Thermodynamics and binding mechanism of polyphenon-60 with human lysozyme elucidated by calorimetric and spectroscopic techniques

Accepted Manuscript Thermodynamics and binding mechanism of polyphenon-60 with human lysozyme elucidated by calorimetric and spectroscopic techniques Shama Yasmeen, Riyazuddeen PII: DOI: Reference:

S0021-9614(17)30046-0 http://dx.doi.org/10.1016/j.jct.2017.02.013 YJCHT 4981

To appear in:

J. Chem. Thermodynamics

Received Date: Revised Date: Accepted Date:

10 September 2016 16 February 2017 18 February 2017

Please cite this article as: S. Yasmeen, Riyazuddeen, Thermodynamics and binding mechanism of polyphenon-60 with human lysozyme elucidated by calorimetric and spectroscopic techniques, J. Chem. Thermodynamics (2017), doi: http://dx.doi.org/10.1016/j.jct.2017.02.013

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Thermodynamics and binding mechanism of polyphenon-60 with human lysozyme elucidated by calorimetric and spectroscopic techniques Shama Yasmeen and Riyazuddeen* Department of Chemistry, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India E-mail: [email protected] *corresponding author

ABSTRACT Protein-drug interaction offer information of the structural features that determine the therapeutic effectiveness of drug and have become an attractive research field in life science, chemistry, and clinical medicine. Interaction of pharmacologically important antioxidant drug polyphenon-60 with human lysozyme (Lys) at physiological pH 7.4 has been studied by using calorimetric and various spectroscopic techniques. UV-visible spectroscopy results indicate the complex formation between Lys and polyphenon-60. The binding constant, quenching mechanism and the number of binding sites were determined by the fluorescence quenching spectra of Lys in presence of polyphenon-60. Fluorescence data calculate that the polyphenon-60 interact with Lys through static quenching mechanism with binding affinity of 2.9 × 104 M-1. The average binding distance between drug and Lys was found to be 2.89 nm on the basis of the theory of Förster's energy transfer. Isothermal titration calorimetry (ITC) data reveals the thermodynamic investigations which suggest that the interaction of Lys and polyphenon-60 through exothermic process and enthalpy driven and also explore that the polyphenon-60 binds in both site of Lys with high and low affinity. Hydrogen bonding (high affinity) and hydrophobic interactions (low affinity) are the major forces in stabilizing the drug protein complex. Far-UV CD and FTIR results deciphere the conformational alterations in the secondary structure of Lys.

Keywords: Circular dichroism; Fluorescence spectroscopy; Human lysozyme; Isothermal titration calorimetry; Polyphenon-60.

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1. Introduction The binding between proteins and drugs has involved a great interest among researchers for several decades. Proteins have the great ability to reversibly bind a large variety of endogenous and exogenous ligands, like nutrients, hormones, fatty acids, and variety of drugs. On the other hand, drugs can cause different changes in protein conformation affecting its physiological functions. Such weakened proteins may be therefore accumulated in body tissues. Consequently, the studies of protein-drug interactions play an important role in pharmacology and pharmacodynamics [1]. Green tea is a highly consumed beverage in Asian countries especially in China and Japan [2]. One of the major active components in green tea is polyphenol [3]. The polyphenon-60 found in the folios of the green tea obtained from the unfermented leaves of the plant Camellia sinensis are thought to be the biochemical compounds [4,5]. Among the diverse cases of tea, green tea contains a comparatively high level of polyphenols, which consist of flavanol monomers (flavan-3-old), likewise known as catechins [6]. Polyphenon-60 contains minimum sixty percent of the catechins. Catechins are water soluble polyphenolic substances that include epigallocatechin (EGC), epicatechin (EC), epigallocatechin-3-gallate (EGCG), and epicatechin-3-gallate (ECG) [7,8]. Epigallocatechin EGCG has been found to be the most abundant and most biologically active compounds of green tea [9,10,11]. Polyphenon-60 is a highly active molecule acting as antioxidant, antidiabetic, antiobesity, antioxidant, antiinflammatory and anticancer agents [12-18]. The antioxidant activity of polyphenon-60 is due to the inherent ability to neutralize free radicals occurring as natural by-products of normal cell. Catechins was also found to be efficacious in cutting down body fat [6,19]. Drinking of green tea potentially decreases the occurrence of breast cancer [3]. Plyphenone-60 has a healing effect on acne by suppressing inflammation [20]. The fields indicated that green tea is a powerful inhibitor of lung tumour development [21,22]. Lysozyme is commonly utilized as a model protein because of its natural abundance, high stability and small size [23]. Human lysozyme has a higher specific activity, it plays significant parts of the human host-defense in various bio-fluids, such as milk, saliva, tears, airway epithelium and blood [24-27]. It is a small protein with molecular weight around 15 kDa and is constituted by 130 amino acid residues, containing 6 tryptophans (Trp), 4 disulfide bonds and 3 tyrosines (Tyr) [28-30]. Its three Trp residues (Trp-62, Trp-63 and Trp-108) are all located near

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the protein's active site, which are known to bind reversibly to a mixture of different molecules [31,32]. Its structure comprises two areas the first domain (residues 40-85) consists mostly of βsheet structure, also known as β-domain whereas the second domain (residues 89–99) is more helical in nature known as α-domain [33]. It has strong ability to carry drugs and functional compounds in mammals. It can cure some illness via the binding with some active compounds [34,35]. Lysozyme acts as an antimicrobial agent, anti-inflammatory agent and anti-HIV and possesses tumouricidal activity [36,37]. Lysozyme has also been reported to be used for wound healing and inhibiting angiogenesis and tumour growth [38,39]. Lysozyme has many physiological and pharmaceutical functions. Therefore, studies on the binding between small molecules and lysozyme have an important significance on realizing the transport and metabolism process of the small molecules. There are some literatures on the interactions between drug molecules and lysozyme. Wang et al. [40] used fluorescence spectroscopy and circular dichroism (CD) to study the interaction between puerarin and lysozyme. Li et al. [41] used spectrophotometric techniques such as steady fluorescence, synchronous fluorescence, CD and UV-vis absorption to study the interaction between nevadensin and lysozyme, and measured the binding constants, binding sites and binding strength for the nevadensin-lysozyme system. Ding [42] investigated the interaction between chloramphenicol and lysozyme by fluorescence, UV/vis and circular dichroism spectra. The primary aim of this study was to discover the nature of the interaction between Lys and polyphenon-60. The presented study is expected to contribute to the current knowledge in the field of protein-drug binding, particularly polyphenon-60-Lys interactions. Thermodynamic parameters can possibly provide valuable information as to the nature of the interactions and the forces that stabilize the complexes. Among many research methods in the area of protein-drug interactions, isothermal titration calorimetry (ITC) has become a versatile technique that would furnish a complete profile of the interaction, including binding constant, stoichiometry, and other important thermodynamic data. Yet, until today, there are no reports where interactions of a Lys with polyphynon-60 have been studied in detail to our best of knowledge. In this project we have under taken the investigation of binding of human lysozyme to polyphenon-60 through UVvisible, fluorescence spectroscopy, isothermal titration calorimetry, circular dichorism and FTIR approaches to understand its binding mechanism. The thermodynamic parameters, such as enthalpy change, entropy change and Gibbs energy change for the binding reaction have been

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calculated. The finding of this study helps in revealing the plausible reason of its its binding with HSA and structural changes which always is the topic of utmost investigation. 2. Experimental 2.1. Materials Recombinant, expressed in rice, lyophilised human lysozyme (L1667, mass fraction ≥0.90) and polyphenon-60 (P1204) were purchased from Sigma-Aldrich. The composition of polyphenon 60 was determined by GC-MS. The components of polyphenon-60 are given in Table 1. Structures of identified catechins are given in Figure 1. The GC-MS chromatogram and MS spectra are given Figures S1 and S2 in the supplementary material. All other reagents and sodium phosphate buffer compounds used were of analytical grade. The 20 mM sodium phosphate buffer (pH= 7.4) was prepared by dissolving the disodium hydrogen orthophosphate dihydrate (0.703 g) and sodium dihydrogen orthophosphate dihydrate (0.16381 g) for 500 ml. Human lysozyme (Lys) was utilized without further purification. The stock solution Lys (2.40×10-4 mol.dm-3) was prepared in 0.020 mol.dm-3 sodium phosphate buffer, pH 7.4 and× its concentration was ୫୑ determined spectrophotometrically using Eୡ୫ of 36 at 280 nm [43]. The stock solution of

polyphenon-60 was prepared by dissolving 5 mg of its crystals in 1 mL of sodium phosphate buffer solution while a working solution was created by diluting it to the desired concentration with the above buffer. The details of the chemical used in the present work are given in Table 1. 2.2. UV-vis absorption measurements UV spectra of Lys in the presence and absence of polyphenon-60 were carried out on a Perkin-Elmer Lambda-25 spectrophotometer (Singapore) over a wavelength range 250-350 nm. The concentration of Lys was 2.0×10-5 mol·dm-3. The concentration of polyphenon-60 was changed from (0 to 6.4×10-5) mol·dm-3 in steps of 8×10-6 mol·dm-3. Quartz cuvettes of 1 cm path length were used for the measurements. For the use of analysing the energy transfer from Lys to polyphenon-60, an absorption spectrum of polyphenon-60 and an emission spectrum of 5×10-6 mol·dm-3 Lys were recorded over the wavelength range (300-400) nm.

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2.3. Fluorescence measurements Fluorescence measurements were performed on Hitachi spectrofluorometer (Model F2500) at temperature T = (298.15 ± 0.01) K with a path length cell of 1 cm. The excitation and emission slits were set at 10 nm. Intrinsic fluorescence was measured by exciting Lys at 295 nm which is the excitation peak for the tryptophan residue. The addition of polyphenon-60 to Lys solution leads to a decrease in the fluorescence intensity indicating that the binding of polyphenon-60 to Lys quenches the intrinsic fluorescence of tryptophan. [44] The decrease in fluorescence intensity at 342 nm by the addition of polyphenon-60 was analysed according to the Stern-Volmer equation [45]

ிబ ி

= ‫ܭ‬௦௩ [ܳ] + 1

(1)

where Fo and F are the fluorescence intensities in absence and presence of quencher (polyphenon-60), respectively; Ksv is the Stern-Volmer quenching constant and Q is quencher concentration. A value of 1.8×10-9 s was used for τo, the fluorescence lifetime of Lys in the absence of the quencher to compute the value of the bimolecular quenching constant, kq using the following equation [46]

݇௤ =

௄ೞೡ

(2)

ఛ೚

The binding constant ka and the number of binding sites can be calculated using the following equation [47]

log

ி೚ ିி ி

= log‫ܭ‬௕ + nlog[ܳ]

(3)

where Kb is the binding constant and n is binding stoichiometry. The change in the Gibbs energy was calculated using the equation

∆‫ = ܩ‬−ܴ݈ܶ݊‫ܭ‬௕

(4)

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2.4. CD measurements The circular dichroism studies of Lys in presence and absence of polyphenon-60 were carried out with JASCO (J-815) CD spectrometer equipped with a Peltier-type temperature controller. All the CD spectra were collected in a cell of 0.5 mm path-length. The scan speed was 100 nm/min and response time of 1 s for all measurements. Each spectrum was the average of 2 scans. The raw data obtained in millidegrees were converted to mean residue ellipticity (MRE) in deg·cm-2·dmol-1, using the following equation [45]

MRE =

ఏ೚್ೞ

(5)

ଵ଴×௡×஼×௟

where Θobs is the observed ellipticity in millidegrees; C is the concentration of Lys in mol·L-1; n is the number of amino acid residues, and l is the length of the light path in cm. All spectra were smoothed by the Savitzky-Golay method with 9 convolution width. Alpha helical content was calculated from the MRE values at 222 nm using the following equation as described by Chen et al. [45]

% ߙ⎼ℎ݈݁݅‫ = ݔ‬ቀ

ெோாమమమ೙೘ ିଶଷସ଴ ଷ଴ଷ଴଴

ቁ × 100

(6)

2.5. Isothermal titration calorimetric (ITC) measurements The interaction between Lys with polyphenon-60 was assessed by ITC using a MicroCal ITC200. ITC titrations were performed at 298 K and reference cell was filled with the 20 mM sodium phosphate buffer (pH 7.4). The Lys solution (7.5 ×10-5 mol·dm-3) was retained in the sample cell and polyphenone-60 (2.5 mg·mL) was filled in the syringe of volume 40 µL. The drug solution was added sequentially in 2 µL aliquots (for a total of 19 injections, 4 s duration each) at 120 s intervals. The reference power and stirring speed were put at 2.5×10-5J·s-1 and 600 RPM, respectively. The ITC experiments were repeated at least two times to make sure reproducibility with background heat adjustments from drug solution titrations into buffer alone to account for the heat of dilution/mixing. The uncertainty reported in terms of standard deviation (i.e. level of confidence = 0.95) from curve fitting and replicate measurements. To correct the heat effects of dilution and mixing, control experiments were performed at the same concentrations of the

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buffer and drugs and subtracted from the respective drug-protein titrations. The heat liberated or taken up upon each injection was evaluated, and the data were plotted as integrated quantities. The titration curves were fitted to a two-site sequential binding model using the MicroCal Origin software. The stoichiometry (n), association constant (Ka), the change in entropy (∆H) and the change in entropy (∆S) were received immediately from the ITC data. While the change in Gibbs energy (∆G) was computed from equation 7 [44]

∆‫ ܩ‬௢ = ∆‫ ܪ‬௢ − ܶ∆ܵ ௢

(7)

The ITC instrument was sometimes calibrated and verified with (water + water) dilution experiments as per the criteria of the manufacturer. The combined standard uncertainty uc(x) for the calorimetric data was calculated using equation ଶ

ଶ

ଶ

ଵ/ଶ

‫ݑ‬௖ (‫ = )ݔ‬ቂ൫‫ݑ‬ଵ (‫) ݔ‬൯ + ൫‫ݑ‬ଶ (‫) ݔ‬൯ + ൫‫ݑ‬ଷ (‫) ݔ‬൯ + … . +݁‫ܿݐ‬ቃ

(8)

where ‫ݑ‬ଵ (‫) ݔ‬, ‫ݑ‬ଵ (‫)ݔ‬, ‫ݑ‬ଵ (‫) ݔ‬, etc. represent the individual uncertainties in the measurements. 2.6. FTIR measurements Fourier transform infrared spectra (FTIR) of the protein solutions were carried out at room temperature and the spectra were collected in the range of 1800-1400 cm−1 using FTIR/FIR spectrometer Frontier (Perkin Elmer) equipped with a Attenuated Total Reflection (ATR) accessory with resolution 4 cm-1 and 40 scans. The concentration of Lys was 7.5×10-5 mol·dm-3 for native and 7.5 ×10-5 mol·dm-3 Lys + 7.5×10-5 g·mL-1 polyphenol-60 for mixture. 3. Results and discussion 3.1. UV-visible absorption study UV absorption measurement is a very simple but effective method for exploring the structural changes and understanding the complex formation. The absorption spectra of Lys in the presence and absence of polyphenon-60 are shown in Figure 2. The strong absorption of Lys at 280 nm resulted from the π–π* transition of phenyl group of tryptophan (Trp) and tyrosines (Tyr) residues in Lys and reflects the frame work conformation of Lys. Absorbance at 280 nm of Lys was increased with the gradual addition of polyphenon-60 (extracted from green tea). The

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increased intensity of the Lys-drug system as compared to the pure Lys is an indication of the complex formation between Lys and polyphenon-60. 3.2. Fluorescence quenching mechanism and determination of binding constant (Kb) Intrinsic fluorescence spectra of Lys in the wavelength range (300-400) nm upon excitation at 295 nm, obtained in the absence and the presence of increasing drug concentrations at room temperature are shown in Figure 3. The appearance of the emission maxima at 342 nm was suggestive of the presence of Trp residues. The fluorescence intensity of Lys decreases gradually with the increase in the concentration of drug in solution. Fall in the fluorescence intensity may be assigned to the extinction of a fluorophore by the change in solution conditions such as temperature, pH or addition of any substance [48]. Presence of polyphenon-60 in the proximity of Lys induces the environment around the protein, which eventually affects the fluorescence spectra. Fluorescence quenching usually proceeds through two mechanisms: static and the dynamic quenching. The static mechanism requires the establishment of the ground state complex between fluorophore and quencher while dynamic quenching proceeds through the shaping of the excited state complex. A broader understanding of the quenching mechanism can be reached from quantitative analysis of the observational data. In order to confirm the quenching mechanism, the procedure of the fluorescence quenching was first assumed to be dynamic quenching and data were analysed according to Stern-Volmer equation. The plots of Fo/F versus polyphenon-60 concentration were found to be linear (Figure 4) with the values of Ksv and kq equal to 1.83×104 M-1 and 1.018×1012 M-1·s-1, respectively. Dynamic and static quenching can be distinguished on the basis of calculated values of the bimolecular rate constant. For dynamic quenching, the maximum scatter collision quenching constant of quencher with the bio molecule is about 2.0 × 1010 M-1·s-1[49]. A larger value of the bimolecular rate constant reflects the engagement of the static quenching mechanism in the present instance. The quenching data were further analysed to evaluate the binding constant (Kb) and the binding stoichiometry (n) (Eq. 3). In this case the binding constant was found to be 2.9 ×104 M-1 and the value of n was approximately equal to 1, which indicates that the equimolar ratio is enough to saturate binding site of Lys with the polyphenon-60 [50]. Further, binding process was observed to be spontaneous and thermodynamically favoured as the ∆‫ ܩ‬଴ binding value was found to be

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negative (-25.4±0.11) kJ·mol-1. All the parameters obtained from fluorescence spectroscopy are given in Table 2. 3.3. Fluorescence resonance energy transfer (FRET) FRET is a non-radiative spectroscopic method that can monitor the proximity and relative angular orientation of fluorophores. Agreeing to this theory in addition to radiation and re absorption, a conveyance of energy could also take place through direct electro dynamic interaction between the primarily excited molecule and its neighbours. Figure 5 depicts a spectral overlap between the fluorescence emission spectra of free Lys and the absorption spectrum of free polyphenon-60 which can be applied for the calculation of Förster’s non-radiative energy transfer theory, FRET, a distance dependent interaction in which excitation energy is transferred non-radiatively from donor to acceptor. According to this theory, the distance r could be calculated by the equation [51,52]

‫ܧ‬୊ୖ୉୘ = ቀ1 −

ி

ிೀ

ቁ=

ோ೚ల

ோ೚ల ି௥ ల

(9)

Here EFRET denotes the efficiency of transfer between the donor and the acceptor; Fo and F are the fluorescence intensities of Lys in absence and presence of polyphenon-60, respectively. The r is the average distance between donor and acceptor; and Ro is the critical distance when the efficiency of transfer is 50%. The value of Ro is calculated utilizing the equation [53]

ܴ௢଺ = 8.79 × 10ିଶହ ‫ ܭ‬ଶ ݊ିସ ߮‫ܬ‬

(10)

Here K2 is the spatial orientation factor of the dipole; n is the refractive index of the medium; φ is the fluorescence quantum yield of the giver; and J is overlap integral of the fluorescence emission spectrum of the giver and the absorption spectrum of the acceptor (Figure 5). The J can be calculated from the following equation ‫׬‬೚ ி(ఒ)ఌ(ఒ)ఒర ௗఒ ∞

‫=ܬ‬

∞

‫׬‬೚ ி(ఒ)ௗ

(11)

10

where F (λ) is the fluorescence intensity of the donor at wavelength λ, and ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ. According to equations (8)–(10), J, R0, and EFRET can be obtained. The J, Ro and EFRET values are applied for the computation of value of r. For Lys, K2 = 2/3 is the spatial orientation factor of the dipole; n is the refractive index of medium (N = 1.36); φ is the fluorescence quantum yield of the donor (φ = 0.14) [51]. The FRET results are summed up in Table 3. The distance between polyphenon-60 and Lys is 2.89 nm, which is less than 7 nm, illustrating that the non-radiation energy transfer from Lys to polyphenon-60 had occurred with high probability. 3.4. Isothermal titration calorimetric (ITC) measurements ITC is a biophysical technique used to determine the thermodynamic parameters for binding of macromolecules (protein) and drug. Its multidimensional approach makes it even more attractive and valuable for protein-drug interactions. Due to more than one binding site in Lys, we analyse ITC data of two binding sites by using sequential binding sites. The primary calorimetric titration data (after appropriate correction for heat of dilution) at a representative temperature (T = 298.15 K) are presented in Figure 6 and the thermodynamic parameters obtained from fitting of the integrated heat data to a two sets of sequential binding sites model are given in Table 4. A representative calorimetric titration profile of Lys with polyphenon-60 at pH 7.4 and 298.15 K is shown in upper panel of Figure 6. Each peak in the isotherm represents a single injection of polyphenon-60 into Lys solution. The negative heat deflection indicates that the binding is an exothermic process. The upper panel of Figure 6 depicts the plot of the amount of heat liberated per injection as a subroutine of the molar ratio of polyphenon-60 to Lys. The polyphenon-60-Lys affinity constant (Ka) value is found to be as (104 and 103) (L·mol-1) for strong and weak binding sites at 298.15 K. The ITC measurements appear to point out that the polyphenon-60-Lys binding phenomenon is characterized by a favourable exothermic enthalpy contribution (∆H<0) with a favourable entropic contribution (∆S>0) for high affinity site. The corresponding negative enthalpy change (∆H) and entropy change (∆S) values suggest that Lys and polyphenon-60 form a complex for low or weak binding sites. The ITC analysis of polyphenon-60 binding to Lys reveals favourable enthalpies of binding, consistent with an extended network of hydrogen-bonding interactions in the complex for both high and low binding affinities. Further, negative ∆G values for both side binding are indicating feasibility of

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the interaction of polyphenon-60 to Lys. This fact is in coherence with our fluorescence results, in which we have also obtained negative value of ∆G. However, on comparing the magnitude of calorimetric parameters with fluorescence binding it is clear that in ITC Kb as well ∆Go are found higher in magnitude than the fluorescence result. The differences in fluorescence and ITC results could be ascertained to the reason that fluorescence measures local change only around the Trp residue whereas ITC measures global change. The dominating binding is received from ITC is enthalpy driven and hydrogen bonding with hydrophobic interaction. 3.5. Circular dichorism (CD) measurements The far-UV CD spectra of Lys are performed in the presence and absence of the polyphenon-60 to obtain an insight into the secondary structural changes of Lys. CD spectra of Lys were obtained in the presence of different concentrations of polyphenon-60. The two peaks are appeared in the Figure 7, one at 208 nm and other at 222 nm both negative peaks are characteristic of α-helical structure. Peak at 208 nm is assigned to n → π* transitions, while peak at 222 nm is allocated to π → π* transitions for the peptide bond of a-helix [53]. The two characteristic peaks are found to decrease with increasing drug concentrations. The polyphenon60 have hydrophobic core that can be interacted with hydrophobic residues of Lys and destabilize it, hence the secondary structure of Lys in the presence of polyphenon-60 deceases which is shown in Table 5. The outcomes indicate that the Lys molecules probably adopt a looser conformation with the extended polypeptide structures that indicate the hydrogen bond alteration in the Lys. Furthermore, it can be concluded that the occupancy of the Trp sites by the binding ligands could actually destabilize the native conformation of the protein. The binding of polyphenon-60 with Lys could not destroy the protein hydrogen bonding networks and induce the secondary structure changes in it. 3.6. FT-IR characterization Fourier transform infrared spectrometry (FT-IR) can furnish valuable insights on the overall secondary structure of proteins in several conditions and has been employed in the determination of ligand-protein interaction. However, FT-IR spectra of proteins exhibit number of amide bands (amide I, II and III) significant to secondary structure, but amide I band is usually considered appropriate, owing to its specific sensitivity towards secondary changes. The

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amide I band is generally due to the vibrations of C=O stretching and prolongs in the range of (1600-1700) cm-1 [54]. The FT-IR spectra of Lys in the absence and presence of polyphenon-60, shown in Figure 8, provide evidence that upon addition of polyphenon-60, the amide-1 band shifts from 1634.28 cm-1 to 1641.82 cm-1, suggesting binding interaction between polyphenon-60 and Lys. 4. Conclusion This study reports spectroscopic and calorimetric studies on the binding of polyphenon60 with Lys using UV-vis absorption, fluorescence, CD, FTIR and ITC techniques. Measurements of fluorescence spectroscopy provide us important information about binding of drug to protein. On the basis of fluorescence results it can be concluded that polyphenon-60 interacts with Lys through static quenching mechanism. The ITC study reveals that polyphenon60 is able to bind to Lys at two different sites and via different modes of interaction and demonstrated that the binding of polyphenon-60 to Lys is enthalpically favoured and is mostly hydrogen bonding in nature with some hydrophobic effects. The binding constant (Kb) which is obtained from both fluorescence and ITC results shows strong binding between Lys and polyphenon-60. Given that binding of polyphenon-60 to Lys is strongly enthalpically favored with an unfavourable entropy change within high affinity site and favorable entropy change with the low affinity site. The distance (r) between Lys and polyphenon-60 was evaluated as r = 2.89 nm, in accordance with Forster non-radioactive resonance energy-transfer theory. The far-UV CD and FTIR results suggest that a conformational alteration takes in the protein on the addition of drug solution. Awareness of the nature and significance of these interactions is vital in assessing the pharmacokinetics and pharmacodynamic properties of drugs during drug design and development. Acknowledgement The authors are thankful to chairman department of chemistry, A.M.U., Aligarh for providing the necessary facilities for the completion of this work. Financial supports from the UGC under SAP DRS II Scheme and the DST under PURSE phase II programme are greatfully acknowledged.

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16

Table 1 Compounds used in this study with their sources and purity.

Material used Recombinant,expressed

Provenance Sigma-Aldrich

in rice, lyophilised

Mass fraction purity ≥0.90 (As stated by the supplier)

human lysozyme (L1667) Polyphenon-60 (P1204)

Sigma-Aldrich

a

0.302 EGCG, 0.205 EGC, 0.083 EC,

0.074 ECG, 0.177 protein, 0.077 caffeine, 0.043 fibres and 0.039 minerals. (by GC-MS) di-sodium

hydrogen Merck

orthophosphate

0.98 (As stated by the supplier)

dihydrate (Na2HPO4.2H2O) Sodium

dihydrogen Merck

orthophosphate

0.98 (As stated by the supplier)

dihydrate (NaH2PO4. 2H2O)

a

EGCG: Epigallocatechin gallate, EGC : Epigallocatechin, EC : Epichatechin (290), ECG : Epicatechine gallate CG.

Table 2

17

Binding parameters such as bimolecular quenching rate constants (Kq), binding constants (Kb), number of binding sites (n) and free energy change (∆G) of Lys and polyphenon-60 obtained from fluorescence quenching experiments at T = 298.15 K, P = 101.13 kPa and pH 7.4. Parameters

Value

Stoichiometry of binding (n)

1.05

Stern-Volmer quenching constant (Ksv)

(1.8329 ±0.012)×104 L·mol-1

bimolecular rate constant (kq)

1.018×1012 L·mol-1·s-1

binding constant (Kb)

(2.9 ±0.018)×104 L·mol-1

Gibbs energy (∆G)

−(25.4±0.11) kJ·mol-1

The standard uncertainty u are u (p) =10 kPa The standard uncertainty u are u (T) = 0.01 K The standard uncertainty u in the molarity of the buffer is u(m) = 0.02 × 10-3 mol·kg-1. The reported uncertainties are combined expanded uncertainties at 0.95 level of confidence (k = 2).

Table 3 The values of overlap integral (J), critical distance (Ro), average distance (r) and efficiency transfer (EFRET) was calculated using fluorescence and UV-Vis spectroscopy measurements performed at T = 298.15 K, P = 101.13 kPa and pH 7.4. Parameters

Value

J /(cm-3·M-1)

(4.11 ± 0.037 )×10-15

Ro /(nm)

2.14±0.004

r /(nm)

2.89±0.006

EFRET

0.14±0.001

Fo

1018

F

874.9

The standard uncertainty u are u (p) =10 kPa The standard uncertainty u are u (T) = 0.01 K

18

The standard uncertainty u in the molarity of the buffer is u(m) = 0.04 × 10-3 mol·kg-1. The reported uncertainties are combined expanded uncertainties at 0.95 level of confidence (k = 2). Table 4 Thermodynamic Parameters: binding constant (Ka), standard Gibbs energy of binding (∆Go), enthalpy of binding (∆Ho), and entropy of binding (∆So) accompanying the titration of 2.5×10-3 g·mL-1 polyphenon-60 with 7.5×10-5 mol·dm-3 Lys obtained from ITC T = 298.15 K, P = 101.13 kPa and pH 7.4. Parameters

Value

High affinity ka /(L·mol-1)

(2.3 ±2.2) ×104

∆Ho /(kJ·mol-1)

-39.33 ± 15.2

∆So /(kJ·mol-1·deg-1)

-0.048 ± 0.002

o

-1

T∆S /(kJ·mol )

-14.03 ± 0.02

∆Go /(kJ·mol-1)

-25.30 ± 0.11

Low affinity ka /(L·mol-1)

(9.19 ± 6.2) ×103

∆Ho /(kJ·mol-1)

-11.48 ± 2.92

∆So/(kJ·mol-1·deg-1)

0.037 ± 0.001

o

-1

T∆S /(kJ·mol )

11.03 ± 0.002

∆Go /(kJ·mol-1)

-22.51± 0.103

The standard uncertainty u are u (p) =10 kPa The standard uncertainty u are u (T) = 0.01 K The standard uncertainty u in the molarity of the buffer is u(m) = 0.04 × 10-3 mol·kg-1. The reported uncertainties are combined expanded uncertainties at 0.95 level of confidence (k = 2).

Table 5

19

Secondary structure analysis from the free Lys and Lys-polyphenon-60 complex at T = 298.15 K, P = 101.13 kPa and pH 7.4. Polyphenon-60 (µg/ml)

α-helix

0

40.9 ± 0.53

5

31.7 ± 0.37

10

25.6 ± 0.22

The standard uncertainty u are u (p) =10 kPa The standard uncertainty u are u (T) = 0.01 K The standard uncertainty u in the molarity of the buffer is u(m) = 0.04 × 10-3 mol·kg-1. The reported uncertainties are combined expanded uncertainties at 0.95 level of confidence (k =2).

20

Epigallocatechin (EGC)

Epicatechin (EC)

Epigallocatechin gallate (EGCG)

Figure 1.Structures of identified catechin compounds.

Epicatechin gallate (ECG)

21

6

5

8

absorbance

4

3

2

1

1

0 250

300

350

wavelength (nm)

Figure 2. Vis spectrum of Lys in the presence of polyphenon-60. The concentration of Lys was fixed at 2.0×10-5 mol·dm-3. Concentration of polyphenon-60 varies from 0. 8 -6.4×10-5 g·mL-1.

1000

0

fluorescence intensity

800

600

12 400

200

0 320

340

360

380

400

420

440

wavelength (nm)

Figure 3. Fluorescence spectrum of Lys in the presence of polyphenon-60. The concentration of Lys was fixed at 5.0×10-6 mol·dm-3. Concentration of polyphenon-60 varies from top to bottom 5×10-6 g·mL-1 to 6.0×10-5 g·mL-1.

22

2.2

2.0

FO/F

1.8

1.6

1.4

1.2

A

1.0

0.00000 0.00001 0.00002 0.00003 0.00004 0.00005 0.00006

[polyphenon-60]

0.2

0.0

log(Fo/F-1)

-0.2

-0.4

-0.6

-0.8

B

-1.0

-1.2 -5.4

-5.2

-5.0

-4.8

-4.6

-4.4

-4.2

log[polyphenon-60]

Figure 4. (A) Stern-Volmer plot for Lys-polyphenon-60 interaction and (B) the modified SternVolmer plot for Lys-polyphenon-60 interaction at 298 K.

23

fluorescence absorbance

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0 300

320

340

360

380

Normalized absorbance

Normalized FI

1.0

0.0 400

wavelength

Figure 5. Spectral overlap of absorption spectra of polyphenon-60 (blue) with fluorescence of Lys (pink). Concentration of Lys and polyphenon-60 equals 5 ×10−6 mol·dm-3 and 5×10-6 g·mL-1, respectively.

24

Time (min) 0

10

20

30

0.00

µWatts

-1.00 -2.00 -3.00 -4.00

-1

kJ mol of injectant

0.0

-8.4

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

Molar Ratio

Figure 6. Representative ITC profile showing the raw data for the integrated heat change (after appropriate correction for heat of dilution) for the binding of Lys with polyphenon-60 at 298 K in phosphate buffer (pH 7.40). The titration curves were fitted to a two site sequential binding model using MicroCal Origin software.

25

-5000

2

-1

MRE (deg cm dmol ))

0

-10000

-15000

0 5µg/ml 10µg/ml -20000 200

210

220

230

240

250

wavelength (nm)

Figure 7. Far-UV CD spectra of interaction of Lys with polyphenon-60 at 298.15 K. Concentration of Lys was fixed at 1.0 ×10-5 mol·dm-3. 0.10 0.09

1641.82

0.08

amide1

absorbance

0.07

1634.28

0.06 0.05 0.04 0.03 0.02 0.01 1500

1600

1700

1800

wavelength

Figure 8. FT-IR spectra of Lys in the absence and presence of polyphenon-60, Lys concentration was fixed at 7.5×10-5 mol·dm-3 and concentration of polyphenon-60 was fixed at 7.5×10-5 g·mL-1

26

Highlights


Thermodynamics of the binding of Lys with polypenone-60 were studied.
The binding was found to be exothermic.
Polyphenon-60 quenches the fluorescence of Lys through static quenching.
Polyphenon-60 binds to Lys through hydrogen binding.
Conformational changes of Lys were studied using circular dichorism.