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Thermodynamic analysis of thymoquinone binding to human serum albumin
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 677–681
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Thermodynamic analysis of thymoquinone binding to human serum albumin Zeyad J. Yasseen a,⇑, Jehad H. Hammad b, Hussein A. ALTalla a a b
Chemistry department, Faculty of Science, Islamic University of Gaza, Palestine Faculty of Medicine, Islamic University of Gaza, Palestine
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Fluorescence method was used to
examine the interaction of human serum albumin with thymoquinone. The thermodynamic parameters of the interaction were calculated. We examined two mathematical models to analyze the interaction occurred. The predominant intermolecular forces affecting the interaction were proposed. Fröster energy transfer theory was used to calculate the distance between the donor macromolecule and the acceptor ligand.
a r t i c l e
i n f o
Article history: Received 28 June 2013 Received in revised form 24 November 2013 Accepted 30 December 2013 Available online 27 January 2014 Keywords: Thermodynamic analysis Thymoquinone Human serum albumin Fluorescence
a b s t r a c t The interaction of thymoquinone (TQ) with human serum albumin (HAS) in physiological buffer (pH = 7.0) was studied at four temperatures in the range 25–50 °C using fluorescence quenching study. The binding parameters were determined by Scatchard and Stern–Volmer models. Fluorescence quenching data revealed that the binding constants (Ksc) are 1.71 104, 1.08 104, 1.03 104 and 0.969 104 M1 at 298, 303, 313 and 323 K, respectively (on the basis of Scatchard model). The thermodynamic parameters DG°, DH° and DS° were calculated the results indicated that the hydrogen bonding and hydrophobic interactions were the predominant intermolecular factors in stabilizing the TQ–HSA complex. The distance between donor (HSA) and acceptor (TQ) was calculated to be 3.26 nm based on Förster’s non-radiative energy transfer theory. Published by Elsevier B.V.
Introduction Human serum albumin (HSA) is the most abundant and highly soluble plasma protein in the blood circulatory system in mammals (approximately 60% of the total protein) [1,2]. It is able to bind and thereby transport various compounds such as fatty ⇑ Corresponding author. Tel.: +970 599928024. E-mail address: [email protected] (Z.J. Yasseen). http://dx.doi.org/10.1016/j.saa.2013.12.112 1386-1425/Published by Elsevier B.V.
acids, bilirubin, tryptophan, steroids and many drugs [3]. HSA concentrations in blood plasma ranges from 3.4 to 5.4 g/dL [4]. HSA plays an important role in transporting metabolites and drugs through the vascular system and also in maintaining the pH and osmotic pressure of the plasma [5]. HSA is a globular single polypeptide chain protein of a molecular mass of about 67 kDa and comprises 585 amino acid residues with one cystein residue at position 34 (in domain I) with free sulfhydryl group [6]. Its structure includes three homologous domains (I, II, and III) that
678
Z.J. Yasseen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 677–681
Fluorescence spectra related to the titration of fixed volume of HAS of 1 lM with concentrated stock ethanolic TQ solution were measured on the Spectrofluorimeter at the desired temperature. The fluorescence intensity measured were corrected for dilution. All samples were measured after two minutes and all binding data reported here correspond to the average values obtained after two different titrations. The fluorescence emission spectra were recorded at four different temperatures in the range 25–50 °C in the wavelength range of 250–500 nm with the excitation wavelength at 278 nm and using slits of 10 nm for both excitation and emission monochromators. Fig. 1. X-ray crystallographic to 2.5 Å resolution of three dimensional Structure of HSA, with its subdomains [10]. The structure corresponds to the ‘‘Protein Data Bank’’.
Results and discussion Calculation of binding constants and data analysis
assemble a heart shaped molecule (Fig. 1). Each domain is formed by two sub-domains (A and B) which possess common structural motifs by various forces such as salt bridges and hydrophobic interactions [7–9]. Thymoquinone (TQ) is a phytochemical compound present in of a plant called Nigella Sativa possesses important properties such as analgestic and anti-flammatory protection of organs against oxidative damage induced by a variety of free radical generating agents. It is a potent anti tumor agent against human colorectal cancer cells [11–13]. Studies on the binding of drugs to protein are of great importance in biological, biomedical and pharmaceutical science. Plasma protein serve as transport carriers for drug bioavailability, distribution in the body, metabolism and excretion. Human serum albumin (HSA) has been used as a model protein for protein folding and ligand – binding studies over many decades [14,15]. The affinity of a drug to a protein would directly influence the concentration of the drug in the binding site and duration of the effectual drug, and consequently contribute to the magnitude of its biological actions in vivo [16]. It is informative to study interaction of bioactive compounds such as TQ with the protein, because the effectiveness of these compounds as pharmaceutical agents based on their binding ability [7]. In the present study, using the spectroflorimetric titration method, the interaction of TQ with HSA has been studied and the results has been interpreted on the basis of different mathematical models leading to estimate the binding parameters. Materials and methods
The fluorescence quenching data were assessed using two distinct mathematical models: Stern–Volmer and Scatchard models. The binding constants (KSV and KSc) obtained at each temperature in the range of 25–50 °C were estimated from the Stern–Volmer and Scatchard plots. Fluorescence quenching of single tryptophan residue in HAS (Trp 214) was used to monitor the TQ/HSA interaction and to measure the binding affinity of the interaction occurred. The addition of TQ to HSA caused a decrease in the intrinsic fluorescence emission intensity of the protein upon excitation at 278 nm (Fig. 2). Fluorescence spectra of HSA alone and complexed with TQ at 25 °C was recorded (Fig. 2). From Fig. 2 it is evident that addition of TQ quenches the fluorescence intensity of HSA and the quenching was accompanied by a small red shift in maximum fluorescence intensity of emission. This behavior could be attributed to an increase in the environmental polarity. The binding curves at different temperatures in the range of 25–50 °C, depecting the change in fluorescence of HSA (DF) as a function of molar ratio [HSA]/[TQ] are shown in Fig. 3. Here, it is clear to see that the binding curve displayed saturation at certain values of the molar ratio at each studied temperature, clearly indicating that the binding occurred at specific binding sites on the HSA. Binding constant and number of binding sites The apparent binding constant (KSV and KSc) and number of binding sites (n) for TQ that binds HSA can be obtained using Eqs. (1) and (2) Stern–Volmer and Scatchard Eqs. (1) and (2) [17,18]:
Materials Human serum albumin (HSA, fatty acid free <0.05%) was purchased from Sigma Aldrich company. Thymoquinone was purchased from Sigma Aldrich and its stock solution was prepared in 5% ethanolic solution. The other substances were of reagent grade, and were used without further purification. phosphate buffer (20 mM, pH = 7.0) was used through the study. All solutions were prepared using doubly distilled water. Fluorescence quenching studies All fluorescence spectra were recorded on an Perkin–Elmer luminescence Spectrofluorimeter, (series no. 70412) equipped with a water-jacketed cuvette holder maintained at a constant temperature by means of a circulatory water bath. A quartz cell of 1.00 cm width was used for the measurements.
Fig. 2. Emission spectra of HSA (1 lM) in 20 mM phosphate buffer (pH = 7.0) and 25 °C. Excitation carried out at 278 nm. The arrow (with its direction) shows that the increasing TQ concentration is accompanied with fluorescence intensity quenching for HSA.
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0.4
1000
0.2 800
0.0
ΔF
r/[TQ] free
600 400
-0.2 -0.4 -0.6 -0.8
200
-1.0 0
-1.2 0.001
0.002
0.003
0.004
0.1
0.005
0.2
0.3
0.4
Fig. 3. Binding curves for spectrofluorimetric titration of TQ with HSA at four different temperatures 25 (s), 30 (d), 40(N) and 50 (.) °C in phosphate buffer solution (pH = 7.0). Change in fluorescence (DF) was plotted as a function of molar ratio [HSA]/[TQ].
F Stern—Volmer : ¼ 1 þ K SV ½TQ free F0 Scatchard :
ð1Þ
r ¼ nK sc rK Sc ½TQfree
ð2Þ
where F and F0 are the emission fluorescence intensities at 334 nm in presence and absence of TQ, respectively. [TQ]free is the free TQ concentration, r is the number of mol of bound TQ per mol of HSA, n is the number of binding sites, KSV and KSc are the Stern–Volmer and Scatchard association constants, respectively. Figs. 4 and 5 show the Stern–Volmer and the Scatchard plots for the interaction TQ/HSA at different temperatures, respectively. Thermodynamic study and nature of the binding forces The fluorescence quenching data accompaining the interaction TQ/HSA in the present study were analyzed on the basis of Stern–Volmer and Scatchard models, Eqs (1) and (2), respectively. The corresponding linear regression equations and results at the four different temperatures are shown in Figs. 4 and 5.
55 50
0.6
0.7
0.8
0.9
Fig. 5. Catchard plots for TQ/HSA at different temperatures: s 25 °C, d 30 °C, N 40 °C and . 50 °C. All in phosphate buffer (pH = 7.0), kex = 278 nm, kem = 334 nm. HSA concentration: 1 lM.
It is evident from Table 1 that the binding TQ/HSA is strong and is affected with temperature, showing a decreasing trend in the value of both binding constants (KSV and KSc) with increasing temperatures in accordance with each other and exhibit a similar dependence on temperature. The binding constant of TQ/HSA decreases with temperature, resulting in a reduction of stability of the formed TQ/HSA complex, The binding constant values for the association TQ/HSA were decreased by the rise in temperature which may indicate the formation of a stable complex at lower temperature. The linearity in Scatchard plots indicates that TQ binds to one class of sites on HSA. The results were discussed in conjunction with the thermodynamic characteristics obtained for TQ binding. And the thermodynamic parameters and nature of binding forces are the main evidence for conforming the binding mode. The thermodynamic parameters at the four temperatures in the range of 25–50° were determined and presented in Table 2. Essentially, the molecular forces contributing to Protein–ligand interactions may be due to a van der Waals interaction, hydrogen bond formation, ionic, electrostatic, or hydrophobic interaction, etc. The signs and magnitudes of thermodynamic parameters forprotein reactions can account for the main forces contributing to protein stability [14]. If the enthalpy changes (DH°) not vary significantly over the temperature range studied, then its value and that of (DS°) can be assessed from the van’t Hoff equation:
ln k ¼
45 40
DH DS þ RT R
ð3Þ
where K is the binding constant at the corresponding temperature calculated from both (Scatchard and Stern–Volmer equations (Eqs. (1) and (2)) and R is the gas constant. The enthalpy change (DH°) and entropy change (DS°) are calculated from the slope and
35
F0 / F
0.5
r
[HSA]/[TQ], M
30 25 20 15
Table 1 Binding parameters for the interaction TQ/HSA.
10 5
Temperature (°C)
0 -5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
[TQ], mM Fig. 4. Stern–Volmer plots for TQ/HSA at different temperatures: s 25 °C, d 30 °C, N 40 °C and . 50 °C. All in phosphate buffer (pH = 7.0), kex = 278 nm, kem = 334 nm. HSA concentration: 1 lM.
20 30 40 50
Binding parameters Stern Volmer
Scatchard
KSV (104)
KSc (104)
N
1.49 1.16 1.03 0.977
1.71 1.08 1.03 0.969
1.7 1.1 1.0 0.96
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Table 2 Thermodynamic parameters for the interactions TQ/HSA at four different temperatures. T (K)
DS° (J mol1 K1)
DG° (kJ mol1)
DH° (kJ mol1)
298 303 313 323
34.08
26.35 26.52 26.89 27.23
16.22
intercept of the van’t Hoff plot of ln K against 1/T, Fig. 6. The Gibbs energy change (DG°) is estimated from Eq. (4).
DG ¼ DH T DS
ð4Þ
The results obtained are listed in Table 2 suggested that the process is both enthalpically and entropically driven. Generally, the positive entropy change occurs because the water molecules that are arrangedin orderly fashion around the ligand and the protein acquire a more random configuration as a result of hydrophobic interactions. Negative enthalpy change is observed whenever there is hydrogen bonding through the binding process [17,18]. The negative DH° and positive DS° values in the interaction TQ/ HSA suggested that both hydrogen bonding and hydrophobic interactions play the major roles in the binding process. In this study, the quenching data were analyzed according to both Stern–Volmer, and Scatchard models Eqs. (1) and (2). Where Ka is the binding constant (on the basis of Scatchard or Stern–Volmer model) at the corresponding temperature, R is the gas constant, and T is the absolute temperature. Binding distance between TQ and the amino acid residue of HSA Fluorescence resonance energy transfer (FRET) is a reliable method for studying protein–ligand interactions and to determine the distance between the donor (HSA) and the acceptor (TQ) can be calculated using (FRET). In this study we used fluorescence resonance energy transfer (FRET) to determine the distance between TQ and the donor amino acid residue [19]. By Föster theory, the efficiency of energy transfer (E) is related to the distance r between the donor and the acceptor by Eq. (6).
E¼1
R60
F ¼ F 0 R60 þ r60
ð6Þ
where F and F0 are the fluorescence intensities of HSA in the presence and absence of TQ; r0 is the distance from the ligand to amino 10.5
Ln K Sc
10.0
9.5
Fig. 7. Spectral overlap of the fluorescence spectra of HSA (solid line) with the absorption spectra of TQ (dashed dot line). [HSA] = 1 lM, and [TQ] = 10 mM , both in buffer solution (pH = 7.0).
acid residue in the HSA, and R is the Förster’s critical distance at which 50% [20] of the excitation energy is transferred to the acceptor. Fig. 7 shows the overlap of the fluorescence spectra of HSA with the absorption spectra of TQ. R0 can be calculated from donor emission and acceptor absorption spectra using the Fröster formula (Eq. (7)):
R60 ¼ 8:79 1025 K 2 N4 /J
ð7Þ
where K is the orientation factor related to the geometry of the donor and acceptor molecules and K2 is the spatial orientation factor of the dipole and has a value of 2/3 f or random orientation as in fluid solution; n is the refractive index of the medium. N is the average refractive index of the medium in the wavelength range where spectral overlap is significant. The symbol / is the fluorescence quantum yield of the donor, J is the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, J is approximated by Eq. (8) and was calculated to be 1.099 1014 cm3 dm3 mol1.
P JðkÞ ¼
FðkÞeðkÞk4 Dk P FðkÞDk
ð8Þ
where F(k) is the fluorescence intensity of the fluorescent donorat wavelength k, and e (k) is the molar absorption coefficient of the acceptor at wavelength k. In the present case, and On the basis of Eqs. (6)–(8) and using the given values, that : K2 = 2/3, n = 1.336, and / = 0.14, we can calculate the following parameters, R0 = 2.56 nm, E = 0.19 and r0 = 3.26 nm. These radii are lower than 7 nm after interaction between TQ and HSA. This is consistent with conditions of Förster’s nonradiative energy transfer theory. The distance r between the donor and the acceptor is less than 7 nm, and the fulfillment of the required condition: 0.5 R0 < r < 1.5 R0 suggests that the energy transfer from donor (HSA) to acceptor (TQ) can occur with high probability. Conclusion
9.0
8.5 0.00310 0.00315 0.00320 0.00325 0.00330 0.00335 0.00340 -1
1/T (K ) Fig. 6. Van’t Hoff plot for the interaction TQ/HSA in buffer solution, pH = 7.0.
The interaction between TQ and HSA at four different temperatures, has been investigated by fluorescence spectroscopy. The intrinsic fluorescence of HSA was quenched as a result of binding with TQ, and the quenching data were analyzed quantitively by two mathematical models (Stern–Volmer and Scatchard). The thermodynamic parameters (DG°, DH°, and DS°) were calculated and asseted on the basis of van t Hoff s equation, with a result indicating that TQ binding with HSA was enthalpically
Z.J. Yasseen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 677–681
and entropically favoured, indicating that both hydrogen bonding and hydrophobic interactions played major roles in stabilizing the TQ-HSA complex formation. The distance (r) between the donor macromolecule and the acceptor (TQ) was calculated to be 3.26 nm according to Fröster energy transfer theory. The experimental results and theoretical data in this study revealed that TQ bind strongly with HSA and the results may take a closer look on new avenues in screening and design of appropriate drugs in the field of medical researches. References [1] U. Kragh-Hansen, Structure and ligand properties of human serum albumin, Dan, Med. Bull. 37 (1990) 57–84. [2] D.C. Carter, J.X. Ho, The structure of serum albumin, Adv. Protein Chem. 45 (1994) 153–203. [3] Wenying He, Ying Li, Xue, Xue, Chunxia, Z. Hu, X. Chen, F. Sheng, Effect of Chinese medicine alpinetin on the structure of human serum albumin, Bioorg. Med. Chem. 13 (2005) 1837–1845. [4] T. Peters, Serum albumin, Adv. Protein Chem. 37 (1985) 161–245. [5] M. Ikeguchi, S. Sugai, M. Fujino, T. Sugawara, K. Kuwajima, Contribution of the 6–120 disulfide bond of a-lactalbumin to the stabilities of its native and molten globule states, Biochemistry 31 (1992) 12695–12700. [6] B. Farruggia, G.A. Picó, Thermodynamic features of the chemical and thermal denaturations of human serum albumin, Int. J. Biol. Macromol. 26 (1999) 317– 323. [7] A. Sulkowska, Interaction of drugs with bovine and human serum albumin, J. Mol. Struct. 614 (2002) 227–232. [8] S. Sugio, A. Kashima, S. Mochizuki, M. Noda, K. Kobayashi, Crystal structure of human serum albumin at 2.5 A resolution, Protein Eng. 12 (1999) 439–446.
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[9] G.A. Picó, Thermodynamic features of the thermal unfolding of human serum albumin, Int. J. Biol. Macromol. 20 (1997) 63–73. [10] W.A. Lim, A. Hodel, R.T. Sauer, F.M. Richards, The crystal structure of a mutant protein with altered but improved hydrophobic core packing, Proc. Natl. Acad. Sci. USA 91 (1994) 423–427. [11] M. El Gazzar, R. El Mezayen, J.C. Marecki, M.R. Nicolls, A. Canastar, S.C. Dreskin, Anti-inflammatory effect of thymoquinone in a mouse model of allergic lung inflammation, Int. Immunopharmacol. 6 (2006) 1135–1142. [12] H. Gali-Muhtasib, M. Diab-Assaf, C. Boltze, J. Al-Hmaira, R. Hartig, A. Roessner, R. Schneider-Stock, Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism, Int. J. Oncol. 25 (2004) 857–866. [13] H. Gali-Muhtasib, A. Roessner, R. Schneider-Stock, Thymoquinone: a promising anti-cancer drug from natural sources, Int. J. Biochem. Cell Biol. 38 (2006) 1249–1253. [14] U. Kragh-Hansen, Molecular aspects of ligand binding to serum albumin, Pharmacol. Rev. 33 (1981) 17–53. [15] K.A. Dill, D.O.V. Alonso, K. Hutchinson, Thermal stabilities of globular proteins, Biochemistry 28 (1989) 5439–5449. [16] G. Lupidi, A. Scire, E. Camaioni, K.H. Khalife, G. DeSanctis, F. Tanfani, E. Damiani, Thymoquinone, apotential therapeutic agent of Nigella sativa, binds to site I of human serum albumin, Phytomedicine 17 (2010) 714–720. [17] P.D. Ross, S. Subramanian, Thermodynamics of protein association reaction: forces contribution to stability, Biochemistry 20 (1981) 3096–3102. [18] H. Aki, M. Yamamoto, Thermodynamic characterization of drug binding to human serum albumin by isothermal titration microcalorimetry, J. Pharm. Sci. 83 (12) (1994) 1712–1716. [19] T. Förster, O. Sinanoglu, Modern Quantum Chemistry, Academic Press, San Diego, 1996. p. 93. [20] Y. Ding, Y. Shu, L. Ge, R. Guo, The effect of sodium dodecyl sulfate on the conformation of bovine serum albumin, Colloids Surf. A, Physicochem. Eng. Asp 298 (2007) 163–169.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Thermodynamic analysis of thymoquinone binding to human serum albumin Zeyad J. Yasseen a,⇑, Jehad H. Hammad b, Hussein A. ALTalla a a b
Chemistry department, Faculty of Science, Islamic University of Gaza, Palestine Faculty of Medicine, Islamic University of Gaza, Palestine
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Fluorescence method was used to
examine the interaction of human serum albumin with thymoquinone. The thermodynamic parameters of the interaction were calculated. We examined two mathematical models to analyze the interaction occurred. The predominant intermolecular forces affecting the interaction were proposed. Fröster energy transfer theory was used to calculate the distance between the donor macromolecule and the acceptor ligand.
a r t i c l e
i n f o
Article history: Received 28 June 2013 Received in revised form 24 November 2013 Accepted 30 December 2013 Available online 27 January 2014 Keywords: Thermodynamic analysis Thymoquinone Human serum albumin Fluorescence
a b s t r a c t The interaction of thymoquinone (TQ) with human serum albumin (HAS) in physiological buffer (pH = 7.0) was studied at four temperatures in the range 25–50 °C using fluorescence quenching study. The binding parameters were determined by Scatchard and Stern–Volmer models. Fluorescence quenching data revealed that the binding constants (Ksc) are 1.71 104, 1.08 104, 1.03 104 and 0.969 104 M1 at 298, 303, 313 and 323 K, respectively (on the basis of Scatchard model). The thermodynamic parameters DG°, DH° and DS° were calculated the results indicated that the hydrogen bonding and hydrophobic interactions were the predominant intermolecular factors in stabilizing the TQ–HSA complex. The distance between donor (HSA) and acceptor (TQ) was calculated to be 3.26 nm based on Förster’s non-radiative energy transfer theory. Published by Elsevier B.V.
Introduction Human serum albumin (HSA) is the most abundant and highly soluble plasma protein in the blood circulatory system in mammals (approximately 60% of the total protein) [1,2]. It is able to bind and thereby transport various compounds such as fatty ⇑ Corresponding author. Tel.: +970 599928024. E-mail address: [email protected] (Z.J. Yasseen). http://dx.doi.org/10.1016/j.saa.2013.12.112 1386-1425/Published by Elsevier B.V.
acids, bilirubin, tryptophan, steroids and many drugs [3]. HSA concentrations in blood plasma ranges from 3.4 to 5.4 g/dL [4]. HSA plays an important role in transporting metabolites and drugs through the vascular system and also in maintaining the pH and osmotic pressure of the plasma [5]. HSA is a globular single polypeptide chain protein of a molecular mass of about 67 kDa and comprises 585 amino acid residues with one cystein residue at position 34 (in domain I) with free sulfhydryl group [6]. Its structure includes three homologous domains (I, II, and III) that
678
Z.J. Yasseen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 677–681
Fluorescence spectra related to the titration of fixed volume of HAS of 1 lM with concentrated stock ethanolic TQ solution were measured on the Spectrofluorimeter at the desired temperature. The fluorescence intensity measured were corrected for dilution. All samples were measured after two minutes and all binding data reported here correspond to the average values obtained after two different titrations. The fluorescence emission spectra were recorded at four different temperatures in the range 25–50 °C in the wavelength range of 250–500 nm with the excitation wavelength at 278 nm and using slits of 10 nm for both excitation and emission monochromators. Fig. 1. X-ray crystallographic to 2.5 Å resolution of three dimensional Structure of HSA, with its subdomains [10]. The structure corresponds to the ‘‘Protein Data Bank’’.
Results and discussion Calculation of binding constants and data analysis
assemble a heart shaped molecule (Fig. 1). Each domain is formed by two sub-domains (A and B) which possess common structural motifs by various forces such as salt bridges and hydrophobic interactions [7–9]. Thymoquinone (TQ) is a phytochemical compound present in of a plant called Nigella Sativa possesses important properties such as analgestic and anti-flammatory protection of organs against oxidative damage induced by a variety of free radical generating agents. It is a potent anti tumor agent against human colorectal cancer cells [11–13]. Studies on the binding of drugs to protein are of great importance in biological, biomedical and pharmaceutical science. Plasma protein serve as transport carriers for drug bioavailability, distribution in the body, metabolism and excretion. Human serum albumin (HSA) has been used as a model protein for protein folding and ligand – binding studies over many decades [14,15]. The affinity of a drug to a protein would directly influence the concentration of the drug in the binding site and duration of the effectual drug, and consequently contribute to the magnitude of its biological actions in vivo [16]. It is informative to study interaction of bioactive compounds such as TQ with the protein, because the effectiveness of these compounds as pharmaceutical agents based on their binding ability [7]. In the present study, using the spectroflorimetric titration method, the interaction of TQ with HSA has been studied and the results has been interpreted on the basis of different mathematical models leading to estimate the binding parameters. Materials and methods
The fluorescence quenching data were assessed using two distinct mathematical models: Stern–Volmer and Scatchard models. The binding constants (KSV and KSc) obtained at each temperature in the range of 25–50 °C were estimated from the Stern–Volmer and Scatchard plots. Fluorescence quenching of single tryptophan residue in HAS (Trp 214) was used to monitor the TQ/HSA interaction and to measure the binding affinity of the interaction occurred. The addition of TQ to HSA caused a decrease in the intrinsic fluorescence emission intensity of the protein upon excitation at 278 nm (Fig. 2). Fluorescence spectra of HSA alone and complexed with TQ at 25 °C was recorded (Fig. 2). From Fig. 2 it is evident that addition of TQ quenches the fluorescence intensity of HSA and the quenching was accompanied by a small red shift in maximum fluorescence intensity of emission. This behavior could be attributed to an increase in the environmental polarity. The binding curves at different temperatures in the range of 25–50 °C, depecting the change in fluorescence of HSA (DF) as a function of molar ratio [HSA]/[TQ] are shown in Fig. 3. Here, it is clear to see that the binding curve displayed saturation at certain values of the molar ratio at each studied temperature, clearly indicating that the binding occurred at specific binding sites on the HSA. Binding constant and number of binding sites The apparent binding constant (KSV and KSc) and number of binding sites (n) for TQ that binds HSA can be obtained using Eqs. (1) and (2) Stern–Volmer and Scatchard Eqs. (1) and (2) [17,18]:
Materials Human serum albumin (HSA, fatty acid free <0.05%) was purchased from Sigma Aldrich company. Thymoquinone was purchased from Sigma Aldrich and its stock solution was prepared in 5% ethanolic solution. The other substances were of reagent grade, and were used without further purification. phosphate buffer (20 mM, pH = 7.0) was used through the study. All solutions were prepared using doubly distilled water. Fluorescence quenching studies All fluorescence spectra were recorded on an Perkin–Elmer luminescence Spectrofluorimeter, (series no. 70412) equipped with a water-jacketed cuvette holder maintained at a constant temperature by means of a circulatory water bath. A quartz cell of 1.00 cm width was used for the measurements.
Fig. 2. Emission spectra of HSA (1 lM) in 20 mM phosphate buffer (pH = 7.0) and 25 °C. Excitation carried out at 278 nm. The arrow (with its direction) shows that the increasing TQ concentration is accompanied with fluorescence intensity quenching for HSA.
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Z.J. Yasseen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 677–681
0.4
1000
0.2 800
0.0
ΔF
r/[TQ] free
600 400
-0.2 -0.4 -0.6 -0.8
200
-1.0 0
-1.2 0.001
0.002
0.003
0.004
0.1
0.005
0.2
0.3
0.4
Fig. 3. Binding curves for spectrofluorimetric titration of TQ with HSA at four different temperatures 25 (s), 30 (d), 40(N) and 50 (.) °C in phosphate buffer solution (pH = 7.0). Change in fluorescence (DF) was plotted as a function of molar ratio [HSA]/[TQ].
F Stern—Volmer : ¼ 1 þ K SV ½TQ free F0 Scatchard :
ð1Þ
r ¼ nK sc rK Sc ½TQfree
ð2Þ
where F and F0 are the emission fluorescence intensities at 334 nm in presence and absence of TQ, respectively. [TQ]free is the free TQ concentration, r is the number of mol of bound TQ per mol of HSA, n is the number of binding sites, KSV and KSc are the Stern–Volmer and Scatchard association constants, respectively. Figs. 4 and 5 show the Stern–Volmer and the Scatchard plots for the interaction TQ/HSA at different temperatures, respectively. Thermodynamic study and nature of the binding forces The fluorescence quenching data accompaining the interaction TQ/HSA in the present study were analyzed on the basis of Stern–Volmer and Scatchard models, Eqs (1) and (2), respectively. The corresponding linear regression equations and results at the four different temperatures are shown in Figs. 4 and 5.
55 50
0.6
0.7
0.8
0.9
Fig. 5. Catchard plots for TQ/HSA at different temperatures: s 25 °C, d 30 °C, N 40 °C and . 50 °C. All in phosphate buffer (pH = 7.0), kex = 278 nm, kem = 334 nm. HSA concentration: 1 lM.
It is evident from Table 1 that the binding TQ/HSA is strong and is affected with temperature, showing a decreasing trend in the value of both binding constants (KSV and KSc) with increasing temperatures in accordance with each other and exhibit a similar dependence on temperature. The binding constant of TQ/HSA decreases with temperature, resulting in a reduction of stability of the formed TQ/HSA complex, The binding constant values for the association TQ/HSA were decreased by the rise in temperature which may indicate the formation of a stable complex at lower temperature. The linearity in Scatchard plots indicates that TQ binds to one class of sites on HSA. The results were discussed in conjunction with the thermodynamic characteristics obtained for TQ binding. And the thermodynamic parameters and nature of binding forces are the main evidence for conforming the binding mode. The thermodynamic parameters at the four temperatures in the range of 25–50° were determined and presented in Table 2. Essentially, the molecular forces contributing to Protein–ligand interactions may be due to a van der Waals interaction, hydrogen bond formation, ionic, electrostatic, or hydrophobic interaction, etc. The signs and magnitudes of thermodynamic parameters forprotein reactions can account for the main forces contributing to protein stability [14]. If the enthalpy changes (DH°) not vary significantly over the temperature range studied, then its value and that of (DS°) can be assessed from the van’t Hoff equation:
ln k ¼
45 40
DH DS þ RT R
ð3Þ
where K is the binding constant at the corresponding temperature calculated from both (Scatchard and Stern–Volmer equations (Eqs. (1) and (2)) and R is the gas constant. The enthalpy change (DH°) and entropy change (DS°) are calculated from the slope and
35
F0 / F
0.5
r
[HSA]/[TQ], M
30 25 20 15
Table 1 Binding parameters for the interaction TQ/HSA.
10 5
Temperature (°C)
0 -5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
[TQ], mM Fig. 4. Stern–Volmer plots for TQ/HSA at different temperatures: s 25 °C, d 30 °C, N 40 °C and . 50 °C. All in phosphate buffer (pH = 7.0), kex = 278 nm, kem = 334 nm. HSA concentration: 1 lM.
20 30 40 50
Binding parameters Stern Volmer
Scatchard
KSV (104)
KSc (104)
N
1.49 1.16 1.03 0.977
1.71 1.08 1.03 0.969
1.7 1.1 1.0 0.96
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Table 2 Thermodynamic parameters for the interactions TQ/HSA at four different temperatures. T (K)
DS° (J mol1 K1)
DG° (kJ mol1)
DH° (kJ mol1)
298 303 313 323
34.08
26.35 26.52 26.89 27.23
16.22
intercept of the van’t Hoff plot of ln K against 1/T, Fig. 6. The Gibbs energy change (DG°) is estimated from Eq. (4).
DG ¼ DH T DS
ð4Þ
The results obtained are listed in Table 2 suggested that the process is both enthalpically and entropically driven. Generally, the positive entropy change occurs because the water molecules that are arrangedin orderly fashion around the ligand and the protein acquire a more random configuration as a result of hydrophobic interactions. Negative enthalpy change is observed whenever there is hydrogen bonding through the binding process [17,18]. The negative DH° and positive DS° values in the interaction TQ/ HSA suggested that both hydrogen bonding and hydrophobic interactions play the major roles in the binding process. In this study, the quenching data were analyzed according to both Stern–Volmer, and Scatchard models Eqs. (1) and (2). Where Ka is the binding constant (on the basis of Scatchard or Stern–Volmer model) at the corresponding temperature, R is the gas constant, and T is the absolute temperature. Binding distance between TQ and the amino acid residue of HSA Fluorescence resonance energy transfer (FRET) is a reliable method for studying protein–ligand interactions and to determine the distance between the donor (HSA) and the acceptor (TQ) can be calculated using (FRET). In this study we used fluorescence resonance energy transfer (FRET) to determine the distance between TQ and the donor amino acid residue [19]. By Föster theory, the efficiency of energy transfer (E) is related to the distance r between the donor and the acceptor by Eq. (6).
E¼1
R60
F ¼ F 0 R60 þ r60
ð6Þ
where F and F0 are the fluorescence intensities of HSA in the presence and absence of TQ; r0 is the distance from the ligand to amino 10.5
Ln K Sc
10.0
9.5
Fig. 7. Spectral overlap of the fluorescence spectra of HSA (solid line) with the absorption spectra of TQ (dashed dot line). [HSA] = 1 lM, and [TQ] = 10 mM , both in buffer solution (pH = 7.0).
acid residue in the HSA, and R is the Förster’s critical distance at which 50% [20] of the excitation energy is transferred to the acceptor. Fig. 7 shows the overlap of the fluorescence spectra of HSA with the absorption spectra of TQ. R0 can be calculated from donor emission and acceptor absorption spectra using the Fröster formula (Eq. (7)):
R60 ¼ 8:79 1025 K 2 N4 /J
ð7Þ
where K is the orientation factor related to the geometry of the donor and acceptor molecules and K2 is the spatial orientation factor of the dipole and has a value of 2/3 f or random orientation as in fluid solution; n is the refractive index of the medium. N is the average refractive index of the medium in the wavelength range where spectral overlap is significant. The symbol / is the fluorescence quantum yield of the donor, J is the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, J is approximated by Eq. (8) and was calculated to be 1.099 1014 cm3 dm3 mol1.
P JðkÞ ¼
FðkÞeðkÞk4 Dk P FðkÞDk
ð8Þ
where F(k) is the fluorescence intensity of the fluorescent donorat wavelength k, and e (k) is the molar absorption coefficient of the acceptor at wavelength k. In the present case, and On the basis of Eqs. (6)–(8) and using the given values, that : K2 = 2/3, n = 1.336, and / = 0.14, we can calculate the following parameters, R0 = 2.56 nm, E = 0.19 and r0 = 3.26 nm. These radii are lower than 7 nm after interaction between TQ and HSA. This is consistent with conditions of Förster’s nonradiative energy transfer theory. The distance r between the donor and the acceptor is less than 7 nm, and the fulfillment of the required condition: 0.5 R0 < r < 1.5 R0 suggests that the energy transfer from donor (HSA) to acceptor (TQ) can occur with high probability. Conclusion
9.0
8.5 0.00310 0.00315 0.00320 0.00325 0.00330 0.00335 0.00340 -1
1/T (K ) Fig. 6. Van’t Hoff plot for the interaction TQ/HSA in buffer solution, pH = 7.0.
The interaction between TQ and HSA at four different temperatures, has been investigated by fluorescence spectroscopy. The intrinsic fluorescence of HSA was quenched as a result of binding with TQ, and the quenching data were analyzed quantitively by two mathematical models (Stern–Volmer and Scatchard). The thermodynamic parameters (DG°, DH°, and DS°) were calculated and asseted on the basis of van t Hoff s equation, with a result indicating that TQ binding with HSA was enthalpically
Z.J. Yasseen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 677–681
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