Spectroscopic and Molecular Docking Studies on the Interaction Between N-Acetyl Cysteine and Bovine Serum Albumin Ali Jahanban-Esfahlan,1,2 Vahid Panahi-Azar,3 Sanaz Sajedi,3 1
Biotechnology Research Centre, Tabriz University of Medical Sciences, Tabriz, Iran
2
Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
3
Drug Applied Research Centre, Tabriz University of Medical Sciences, Tabriz, Iran
Received 18 May 2015; accepted 22 June 2015 Published online 3 July 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22697
ABSTRACT: The interaction between N-acetyl cysteine (NAC) and bovine serum albumin (BSA) was investigated by UV–vis, fluorescence spectroscopy, and molecular docking methods.
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of any preprints from the past two calendar years by emailing the Biopolymers editorial office at [email protected].
Fluorescence study at three different temperatures indicated that the fluorescence intensity of BSA was reduced upon the addition of NAC by the static quenching mechanism. Binding constant (Kb) and the number of binding sites (n) were determined. The binding constant for the interaction of NAC and BSA was in the order of 103 M21, and the number of binding sites was obtained to be equal to 1. Enthalpy (DH), entropy (DS), and Gibb’s free energy (DG) as thermodynamic values were also achieved by van’t Hoff equation. Hydrogen bonding and van der Waals force were the major intermolecular forces in the interaction process and it was spontaneous. Finally, the binding mode and the binding sites were clarified using molecular docking which were in good agreement with the results of C 2015 Wiley Periodicals, Inc. spectroscopy experiments. V
Biopolymers 103: 638–645, 2015. Keywords: bovine serum albumin; fluorescence; spectroscopy; molecular docking; N-acetyl cysteine; protein; interaction
Correspondence to: Ali Jahanban-Esfahlan; e-mail: [email protected]; [email protected] Contract grant sponsor: Student Research Committee of Tabriz University of Medical Sciences C 2015 Wiley Periodicals, Inc. V
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INTRODUCTION
S
erum albumins are known as the most abundant proteins in the blood and act as the main carriers in the circulatory system.1 Thus, they play a fundamental role in the transportation and disposition of numerous exogenous and endogenous molecules including drugs.2 Bovine serum albumin (BSA) is a common protein used for investigating the protein–ligand interactions due to its availability, low cost, stability, and high structural homology with human serum albumin (HSA).3 It is a globular heart-shaped protein which consists of 583 amino acid residues. Molecular mass of this macromolecule is 66.4 kDa. BSA contains three homologous domains (I, II, and III) and each domain is composed of two subdomains of A and B. The intrinsic fluorescence of BSA makes it possible to investigate the interaction between various ligands and albumin using fluorescence spectroscopic technique. The intrinsic fluorescence of BSA originates principally from the presence of two tryptophan amino acid residues. The first one as Trp212 is placed within a hydrophobic binding pocket of Domain II and the other one as Trp-134 is located on the surface of Domain I.4–6 The fluorescence intensity of BSA may be altered upon the addition of a substance and its interaction with protein. Hence, it would be possible to study the binding mechanisms of various molecules with BSA and achieve valuable information about the interaction process.7–12
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Interaction of NAC With BSA
N-Acetyl cysteine (NAC) is a water soluble antioxidant compound which can suppress the generation of reactive oxygen species (ROS).13–15 Thus, it acts as a powerful scavenger of ROS and decreases the inflammation.16 The chemical structure of NAC is shown in Figure 1. Various investigations have revealed that pre-treatment with NAC significantly protects cells and tissues from oxidative damage.17–19 NAC shows a pro-oxidant property because it facilitates the synthesis of glutathione.20 Treatment of NAC has a neuro-protective effect in various neurodegenerative diseases such as Alzheimer, spinal cord injury, endotoxemia, and schizophrenia.21–26 Furthermore, it is suitable for treatment of pulmonary diseases27–30 and bronchitis.31–33 NAC is known as a safe drug with minor side-effects.34 Acetaminophen overdose is one of the most common clinical uses of NAC.35 In an investigation, Sun et al.36 studied the effects of N-acetyl-L-cysteine-capped CdTe quantum dots on BSA and bovine hemoglobin. Recently, Sun et al.37 considered the effect of N-acetyl-L-cysteine capped CdTe quantum dots on the structure and activity of human serum albumin by spectroscopic techniques. However, to the best of our knowledge the interaction of NAC with proteins particularly serum albumins have not been studied comprehensively. Accordingly, in the current investigation, we wanted to characterize the interaction between NAC and BSA in vitro under physiological conditions, using ultraviolet and fluorescence spectroscopic techniques. Additionally, the molecular docking was performed for further clarify the interaction of NAC with BSA.
MATERIALS AND METHODS Materials BSA and NAC were purchased from Sigma (Steinheim, Germany) and used as supplied. All reagents and solvents were analytical grade and used without further purification. Ultra-pure water used in all experiments was obtained from Milli-Q gradient water purification system (Millipore, USA).
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FIGURE 1 Chemical structure of NAC.
the set values ( 6 1 8C) using a temperature controller apparatus and a water jacket cell holder equipped with stirrer. The excitation wavelength was set at 290 nm and the fluorescence intensity was measured using the peak height at 348 nm at three different temperatures (280, 290, and 300 K). The fluorescence measurements were recorded at pH 7.4 by keeping the concentration of BSA constant (25 mM), while various concentrations of the NAC (0–250 mM) were used. The synchronous fluorescence spectra were recorded in the synchronous scan mode with an offset of 15 or 60 nm (Dk 5 kem 2 kex 5 15 or 60 nm).
UV–Vis Absorption Spectroscopy The UV–vis absorption spectrum of BSA in the presence and absence of NAC was recorded in the range of 200–350 nm at room temperature using T70 UV/VIS spectrophotometer (PG Instrument Ltd, UK). The concentration of BSA and NAC was 50 mM.
Docking Studies The preparation of chemical structure of NAC and molecular docking were done with ArgusLab 4.0.1 program (Mark A. Thompson, Planaria Software LLC, Seattle, WA, http://www.arguslab.com). The crystal structure of free BSA with PDB ID: 3VO3 was downloaded from the Protein Data Bank (http://www.rcsb.org). Water molecules were removed, and hydrogen atoms were added to the crystal structure of BSA. The docking runs were performed on the Argus Dock docking engine using with a maximum of 200 candidate poses and ligand was selected as flexible. The conformations were ranked using the Ascore scoring function, which estimates the free binding energy. The applied box size was 80 3 80 3 80 A˚ with grid resolution of 0.4 A˚.
Preparation of Stock Solutions The appropriate amount of BSA protein was dissolved in 10 mM phosphate buffer (pH 7.4) and fresh solution was used. A stock solution of 10 mM NAC was prepared by dissolving certain amount of its powder in water and then appropriately diluting to prepare working solutions for fluorescence and UV–vis experiments.
Fluorescence Spectroscopy The fluorescence investigations were performed with a Jasco FP-750 spectrofluorimeter (Kyoto, Japan) equipped with a 150 W Xenon lamp using 1.0 cm quarts cell. The excitation and emission monochromator band widths were set to 5 nm. Temperature was controlled at
Biopolymers
RESULTS AND DISCUSSION Analysis of Fluorescence Quenching of BSA by NAC The measurement of intrinsic fluorescence intensity of protein before and after the addition of NAC was used in order to investigate the interaction mechanism of NAC with BSA. The measurement of fluorescence intensity can give information about the molecular environment area surrounding the fluorophore molecules. Figure 2 shows the effect of NAC on the fluorescence intensity of BSA. When different concentrations of
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FIGURE 2 The fluorescence spectra of 25 lM BSA mixed with various concentrations of NAC (kex 5 290 nm) at 37 6 1 8C. The concentration of NAC was 0–250 lM from the top to the bottom.
NAC solution were added to a fixed concentration of BSA, a significant reduction in the fluorescence intensity of BSA was achieved, which indicated that NAC could quench the fluorescence intensity of BSA. Furthermore, after the addition of NAC, the maximum wavelength of BSA shifted from 348 to 351 nm. Consequently, a small shift to higher wavelengths in the maximum emission was seen and it could be rational that the fluorophore of protein was placed in a more hydrophobic environment after the addition of NAC. These observations have previously been reported by other authors4,10,38–41 and were related to the loss of the compact structure of hydrophobic sub-domain IIA of BSA where tryptophan residue is placed.3 The quantitative analysis for the interaction of NAC and BSA was performed by the fluorescence quenching at 348 nm and 37 6 1 8C.
Binding Parameters and Mechanism The fluorescence quenching can occur by different mechanisms, which were generally classified as dynamic and static quenching. Usually, dynamic quenching refers to a collisional encountering between the quencher and excited state molecule of the fluorophore but static quenching is resulted from the formation of a non-fluorescent ground state complex between the fluorophore and the quencher. A well-known Stern– Volmer equation could be used to obtain the possible quench-
FIGURE 3 The Stern–Volmer plots of BSA fluorescence quenching by NAC at three different temperatures.
ing mechanism.9 The Stern–Volmer equation for static quenching could be presented by9: F0 511KSV ½Q511Kq s0 ½Q F Kq 5KSV =s0
(1) (2)
in this equation, F and F0 show the fluorescence intensities with and without the quencher, respectively. Kq, KSV, s0, and [Q] are the quenching rate constant, the Stern–Volmer quenching constant, the average lifetime of the biomolecule without the quencher, and the concentration of quencher, respectively. Figure 3 shows the Stern–Volmer plots for NAC– BSA complex and observed to be linear at three different considered temperatures. Table I summarizes the calculated values of KSV at those temperatures. The linear Stern–Volmer plots for NAC–BSA complex showed the presence of static Table I Stern–Volmer Quenching Constants (KSV) and Bimolecular Quenching Rate Constant (kq) for the Interaction of NAC With BSA at Three Different Temperatures T (K) 280 290 300 a
KSV (3103 M21)
Kq (31011 M21 s21)
Ra
6.965 5.462 3.658
6.965 5.462 3.658
0.9957 0.9954 0.9935
R is the linear correlated coefficient.
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Table II Modified Stern–Volmer Association Constant (Ka) for NAC–BSA at Three Different Temperatures T (K) Ka (3103 M21) 280 290 300 a
4.218 2.729 1.471
Ra 0.9974 0.9966 0.9987
DH DG DS (kJ mol21) (kJ mol21) (J mol21 K21) 236.70
219.51 218.90 218.28
261.39
R is the linear correlated coefficient.
rephore. The plot of F0/(F0 2 F) vs. 1/[Q] yields 1/fa as the intercept, and 1/(faKa) as the slope (Figure 4). Table II shows the corresponding results of Ka values for the interaction of NAC with BSA. As it was observed from Table II, the decreasing trend of Ka with increasing temperature was in accordance with KSV’s dependence on temperature.
Determination the Binding Constant and the Number of Binding Sites
FIGURE 4 Modified Stern–Volmer plots for NAC–BSA at three different temperatures.
quenching mechanism for the binding of NAC to BSA. Moreover, the results demonstrated that the Stern–Volmer quenching constant, Ksv is inversely correlated with temperature, which approved that the probable quenching mechanism of fluorescence of BSA by NAC was not initiated by dynamic collision, instead it came from the complex formation. Since the fluorescence lifetime of the biopolymer is thought to be 1028 s,42 the quenching rate constants (Kq) were calculated by means of Eq. (2) and are given in Table I. The maximum scatter collision quenching constant, Kq, of various quenchers with the biopolymer43 is determined to be 1010 M21 S21. In this work, the Kq values were in the order of 1011 M21 S21 for the interaction of NAC with BSA. Hence, the rate constant of the protein quenching procedure caused by NAC was higher than the value of Kq for the scatter mechanism. This demonstrated that the fluorescence quenching of BSA was not originated from dynamic collision however initiated by the formation of a complex between NAC and BSA. Using another modified form of Stern–Volmer equation, the fluorescence data could be more evaluated44: F0 1 1 5 1 ðF0 2FÞ fa fa Ka ½Q
(3)
in this equation, Ka is the effective quenching constant for the accessible fluorophores and fa is the fraction of accessible fluoBiopolymers
For the static quenching mechanism, if it is assumed that there are independent and similar binding sites on a macromolecule, the number of binding sites, n and the binding constant, k can be determined by the another modified Stern–Volmer equation45: log
ðF0 2FÞ 5log Kb 1n log ½Q F
(4)
in this equation, Kb and n show the binding constant and the number of binding sites, respectively. The values of Kb and n for NAC–BSA were determined from the intercept and slope of the plot of log (F0 2 F)/F vs. log [Q], respectively. The plots for NAC–BSA at three different temperatures are displayed in Figure 5. From pharmacokinetics point of view, the value of Kb for a drug is important because it determines the distribution of a drug in the blood and therefore influences the efficacy of a drug. Thus, the weak binding can lead to a short lifetime or poor distribution, but strong binding can reduce the concentrations of free drug in the blood. As it can be understood from the values of Kb and n (given in Table III) the values of n obtained at three investigated temperatures were nearly equal to 1, which showed the presence of only one binding site for NAC on BSA. The value of n is advantageous to obtain the number of binding sites and determine the binding sites on serum proteins for a molecule. The Kb values were in the order of 103 M21, demonstrating that there was a strong interaction between NAC and BSA. These findings along with the achieved effective quenching constants proposed a strong affinity between NAC and BSA. Additionally, similar binding constants in the order of 104 or 103 M21 were described for numerous
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Jahanban-Esfahlan et al. Table III The Binding Constants and the Number of Binding Sites, n for the Interaction of NAC With BSA at Three Different Temperatures T (K) 280 290 300 a
Kb ( 3 103 M21)
n
Ra
1.616 1.516 1.186
1.10 1.13 1.15
0.9965 0.9944 0.9938
R is the linear correlated coefficient.
DH DS 1 lnK 52 RT R
(5)
DG5DH2T DS52RT ln K
(6)
in this equation, K is the effective quenching constants Ka at the analogous temperatures and R is the gas constant. DH and DS are the enthalpy and entropy changes, respectively. From the temperature dependence of the binding constants (Figure 6), the thermodynamic parameters involved in the interaction procedure were determined (Table II). As shown in Table II, DH and DS for the binding of NAC and BSA were 36.70 kJ 21 21 21 FIGURE 5 The plot of log(F0 2 F)/F as a function of log[Q] for mol and 61.39 J mol K . The negative values obtained the determination of the number of bound NAC molecules (n) per for DH and DS demonstrated that the binding of NAC to BSA BSA molecule. was principally enthalpy driven, while the entropy was not ligand–protein complexes via fluorescence spectroscopic technique.11,12,46 In an investigation, Li et al.47 considered the binding of three different phenolic compounds namely as caffeic acid, chlorogenic acid, and ferulic acid with BSA. They reported that the size of molecule and the number of functional groups were the main important factors in a strong interaction. Those authors showed that chlorogenic acid had strong interaction compared to the two other phenolic compounds due to its big molecular structure. Consequently, the strong interaction of NAC and BSA could be related to its big chemical structure or more functional groups.
Thermodynamic Parameters Commonly, four types of non-covalent interactions play important role in the binding of different ligands to proteins. Theses interactions are hydrogen bonds, van der Waals, electrostatic, and hydrophobic forces.48 In this study, the considered temperatures for the calculation of thermodynamic parameters were 280, 290, and 300 K which BSA does not undergo any structural degradation. If the enthalpy changes (DH) not fluctuate significantly over a temperature range investigated, then its value and the entropy change (DS) could be calculated from the van’t Hoff equation:
FIGURE 6 Van’t Hoff plot for the interaction of BSA with NAC in phosphate buffer, pH 7.4.
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favorable for it. Also, the negative value for Gibb’s free energy (DG) indicated that the interaction between NAC and BSA was spontaneous. In a study by Ross and Subramanian,49 it has been shown that the signs and amounts of DH and DS as thermodynamic parameters for protein–ligand interactions can be used for determining the major forces contributing to protein– ligand stability. From the thermodynamic stand-point, DH > 0 and DS > 0 suggests a hydrophobic interaction, DH < 0 and DS < 0 shows the van der Waals force or hydrogen bond formation, and DH < 0 and DS > 0 proposes an electrostatic force. The negative DH and DS values for the binding of NAC to BSA showed that the interaction procedure considered in the present study was principally due to hydrogen bonding and van der Waals forces.
Investigation of BSA Conformation Changes
FIGURE 7 UV–vis absorption spectra of BSA in the absence and presence of NAC. The concentration of NAC and BSA was 50 lM. The dashed line is BSA and the dotted line is BSA in the presence of NAC.
In order to investigate the effects of NAC on the conformation changes of BSA, UV–vis absorption spectroscopy and the synchronous fluorescence measurements were performed. Figure 7 shows the UV–vis absorption spectra of free BSA and BSA in the presence of NAC obtained at room temperature. It was found that the absorbance of BSA reduced remarkably after the addition of NAC. Additionally, the maximum wavelength of NAC–BSA was shifted slightly toward higher wavelengths. These results indicated that there was an interaction between
FIGURE 8 The synchronous fluorescence spectra of BSA-NAC. Dk 5 60 nm (A) and Dk 5 15 nm (B). The concentration of BSA was 25 lM while the concentrations of NAC were from 0–250 lM.
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FIGURE 9 Docked NAC with BSA (A). NAC molecule is shown as cylinder mode and BSA is represented as magenta cartoon ribbon. Hydrogen bonds between docked NAC and amino acids residues of BSA (B). H-bonds are shown as yellow dashed lines. The structure of NAC and amino acids is represented as cylinder model. Hydrogen atoms are not displayed.
NAC and BSA. Moreover, it is well-established that formation of a complex between protein and substance in the static quenching could increase or decrease the UV–vis spectrum of protein.50 Consequently, the fluorescence quenching of BSA in this work was principally initiated by complex formation between NAC and BSA. Reduction of UV–vis spectra after the addition of NAC approved that the mechanism of binding was predominantly a complex formation procedure. Additionally, it is well-known that the UV–vis absorbance of BSA at 280 nm is due to the presence of aromatic amino acids phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp). The reduction in the peak intensity at 280 nm demonstrated that the binding of NAC to BSA could lead to the loosening and unfolding of BSA conformation while increasing the hydrophobicity of the micro-environment of the aromatic amino acid residues.51 Synchronous fluorescence spectroscopy is a useful method because it gives characteristic information about the microenvironmental changes in the vicinity of the chromophores. Sensitivity, spectral simplification, and spectral bandwidth reduction and avoiding different perturbing effects are the main advantages of this technique.51 In the current investigation, the spectrum was recorded through the simultaneous scanning of excitation and emission monochromators of a spectrofluorimeter, with a fixed wavelength difference (Dk) between them. In the case of BSA, if Dk 5 15 nm, the synchronous fluorescence spectra shows the spectral character for tyrosine residues only, and if Dk 5 60 nm, it shows that of the tryptophan residues only.52 The synchronous fluorescence spectra of BSA upon the addition of different concentrations of NAC at Dk 5 15 and 60 nm are presented in Figures 8A and
8B. It was understandable that the quenching of the fluorescence intensity of tryptophan residues was stronger than that of tyrosine residues, suggesting that tryptophan residues contributed significantly to the quenching of intrinsic fluorescence of BSA. Additionally, a slight blue shift in the maximum emission wavelength of tryptophan residue was detected upon the addition of NAC, demonstrating that the conformation of BSA was altered. Consequently, the polarity around tryptophan residue was reduced and it was located in a less hydrophobic environment. These observations recommended that NAC induces a conformational change in BSA. Yet the microenvironment around the Tyr residues has no considerable change.
Docking Studies In the current investigation, ArgusLab docking software was used to understand the interaction of NAC with BSA. Our UV–vis and the fluorescence spectroscopy results were complemented with the computational molecular docking studies in which NAC was docked to BSA in order to determine the favourite binding sites. The best conformation of the binding mode between NAC and BSA is shown in Figure 9A. The binding sites of BSA for exogenous or endogenous ligands may be located in subdomains IIA and IIIA, known as Sudlow’s sites I and II, respectively, and the drug binding sites are often located in these subdomains.2The docking results showed that NAC binds within the binding pocket of sub-domain IIA or Sudlow’s site I. Figure 9B presents hydrogen bonds for NAC–BSA complex. NAC forms H-bonds with His-241, Arg-217, Tyr-149, and Arg-256 residues. In addition, NAC is surrounded by Asn–482, Val–481, Pro–485, Arg– Biopolymers
Interaction of NAC With BSA
484, Arg– 347, Ser–343, Glu–449, Ser–453, Trp–213, Leu–197, Leu–480, and Arg–483 amino acid residues with the binding energy of 9.58 kcal/mol (amino acids residues were not shown). It is understandable that various hydrophobic and hydrophilic amino acids are in contact with NAC in NAC–BSA complex. Collectively, these results could explain the effective fluorescence quenching of BSA in the presence of NAC.
CONCLUSIONS In the present investigation, the interaction of NAC with BSA was studied by spectroscopic methods under physiological conditions and several binding parameters have been determined. The results of fluorescence spectroscopy showed a static quenching mechanism for the binding of NAC to BSA. Also, a single class of binding site on BSA for NAC was determined according to the values of n. Finally, docking studies showed that NAC could bind to BSA at site I in subdomain IIA.
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21. Zhang, H.; Spapen, H.; Nguyen, D. N.; Benlabed, M.; Buurman, W.; Vincent, J.-L. Am J Physiol 1994, 266, 46–54. 22. Adair, J. C.; Knoefel, J. E.; Morgan, N. Neurology 2001, 57, 1515–1517. 23. Fukami, G.; Hashimoto, K.; Koike, K.; Okamura, N.; Shimizu, E.; Iyo, M. Brain Res 2004, 1016, 90–95. 24. Mayer, M.; Noble, M. Proc Natl Acad Sci 1994, 91, 7496–7500. 25. Mohammadi, S.; Movahedin, M.; Mowla, S. J. Reprod Biol Endocrinol 2009, 7, 1–6. 26. Andreassen, O. A.; Dedeoglu, A.; Klivenyi, P.; Beal, M. F.; Bush, A. I. Neuroreport 2000, 11, 2491–2493. 27. Decramer, M.; Rutten-van M€ olken, M.; Dekhuijzen, P. R.; Troosters, T.; van Herwaarden, C.; Pellegrino, R.; van Schayck, C. O.; Olivieri, D.; Del Donno, M.; De Backer, W. Lancet 2005, 365, 1552–1560. 28. Hansen, N.; Skriver, A.; Brorsen-Riis, L.; Balsløv, S.; Evald, T.; Maltbaek, N.; Gunnersen, G.; Garsdal, P.; Sander, P.; Pedersen, J. Respir Med 1994, 88, 531–535. 29. Dekhuijzen, P. Eur Respir J 2004, 23, 629–636. 30. Poole, P. J.; Black, P. N. BMJ 2001, 322, 1271. 31. Stey, C.; Steurer, J.; Bachmann, S.; Medici, T.; Tramer, M. Eur Respir J 2000, 16, 253–262. 32. Dueholm, M.; Nielsen, C.; Thorshauge, H.; Evald, T.; Hansen, N.-C.; Madsen, H.; Maltb, N. Respir Med 1992, 86, 89–92. 33. Parr, G.; Huitson, A. Br J Dis Chest 1987, 81, 341–348. 34. Kumar, B. A.; Reddy, A. G.; Kumar, P. R.; Reddy, Y. R.; Rao, T. M.; Haritha, C. J Natural Sci Biol Med 2013, 4, 414. 35. Dodd, S.; Dean, O.; Copolov, D. L.; Malhi, G. S.; Berk, M. Expert Opin Biol Ther 2008, 8, 1955–1962. 36. Sun, H.; Cui, E.; Tan, Z.; Liu, R. J Biochem Mol Toxicol 2014, 28, 549–557. 37. Sun, H.; Yang, X.; Li, M.; Han, S.; Liu, Y.; Tan, X.; Liu, C.; Liu, R. J Luminescence, 2015, 167, 1–7. 38. Wang, N.; Ye, L.; Yan, F.; Xu, R. Int J Pharm 2008, 351, 55–60. 39. Hu, Y.-J.; Liu, Y.; Shen, X.-S.; Fang, X.-Y.; Qu, S.-S. J Mol Struct 2005, 738, 143–147. 40. Naik, P.; Chimatadar, S.; Nandibewoor, S. Spectrochim Acta A Mol Biomol Spectrosc 2009, 73, 841–845. 41. Belatik, A.; Hotchandani, S.; Bariyanga, J.; Tajmir-Riahi, H. Eur J Med Chem 2012, 48, 114–123. 42. Chen, G.-Z.; Huang, X.-Z.; Xu, J.-G.; Zheng, Z.; Wang, Z. The methods of fluorescence analysis. Science Press: Beijing, 1990. 43. Lakowicz, J. R.; Weber, G. Biochemistry 1973, 12, 4161–4170. 44. Lehrer, S. Biochemistry 1971, 10, 3254–3263. 45. Ulrich, K.-H. Danish Med Bull 1990, 37, 57–84. 46. Mandeville, J.; Tajmir-Riahi, H. Biomacromolecules 2010, 11, 465–472. 47. Li, S.; Huang, K.; Zhong, M.; Guo, J.; Wang, W.-z.; Zhu, R. Spectrochim Acta A Mol Biomol Spectrosc 2010, 77, 680–686. 48. Leckband, D. Annu Rev Biophys Biomol Struct 2000, 29, 1–26. 49. Ross, P. D.; Subramanian, S. Biochemistry 1981, 20, 3096–3102. 50. Sahoo, B. K.; Ghosh, K. S.; Dasgupta, S. Biopolymers 2009, 91, 108–119. 51. Samari, F.; Shamsipur, M.; Hemmateenejad, B.; Khayamian, T.; Gharaghani, S. Eur J Med Chem 2012, 54, 255–263. 52. Zhou, X. M.; Lu, W. J.; Su, L.; Shan, Z. J.; Chen, X. G. J Agric Food Chem 2012., 60, 1135–1145.
Reviewing Editor: David Case
Biotechnology Research Centre, Tabriz University of Medical Sciences, Tabriz, Iran
2
Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
3
Drug Applied Research Centre, Tabriz University of Medical Sciences, Tabriz, Iran
Received 18 May 2015; accepted 22 June 2015 Published online 3 July 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22697
ABSTRACT: The interaction between N-acetyl cysteine (NAC) and bovine serum albumin (BSA) was investigated by UV–vis, fluorescence spectroscopy, and molecular docking methods.
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of any preprints from the past two calendar years by emailing the Biopolymers editorial office at [email protected].
Fluorescence study at three different temperatures indicated that the fluorescence intensity of BSA was reduced upon the addition of NAC by the static quenching mechanism. Binding constant (Kb) and the number of binding sites (n) were determined. The binding constant for the interaction of NAC and BSA was in the order of 103 M21, and the number of binding sites was obtained to be equal to 1. Enthalpy (DH), entropy (DS), and Gibb’s free energy (DG) as thermodynamic values were also achieved by van’t Hoff equation. Hydrogen bonding and van der Waals force were the major intermolecular forces in the interaction process and it was spontaneous. Finally, the binding mode and the binding sites were clarified using molecular docking which were in good agreement with the results of C 2015 Wiley Periodicals, Inc. spectroscopy experiments. V
Biopolymers 103: 638–645, 2015. Keywords: bovine serum albumin; fluorescence; spectroscopy; molecular docking; N-acetyl cysteine; protein; interaction
Correspondence to: Ali Jahanban-Esfahlan; e-mail: [email protected]; [email protected] Contract grant sponsor: Student Research Committee of Tabriz University of Medical Sciences C 2015 Wiley Periodicals, Inc. V
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INTRODUCTION
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erum albumins are known as the most abundant proteins in the blood and act as the main carriers in the circulatory system.1 Thus, they play a fundamental role in the transportation and disposition of numerous exogenous and endogenous molecules including drugs.2 Bovine serum albumin (BSA) is a common protein used for investigating the protein–ligand interactions due to its availability, low cost, stability, and high structural homology with human serum albumin (HSA).3 It is a globular heart-shaped protein which consists of 583 amino acid residues. Molecular mass of this macromolecule is 66.4 kDa. BSA contains three homologous domains (I, II, and III) and each domain is composed of two subdomains of A and B. The intrinsic fluorescence of BSA makes it possible to investigate the interaction between various ligands and albumin using fluorescence spectroscopic technique. The intrinsic fluorescence of BSA originates principally from the presence of two tryptophan amino acid residues. The first one as Trp212 is placed within a hydrophobic binding pocket of Domain II and the other one as Trp-134 is located on the surface of Domain I.4–6 The fluorescence intensity of BSA may be altered upon the addition of a substance and its interaction with protein. Hence, it would be possible to study the binding mechanisms of various molecules with BSA and achieve valuable information about the interaction process.7–12
Biopolymers Volume 103 / Number 11
Interaction of NAC With BSA
N-Acetyl cysteine (NAC) is a water soluble antioxidant compound which can suppress the generation of reactive oxygen species (ROS).13–15 Thus, it acts as a powerful scavenger of ROS and decreases the inflammation.16 The chemical structure of NAC is shown in Figure 1. Various investigations have revealed that pre-treatment with NAC significantly protects cells and tissues from oxidative damage.17–19 NAC shows a pro-oxidant property because it facilitates the synthesis of glutathione.20 Treatment of NAC has a neuro-protective effect in various neurodegenerative diseases such as Alzheimer, spinal cord injury, endotoxemia, and schizophrenia.21–26 Furthermore, it is suitable for treatment of pulmonary diseases27–30 and bronchitis.31–33 NAC is known as a safe drug with minor side-effects.34 Acetaminophen overdose is one of the most common clinical uses of NAC.35 In an investigation, Sun et al.36 studied the effects of N-acetyl-L-cysteine-capped CdTe quantum dots on BSA and bovine hemoglobin. Recently, Sun et al.37 considered the effect of N-acetyl-L-cysteine capped CdTe quantum dots on the structure and activity of human serum albumin by spectroscopic techniques. However, to the best of our knowledge the interaction of NAC with proteins particularly serum albumins have not been studied comprehensively. Accordingly, in the current investigation, we wanted to characterize the interaction between NAC and BSA in vitro under physiological conditions, using ultraviolet and fluorescence spectroscopic techniques. Additionally, the molecular docking was performed for further clarify the interaction of NAC with BSA.
MATERIALS AND METHODS Materials BSA and NAC were purchased from Sigma (Steinheim, Germany) and used as supplied. All reagents and solvents were analytical grade and used without further purification. Ultra-pure water used in all experiments was obtained from Milli-Q gradient water purification system (Millipore, USA).
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FIGURE 1 Chemical structure of NAC.
the set values ( 6 1 8C) using a temperature controller apparatus and a water jacket cell holder equipped with stirrer. The excitation wavelength was set at 290 nm and the fluorescence intensity was measured using the peak height at 348 nm at three different temperatures (280, 290, and 300 K). The fluorescence measurements were recorded at pH 7.4 by keeping the concentration of BSA constant (25 mM), while various concentrations of the NAC (0–250 mM) were used. The synchronous fluorescence spectra were recorded in the synchronous scan mode with an offset of 15 or 60 nm (Dk 5 kem 2 kex 5 15 or 60 nm).
UV–Vis Absorption Spectroscopy The UV–vis absorption spectrum of BSA in the presence and absence of NAC was recorded in the range of 200–350 nm at room temperature using T70 UV/VIS spectrophotometer (PG Instrument Ltd, UK). The concentration of BSA and NAC was 50 mM.
Docking Studies The preparation of chemical structure of NAC and molecular docking were done with ArgusLab 4.0.1 program (Mark A. Thompson, Planaria Software LLC, Seattle, WA, http://www.arguslab.com). The crystal structure of free BSA with PDB ID: 3VO3 was downloaded from the Protein Data Bank (http://www.rcsb.org). Water molecules were removed, and hydrogen atoms were added to the crystal structure of BSA. The docking runs were performed on the Argus Dock docking engine using with a maximum of 200 candidate poses and ligand was selected as flexible. The conformations were ranked using the Ascore scoring function, which estimates the free binding energy. The applied box size was 80 3 80 3 80 A˚ with grid resolution of 0.4 A˚.
Preparation of Stock Solutions The appropriate amount of BSA protein was dissolved in 10 mM phosphate buffer (pH 7.4) and fresh solution was used. A stock solution of 10 mM NAC was prepared by dissolving certain amount of its powder in water and then appropriately diluting to prepare working solutions for fluorescence and UV–vis experiments.
Fluorescence Spectroscopy The fluorescence investigations were performed with a Jasco FP-750 spectrofluorimeter (Kyoto, Japan) equipped with a 150 W Xenon lamp using 1.0 cm quarts cell. The excitation and emission monochromator band widths were set to 5 nm. Temperature was controlled at
Biopolymers
RESULTS AND DISCUSSION Analysis of Fluorescence Quenching of BSA by NAC The measurement of intrinsic fluorescence intensity of protein before and after the addition of NAC was used in order to investigate the interaction mechanism of NAC with BSA. The measurement of fluorescence intensity can give information about the molecular environment area surrounding the fluorophore molecules. Figure 2 shows the effect of NAC on the fluorescence intensity of BSA. When different concentrations of
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FIGURE 2 The fluorescence spectra of 25 lM BSA mixed with various concentrations of NAC (kex 5 290 nm) at 37 6 1 8C. The concentration of NAC was 0–250 lM from the top to the bottom.
NAC solution were added to a fixed concentration of BSA, a significant reduction in the fluorescence intensity of BSA was achieved, which indicated that NAC could quench the fluorescence intensity of BSA. Furthermore, after the addition of NAC, the maximum wavelength of BSA shifted from 348 to 351 nm. Consequently, a small shift to higher wavelengths in the maximum emission was seen and it could be rational that the fluorophore of protein was placed in a more hydrophobic environment after the addition of NAC. These observations have previously been reported by other authors4,10,38–41 and were related to the loss of the compact structure of hydrophobic sub-domain IIA of BSA where tryptophan residue is placed.3 The quantitative analysis for the interaction of NAC and BSA was performed by the fluorescence quenching at 348 nm and 37 6 1 8C.
Binding Parameters and Mechanism The fluorescence quenching can occur by different mechanisms, which were generally classified as dynamic and static quenching. Usually, dynamic quenching refers to a collisional encountering between the quencher and excited state molecule of the fluorophore but static quenching is resulted from the formation of a non-fluorescent ground state complex between the fluorophore and the quencher. A well-known Stern– Volmer equation could be used to obtain the possible quench-
FIGURE 3 The Stern–Volmer plots of BSA fluorescence quenching by NAC at three different temperatures.
ing mechanism.9 The Stern–Volmer equation for static quenching could be presented by9: F0 511KSV ½Q511Kq s0 ½Q F Kq 5KSV =s0
(1) (2)
in this equation, F and F0 show the fluorescence intensities with and without the quencher, respectively. Kq, KSV, s0, and [Q] are the quenching rate constant, the Stern–Volmer quenching constant, the average lifetime of the biomolecule without the quencher, and the concentration of quencher, respectively. Figure 3 shows the Stern–Volmer plots for NAC– BSA complex and observed to be linear at three different considered temperatures. Table I summarizes the calculated values of KSV at those temperatures. The linear Stern–Volmer plots for NAC–BSA complex showed the presence of static Table I Stern–Volmer Quenching Constants (KSV) and Bimolecular Quenching Rate Constant (kq) for the Interaction of NAC With BSA at Three Different Temperatures T (K) 280 290 300 a
KSV (3103 M21)
Kq (31011 M21 s21)
Ra
6.965 5.462 3.658
6.965 5.462 3.658
0.9957 0.9954 0.9935
R is the linear correlated coefficient.
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Table II Modified Stern–Volmer Association Constant (Ka) for NAC–BSA at Three Different Temperatures T (K) Ka (3103 M21) 280 290 300 a
4.218 2.729 1.471
Ra 0.9974 0.9966 0.9987
DH DG DS (kJ mol21) (kJ mol21) (J mol21 K21) 236.70
219.51 218.90 218.28
261.39
R is the linear correlated coefficient.
rephore. The plot of F0/(F0 2 F) vs. 1/[Q] yields 1/fa as the intercept, and 1/(faKa) as the slope (Figure 4). Table II shows the corresponding results of Ka values for the interaction of NAC with BSA. As it was observed from Table II, the decreasing trend of Ka with increasing temperature was in accordance with KSV’s dependence on temperature.
Determination the Binding Constant and the Number of Binding Sites
FIGURE 4 Modified Stern–Volmer plots for NAC–BSA at three different temperatures.
quenching mechanism for the binding of NAC to BSA. Moreover, the results demonstrated that the Stern–Volmer quenching constant, Ksv is inversely correlated with temperature, which approved that the probable quenching mechanism of fluorescence of BSA by NAC was not initiated by dynamic collision, instead it came from the complex formation. Since the fluorescence lifetime of the biopolymer is thought to be 1028 s,42 the quenching rate constants (Kq) were calculated by means of Eq. (2) and are given in Table I. The maximum scatter collision quenching constant, Kq, of various quenchers with the biopolymer43 is determined to be 1010 M21 S21. In this work, the Kq values were in the order of 1011 M21 S21 for the interaction of NAC with BSA. Hence, the rate constant of the protein quenching procedure caused by NAC was higher than the value of Kq for the scatter mechanism. This demonstrated that the fluorescence quenching of BSA was not originated from dynamic collision however initiated by the formation of a complex between NAC and BSA. Using another modified form of Stern–Volmer equation, the fluorescence data could be more evaluated44: F0 1 1 5 1 ðF0 2FÞ fa fa Ka ½Q
(3)
in this equation, Ka is the effective quenching constant for the accessible fluorophores and fa is the fraction of accessible fluoBiopolymers
For the static quenching mechanism, if it is assumed that there are independent and similar binding sites on a macromolecule, the number of binding sites, n and the binding constant, k can be determined by the another modified Stern–Volmer equation45: log
ðF0 2FÞ 5log Kb 1n log ½Q F
(4)
in this equation, Kb and n show the binding constant and the number of binding sites, respectively. The values of Kb and n for NAC–BSA were determined from the intercept and slope of the plot of log (F0 2 F)/F vs. log [Q], respectively. The plots for NAC–BSA at three different temperatures are displayed in Figure 5. From pharmacokinetics point of view, the value of Kb for a drug is important because it determines the distribution of a drug in the blood and therefore influences the efficacy of a drug. Thus, the weak binding can lead to a short lifetime or poor distribution, but strong binding can reduce the concentrations of free drug in the blood. As it can be understood from the values of Kb and n (given in Table III) the values of n obtained at three investigated temperatures were nearly equal to 1, which showed the presence of only one binding site for NAC on BSA. The value of n is advantageous to obtain the number of binding sites and determine the binding sites on serum proteins for a molecule. The Kb values were in the order of 103 M21, demonstrating that there was a strong interaction between NAC and BSA. These findings along with the achieved effective quenching constants proposed a strong affinity between NAC and BSA. Additionally, similar binding constants in the order of 104 or 103 M21 were described for numerous
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Jahanban-Esfahlan et al. Table III The Binding Constants and the Number of Binding Sites, n for the Interaction of NAC With BSA at Three Different Temperatures T (K) 280 290 300 a
Kb ( 3 103 M21)
n
Ra
1.616 1.516 1.186
1.10 1.13 1.15
0.9965 0.9944 0.9938
R is the linear correlated coefficient.
DH DS 1 lnK 52 RT R
(5)
DG5DH2T DS52RT ln K
(6)
in this equation, K is the effective quenching constants Ka at the analogous temperatures and R is the gas constant. DH and DS are the enthalpy and entropy changes, respectively. From the temperature dependence of the binding constants (Figure 6), the thermodynamic parameters involved in the interaction procedure were determined (Table II). As shown in Table II, DH and DS for the binding of NAC and BSA were 36.70 kJ 21 21 21 FIGURE 5 The plot of log(F0 2 F)/F as a function of log[Q] for mol and 61.39 J mol K . The negative values obtained the determination of the number of bound NAC molecules (n) per for DH and DS demonstrated that the binding of NAC to BSA BSA molecule. was principally enthalpy driven, while the entropy was not ligand–protein complexes via fluorescence spectroscopic technique.11,12,46 In an investigation, Li et al.47 considered the binding of three different phenolic compounds namely as caffeic acid, chlorogenic acid, and ferulic acid with BSA. They reported that the size of molecule and the number of functional groups were the main important factors in a strong interaction. Those authors showed that chlorogenic acid had strong interaction compared to the two other phenolic compounds due to its big molecular structure. Consequently, the strong interaction of NAC and BSA could be related to its big chemical structure or more functional groups.
Thermodynamic Parameters Commonly, four types of non-covalent interactions play important role in the binding of different ligands to proteins. Theses interactions are hydrogen bonds, van der Waals, electrostatic, and hydrophobic forces.48 In this study, the considered temperatures for the calculation of thermodynamic parameters were 280, 290, and 300 K which BSA does not undergo any structural degradation. If the enthalpy changes (DH) not fluctuate significantly over a temperature range investigated, then its value and the entropy change (DS) could be calculated from the van’t Hoff equation:
FIGURE 6 Van’t Hoff plot for the interaction of BSA with NAC in phosphate buffer, pH 7.4.
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favorable for it. Also, the negative value for Gibb’s free energy (DG) indicated that the interaction between NAC and BSA was spontaneous. In a study by Ross and Subramanian,49 it has been shown that the signs and amounts of DH and DS as thermodynamic parameters for protein–ligand interactions can be used for determining the major forces contributing to protein– ligand stability. From the thermodynamic stand-point, DH > 0 and DS > 0 suggests a hydrophobic interaction, DH < 0 and DS < 0 shows the van der Waals force or hydrogen bond formation, and DH < 0 and DS > 0 proposes an electrostatic force. The negative DH and DS values for the binding of NAC to BSA showed that the interaction procedure considered in the present study was principally due to hydrogen bonding and van der Waals forces.
Investigation of BSA Conformation Changes
FIGURE 7 UV–vis absorption spectra of BSA in the absence and presence of NAC. The concentration of NAC and BSA was 50 lM. The dashed line is BSA and the dotted line is BSA in the presence of NAC.
In order to investigate the effects of NAC on the conformation changes of BSA, UV–vis absorption spectroscopy and the synchronous fluorescence measurements were performed. Figure 7 shows the UV–vis absorption spectra of free BSA and BSA in the presence of NAC obtained at room temperature. It was found that the absorbance of BSA reduced remarkably after the addition of NAC. Additionally, the maximum wavelength of NAC–BSA was shifted slightly toward higher wavelengths. These results indicated that there was an interaction between
FIGURE 8 The synchronous fluorescence spectra of BSA-NAC. Dk 5 60 nm (A) and Dk 5 15 nm (B). The concentration of BSA was 25 lM while the concentrations of NAC were from 0–250 lM.
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FIGURE 9 Docked NAC with BSA (A). NAC molecule is shown as cylinder mode and BSA is represented as magenta cartoon ribbon. Hydrogen bonds between docked NAC and amino acids residues of BSA (B). H-bonds are shown as yellow dashed lines. The structure of NAC and amino acids is represented as cylinder model. Hydrogen atoms are not displayed.
NAC and BSA. Moreover, it is well-established that formation of a complex between protein and substance in the static quenching could increase or decrease the UV–vis spectrum of protein.50 Consequently, the fluorescence quenching of BSA in this work was principally initiated by complex formation between NAC and BSA. Reduction of UV–vis spectra after the addition of NAC approved that the mechanism of binding was predominantly a complex formation procedure. Additionally, it is well-known that the UV–vis absorbance of BSA at 280 nm is due to the presence of aromatic amino acids phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp). The reduction in the peak intensity at 280 nm demonstrated that the binding of NAC to BSA could lead to the loosening and unfolding of BSA conformation while increasing the hydrophobicity of the micro-environment of the aromatic amino acid residues.51 Synchronous fluorescence spectroscopy is a useful method because it gives characteristic information about the microenvironmental changes in the vicinity of the chromophores. Sensitivity, spectral simplification, and spectral bandwidth reduction and avoiding different perturbing effects are the main advantages of this technique.51 In the current investigation, the spectrum was recorded through the simultaneous scanning of excitation and emission monochromators of a spectrofluorimeter, with a fixed wavelength difference (Dk) between them. In the case of BSA, if Dk 5 15 nm, the synchronous fluorescence spectra shows the spectral character for tyrosine residues only, and if Dk 5 60 nm, it shows that of the tryptophan residues only.52 The synchronous fluorescence spectra of BSA upon the addition of different concentrations of NAC at Dk 5 15 and 60 nm are presented in Figures 8A and
8B. It was understandable that the quenching of the fluorescence intensity of tryptophan residues was stronger than that of tyrosine residues, suggesting that tryptophan residues contributed significantly to the quenching of intrinsic fluorescence of BSA. Additionally, a slight blue shift in the maximum emission wavelength of tryptophan residue was detected upon the addition of NAC, demonstrating that the conformation of BSA was altered. Consequently, the polarity around tryptophan residue was reduced and it was located in a less hydrophobic environment. These observations recommended that NAC induces a conformational change in BSA. Yet the microenvironment around the Tyr residues has no considerable change.
Docking Studies In the current investigation, ArgusLab docking software was used to understand the interaction of NAC with BSA. Our UV–vis and the fluorescence spectroscopy results were complemented with the computational molecular docking studies in which NAC was docked to BSA in order to determine the favourite binding sites. The best conformation of the binding mode between NAC and BSA is shown in Figure 9A. The binding sites of BSA for exogenous or endogenous ligands may be located in subdomains IIA and IIIA, known as Sudlow’s sites I and II, respectively, and the drug binding sites are often located in these subdomains.2The docking results showed that NAC binds within the binding pocket of sub-domain IIA or Sudlow’s site I. Figure 9B presents hydrogen bonds for NAC–BSA complex. NAC forms H-bonds with His-241, Arg-217, Tyr-149, and Arg-256 residues. In addition, NAC is surrounded by Asn–482, Val–481, Pro–485, Arg– Biopolymers
Interaction of NAC With BSA
484, Arg– 347, Ser–343, Glu–449, Ser–453, Trp–213, Leu–197, Leu–480, and Arg–483 amino acid residues with the binding energy of 9.58 kcal/mol (amino acids residues were not shown). It is understandable that various hydrophobic and hydrophilic amino acids are in contact with NAC in NAC–BSA complex. Collectively, these results could explain the effective fluorescence quenching of BSA in the presence of NAC.
CONCLUSIONS In the present investigation, the interaction of NAC with BSA was studied by spectroscopic methods under physiological conditions and several binding parameters have been determined. The results of fluorescence spectroscopy showed a static quenching mechanism for the binding of NAC to BSA. Also, a single class of binding site on BSA for NAC was determined according to the values of n. Finally, docking studies showed that NAC could bind to BSA at site I in subdomain IIA.
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Reviewing Editor: David Case
