1.2×10 -3

A

4

Vo (mM min-1)

5 6 7

9.0×10 -4 6.0×10 -4 1:0 1:5 1:10

3.0×10 -4

8

0 0.0

9

0.3

0.6

0.9

S [mM]

10 11 12

6000 B

13

5000

14

1/vo (min mM-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 44

15 16 17 18

4000 3000 2000

19

0

20

-6

-3

0

3

6

9

12

1/[S] (mM-1)

21 22

1:0 1:5 1:10

1000

Figure 8:

23 24 25 36 ACS Paragon Plus Environment

Page 37 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Figure 9:


Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 38 of 44

Table 1: The quenching constant (Ksv), binding constant (Kb), binding stoichiometry (n), and thermodynamic parameters between HSA

and TH at 25, 30, 37 and 42 °C obtained from fluorescence quenching experiments. Parameter

25 °C

n (binding stoichiometry, HSA:TH) -1

30 °C

0.93±0.16

37 °C

0.93±0.11 4

42 °C

0.92±0.12

0.93±0.17

KSV (Stern-Volmer constant, M )

4.9±0.34×10

4.0±0.29×10

3.5±0.24×10

3.0±0.28×104

Kb (binding constant, M-1)

2.3±0.13×104

1.8±0.11×104

1.5±0.16×104

1.4±0.26×104

kq (bimolecular quenching rate constant, M-1 s-1)

8.4±0.19×1012

6.9±0.12×1012

6.0±0.13×1012

5.1±0.23×1012

∆Hº (binding enthalpy, kcal mol-1) -1

4

4

-4.9±0.11

T∆Sº (entropy change, kcal mol )

1.02±0.12

1.03±0.11

1.06±0.13

1.07±0.15

∆Gº (Gibbs free energy change, kcal mol-1)

-5.92±0.14

5.93±0.13

5.96±0.16

5.97±0.17

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


Page 39 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2: FRET from steady state measurements performed at 25 °C. Parameter

Value

J (cm-3 M-1)

2.45×10-15

Ro (nm) r (nm) EFRET Fo 3667 F 2109

2.01 2.11 0.42



Page 40 of 44

Table 3: Thermodynamic parameters for the thermal unfolding of HSA and HSA-TH complex obtained by differential scanning calorimetry at pH 7.4 Transition

Parameters

Native HSA

Modified HSA

Tm1

7.41±0.033

59±0.02

∆H1cal

225.9±1.85

91.78±1.14

∆H 1vH 1

74.2±0.49 0.32

102.7±0.71 1.11

Tm2 2 ∆H cal

70.28±0.21 47.39±1.95

68.23±0.13 66.13±1.29

∆H 2vH R1

67.39±2.7 1.42

55.19±0.96 0.83

1st transition

R

2nd transition

R1 or R2=∆HvH/∆Hcal Tm is expressed in °C ∆H is expressed in kcal mol-1


Page 41 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 4: Kinetic parameters describing Michaelis-Menten constant, Vmax, catalytic constant and catalytic efficiency of HSA in the absence and presence of increasing TH concentrations. HSA:TH

RA

Vmax

Km

kcat

kcat/Km

(%)

(mM min-1)

(mM)

(min-1)

(mM-1 min-1)

1:0

100

15.0×10-4

26.7×10-2

30.0×10-2

1.12

1:5

96

16.4×10-4

53.0×10-2

32.8×10-2

0.62

1:10

92

17.3×10-4

91.2×10-2

34.6×10-2

0.38

All measurements were carried out in 20 mM sodium phosphate buffer pH 7.4 at 37 °C Values of Vmax and Km were derived from Lineweaver-Burk equation (10) RA; relative activity kcat/Km; catalytic efficiency kcat; catalytic constant (Vmax= kcat × Enzyme concentration) The concentration of HSA was 5 µM



Page 42 of 44

Table 5: Docking results showing detailed analysis of the binding free energy, major residues involved in hydrogen bonding and hydrophobic interactions.

System

HSA-TH

Hydrogen bond (length) K199--O1 TH (2.94 Å) H242--O1 TH (2.42 Å)

Hydrophobic interaction

Y150, E153, L238, R257, A261, I264, S287, I290, A291

Binding free energy (kcal mol-1) -7.0


Page 43 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 6: Changes in the ASA (Å2) values of the interacting residues of HSA and HSA-TH complex Residues

ASA (Å2) of HSA

ASA (Å2) of HSA

∆ASA (Å )

2

Y150

17.30

1.49

15.81

E153

11.65

2.40

9.25

K199

32.00

14.81

17.19

L238

30.63

6.66

23.97

H242

5.23

0

5.23

R257

19.64

5.39

14.25

A261

4.27

0.13

4.14

I264

10.89

8.31

2.58

S287

8.87

4.23

4.64

I290

13.68

2.86

10.82

A291

36.98

3.66

33.32



Page 44 of 44

For table of contents use only

Binding of tolperisone hydrochloride with human serum albumin: effects on the conformation, thermodynamics, and activity of HSA Gulam Rabbani*, Eun Ju Lee, Khurshid Ahmad, Mohammad Hassan Baig and Inho Choi*

Department of Medical Biotechnology, Yeungnam University, 280 Daehak-ro, Gyeongsan, Gyeongbuk-38541, Republic of Korea

*Address for correspondence Department of Medical Biotechnology, Yeungnam University, 280 Daehak-ro, Gyeongsan, Gyeongbuk-38541, Republic of Korea Fax: +82 53 810 4769; E-mail: [email protected] [email protected]


Binding of Tolperisone Hydrochloride with Human Serum Albumin

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