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Kinetic studies of bovine serum albumin interaction with PG and TBHQ using surface plasmon resonance
Accepted Manuscript Title: Kinetic studies of bovine serum albumin interaction with PG and TBHQ using surface plasmon resonance Author: Farzaneh Fathi Jafar Ezzati Nazhad Dolatabadi Mohammad-Reza Rashidi Yadollah Omidi PII: DOI: Reference:
S0141-8130(16)30592-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.06.054 BIOMAC 6227
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
26-5-2016 15-6-2016 17-6-2016
Please cite this article as: Farzaneh Fathi, Jafar Ezzati Nazhad Dolatabadi, MohammadReza Rashidi, Yadollah Omidi, Kinetic studies of bovine serum albumin interaction with PG and TBHQ using surface plasmon resonance, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.06.054 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Kinetic studies of bovine serum albumin interaction with PG and TBHQ using surface plasmon resonance
Farzaneh Fathi1,2,3, Jafar Ezzati Nazhad Dolatabadi1*, Mohammad-Reza Rashidi1*, Yadollah Omidi1
1
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz,
Iran. 2
Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
3
Student research committee, Tabriz University of Medical Sciences, Tabriz, Iran.
*Corresponding authors E-mail: [email protected] & [email protected], Tel: +98 41 33367914, Fax: +98 41 33367929.
1
Abstract Propyl gallate (PG) and tert-butylhydroquinone (TBHQ) are examples of phenolic antioxidant agents, which have widespread use in food industry. In this study, for the first time, we report on the interaction of PG and TBHQ with bovine serum albumin (BSA) using surface plasmon resonance (SPR). In order to modify Au slide with carboxyl functional group, 11mercaptoundecanoic acid (MUA) was used. After activation of carboxylic groups, BSA was immobilized onto the MUA through both covalent amide and electrostatic binding formation. The SPR analysis showed dose-response sensograms of BSA upon increasing concentration of PG and TBHQ. At pH 4.5 the equilibrium dissociation constant or affinity unit (KD) for PG and TBHQ were 1.89e-10 and 1.49e-10 and at pH 7.5were 4.74e-10 and 1.83e-9, respectively. The smaller amount of KD demonstrated high food additive molecules affinity to BSA. Based on these findings, it can be concluded that PG and TBHQ molecules can interact with BSA and effectively distributed within the body. Besides, SPR can be considered as useful automatic tool for quantification of PG and TBHQ interaction with serum albumin and it can deliver precise real-time kinetic data.
Keywords: Food additive; bovine serum albumin; surface plasmon resonance.
2
1. Introduction Serum albumins such as bovine (BSA) and human (HSA) serum albumins are abundant serum proteins, which act as transport carrier in blood plasma with abundance 52-60% [1]. Because of having multiple lipophilic binding sites located in subdomains IIA and IIIA, serum albumins are capable of transporting and distributing of endogenous and exogenous compounds like various ligands, vitamins, hormones, drugs, nutrients, food additives and other physiological substances [2-7]. With increasing the amount of binding affinity between these proteins and compounds, free concentration and the physiological activity of various materials such as drugs decreased in blood plasma. Therefore, the investigation of binding affinity between serum albumins and various compounds are essential. BSA is large monomeric protein (66kDa) with a single chain of 583 amino acid residues and isoelectric point around 4.7–5.2 that share around 76% structural homology with HSA [8]. Food additives have been widely used in food industry in order to preserve flavor of foods or improve its taste and appearance in recent decades. Propyl gallate (PG) and tertbutylhydroquinone (TBHQ) are examples of phenolic antioxidant agents that added to unsaturated vegetable oils, fats and meat products for prevention from oxidation process [9-13]. A number of studies have been reported the interaction of the food additives with serum albumins using spectroscopic (UV/VIS, fluorescence), microcalorimetric techniques, and FT-IR technique [14-17]. In addition to these procedures, surface plasmon resonance (SPR) technique has been developed during recent two decades for investigation of interaction between two ingredients like antigen-antibody [18], cell-drug [19], drug-protein [20-22], DNA-RNA and etc [23]. Also calculation of reliable affinity and kinetics binding constants like association rate
3
constant (ka), dissociation rate constant (kd) and affinity (KD) has been reported using SPR [2426]. To the best of our knowledge, the interaction of PG and TBHQ with BSA has not been studied by SPR. Given the widespread application of these food additives, their interaction with the main protein of plasma, albumin, needs to be addressed. To this end, in the current study, we describe a new SPR method for calculation of kinetic parameters such as binding constants and affinity (Ka, Kd, KD) in the interaction of PG and TBHQ with BSA.
2. Materials and methods 2.1. Materials TBHQ, PG, BSA, 11-mercaptoundecanoic acid (11-MUA), ethanolamine-HCl, sodium hydroxide (NaOH), N-hydroxysuccinimide (NHS), and N-ethyl- N’-(3-diethylaminopropyl) carbodiimide (EDC) were purchased from Sigma–Aldrich. Pure gold chips were prepared from bionavis company (Tampere-region, Finland). All other chemicals were obtained from SigmaAldrich (Steinheim, Germany).
2.2. SPR Measurements A multi-parameter SPR device (MP-SPR Navi 210A, BioNavis Ltd, Tampere-region, Finland) that uses the Kretscheman prism configuration having a goniometer with dual flow channels and cohesive peristaltic pump with 100 μL sampler loops were applied for investigation of kinetic parameters. Briefly, the experiments were carried out in fixed angle mode. A flow rate of 30 μL min−1 was used throughout the experiments with a sensor temperature fixed at 27 °C. A laser with a wavelength of 670 nm was used as a light source to excite the surface plasmon at the
4
dielectric gold interface. In order to correct bulk effects, the response of a blank reference spot has been subtracted from the response of the ligand spot [27].
2.3. Cleaning of the bare gold sensor surface SPR-Navi Au-slides are made of BK7- glass with 240 mm2 surface area with deposition of 50 nm gold layers on it. For elimination of contamination on the Au slides surface, we used ammonia/hydrogen peroxide solution. Briefly, each gold slide was immersed in boiling solution of ammonia (NH4OH, 30%, 1 part) and hydrogen peroxide (H2O2, 30%, 1 part) in Milli-Q-water (5 parts) for about 10 minutes while the temperature of the heater-plate was set to 95°C. Then, the slides were rinsed thoroughly with Milli-Q-water several times and dried under a stream of nitrogen [28].
2.4. Preparation of self-assemble monolayer (SAM) on Au-sensor slide In order to modify the Au surface with carboxyl functional group, a bare Au slide was immersed in the mixed solution consisting of 1mM MUA in ethanol and Milli-Q-water at the ratio of 7:3, respectively at 25°C [29]. After 20 h, SAM on Au slide was formed and then MUA-slide was completely rinsed 3 times with ethanol and 3 times with PBS buffer. Finally the gold slides were dried with a stream of nitrogen and this modified slide insert into the sensor-slide-holder for attachment of BSA in SPR instrument. It is noteworthy to mention that touching of the modified slide must be avoided due to prevention of the gold surface from scratches or contamination [30, 31].
5
2.5. BSA immobilization through amino coupling Several phases successively have been done for immobilization BSA on MUA-sensor slides. Briefly, after inserting MUA-sensor on SPR instrument, reaching a stable baseline with straightline form in sensogram has been attained after 15 min. During baseline phase only PBS buffer is running to the modified gold surface. After achieving a steady baseline, the sensor surface cleaned with NaCL (2M) and NaOH (0.1M) by running them to both channels for 3 minutes. A typical biomolecule immobilization procedure was accomplished by using traditional carbodiimide strategy. For activation of slide surface, EDC: NHS 1:1 (NHS 0.05M + EDC 0.2M) solution was injected to the carboxylated surface for 3 min and then rinsed with buffer solution for 5 min. The cleaned slides were immediately used for injection of BSA solution (0.25mg/ml) in PBS buffer at flow rate of 30µl/min for 7 min. The immobilization procedure has been performed in two PH (4.5 and 7.5). After the immobilization of BSA, ethanolamine-HCl (1.0M) solution was injected to the slide surface for blocking of the non-specific binding [32, 33].
2.6. Kinetic analysis of PG and TBHQ to immobilized BSA Various concentration of PG and TBHQ (100- 800 µM) in PBS buffer were injected using a flow rate of 30 µl/min for 3 min. Since BSA was immobilized at two flow cells, one of them was used for sample injection and the other one was used as reference. Regeneration process was not crucial due to rapid dissociation of both food additives from BSA surface. But BSA immobilized surface was regenerated through 1 minute injection of glycin- HCl (10mM, PH = 2.0) before investigation of second food additive. All the above mentioned steps were performed at two PH (4.5 and 7.5) of running buffer.
6
Trace DrawerTM for SPR NaviTM was applied for calculations of affinity and kinetic of the measured interaction. Before calculations in Trace Drawer data is extracted with SPR NaviTM Data viewer software.
3. Results and discussion 3.1. Immobilization of BSA through amine coupling Schematic illustration of BSA immobilization by amine coupling was shown in Figure 1. In the first stage, a SAM of MUA was formed on a gold surface by immersing the surface into a 1m ethanolic solution for at least 20 h, followed by a thorough rinsing with both ethanol and water [34, 35]. After activation of carboxylic groups via EDC/NHS, BSA was adsorbed onto the MUA through both covalent amide and electrostatic binding formation as described previously [31]. Finally for blocking unreacted sites of surface ethanolamine was used. Sensogram measurements of these steps were carried out at two pH (4.5 and 7.4), and were shown in figure 2. Immobilization level and response unit (RU) of BSA at pH 4.5 was three times more than that of pH 7.5 due to isoelectric point (PI) of the BSA. The PI of the BSA is 4.7 and amino groups of the BSA below the 4.7 have positive charge [36]. Therefore, negative charge of carboxylic groups and positive charge of amino groups in injected BSA may lead in high electrostatic binding formation and high BSA molecules attachment to sensor surface at pH 4.5 (RU = 0.065). On the other hand at pH above the PI of the BSA, the carboxyl groups on the BSA caused electrostatic repulsion in interaction with negative charge of MUA surface. Thus, less RU for immobilization BSA was obtained at pH 7.5 (0.022RU). The SPR curve shift angle of MUA-Au sensor slide surface compared to BSA-immobilized sensor slide were shown at figure 3 and Table. 1. When a new layer was formed on Au sensor
7
slide, the resonance curves shift to higher incident angles and become wider because of the increase in the thickness of the adsorbed layer on the surface. The SPR curve exhibits the steepest falling slope position during binding of analytes to the surface layer and shift of total internal reflection (TIR). In this study shift angle of BSA attachment on the gold surface at pH 4.5 was more than pH 7.5, which confirmed the results arise from immobilization sensogram. From the above results, it can be concluded that the formation of MUA SAM and BSA attachment was performed properly on the Au sensor slide.
3.2. Food additives-BSA interaction Figure 4 shows the SPR curve of PG and TBHQ interaction with BSA at concentrations ranging from 100 to 800 µM, which injected to BSA immobilized surface after reference subtraction. For minimizing mass transport effect that lead to inaccurate data, not only the BSA molecule was immobilized at the low concentration but also the flow rate of kinetic tests set at a higher rate of 30 µL/min [37]. Dose-response sensograms obtained for both PG and TBHQ and confirmed previous report using other methods such as spectroscopic (UV/VIS, fluorescence) [17, 38]. Based upon the previously reported thermodynamic parameters, PG and TBHQ interaction process with BSA is spontaneous. In addition, hydrophobic forces and hydrogen bonds may play the main role in binding of TBHQ to BSA, while the main interaction between PG and albumin seems to be due to the hydrophobic interaction [17, 38, 39].
3.3. Kinetics of BSA interaction with food additives Kinetic parameters give information about how fast things happen. ka is number of AB formed per second and kd is fraction of complexes decay per second (eq. 1). Affinity determines how
8
much complex is formed at equilibrium and its unit is KD, which can be calculated through eq. 2 [40].
A B
ka
AB
(Eq. 1) kd
KD
kd ka
(Eq. 2)
The results of kinetic rate constants (ka and kd), as well as KD were summarized in Table 1. High ka constant has been obtained for both PG and TBHQ due to more BSA immobilization on the slide surface at pH 4.5 in comparison with pH 7.5, which is indicative of less time requirement for formation of complex. But the amount of Kd constant at pH 4.5 has been less than pH 7.5 that suggested more time requirement for decaying PG and TBHQ from BSA when more BSA immobilized on the surface. The smaller amounts of KD confirmed high food additives molecule affinity to BSA [41, 42]. As SPR-based detection is heavily dependent on the mass change of the analytes in the sensing medium, stronger complexes of PG with BSA may due to the higher molecular weight and larger structure of PG compared to TBHQ [43]. Finally, it should be noted that SPR not only can be used as automatic tool for quantification of these food additives interaction with serum albumin but also for determination of other food additives and antioxidants and it can estimate precise real-time kinetic data and binding information. Conclusions In the current study, the interaction of PG and TBHQ with BSA was investigated by employing SPR method under physiological conditions. The attained results demonstrate that PG and TBHQ can dynamically bind to BSA with high affinity, by which the dose-response sensograms obtained for both PG and TBHQ and verified previous reports. The high affinity of both PG and TBHQ to BSA confirmed by low amount of KD. The binding of these food additives to albumins is greatly important in food chemistry and industry. Therefore, it is advisable to make a thorough analysis on the widespread use of PG and TBHQ in food industry. Acknowledgment The authors are grateful for financial support from Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences.
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[32] S. Löfås, M. Malmqvist, I. Rönnberg, E. Stenberg, B. Liedberg, I. Lundström, Bioanalysis with surface plasmon resonance, Sensors and Actuators B: Chemical 5(1-4) (1991) 79-84. [33] B. Johnsson, S. Löfås, G. Lindquist, Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors, Analytical biochemistry 198(2) (1991) 268-277. [34] J. Homola, M. Piliarik, Surface plasmon resonance (SPR) sensors, Springer2006. [35] M. Tachibana, K. Yoshizawa, A. Ogawa, H. Fujimoto, R. Hoffmann, Sulfur-gold orbital interactions which determine the structure of alkanethiolate/Au (111) self-assembled monolayer systems, The Journal of Physical Chemistry B 106(49) (2002) 12727-12736. [36] B.D. Lang, M. Delmar, W. Coombs, Surface plasmon resonance as a method to study the kinetics and amplitude of protein-protein binding, Practical Methods in Cardiovascular Research, Springer2005, pp. 936-947. [37] P. Schuck, H. Zhao, The Role of Mass Transport Limitation and Surface Heterogeneity in the Biophysical Characterization of Macromolecular Binding Processes by SPR Biosensing, in: J.N. Mol, E.M.J. Fischer (Eds.), Surface Plasmon Resonance: Methods and Protocols, Humana Press, Totowa, NJ, 2010, pp. 15-54. [38] J. Ezzati Nazhad Dolatabadi, V. Panahi-Azar, A. Barzegar, A.A. Jamali, F. Kheirdoosh, S. Kashanian, Y. Omidi, Spectroscopic and molecular modeling studies of human serum albumin interaction with propyl gallate, RSC Advances 4(110) (2014) 64559-64564. [39] L. Bekale, D. Agudelo, H.A. Tajmir-Riahi, The role of polymer size and hydrophobic end-group in PEG–protein interaction, Colloids and Surfaces B: Biointerfaces 130 (2015) 141-148. [40] T. Rispens, H. te Velthuis, P. Hemker, H. Speijer, W. Hermens, L. Aarden, Label-free assessment of high-affinity antibody–antigen binding constants. Comparison of bioassay, SPR, and PEIA-ellipsometry, Journal of Immunological Methods 365(1–2) (2011) 50-57.
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Fig. 1. Schematic illustration of BSA immobilization by amine coupling: 1) Formation of selfassembled monolayers (SAM) of MUA. 2) Activation of –COOH in MUA by EDC/NHS. 3) Immobilization of the BSA. 4) Deactivation of unreacted activated ester sites by ethanolamines. 5) Interaction of food additives (FA) with BSA.
15
Fig. 2. SPR sensogram of BSA immobilization on a MUA-modified Au-sensor slide at pH 4.5 (A) and pH 7.5 (B). Cleaning: cleaning of the MUA with NaCL (2M ) + NaOH (0.01M) (injection time: 2 min, flow rate: 20 µl/min); activation: activation of the MUA with a mixture of EDC/NHS (injection time: 3 min, flow rate: 30 µl/min); immobilization: immobilization of 0.25 mg/ml BSA (injection time: 7 min, flow rate: 30 µl/min); deactivation: deactivation of remaining reactive NHS esters with ethanolamine (injection time:3 min, flow rate: 20 µl/min).
16
Fig. 3. SPR curve before and after 0.25mg/ml BSA injection. A) & C) SPR curve before BSA immobilization at pH 4.5 and 7.5, respectively. B) & D) SPR curve after BSA immobilization pH 4.5 and 7.5, respectively. (FC1 and FC2 demonstrate flow cell 1(blue curve) and flow cell 2 (red curve)).
17
Fig.4. Sensogram of PG and TBHQ interaction with immobilized BSA. A) and B) PG and TBHQ interaction with BSA at pH 4.5, respectively. C) and D) PG and TBHQ interaction with BSA at pH 7.5, respectively. (Five concentrations of food additives have been used: 100, 200, 400, 600 and 800 µM).
18
Table. 1. SPR angle shift values for BSA immobilization on MUA functionalized Au slide surface. Gold surface
Channel 1
Channel 2
Running buffer PH
Before BSA immobilization
70.18
69.53
4.5
After BSA immobilization
70.38
69.79
4.5
Angle shift (∆Ө)
0.20
0.26
4.5
Before BSA immobilization
69.65
69.83
7.5
After BSA immobilization
69.73
69.92
7.5
Angle shift (∆Ө)
0.08
0.09
7.5
19
Table.2. Kinetic measurements data of PG and TBHQ interaction with BSA.
Food additive
Running buffer pH
Ka (1/ (M×S))
Kd (1/S)
KD (M)
PG
4.5
2.53e5
4.78e-5
1.89e-10
8.83e4
1.32e-5
1.49e-10
7.37e4
3.49e-4
4.74e-10
4.10e4
7.50e-5
1.83e-9
TBHQ PG TBHQ
7.5
20
S0141-8130(16)30592-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.06.054 BIOMAC 6227
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
26-5-2016 15-6-2016 17-6-2016
Please cite this article as: Farzaneh Fathi, Jafar Ezzati Nazhad Dolatabadi, MohammadReza Rashidi, Yadollah Omidi, Kinetic studies of bovine serum albumin interaction with PG and TBHQ using surface plasmon resonance, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.06.054 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Kinetic studies of bovine serum albumin interaction with PG and TBHQ using surface plasmon resonance
Farzaneh Fathi1,2,3, Jafar Ezzati Nazhad Dolatabadi1*, Mohammad-Reza Rashidi1*, Yadollah Omidi1
1
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz,
Iran. 2
Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
3
Student research committee, Tabriz University of Medical Sciences, Tabriz, Iran.
*Corresponding authors E-mail: [email protected] & [email protected], Tel: +98 41 33367914, Fax: +98 41 33367929.
1
Abstract Propyl gallate (PG) and tert-butylhydroquinone (TBHQ) are examples of phenolic antioxidant agents, which have widespread use in food industry. In this study, for the first time, we report on the interaction of PG and TBHQ with bovine serum albumin (BSA) using surface plasmon resonance (SPR). In order to modify Au slide with carboxyl functional group, 11mercaptoundecanoic acid (MUA) was used. After activation of carboxylic groups, BSA was immobilized onto the MUA through both covalent amide and electrostatic binding formation. The SPR analysis showed dose-response sensograms of BSA upon increasing concentration of PG and TBHQ. At pH 4.5 the equilibrium dissociation constant or affinity unit (KD) for PG and TBHQ were 1.89e-10 and 1.49e-10 and at pH 7.5were 4.74e-10 and 1.83e-9, respectively. The smaller amount of KD demonstrated high food additive molecules affinity to BSA. Based on these findings, it can be concluded that PG and TBHQ molecules can interact with BSA and effectively distributed within the body. Besides, SPR can be considered as useful automatic tool for quantification of PG and TBHQ interaction with serum albumin and it can deliver precise real-time kinetic data.
Keywords: Food additive; bovine serum albumin; surface plasmon resonance.
2
1. Introduction Serum albumins such as bovine (BSA) and human (HSA) serum albumins are abundant serum proteins, which act as transport carrier in blood plasma with abundance 52-60% [1]. Because of having multiple lipophilic binding sites located in subdomains IIA and IIIA, serum albumins are capable of transporting and distributing of endogenous and exogenous compounds like various ligands, vitamins, hormones, drugs, nutrients, food additives and other physiological substances [2-7]. With increasing the amount of binding affinity between these proteins and compounds, free concentration and the physiological activity of various materials such as drugs decreased in blood plasma. Therefore, the investigation of binding affinity between serum albumins and various compounds are essential. BSA is large monomeric protein (66kDa) with a single chain of 583 amino acid residues and isoelectric point around 4.7–5.2 that share around 76% structural homology with HSA [8]. Food additives have been widely used in food industry in order to preserve flavor of foods or improve its taste and appearance in recent decades. Propyl gallate (PG) and tertbutylhydroquinone (TBHQ) are examples of phenolic antioxidant agents that added to unsaturated vegetable oils, fats and meat products for prevention from oxidation process [9-13]. A number of studies have been reported the interaction of the food additives with serum albumins using spectroscopic (UV/VIS, fluorescence), microcalorimetric techniques, and FT-IR technique [14-17]. In addition to these procedures, surface plasmon resonance (SPR) technique has been developed during recent two decades for investigation of interaction between two ingredients like antigen-antibody [18], cell-drug [19], drug-protein [20-22], DNA-RNA and etc [23]. Also calculation of reliable affinity and kinetics binding constants like association rate
3
constant (ka), dissociation rate constant (kd) and affinity (KD) has been reported using SPR [2426]. To the best of our knowledge, the interaction of PG and TBHQ with BSA has not been studied by SPR. Given the widespread application of these food additives, their interaction with the main protein of plasma, albumin, needs to be addressed. To this end, in the current study, we describe a new SPR method for calculation of kinetic parameters such as binding constants and affinity (Ka, Kd, KD) in the interaction of PG and TBHQ with BSA.
2. Materials and methods 2.1. Materials TBHQ, PG, BSA, 11-mercaptoundecanoic acid (11-MUA), ethanolamine-HCl, sodium hydroxide (NaOH), N-hydroxysuccinimide (NHS), and N-ethyl- N’-(3-diethylaminopropyl) carbodiimide (EDC) were purchased from Sigma–Aldrich. Pure gold chips were prepared from bionavis company (Tampere-region, Finland). All other chemicals were obtained from SigmaAldrich (Steinheim, Germany).
2.2. SPR Measurements A multi-parameter SPR device (MP-SPR Navi 210A, BioNavis Ltd, Tampere-region, Finland) that uses the Kretscheman prism configuration having a goniometer with dual flow channels and cohesive peristaltic pump with 100 μL sampler loops were applied for investigation of kinetic parameters. Briefly, the experiments were carried out in fixed angle mode. A flow rate of 30 μL min−1 was used throughout the experiments with a sensor temperature fixed at 27 °C. A laser with a wavelength of 670 nm was used as a light source to excite the surface plasmon at the
4
dielectric gold interface. In order to correct bulk effects, the response of a blank reference spot has been subtracted from the response of the ligand spot [27].
2.3. Cleaning of the bare gold sensor surface SPR-Navi Au-slides are made of BK7- glass with 240 mm2 surface area with deposition of 50 nm gold layers on it. For elimination of contamination on the Au slides surface, we used ammonia/hydrogen peroxide solution. Briefly, each gold slide was immersed in boiling solution of ammonia (NH4OH, 30%, 1 part) and hydrogen peroxide (H2O2, 30%, 1 part) in Milli-Q-water (5 parts) for about 10 minutes while the temperature of the heater-plate was set to 95°C. Then, the slides were rinsed thoroughly with Milli-Q-water several times and dried under a stream of nitrogen [28].
2.4. Preparation of self-assemble monolayer (SAM) on Au-sensor slide In order to modify the Au surface with carboxyl functional group, a bare Au slide was immersed in the mixed solution consisting of 1mM MUA in ethanol and Milli-Q-water at the ratio of 7:3, respectively at 25°C [29]. After 20 h, SAM on Au slide was formed and then MUA-slide was completely rinsed 3 times with ethanol and 3 times with PBS buffer. Finally the gold slides were dried with a stream of nitrogen and this modified slide insert into the sensor-slide-holder for attachment of BSA in SPR instrument. It is noteworthy to mention that touching of the modified slide must be avoided due to prevention of the gold surface from scratches or contamination [30, 31].
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2.5. BSA immobilization through amino coupling Several phases successively have been done for immobilization BSA on MUA-sensor slides. Briefly, after inserting MUA-sensor on SPR instrument, reaching a stable baseline with straightline form in sensogram has been attained after 15 min. During baseline phase only PBS buffer is running to the modified gold surface. After achieving a steady baseline, the sensor surface cleaned with NaCL (2M) and NaOH (0.1M) by running them to both channels for 3 minutes. A typical biomolecule immobilization procedure was accomplished by using traditional carbodiimide strategy. For activation of slide surface, EDC: NHS 1:1 (NHS 0.05M + EDC 0.2M) solution was injected to the carboxylated surface for 3 min and then rinsed with buffer solution for 5 min. The cleaned slides were immediately used for injection of BSA solution (0.25mg/ml) in PBS buffer at flow rate of 30µl/min for 7 min. The immobilization procedure has been performed in two PH (4.5 and 7.5). After the immobilization of BSA, ethanolamine-HCl (1.0M) solution was injected to the slide surface for blocking of the non-specific binding [32, 33].
2.6. Kinetic analysis of PG and TBHQ to immobilized BSA Various concentration of PG and TBHQ (100- 800 µM) in PBS buffer were injected using a flow rate of 30 µl/min for 3 min. Since BSA was immobilized at two flow cells, one of them was used for sample injection and the other one was used as reference. Regeneration process was not crucial due to rapid dissociation of both food additives from BSA surface. But BSA immobilized surface was regenerated through 1 minute injection of glycin- HCl (10mM, PH = 2.0) before investigation of second food additive. All the above mentioned steps were performed at two PH (4.5 and 7.5) of running buffer.
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Trace DrawerTM for SPR NaviTM was applied for calculations of affinity and kinetic of the measured interaction. Before calculations in Trace Drawer data is extracted with SPR NaviTM Data viewer software.
3. Results and discussion 3.1. Immobilization of BSA through amine coupling Schematic illustration of BSA immobilization by amine coupling was shown in Figure 1. In the first stage, a SAM of MUA was formed on a gold surface by immersing the surface into a 1m ethanolic solution for at least 20 h, followed by a thorough rinsing with both ethanol and water [34, 35]. After activation of carboxylic groups via EDC/NHS, BSA was adsorbed onto the MUA through both covalent amide and electrostatic binding formation as described previously [31]. Finally for blocking unreacted sites of surface ethanolamine was used. Sensogram measurements of these steps were carried out at two pH (4.5 and 7.4), and were shown in figure 2. Immobilization level and response unit (RU) of BSA at pH 4.5 was three times more than that of pH 7.5 due to isoelectric point (PI) of the BSA. The PI of the BSA is 4.7 and amino groups of the BSA below the 4.7 have positive charge [36]. Therefore, negative charge of carboxylic groups and positive charge of amino groups in injected BSA may lead in high electrostatic binding formation and high BSA molecules attachment to sensor surface at pH 4.5 (RU = 0.065). On the other hand at pH above the PI of the BSA, the carboxyl groups on the BSA caused electrostatic repulsion in interaction with negative charge of MUA surface. Thus, less RU for immobilization BSA was obtained at pH 7.5 (0.022RU). The SPR curve shift angle of MUA-Au sensor slide surface compared to BSA-immobilized sensor slide were shown at figure 3 and Table. 1. When a new layer was formed on Au sensor
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slide, the resonance curves shift to higher incident angles and become wider because of the increase in the thickness of the adsorbed layer on the surface. The SPR curve exhibits the steepest falling slope position during binding of analytes to the surface layer and shift of total internal reflection (TIR). In this study shift angle of BSA attachment on the gold surface at pH 4.5 was more than pH 7.5, which confirmed the results arise from immobilization sensogram. From the above results, it can be concluded that the formation of MUA SAM and BSA attachment was performed properly on the Au sensor slide.
3.2. Food additives-BSA interaction Figure 4 shows the SPR curve of PG and TBHQ interaction with BSA at concentrations ranging from 100 to 800 µM, which injected to BSA immobilized surface after reference subtraction. For minimizing mass transport effect that lead to inaccurate data, not only the BSA molecule was immobilized at the low concentration but also the flow rate of kinetic tests set at a higher rate of 30 µL/min [37]. Dose-response sensograms obtained for both PG and TBHQ and confirmed previous report using other methods such as spectroscopic (UV/VIS, fluorescence) [17, 38]. Based upon the previously reported thermodynamic parameters, PG and TBHQ interaction process with BSA is spontaneous. In addition, hydrophobic forces and hydrogen bonds may play the main role in binding of TBHQ to BSA, while the main interaction between PG and albumin seems to be due to the hydrophobic interaction [17, 38, 39].
3.3. Kinetics of BSA interaction with food additives Kinetic parameters give information about how fast things happen. ka is number of AB formed per second and kd is fraction of complexes decay per second (eq. 1). Affinity determines how
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much complex is formed at equilibrium and its unit is KD, which can be calculated through eq. 2 [40].
A B
ka
AB
(Eq. 1) kd
KD
kd ka
(Eq. 2)
The results of kinetic rate constants (ka and kd), as well as KD were summarized in Table 1. High ka constant has been obtained for both PG and TBHQ due to more BSA immobilization on the slide surface at pH 4.5 in comparison with pH 7.5, which is indicative of less time requirement for formation of complex. But the amount of Kd constant at pH 4.5 has been less than pH 7.5 that suggested more time requirement for decaying PG and TBHQ from BSA when more BSA immobilized on the surface. The smaller amounts of KD confirmed high food additives molecule affinity to BSA [41, 42]. As SPR-based detection is heavily dependent on the mass change of the analytes in the sensing medium, stronger complexes of PG with BSA may due to the higher molecular weight and larger structure of PG compared to TBHQ [43]. Finally, it should be noted that SPR not only can be used as automatic tool for quantification of these food additives interaction with serum albumin but also for determination of other food additives and antioxidants and it can estimate precise real-time kinetic data and binding information. Conclusions In the current study, the interaction of PG and TBHQ with BSA was investigated by employing SPR method under physiological conditions. The attained results demonstrate that PG and TBHQ can dynamically bind to BSA with high affinity, by which the dose-response sensograms obtained for both PG and TBHQ and verified previous reports. The high affinity of both PG and TBHQ to BSA confirmed by low amount of KD. The binding of these food additives to albumins is greatly important in food chemistry and industry. Therefore, it is advisable to make a thorough analysis on the widespread use of PG and TBHQ in food industry. Acknowledgment The authors are grateful for financial support from Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences.
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Fig. 1. Schematic illustration of BSA immobilization by amine coupling: 1) Formation of selfassembled monolayers (SAM) of MUA. 2) Activation of –COOH in MUA by EDC/NHS. 3) Immobilization of the BSA. 4) Deactivation of unreacted activated ester sites by ethanolamines. 5) Interaction of food additives (FA) with BSA.
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Fig. 2. SPR sensogram of BSA immobilization on a MUA-modified Au-sensor slide at pH 4.5 (A) and pH 7.5 (B). Cleaning: cleaning of the MUA with NaCL (2M ) + NaOH (0.01M) (injection time: 2 min, flow rate: 20 µl/min); activation: activation of the MUA with a mixture of EDC/NHS (injection time: 3 min, flow rate: 30 µl/min); immobilization: immobilization of 0.25 mg/ml BSA (injection time: 7 min, flow rate: 30 µl/min); deactivation: deactivation of remaining reactive NHS esters with ethanolamine (injection time:3 min, flow rate: 20 µl/min).
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Fig. 3. SPR curve before and after 0.25mg/ml BSA injection. A) & C) SPR curve before BSA immobilization at pH 4.5 and 7.5, respectively. B) & D) SPR curve after BSA immobilization pH 4.5 and 7.5, respectively. (FC1 and FC2 demonstrate flow cell 1(blue curve) and flow cell 2 (red curve)).
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Fig.4. Sensogram of PG and TBHQ interaction with immobilized BSA. A) and B) PG and TBHQ interaction with BSA at pH 4.5, respectively. C) and D) PG and TBHQ interaction with BSA at pH 7.5, respectively. (Five concentrations of food additives have been used: 100, 200, 400, 600 and 800 µM).
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Table. 1. SPR angle shift values for BSA immobilization on MUA functionalized Au slide surface. Gold surface
Channel 1
Channel 2
Running buffer PH
Before BSA immobilization
70.18
69.53
4.5
After BSA immobilization
70.38
69.79
4.5
Angle shift (∆Ө)
0.20
0.26
4.5
Before BSA immobilization
69.65
69.83
7.5
After BSA immobilization
69.73
69.92
7.5
Angle shift (∆Ө)
0.08
0.09
7.5
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Table.2. Kinetic measurements data of PG and TBHQ interaction with BSA.
Food additive
Running buffer pH
Ka (1/ (M×S))
Kd (1/S)
KD (M)
PG
4.5
2.53e5
4.78e-5
1.89e-10
8.83e4
1.32e-5
1.49e-10
7.37e4
3.49e-4
4.74e-10
4.10e4
7.50e-5
1.83e-9
TBHQ PG TBHQ
7.5
20