Biophysical investigation of thymoquinone binding to ‘N’ and ‘B’ isoforms of human serum albumin: exploring the interaction mechanism and radical scavenging activity

Abstract

Thymoquinone (TQ) is the main constituent of Nigella sativa and is traditionally used as a folk medicine. Our aim was to investigate the binding mechanism of TQ to human serum albumin (HSA) isoforms (‘N’ form at pH 7.4 and ‘B’ form at pH 9.0) using biophysical methods such as intrinsic tryptophan fluorescence quenching, isothermal titration calorimetry (ITC), circular dichroism (CD), dynamic light scattering (DLS), Förster resonance energy transfer (FRET) and antioxidant activity in the absence and presence of TQ. We have calculated the binding and thermodynamic parameters from spectroscopic and calorimetric methods. CD and DLS were respectively used to monitor the changes in the secondary structure and hydrodynamic radii of HSA as a result of its interaction with TQ. The esterase and antioxidant or radical scavenging activities of both the isoforms of HSA were investigated in the absence/presence of TQ. The antioxidant activity of TQ was remarkably enhanced upon its interaction with HSA. Therefore, the efficiency of HSA to scavenge the free radical ions was increased in the presence of TQ which is generated in the body by various metabolic processes.

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Introduction

Thymoquinone (TQ), a major constituent of Nigella sativa (Ranunculaceae) essential oil, has been traditionally employed in folk medicine and is now recognized as a herbal remedy by a number of pharmacopoeias.¹ It is an annual plant that grows in the mediterranean area of India and Pakistan. The biochemical activities of N. sativa has been ascribed to quinones specifically TQ or 2-isopropyl-5-methyl-1, 4-benzoquinone.² TQ shows antimicrobial,³ anti-inflammatory,⁴ neuroprotective,⁵ antidiabetic, anticancerous,⁶ antihypertensive, and mast cell stabilizing effects,⁷ in addition to a protective role against in vitro induced ischemia.^8,9 Moreover, the therapeutic potential of TQ has been confirmed in cancer research also.^10–15 TQ has a promising role as antineoplastic growth inhibitor against various tumor cell lines.^16,17 It has protective effects against rheumatoid arthritis,¹⁸ induces telomere shortening, DNA damage, apoptosis in human glioblastoma cells¹⁹ and triple-negative breast cancer (TNBC) cells.²⁰ TQ also shows anti-oxidative properties against oxidative damage induced by a variety of free radical generating agents (including carbon tetrachloride, cis-platin, doxorubicin and recently HIV-1 protease inhibitor),^2,21 analgesic and anti-inflammatory role against renal injury.²² Because of its immense biological importance, there is an escalating interest to test it in pre-clinical and clinical researches for assessing its health benefits.

Human serum albumin (HSA) is a highly abundant serum protein that comprises 50–60% of the total plasma protein in humans.²³ Albumin is responsible for the transport, storage and metabolism of many therapeutic drugs in the blood stream thereby restricting their free, active concentrations and therefore can significantly affect their pharmacokinetics.²⁴ There are four pH dependent isoforms of HSA that have been characterized in the past. At physiological pH 7.4, HSA assumes the native form (N) which changes to fast migrating form (F) at pH < 4.3 and at pH < 2.7 it changes to the fully extended form (E). Whereas on the basic side at pH > 8 the ‘N’ form changes to basic form (B).²⁵ Polyphenols interact with HSA through its binding sites at different domains. HSA has two primary binding sites for various ligands commonly referred as binding site I and II which are located in subdomain IIA and IIIA, respectively.¹⁰ HSA binds a variety of molecules, a property that can have profound effects on their pharmacokinetics and pharmacodynamics.²⁶ Binding of polyphenols to albumin alters the pattern and volume of distribution, lowers the rate of clearance, and increases the plasma half-life of the drug.

The work presented here was focused on dissecting the spectroscopic and thermodynamic basis of HSA–polyphenols interactions investigating the mode and forces responsible for binding. Specifically, the aim of this study was to explore the binding of polyphenols to HSA under normal as well as alkaline conditions. Trp fluorescence quenching was monitored at different temperatures to elucidate the mechanism of TQ binding to HSA. CD and DLS were used to study the effect of TQ binding on the overall conformation of HSA, while ITC was used to determine the thermodynamic of TQ–HSA interaction. We have also monitored the esterase activity and radical scavenging activity of HSA in the presence of TQ. Antioxidant property of TQ plays an important role in various types of diseases and it also dependent on condition such as alkalosis where it's antioxidant activity increases and esterase activity reduces. Therefore, the binding and thermodynamics studies of TQ–HSA interaction shall provide useful information on the structural features that determine the therapeutic efficacy of TQ.

Materials and methods

Materials

Fatty acid free human serum albumin (A1887), thymoquinone (274666), glycine (G8898), p-nitrophenyl acetate, 4-p-NPA (N8130), 2,2′-azino-bis (3-ethyl benzothiazoline-6-sulfonic acid) diammonium salt, ABTS (A1888), potassium peroxodisulfate, K₂S₂O₈ (P5592), trolox (238813) were purchased from Sigma-Aldrich and MOPS (134894) buffer was purchased from SRL.

Sample preparation

A stock solution of HSA was made in 20 mM MOPS pH 7.4 and glycine–NaOH pH 9.0 buffers and the protein concentrations was determined spectrophotometrically using E^1%_{280 nm} of 5.30 at 280 nm (ref. 7 and 8) on a Perkin-Elmer Lambda 25 spectrophotometer. Buffers used throughout the experiments were filtered by 0.45 μm Millipore Millex-HV PVDF filter and pH was measured using Mettler Toledo (model S20) pH meter. The stock solution of TQ (5 mM) was prepared in 10% ethanol and finally adjusted to 1.0 ml by diluting with respective buffers.

UV-visible absorption measurements

UV-visible spectra were recorded between 250 and 350 nm on Perkin-Elmer Lambda 25 double beam spectrophotometer attached with Peltier temperature programmer (PTP-1) to maintain temperature at 37 °C throughout the experiments. HSA (6 μM) was titrated by 0–30 μM TQ in a 1 cm path length cuvette of 3 ml. All HSA–TQ absorbance spectra were corrected with respective blank, which consist same concentration of TQ in buffer in the absence of HSA.

Fluorescence quenching measurements

All the fluorescence measurements were carried out on Schimadzu (RF-5301PC) fluorescence spectrophotometer equipped with a constant temperature holder attached with Neslab RTE-110 water bath with an accuracy of ±0.1 °C. Intrinsic fluorescence was measured by exciting HSA (2 μM) at 295 nm and the emission spectrum was measured in the range of 300–450 nm, because tryptophan fluorescence is used as a probe of local environment in a protein for determination of protein structure, dynamics as well as ligand binding. The decrease in fluorescence intensity at particular wavelength was analyzed according to the Stern–Volmer eqn (1):⁸where F_o and F were the fluorescence intensities in the absence and presence of quencher (TQ), K_SV is the Stern–Volmer quenching constant. Binding constants and binding stoichiometry were obtained from eqn (2):²⁷

K_SV = k_q·τ_o (2)

where k_q is the bimolecular rate constant of the quenching reaction and τ_o is the average integral fluorescence life time of tryptophan which is ∼5.78 × 10⁻⁹ s.²⁸where K_b is the binding constant and n is binding stoichiometry.

The thermodynamic parameters i.e. change in enthalpy (ΔH°) and change in entropy (ΔS°) were determined after measuring K_b at different temperatures and the results were analyzed according to van't Hoff eqn (4):

where R is universal gas constant (1.987 cal mol⁻¹ K⁻¹).

The change in Gibbs free energy (ΔG°) can be further determined from separate terms of enthalpy change (ΔH°) and entropy change (ΔS°) according to the eqn (5):

ΔG° = ΔH° − TΔS° (5)

The three-dimensional fluorescence measurement were performed on Hitachi F-4500 spectrofluorometer under the following condition: the emission wavelength was recorded between 200 and 600 nm, the initial excitation wavelength was set to 200 nm with increment of 5 nm, the excitation and emission slit widths were fixed at 10 nm.²⁹

Isothermal titration calorimetric measurements (ITC)

The energetics of the binding of TQ to HSA at 37 °C was measured by using a VP-ITC titration microcalorimeter (MicroCal Inc., Northampton, MA). Prior to begin the titration experiment, all samples were degassed on a thermovac. The sample cell loaded with 20 μM HSA dissolved in pH 7.4 and pH 9.0 and reference cell filled with the respective buffer. Multiple injections of 10 μL of TQ solution (3.0 mM) were made into the sample cell containing serum albumin. Each injection was made over 20 s with an interval of 180 s between successive injections. The reference power and stirring speed were set at 16 μcal s⁻¹ and 307 rpm, respectively. Heats of dilution for the ligands were determined by the control experiments, and these were subtracted from the integrated data before curve fitting. The first derivative of temperature dependence of enthalpy change is used for the calculation of experimental heat capacity change calculated from eqn (6):³⁰

Temperature dependent van't Hoff enthalpy (ΔH_vH) at particular temperature is calculated by the equation:

where, T₁ and T₂ are the maximum and minimum experimental temperature, K(T₁) and K(T₂) are the values of binding constant at respective temperatures.

Circular dichroism (CD) measurements

CD measurements were carried out with a Jasco spectropolarimeter (J-815) attached with a Peltier-type temperature controller. The instrument was calibrated with D-10-camphorsulfonic acid. All The CD measurements were carried at pH 7.4 and pH 9.0 at physiological temperature 37 °C. Spectra were collected with a scan speed of 50 nm min⁻¹, data pitch 0.1 nm and a response time of 2 s. Each spectrum was the average of 2 scans. Far-UV CD spectra (190–250 nm) were taken at TQ concentrations from 0–50 μM with protein concentrations of 2 μM and 0.1 cm path length cells. The results were expressed as mean residue ellipticity (MRE) in degree cm² dmol⁻¹, which is defined as:where θ_obs is the observed ellipticity in degrees, C is the molar concentration of HSA, n is the number of amino acid residues (585 − 1 = 584) and l is the pathlength of cuvette in centimeter. Helical content of HSA was calculated from the MRE values at 222 nm using the following equation as described by Chen et al.:³¹

The thermal denaturation experiments were carried between 25 and 90 °C with 1 °C min⁻¹ temperature slope probed by far-UV CD at 222 nm. The curves were normalized, assuming a linear temperature dependence of the base lines of native and denatured states.

Data analysis of thermal denaturation

Thermal denaturation data from CD spectroscopy were analyzed on the basis of two-state unfolding model. For a single step unfolding process, N ⇄ U, where N is the native state and U is the unfolded state, the equilibrium constant K_u is:with f_u and f_n being the molar fraction of U and N, respectively.where Y_obs, Y_n and Y_u represent the observed property, the property of the native state, and the property of unfolded state, respectively.

Dynamic light scattering measurements

DLS measurements were carried out at 830 nm by using DynaPro-TC-04 dynamic light scattering equipment (Protein Solutions, Wyatt Technology, Santa Barbara, CA) equipped with a temperature-controlled micro sampler. HSA (30 μM) was incubated with the different concentration of TQ for 8 h, before scanning the samples were spun at 10 000 rpm for 10 min and were filtered serially through 0.22 and 0.02 μm Whatman syringe filters directly into a 12 μl quartz cuvette. For each experiment, 20 measurements were taken. Mean hydrodynamic radius (R_h) and polydispersity were analyzed using Dynamics 6.10.0.10 software at optimized resolution. The R_h was estimated on the basis of an autocorrelation analysis of scattered light intensity data based on translation diffusion coefficient by Stoke's–Einstein relationship:where R_h is the hydrodynamic radius, k is Boltzmann constant, T is the absolute temperature, η is the viscosity of water and D is the diffusion coefficient.³²

Tryptophan fluorescence resonance energy transfer (FRET) to TQ

The fluorescence spectra of HSA (2 μM) and absorption spectra of TQ (2 μM) between 300 and 400 nm were scanned in similar way as given in method sections ‘Fluorescence quenching’ and ‘UV-visible’ experiments at 37 °C. If the emission spectrum of donor (Trp214) significantly overlaps with the absorption spectrum of acceptor (TQ), these donor–acceptor pairs will be considered in Förster distance and then we could ascertain the possibility of energy transfer.³³ Therefore, the degree of energy transfer depends upon the area of overlap and the distance between these donor–acceptor molecules. The efficiency of energy transfer (E) is calculated using the following equation:³⁴where F_o and F were the fluorescence intensities of HSA in absence and presence of TQ, respectively; r is the distance between donor and acceptor and R_o is the critical distance at which transfer efficiency equals to 50% which can be calculated from the following equation:

R_o⁶ = 8.79 × 10⁻²⁵K²n⁻⁴ϕJ (14)

where K² is the orientation factor related to the geometry of the donor and acceptor of dipoles, n is the refractive index of the medium, ϕ is the fluorescence quantum yield of the donor in absence of acceptor; and J expresses the degree of spectral overlap between the donor emission and the acceptor absorption which can be evaluated by integrating the overlap spectral area in between 300 and 400 nm from following equation:where F(λ) is the fluorescence intensity of the donor at wavelength range λ which is dimensionless, and ε(λ) is the molar absorptivity (extinction coefficient) of the acceptor at wavelength λ in M⁻¹ cm⁻¹. In the present study, K², n and ϕ were taken as 2/3, 1.336 and 0.118, respectively.³⁵

Molecular docking parameters

The three dimensional X-ray crystal structure of HSA (PDB ID: 1AO6, resolution 2.5 Å) was downloaded from the RCSB Protein Data Bank. The three dimensional structure of TQ was retrieved from pubchem [CID: 10281]. We performed docking studies using docking program AutoDock version 4.0.^36,37 AutoDock works on Lamarkian genetic algorithm and calculate all possible conformations of the ligand that binds to the protein. Polar hydrogen atoms, Kollman charges were merged to the protein and Gasteiger charges were added to the ligands using graphical user interface program AutoDock Tools (ADT) and then prepared file was saved in PDBQT format. For the preparation of the grid map using a grid box Auto-Grid was used. Size of grid was set to 70 Å × 70 Å × 70 Å xyz points with spacing of 0.375 Å, which covers all the available active site residues. To encompass two binding sites (subdomain IIA and IIIA, respectively) during the docking process, the two different grid centers along the x-, y-, z-axes were set for subdomain IIA and for subdomain IIIA, respectively. To achieve our goal the complex showing lowest binding energy with best fitness score was used. For visualization purpose we used Pymol version 1.3 and chimera version 1.8.³⁸

Enzyme activation kinetics by esterase activity (determination of esterase-like activity)

The reaction of p-nitrophenyl acetate with HSA was followed spectrophotometrically by monitoring the appearance of p-nitrophenol³⁹ at 405 nm for time duration of 2 min on Perkin-Elmer Lambda 25 double beam spectrophotometer attached with Peltier temperature programmer (PTP-1) to maintain temperature at 25 °C throughout the experiments. The reaction mixtures contained 5 μM p-nitrophenyl acetate and 5 μM protein in 20 mM MOPS (pH 7.4) and 20 mM glycine–NaOH (pH 9.0) buffers. The Michaelis–Menten equation was used to get the rectangular hyperbolic pattern of a typical enzyme-substrate reaction:where V_o and V_max is the initial and maximum velocity, respectively; [S] is the substrate concentration, K_m is the Michaelis–Menton constant. The reciprocal of catalytic velocity was plotted against the reciprocal of substrate concentration at a constant activator concentration according to the eqn (17) (Lineweaver–Burk plot):

Antioxidant or free radical scavenging activity (a decolorization assay)

The antioxidant activity experiments were performed on the Perkin-Elmer Lambda 25 double beam spectrophotometer attached with Peltier temperature programmer (PTP-1) to maintain temperature at 37 °C throughout the experiments. The TEAC assay was performed as described by Re et al.⁴⁰ with minor modifications. ABTS was dissolved in respective buffers (pH 7.4 and pH 9.0) to a 7 mM concentration. ABTS radical cation (ABTS˙⁺) was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. Because ABTS and potassium persulfate react stoichiometrically at a ratio of 1 : 0.5, this will result in incomplete oxidation of the ABTS. Oxidation of the ABTS commenced immediately, but the absorbance was not maximal and stable until more than 6 h. The radical was stable in this form for more than two days when stored in dark condition at room temperature. For the study of phenolic compounds, the ABTS˙⁺ solution was diluted with respective buffers (pH 7.4 and pH 9.0) to an absorbance of 0.71 (±0.02) at 734 nm and equilibrated at 37 °C. After addition of antioxidant the absorbance was measured at 734 nm at 37 °C exactly 30 min after initial mixing. Appropriate solvent blanks were run in each assay and subtracted, respectively. All determinations were carried out at least three times, and in triplicate, on each occasion and at each separate concentration of the standard and samples.

Results and discussion

UV-visible absorption studies

Ultraviolet/visible absorption spectroscopy is a powerful tool for steady-state studies of protein–ligand interaction. In proteins, we distinguish different internal chromophores that give rise to electronic absorption bands. From Fig. 1 we can see that the absorption peak of HSA centers at ∼280 nm mainly due to absorption of tryptophan residue. However, after addition of the TQ, the maximal absorption peak as well as absorption intensity of HSA is slightly affected. We observed that upon increasing the concentration of TQ, the conformation of HSA was slightly affected. It was evident from the disrupted absorption spectra of HSA around 280 nm (corresponding to Trp residue) and 256 nm (corresponding to transition region of disulphide bond and Phe residues absorption) in the presence of TQ.

Fig. 1 UV-visible absorption spectra of HSA (6 μM) in absence and presence (00–30 μM) of TQ at (A) pH 7.4; (B) pH 9.0, respectively.

Tryptophan fluorescence quenching by TQ

To avoid the effect of phenylalanine, we explored Trp fluorescence quenching experiments to determine the interaction of TQ with ‘N’ and ‘B’ isoforms (at pH 7.4 and pH 9.0, respectively) of HSA at 37 °C. We observed a strong fluorescence peak of HSA around 340 nm when excited at 295 nm in the absence of TQ. The fluorescence of HSA was quenched in the presence of increasing concentrations of TQ, clearly indicating an interaction between TQ and HSA (ESI Fig. S1†). The intensity of tryptophan fluorescence emission decreases continuously and gets saturated at higher TQ concentrations, proving that TQ binding sites on HSA was fully occupied. The decrease in fluorescence intensity upon addition of polyphenols was analyzed according to the Stern–Volmer eqn (1). There is a linear dependence between F_o/F and molar concentration of the TQ (1 : 1). The qualitative emission spectral features were slightly affected upon interaction of TQ to HSA which suggests about the minor ligand-induced conformational changes in the protein occurs due to increase in molecular closeness of TQ. The same experimental procedures were also followed at 15, 25 and 45 °C where we found that upon increasing the temperature, the quenching also decreases, or in other words, the extent of lowering in fluorescence emission was higher at lower temperatures (Fig. 2A-I and B-II). The K_SV values for TQ at different temperature as well as at different pH are given in the Table 1.

Fig. 2 The Stern–Volmer plots for the HSA–TQ interaction of ‘N’ isoform pH 7.4 and ‘B’ isoform pH 9.0 at 15, 25, 37, 45 °C and in the presence of 0.15 N NaCl at 37 °C (A-I and B-I). Plot of log [(F_o/F) − 1] vs. log[Q] for the determination of binding constants and binding stoichiometry for HSA–TQ interaction at pH 7.4 and pH 9.0 at 15, 25, 37, 45 °C and in the presence of 0.15 N NaCl at 37 °C at pH 7.4 (A-II and B-II).

Table 1 Binding and thermodynamic parameters of HSA and TQ at different temperatures obtained from fluorescence quenching experiments and ITC^a

pH/isoform		Temp. (°C)	KSV × 10^4 (M^−1)	kq × 10^12 (M^−1 s^−1)	n	Kb × 10^4 (M^−1)	ΔG° (kcal mol^−1)	ΔH° (kcal mol^−1)	TΔS° (kcal mol^−1 K^−1)	Dominating forces involve (inferred)
a KSV = Stern–Volmer constant; kq = bimolecular rate constant; n = stoichiometry of binding; Kb = binding constant; ΔG° = change in free energy; ΔH° = change in enthalpy; TΔS° = change in entropy.b kcal mol^−1 deg^−1.
pH 7.4/N		15	2.45	4.23	1.01	3.08	−5.90	−5.10	0.80	H-bonding, hydrophobic interactions
		25	2.11	3.65	1.00	2.30	−5.93		0.83
		37	1.15	1.98	1.02	1.63	−5.97		0.87
		45	0.85	1.47	0.99	0.82	−5.99		0.89
NaCl (0.15 M)		37	0.98	1.69	1.01	1.24	−5.80
pH 9.0/B		15	0.55	0.09	0.98	0.44	−4.79	−3.71	1.08
		25	0.35	0.06	0.99	0.34	−4.82		1.11
		37	0.27	0.04	1.01	0.28	−4.87		1.16
		45	0.08	0.01	1.08	0.27	−4.90		1.19
Values obtained from ITC	pH 7.4/N	37				1.01 ± 0.14	−127.55	−63.50 ± 2.48	−63.55^b	H-bonding, conformational changes
	pH 9.0/B	37				0.58 ± 0.07	−75.01	−37.05 ± 1.58	−37.51^b

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Determination of binding constant and binding stoichiometry

The binding constant and the binding stoichiometry can be calculated using the eqn (2). A plot of log[(F_o/F) − 1] vs. log[TQ] gives a straight line, whose slope equals to binding stoichiometry (n) and the intercept on y-axis equals to binding constant (K_b), respectively (Fig. 2A-II and B-II). The values of K_b and n at 15, 25, 37 and 45 °C are listed in Table 1. For TQ, the values of K_b and n at pH 7.4 and 9.0 were calculated at different temperatures as well as in presence of salt (NaCl). The data shows that K_b decreases on increasing the temperature in both ‘N’ and ‘B’ isoforms but these values are greater for ‘N’ (1.63 × 10⁴ M⁻¹) than the ‘B’ (0.28 × 10⁴ M⁻¹) form at physiological temperature. It implies that under basic conditions the binding capacity of TQ reduces up to ∼6 times than that under neutral (physiological) conditions. Conclusively, it shows that pH induced conformational change in the protein affects the mode and mechanism of quenching and hence TQ binding to the HSA molecules. Moreover, in the presence of NaCl, the extent of binding was not significantly changed thus its giving the clues that the electrostatic force doesn't play any role in TQ–HSA interactions.

Mechanism of HSA–TQ interaction

Fluorescence quenching can be either dynamic or static in nature. To know about the quenching mechanism of HSA by TQ, the values of k_q obtained from eqn (3) was closely observed and found that it was of the order of 10¹², which was 100 times higher than the maximum scatter collision quenching constant of various quenchers with biopolymers (2 × 10¹⁰ M⁻¹ s⁻¹).⁴¹ This shows that quenching is not initiated by dynamic diffusion but occurs by formation of a strong ground state complex between HSA and TQ. Further, the temperature dependency of K_SV was studied and we observed that the slopes (K_SV values) decreased with increase in temperature,⁴² further confirming that the binding of TQ to HSA was due to complex formation (static quenching). In static quenching, K_SV decreases due to the formation of complex between ligand and protein, which undergoes dissociation on increasing temperature.²⁹

Thermodynamics of HSA–TQ interaction

According to the binding constants of TQ to HSA at all the four temperatures, the thermodynamic parameters were determined from linear van't Hoff plot (eqn (4)) and the observed data are shown in Table 1 and ESI Fig. S2.† For the determination of enthalpy–entropy relation in protein–ligand interaction we considered only three temperatures viz. 15, 25 and 37 °C (NOT 42 °C) to ensure that the integrity of protein conformation was not affect, otherwise it would to lead false interpretation of thermodynamic parameters for the interaction studies. It is very well documented that at 42 °C, the domain III of HSA starts to melt, hence, major structural changes occurs beyond this temperature. But we had included the data of 45 °C to assumed the property of protein unchanged.²⁷ In other words, obtained enthalpy–entropy changes are mainly caused by the binding of the TQ molecule to HSA. The negative values of ΔG° manifested in each condition suggest that the interaction was spontaneous. ΔH° and ΔS° for the complex formation between TQ and HSA were found to be −5.10 kcal mol⁻¹ and 0.80, 0.83, 0.87 and 0.89 kcal mol⁻¹ K⁻¹, respectively at 15, 25, 37 and 45 °C at pH 7.4 and a similar pattern was also obtained at pH 9.0. Thus, the formation of TQ–HSA complexation is an exothermic reaction accompanied by positive ΔS° value. The role of bound water to the protein molecule in or near the binding pockets may be disturbed as a positive TΔS° value is a strong indication that water molecules have been excluded from the binding site interface. From the point of view of water structure, these thermodynamic signatures of protein–ligand interactions impersonate the type of forces responsible for ligand association. A positive ΔS° value is frequently taken as a typical evidence for hydrophobic interaction whereas a negative ΔH° is taken for hydrogen binding.⁴³ Therefore, binding of TQ to HSA might involve H-bonding as well as hydrophobic interaction as evidenced by the above thermodynamic signatures (ESI Fig. S2†). Furthermore, it was found that the major contribution to ΔG° arises from the ΔH° rather than from ΔS°, so binding process was enthalpy driven.

Isothermal titration calorimetric measurements

The associated thermodynamic and binding parameters were further investigated through ITC measurements. Representative calorimetric measurements to determine the mode of binding of TQ with HSA isoforms at 37 °C are shown in Fig. 3. In the ITC profiles, the lower panel shows the plot of heat librated per injection as a function of molar ratio of the drug to the protein and upper panel, each peak represents the binding isotherm of a single injection of the drug into the protein solution. The titration of TQ to HSA shows negative heat deflection indicating that the reactions were mainly exothermic. The association constant (K_a) and enthalpy change (ΔH°) were directly obtained after the best fitting for the integrated heats was obtained using single set of binding model with lowest χ² value. The Gibbs free energy and entropy changes were calculated from eqn (6) and (7), respectively and obtained thermodynamic or binding parameters are summarized in Table 1. The binding of TQ shows exothermic process that are the characteristics of hydrogen bond and conformational changes⁴⁴ and the values of binding constant were varying in the range of 10³ to 10⁴. Moreover, the negative value of ΔG° suggests that the TQ–HSA complex formation was spontaneous at both pH values (pH7.4 and pH 9.0). The negative values of ΔH° and positive value of ΔS° values advocate that the involvement of hydrogen bond and hydrophobic interaction in the formation of the protein–TQ complex,⁴⁵ which indicates the occurrence of enthalpy–entropy compensation effect in which enthalpy loss due to the deformation of H-bond is counter balanced by entropic penalty due to the burial of involved groups. This effect is common in protein–ligand interactions.⁴⁶

Fig. 3 Isothermal titration calorimetric profile of HSA in presence of TQ at pH 7.4 (A) and pH 9.0 (B) at 37 °C. Titration of TQ with HSA shows calorimetric response as successive titrations of TQ to the sample cell.

Circular dichroism measurement

It is possible to estimate the contents of secondary structure of protein using far-UV CD spectra (190–250 nm). A positive peak near 195 nm and two negative peaks near 208 and 222 nm is characteristic feature of α-helical protein. The far-UV CD studies were performed on the protein and protein–TQ complexes in order to investigate the possibility of any structural change of the protein upon complexation with the TQ. Fig. 4-I shows that the CD spectra of HSA with various TQ concentrations at pH 7.4 and 9.0. From Fig. 4-IA (pH 7.4) and 4-IB (pH 9.0) the spectra of HSA in the absence and presence of TQ, as the TQ concentration increases a notable spectral rearrangement occurs in HSA with increase in major minima (208 nm) as well as slight changes in the shape of spectra due to intramolecular H-bonding rearrangement as justified by fluorescence quenching experiments. The TQ induced alterations in secondary structures of HSA were quantified by Chen et al. method,³¹ and the calculated values are summarized in Table 2. In the presence of TQ, a significant increase in the α-helical content of both HSA isoforms was observed. Using eqn (9), the α-helical content in ‘N’ isoform of HSA was calculated. It increased from 4% and 9% while in case of ‘B’ isoform of HSA it increased from 9% and 18% as compared to native structure, correspondingly. Overall, the ‘B’ isoform of HSA is more stable in the presence of TQ.

Fig. 4 Far-UV CD spectra of HSA (2 μM) in absence and presence of TQ at pH 7.4 (4-IA) and pH 9.0 (4-IB). Far-UV CD thermal unfolding spectra of HSA (2 μM) in the absence and presence of TQ in molar ratio of 1 : 05 and 1 : 25 at pH 7.4 (4-IIA) and pH 9.0 (4-IIB), respectively.

Table 2 Secondary structural analysis to determine the percent structural change in the isoforms of HSA and HSA–TQ complexed

pH/isoform	HSA (μM)	TQ (μM)	% increment in α-helix	Tm (°C)
pH 7.4/N	02	00	0	68.18
	02	10	4	72.35
	02	50	9	72.66
pH 9.0/B	02	00	0	72.81
	02	10	9	74.24
	02	50	18	75.02

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Thermal stability of HSA was enhanced by TQ

The existence of intermediates in the thermal unfolding pathway of a protein can also be evidenced by observing the changes in its secondary structure. Fig. 4-II shows the change in the ellipticity at 222 nm of the HSA : TQ at molar ratios 1 : 0, 1 : 5 and 1 : 25, respectively as obtained from CD experiments. The mid-point temperature (T_m) was determined by fitting the ellipticity values in to a two-state folding-unfolding model eqn (10) and (11). This shows that the protein loses a considerable fraction of its secondary structure during thermal denaturation. The decrease in ellipticity at 222 nm of HSA is shown as a function of temperature and the denaturation profile is found to be consistent with the earlier reports.⁴⁷ It is noted that the presence of intermediate unfolded states in the thermal denaturation of bovine serum albumin (BSA), which is structurally similar to HSA, has already been indicated by calorimetric studies.⁴⁸ The CD thermal profiles, including the 1 : 25 molar ratio of HSA : TQ showed more thermal stability, indicating that the protein is more stable in the presence of TQ as compared to the native condition. Thus, the higher stability of HSA in the presence of 1 : 25 molar ratio corresponds to increase in the secondary structure. The obtained T_m values of TQ-bound HSA are significantly higher than the native one (Table 2). Denaturation of HSA at high temperature occurs by weakening of hydrophobic as well as polar interactions, which may also facilitate the TQ binding property of HSA. Here, enhancement in thermal stability is also implied by better interaction of TQ at high temperature which induces to more helical structure formation in unordered protein segments of ‘B’ isoform as compare to ‘N’ isoform of HSA.

Dynamic light scattering studies

Unfolding of a protein is usually marked by a change in the secondary and globular structure of the protein. The change in the globular structure of a protein can be studied by DLS measurements. It is clear from the above findings that the interaction of TQ with HSA causes conformational changes. So, we decided to measure the molecular sizes of HSA in the absence and presence of TQ by determining the hydrodynamic radii using DLS. The Fig. 5-I and II shows that the change in the globular structure of the protein at 25 °C. The hydrodynamic radii (R_h) of native HSA and HSA in the presence of TQ were calculated and values are shown in Table 3. The R_h values of native HSA were 4.0 nm and 3.6 nm at pH 7.4 and 9.0, respectively. These results are in excellent agreement with previous observations at pH 7.4 and pH 9.0, respectively.⁴⁹ The R_h values of HSA complexed with TQ were higher than the native one. The increase in hydrodynamic radii upon ligand binding may be due to the “expansion of domains” which may lead to an increase in the molecular volume as a result of conformational changes. Similar results were also observed previously in the presence of atropine (4.1%), propranolol (11.1%), clonidine (14.4%), phenylephrine (16.6%) and carbachol (15.5%).⁵⁰ The lower values of polydispersity (<20) were indicative of homogenous species in the solution. Fig. 5-II shows the characteristic examples of the dependencies of globule size and compressibility upon the HSA : drug molar ratios. The principal structural rearrangements were displayed at each HSA : drug molar ratios and it increased with an increase in HSA : drug ratio. We also observed that at extremely higher molar ratio of HSA : drug (1 : 25), the hydrodynamic radii were either slightly changed or remain unaffected (Fig. 5-II). It suggested that the structural changes in HSA occurred only by the molecules which were bound to the protein and affected its secondary structures near the binding site.⁵¹

Fig. 5 Dynamic light scattering of HSA–TQ complex. (5-I) Determination of hydrodynamic radii (R_h) of HSA in the absence and presence of TQ (HSA : TQ molar ratio 1 : 0, 1 : 5 and 1 : 25); A-I, A-II, A-III and B-I, B-II, B-III for pH 7.4 and pH 9.0, respectively. (5-II) TQ concentration dependent changes in hydrodynamic radii of HSA at both pH, shows that hydrodynamic radii increases on increasing the concentration of TQ at both pH.

Table 3 Characteristics of hydrodynamic radii (R_h) and polydispersity (P_d) distribution of HSA solution in absence and presence of TQ at pH 7.4 and pH 9.0

pH/isoforms	HSA : TQ	Rh (nm)	Change in Rh (%)	Pd (%)
pH 7.4/N	1 : 00	4.0	100	16.0
	1 : 05	4.2	105	16.2
	1 : 25	4.4	110	19.6
pH 9.0/B	1 : 00	3.6	100	17.4
	1 : 05	3.8	106	13.9
	1 : 25	4.0	111	17.6

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Energy transfer between TQ and HSA

In order to estimate the binding of TQ to the model transporter protein HSA, we have also explored the possibility of energy transfer between donor to acceptor using Förster's resonance energy transfer (FRET) method. In Fig. 6, the emission spectrum from the single tryptophan (Trp214) of HSA and the absorption spectrum of TQ are shown. According to FRET theory as describe in the method section, efficiency of energy transfer (E_FRET), spectral overlap (J), Förster's distance (R_o) of the donor (Trp214), and r value were derived from the overlaid spectra and the value of energy transfer between TQ and HSA was calculated from the eqn (13)–(15). At pH 7.4, the energy was efficiently transferred from Trp214 of HSA to the bound TQ as indicated by a large spectral overlap between the emission spectra of Trp214 and the absorption spectrum of TQ. Hence, the probability of TQ binding was stronger with the ‘N’ isomer of HSA at pH 7.4. Further, it is evident from the Fig. 6B that the spectral overlap of the HSA–TQ system was significantly lower at pH 9.0 due to minimum contribution from the Trp214. We calculated the energy transfer parameters and found that E_FRET = 0.3048, J = 3.24 × 10⁻¹⁴ cm³ M⁻¹, R_o = 2.981 nm, r = 3.42 nm for HSA at pH 7.4 and E_FRET = 0.1400, J = 2.72 × 10⁻¹⁵ cm³ M⁻¹, R_o = 1.97 nm, r = 2.66 nm for HSA at pH 9.0, respectively (Table 4). The average distance between the donor and acceptor fluorophores was on the 2–8 nm scale and 0.5R_o < r < 1.5R_o. In our study, the donor to acceptor distance was less than 8 nm, indicating that the energy transfer from HSA to TQ occurred with high probability. These results were also in accordance with a static quenching mechanism.

Fig. 6 Tryptophan fluorescence resonance energy transfer. Spectral overlap of the fluorescence emission of HSA (λ_ex = 295 nm) and absorption spectra of TQ [HSA = TQ = 2 μM] at (A) pH 7.4; (B) pH 9.0, respectively.

Table 4 FRET data obtained from spectral overlap of HSA emission and TQ absorption at 37 °C

Variables	pH 7.4	pH 9.0
F	142.862	200.044
Fo	205.504	232.628
EFRET	0.3048	0.1400
J (cm^3 M^−1)	3.24 × 10^−14	2.702 × 10^−15
Ro (nm)	2.981	1.970
r (nm)	3.42	2.66

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Three-dimensional conformational investigation of TQ binding with HSA

Three-dimensional fluorescence spectroscopy is a new analytical technique that is applied to investigate the conformational changes of the proteins. The excitation and emission wavelength of the fluorescence intensity can be used as the axes rendering the investigation of the characteristic conformational changes of proteins more scientific and credible.²⁹ The maximum fluorescence emission wavelength of amino acid residues in a protein is related to the polarity of the environment. Experiments have suggested that the fluorescence emission spectrum wavelength and the synchronous fluorescence spectrum wavelength of HSA in the absence and presence of drugs show distinct differences and sharp changes, which provide relative information on the configuration of the protein.⁵² The three-dimensional spectra and contour maps of HSA and HSA–TQ complexes are presented in Fig. 7 and ESI Fig. S3† and the obtain values are listed in ESI Table T-1.† In the figure of three-dimensional spectra, two characteristic fluorescence peaks of HSA (peak 1 and peak 2) were clearly observed, while peak 3 and peak 4 represents Rayleigh scattering peak (λ_ex = λ_em) and second-order scattering peak (λ_em = 2λ_ex),^29,52 respectively. The peak 1 represents fluorescence arising mainly from tryptophan and tyrosine (negligible contribution of phenylalanine fluorescence)⁵³ when the protein is excited at 280 nm. On the other hand, peak 2 is the characteristic fluorescence peak representing polypeptide backbone structure. In our study, the decrease in the intensities of peaks 1 and 2 clearly indicated that the fluorescence of HSA was quenched as a result of TQ binding and the conformation of the protein was also altered as a result of it.

Fig. 7 Three-dimensional fluorescence spectra of HSA (2 μM) in absence (A and B) and presence (A-I and B-I) of TQ at pH 7.4 and pH 9.0, respectively. [HSA : TQ = 1 : 1].

Molecular docking

Main aim of the study was to presumptive binding site of TQ in HSA as reported earlier by G. Lupidi et al.¹³ We used X-ray structure of HSA with high resolution (PDB ID: 1AO6, res. 2.5 Å) as a template and the prediction of binding mode of TQ was done using AutoDock software package. To comprise the subdomain IIA and IIIA of HSA, the two regions of interest used for molecular docking were defined to determine the binding residues and their positions and their energy score was compared. The docking results showed that TQ binds to HSA within the binding pocket of subdomain IIA with an estimated docking energy of about −5.83 kcal mol⁻¹, while for the second binding site subdomain IIIA the free energy of binding was found to be −5.62 kcal mol⁻¹.

Therefore we can argue that TQ has a better binding preference for the drug binding site I (subdomain IIA) of HSA. This result is in agreement with earlier published data, as it is reported that quinone-related derivatives bind to subdomain IIA of HSA. The binding of TQ to subdomain IIA of HSA was also confirmed by the drug-displacement experiments.^54,55 This binding site for TQ at subdomain IIA was located predominantly in a hydrophobic cleft walled by the amino acid residues Tyr150, Leu199, Trp214, Leu219, Arg222, Leu238, Arg257, Leu260, Ala261, Ile264, Ser287, Ile290, and Ala291 (as shown in Fig. 8A). One of the oxygen from TQ was interacting with Tyr150 through hydrogen bonding, and its methyl group was found to interact with Trp214 through hydrophobic interactions (Fig. 8B).

Fig. 8 Molecular docking results (A) TQ represented as ball and stick model and HSA in ribbon, (B) hydrogen bonding between Tyr150 and Trp214 with TQ in the region of binding pocket.

Esterase-like activity of HSA-isoforms in the presence of TQ

HSA has an esterase-like activity for the deglucuronidation of acyl-glucuronide of fenoprofen, etodolac, ketoprofen and gemfibrozil.^56–59 The double-reciprocal plots for substrates were characterized by a family of linear, nonparallel lines that converged to the left of the y-axis (ESI Fig. S4†). In presence of TQ, the K_m increased at pH 7.4 from 6.66 × 10⁻⁶ M to 9.37 × 10⁻⁶ M. The higher value of K_m obtained for the HSA incubated at pH 7.4 in the presence of TQ showed a lower affinity for the substrate. The incubated enzyme besides showing higher K_m for substrate also showed a higher k_cat value yielding overall a decrement in catalytic efficiency (k_cat/K_m) relative to the HSA in the absence of TQ. Altogether, our results indicated that addition of TQ alters both K_m and V_max values of HSA. Increase in K_m and V_max indicated that the esterase activity of HSA was inhibited in a competitive manner because TQ was directly competing with the substrate for a fixed number of active sites on enzymes. The large increase in K_m upon the binding of TQ indicated changes in the tertiary structure of HSA that might lead to steric effects resulting from limitation of the accessibility of substrate to the active site. The catalytic efficiency value, which is the ratio of k_cat over K_m was also different for free HSA and HSA–TQ complex. As shown in Table 5, the catalytic efficiency of HSA–TQ was lower than the free enzyme indicating that the enzyme was poorer on the substrate in the presence of TQ.

Table 5 Kinetic parameters for the hydrolysis of p-nitrophenyl acetate by HSA

pH/isoform	System	RA (%)	Vmax (M min^−1)	Km (M)	kcat (min^−1)	kcat/Km (M^−1 min^−1)
pH 7.4/N	HSA	100	4.27 × 10^−8	6.66 × 10^−6	0.854 × 10^−2	1.28 × 10^3
	HSA + TQ	127	5.42 × 10^−8	9.37 × 10^−6	1.048 × 10^−2	1.11 × 10^3
pH 9.0/B	HSA	100	6.70 × 10^−7	8.55 × 10^−5	13.41 × 10^−2	1.56 × 10^3
	HSA + TQ	113	5.12 × 10^−7	4.09 × 10^−5	10.24 × 10^−2	2.50 × 10^3

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We have obtained opposite pattern of K_m and V_max at pH 9.0, decrease in K_m from 8.55 × 10⁻⁵ M to 4.09 × 10⁻⁵ M and V_max 6.70 × 10⁻⁷ to 5.12 × 10⁻⁷ M min⁻¹ revealed that HSA in the presence of TQ at pH 9.0 had an improved affinity and tighter substrate binding capability as compared to that at pH 7.4 (Table 5). The second order rate constant k_cat/K_m ratio indicates the catalytic efficiency and kinetic perfection of the enzyme in transforming substrates. The higher the k_cat/K_m ratio, the better the enzyme works on that substrate. A comparison of k_cat/K_m ratio for the same enzyme with substrates in different conditions is widely used as a measure of enzyme effectiveness. TQ induced the catalytic activation of HSA at pH 9.0 and allowed the reaction to approach the limit of maximum diffusion just like in an ideal enzyme (acetyl cholinesterase) where every interaction with substrate yields a product and for these enzymes, from the diffusion theory, the value of k_cat/K_m ranges 6 × 10⁹ to 6 × 10¹⁰ M⁻¹ min⁻¹. Our enzyme kinetics results suggested that the TQ acts as an activator of the esterase activity of HSA in alkaline conditions. The k_cat values were strikingly dependent on pH and showed that the susceptibility of the active sites to nucleophilic attack increases with pH. It has been reported that pH dependent conformational changes (‘N’↔‘B’ transition) occur in albumin when going from native to slightly alkaline pH.²⁵ Therefore, alteration in nucleophilic attack in active sites and in the affinity to p-NPA could be due, totally, or partly, to changes in the tertiary structure of albumin, which convoy the pH-dependent ‘N’-‘B’ transition. From all of our experiments dealing with the secondary and tertiary structure of HSA in the presence of TQ molecules, it becomes apparent that upon interaction between TQ and HSA, the affinity of the protein for its substrate is enhanced in alkaline condition.

Antioxidant or radical scavenging activity of HSA in presence of TQ

TQ has several important functional properties, out of them we have evaluated the free radical scavenging activity because of its deleterious job in the food and biological systems.⁶⁰ Some of its properties were previously demonstrated by several pharmacological studies as membrane lipid peroxidation, reduction of eicosanoid generation,⁶¹ anti-inflammatory and analgesic,^2,62 protection of body organs against oxidative damage induced by various type free radical generating agents.^63–65 Oral intake of TQ is capable of protecting numerous organs against oxidative damage induced by free radical-generating agents including doxorubicin-induced cardiotoxicity.^66,67 TQ act as scavenger of superoxide, hydroxyl radical and singlet molecular oxygen.⁶⁸ HSA itself have a very good antioxidant activity that plays an important role in human health. In alkaline condition, antioxidant property of HSA increases due to its conformational change and the activation of antioxidant activity is also thiol-dependent.⁶⁹ The carboxyl group modification of HSA causes approximately 40-fold increase in the antioxidant activity. These chemical modification studies indicate that the addition to functional cysteine(s) or cationic amino acid residues such as arginine, histidine and lysine involve in antioxidant reactions. These results recommend that the activation of thiol-dependent antioxidant activity of HSA at alkaline pH is due to the conformational change which was favorable for the functional cysteine(s)-mediated catalysis. HSA shows specific antioxidant property due to its multiple ligand-binding and free radical-trapping properties and are directly connected to the conformational change in structure and the redox state of molecule.^70,71 Currently various methods are used to described the antioxidant activity of plant derived phenolic compounds. These chemical assays are based on the ability to scavenge the free radical by various radical generating system and method for decolorization. ABTS˙⁺ radical scavenging method is most appropriate format for decolorization assay and very common spectrophotometric procedure to determine the antioxidant capacity of plant derived components due to its sensitive, simple, rapid and reproducible procedure.⁷² Biochemical assay are based on the scavenging ability of synthetic free radicals which are generated by different radical-generating systems. Free radicals are generating prior to reactions that involve in the production of blue/green ABTS˙⁺ chromophores that was formed due to reaction between ABTS and potassium persulphate.

ABTS˙ + AH → ABTS⁺ + A˙

The extent of inhibition of the absorbance of the ABTS˙⁺ is plotted as a function of concentration in order to determine the TEAC, that can be assessed as a function of time. The dose–response curve obtained by analysis of a range of concentrations of antioxidant compounds, was plotted as the percentage inhibition of the absorbance of the ABTS˙⁺ solution as a function of concentration of antioxidant (Fig. 9A). Trolox and BHA were used as standard reference compounds (Fig. 9B). To calculate the TEAC, the gradient of the plot of the percentage inhibition of absorbance vs. concentration plot for the antioxidant in question is divided by the gradient of the plot for Trolox and BHA. The scavenging capability of ABTS⁺ radical was calculated using the following equation:

where, A_c is the initial concentration of the ABTS⁺ and A_s is absorbance of the remaining concentration of ABTS⁺ in the presence of TQ.⁷³

Fig. 9 The effects of concentration of the antioxidant on the inhibition of the ABTS˙⁺ by trolox and BHA at pH 7.4 and pH 9.0. Absorbance of ABTS radical scavenging activity of different concentrations of HSA, TQ and reference antioxidants as trolox and BHA (ABTS⁺: 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) at pH 7.4 (9-IA), pH 9.0 (9-IB). Percent inhibition of standard reference compounds trolox and BHA (9-II) as well as in presence of phenolic compound TQ and protein at particular concentrations at pH 7.4 (9-IIIA) and pH 9.0 (9-IIIB), respectively.

This gives the TEAC at the specific time point and the calculated results for the flavonoids are given in ESI Table T-2.† All the tested compounds exhibited admirable radical cation scavenging activity. As seen in Fig. 9C, TQ had effective radical scavenging activity in a concentration-dependent manner (16.42–164.2 μg ml⁻¹). TQ undergoes reduction process and gets converted into its more anti-oxidative form i.e. dihydrothymoquinone (DHTQ). TQ and DHTQ inhibited non-enzymatic process in liver that was also dose dependent.⁶⁷ There was a significant increase in the overall concentration of ABTS˙⁺ due to the scavenging capacity of TQ concentrations. Also, the scavenging effect of TQ and standards, on the ABTS˙⁺ decreased in that order: at pH 7.4 HSA–TQ > HSA (100 μM) > TQ (2500 μM) > trolox (100 μM), which were 81.39%, 50.37%, 47.45% and 43.76%, and at pH 9.0 HSA–TQ > HSA (100 μM) > TQ (2500 μM) > trolox (100 μM), which were 100%, 99.97%, 96.19% and 88.39% at the concentration of 100 μL ml⁻¹ respectively.

Conclusion

In the present study, we evaluated the binding properties of ‘N’ and ‘B’ isoform of HSA (at pH 7.4 and pH 9.0, respectively) with TQ, an important constituent of Nigella sativa. The binding affinity and thermodynamic parameters were higher for ‘N’ isoform as compared to ‘B’ isoform of HSA. The interaction of TQ with HSA was favored by H-bonding and hydrophobic interactions. The molecular size and thermal stability of HSA were increased in the presence of TQ. We found that the esterase activity of HSA is enhanced in ‘N’ isoform in the presence of TQ as compared to ‘B’ isoform, while the antioxidant activity is quite significant in ‘B’ isoform. The overall antioxidant activity of HSA is enhanced in the presence of TQ. Thus, the phenolic compound TQ which is a component of Nigella sativa has a great potential to bind HSA and induces its free radical scavenging activity. This study provides insight into HSA–TQ interaction, which is of great importance in understanding the chemico–biological interactions for drug-designing, pharmacy and biochemistry.

Abbreviations

HAS	Human serum albumin
TQ	Thymoquinone
ABTS	2,2′-Azino-bis (3-ethyl benzothiazoline-6-sulfonic acid) diammonium salt
DLS	Dynamic light scattering
FRET	Förster resonance energy transfer
BHA	Butylated hydroxyanisole
MRE	Mean residual ellipticity.

Acknowledgements

MI is highly thankful to Indian Council of Medical Research (ICMR), New Delhi, for financial support in the form of senior research fellowship (BIC/11(12)/2013) and Grant (no. BMS-58/14/2006). GR is highly thankful to Council of Scientific and Industrial Research, New Delhi, India for financial support in the form of Research Associateship. The authors would like thank to Interdisciplinary Biotechnology Unit, Aligarh Muslim University for providing instrumental facilities.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra09892g

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