Kinetic and thermodynamic analysis of ultra-high pressure and heat-induced denaturation of bovine serum albumin by surface plasmon resonance

Purpose: To undertake comparative kinetic and thermodynamic analyses of the interaction of bovine serum albumin (BSA) with IgG pre-treated with ultra-high pressure (UHP) and moderate heat. Methods: BSA solutions were processed at 100 – 600 MPa and 25 – 40 °C. We applied an optical biosensor based on surface plasmon resonance (SPR). The dissociation and association kinetics of antigen-antibody complexes were measured at different temperatures. By analyzing the resultant sensograms, the association rate constant (ka), dissociation rate constant (kd), equilibrium dissociation constant (KD), and thermodynamic parameters were calculated. Results: The equilibrium disassociation constant, KD, ranged from a low value of 3.15 × 10 M (0.1 MPa, 25 °C) to a high value of 66.42 × 10 M (600 MPa, 55 °C). Increase in pressure and temperature led to decrease in the affinity of BSA for IgG. Pressure levels above 300 MPa promoted interactions between breakage of disulfide bonds, and the unfolding and aggregation of BSA. Conclusions: These results show that the combination of UHP and moderate heat treatment cdecrease the allergenicity of BSA by changing their protein conformation.


INTRODUCTION
Food allergies have become increasingly prevalent and occur especially in infants or children [1]. Approximately 1 -2 % of infants and 8 % of children suffer from food allergies [2]. Meat and meat products are particularly important foods due to nutritional and functional properties. It has been reported that allergy to meat is rare; positive skin prick tests to beef were described in only 7.69 % of children with atopic dermatitis [3,4]. However, BSA has been identified as one of the most important allergens in bovine meat, with a high degree of cross-reactions due to the significant sequence and structural similarities of serum albumin from different organisms [5,6]. In recent years, ultrahigh pressure (UHP) has been more frequently applied instead of traditional thermal treatment, especially for pasteurization [7]. UHP technology has negligible effects on small nutrients, preserving the nutritional and gustatorial properties. Previous research have reported the influence of high pressure on the allergenicity of beef extracts. It has been discovered that the structural changes induced by pressure may reduce or eliminate the antigencity of BSA and pig serum albumin (PSA) [8]. However, there are a few papers published on kinetic and thermodynamic changes in the binding properties of BSA treated by UHP.
Surface plasmon resonance (SPR) is a resonant charge density oscillation, which arises at the surface of a metallic film when light is reflected at the film under specific conditions. SPR could measure extremely small variations in mass on the metal surface due to interactions between a receptor on the metal and its target molecule. Thus, a receptor such as an antibody could be immobilized on the surface of the metal. Compared with other techniques, SPR technology permits a label-free and real-time analysis [9,10]. Recently, SPR was widely used in the study of interactions of various biological molecules, ranging from proteins, oligonucleotides and lipids, to small particles [11]. In the field of allergies, SPR has been applied to detect specific antibodies, to measure their binding affinities to their allergens, and to estimate enthalpy and entropy changes in antigen-antibody complex formation [12,13].
The main goal of the present study consisted of comparative kinetic and thermodynamic analyses of the interaction of BSA with IgG pre-treated with UHP and moderate heat. For this purpose, we applied an optical biosensor, Biacore™ T200 [13], based on SPR. The dissociation and association kinetics of antigen-antibody complexes were measured at different temperatures. By analyzing the resultant sensograms, the association rate constant (k a ), dissociation rate constant (k d ), equilibrium dissociation constant (K D ), and thermodynamic parameters were calculated. Some researchers have reported that UHP treatment induces changes in tertiary structure. The secondary structure has always been shown to experience no significant change. Thus, structural changes in BSA after the application of pressure and moderate heat treatments were also assessed in order to determine the changes in free sulfhydryl (SH) contents.

UHP treatment
A high-pressure machine unit (model S-FL8509-W， Stansted Fluid Power, Stansted, UK) with chamber dimension of 300 mm height and 37 mm diameter was used in the experiments involving UHP treatment. The system can be operated over a pressure range from 0 MPa to 900 MPa under controlled temperature conditions (20 -100 °C). Prior to the UHP treatment, BSA was dissolved in PBS (pH 7.4) and was 5 % (w/v) in the polyethylene bags. Samples were subjected to UHP treatment at 100, 200, 300, 400, 500, and 600 MPa for 10 min, and the temperature was separately stabilized at 25, 40, and 55 °C during UHP treatment. After UHP treatment, the BSA solution was removed from the vessel and immediately cooled in an ice bath. Unpressurized samples served as the experimental control group.

Surface modification of SPR chip
Acidic piranha solution (H 2 O 2 :H 2 SO 4 in a 1:3 ratio) was applied to clean the SPR surface. After rinsing with pure water, the chip was washed three times with absolute ethanol and then dried in a vacuum oven. The surface of the chip, used for immobilization, was chemically modified by reaction with a 3:1 v/v ratio of a mixture of EDC and NHS (15 μL/min, 420 s). Goat anti-mouse IgG (100 μg/mL in PBS, pH 5.0, 420 s) was immobilized on a separately designated sensor surface by amine coupling at 25 °C. Following deactivation, hydroxyethylammonium chloride (1 mol/L, 25 μL/min, 360 s) was applied to remove the unreacted activated groups. The chip was stabilized by washings with PBST buffer to remove excess antibody adsorbed on the chip.

SPR-based assays and binding kinetics of BSA
Binding kinetics and affinity assays were determined at different temperatures based on the Biacore™ T200 biosensor (GE Healthcare, Uppsala, Sweden). The BSA solution, at different concentrations, was injected after the surface was equilibrated by injection of blank buffer. Time-dependent binding curves were simultaneously monitored. The limit of detection (LOD) was calculated using Eq 1.
where S is the standard deviation of the intercept and m is the slope of the regression line [15].
Affinity constants were measured by Biacore™ T200, and a 1:1 Langmuir binding model was applied to the resulting graphs. Association and dissociation curves were fitted globally. The rate of complex formation during analyte injection was calculated according to Eq 2.
where R is the SPR signal in response units (RU), C is the analyte concentration, R max is the maximum analyte binding capacity in RU, and dR/dt is the rate of SPR signal change.
Multiple curves indicate the different concentrations of the protein antigen used to obtain the equilibrium dissociation constant. k a and k d were measured according to the RU differences between flow buffer and injected sample buffer. The equilibrium disassociation constant was defined as K D; where

Determination of thermodynamic parameters by SPR
The interaction between the immobilized antimouse IgG and BSA samples was examined using biosensor analysis at different pressures. Analyte solutions were obtained by serial dilutions under a buffer stream (PBST, pH 7.4). To regenerate the surface, Gly-HCl (10 mM, pH 2.1, 30 s) was injected between each analyte injection. The Gibbs free energy changes ( G  ) were calculated by the K D values according to Eq 3.
where T is absolute temperature, R is the universal gas constant, and K D is the equilibrium dissociation constant of the antibody-BSA complex.  (5) where A 412 is the absorbance at 412 nm, C is the sample concentration, and D is the dilution factor.

Statistics
Statistical analysis were performed using SPSS software (SPSS 15.0 K, Chicago, IL, USA). All data were expressed as the mean ± standard error of the mean. Comparisons of mean values were performed by one-way ANOVA. Significant difference was determined at the 0.05 probability level.

Coupling
Antibodies (100 μg/mL) were directly immobilized on a CM5 sensor chip using a standard amine coupling protocol to a level of 18000 RU. The coupling curve is shown in Figure 1. Figure 2A shows the effect of BSA concentration on the SPR response of the BSA sensor. BSA samples (5 mg/mL, pH 7.4) were dissolved in PBS buffer (pH 7.4) to concentrations ranging from 0.5 to 222 nM. These were then used to generate a curve in order to get the lowest working concentrations of BSA in the PBS buffer.

Linearity range
A typical dose-response curve and sensogram at the different concentration gradients of BSA is illustrated in Figure 2A. The initial binding rate depended on the concentration and flow rate of BSA. As shown in Figure 2B, the SPR sensor signal increased linearly with the concentration of BSA. The SPR responses were linearly correlated with the concentration of BSA. The regression equation obtained is shown in Eq 6.

Y = 0.10462x + 8.01897 (R 2 = 0.99759) ……………… (6)
The LOD value for BSA was 1.6 nM. Since the analyte concentrations range between 20% and 80 % of ligand saturation, or 0.1 -10 times the K D, the analyte concentrations should range from 30 -66,000 nM. In this study, binding affinity was determined at the concentrations of 0.156, 0.312, 0.625, 1.2, 2.5 and 5 μM.    , 55 °C). The results showed that the observed decrease in affinity was attained by a decrease in k a, and an increase in k d . The decrease in k a provided a negative effect on the affinity. On the other hand, the decrease in k d was positive. The lowest k a and the highest k d suggested that the interaction of the sample (600 MPa, 55 °C) with IgG was the lowest in affinity. In the affinity measurement of antibody-antigen interaction, it is often advantageous to have a high k d , manifested as an index of short residency time on the receptor (residency time=1/k d ).

Thermodynamic data
The analyte protein (BSA) interacted with the ligand IgG by non-covalent bonds such as Van der Waals forces, electrostatic interactions and hydrophobic interactions [17]. During the binding process, hydrogen bonds between the ligand and water molecules were disturbed by the effect of de-solvation.
Simultaneously, de-solvation induced reactions between the non-polar groups of the analyte and the ligand [18]. The values of thermodynamic parameters are shown in Figure  3. For all the treated samples, the antibodyantigen binding interactions were driven by enthalpy and entropy. The experimental data suggest that increased pressure induced enthalpy changes from positive to negative. Enthalpy changes were caused by the breakage and formation of hydrogen bonds, Van der Waals interactions and hydrophobic effects [19]. ΔH < 0 indicated that the increase in temperature was negative to the binding process. The thermodynamics results were consistent with dynamic results. It was observed that increases in temperature and pressure led to decreases in values of ΔH and ΔS. Pressure levels of 500 and 600 MPa changed the value of ΔS from positive to negative.
The total free energy change of the interactions was determined by free energy of association (ΔG ass ) and free energy of dissociation (ΔG diss ) where ΔG = ΔG ass -ΔG diss . Values of ΔG ass and ΔG diss were calculated from k a and k d at different temperatures. The data showed that significant changes in Gibbs free energy did not occur during complex formation. As shown in Figure 3, the decrease in affinity received a larger contribution from ΔH or ΔS than from ΔG during antigen-antibody binding.

Structural analysis data
To better understand the kinetic and thermodynamic analyses conducted by SPR, the SH contents of BSA were determined (Figure 4). The results suggest that the conformational changes induced by pressure (100 -300 MPa) on BSA exposed its free SH groups. However, at pressures above 300 MPa, the free SH groups followed a reverse trend and the content of SH decreased compared with the control samples (0.1 MPa). A significant amount of free SH groups in the native globular conformation were masked to attack, due to their location in poorly accessible regions of the folded structure. Therefore, the level of SH groups could be considered as an indication of protein unfolding or denaturation [20].

DISCUSSION
The kinetic results showed that as the temperature of measurement increased, the value of the K D decreased, and the affinity of the antibody and BSA decreased. The data also indicated that the increasing pressure and temperature resulted in quicker dissociation and weaker affinity, and that higher pressure and temperature resulted in reduction of BSA allergenicity. Thermodynamic analysis results suggest that the changes in the entropy of the system were negative and adverse to the Generally, antigen-antibody interaction is accompanied by a conformational change such as rotation. In this study, entropy changes were obtained mainly from protein-solvent interactions, and from the protein conformational changes after ligand binding. The formation of BSA-IgG complex is actually a process driven by enthalpy and solvent entropy. Similar to most other antigen-antibody interactions, the binding of an antigen is enthalpically driven throughout the physiological temperature range. Structural flexibility of the antigen-antibody site is key to the antigen binding activity [20]. Burgen et al [21] suggested that such a conformational flexibility would result in a faster rate of association and dissociation, a mechanism of recognition tagged the "induced fit" model. The change of the SH content demonstrated that UHP (at 100 -300 MPa) was able to induce breakage of disulfide bonds in BSA, as well as unfolding of BSA. On the other hand, pressure levels above 300 MPa enhanced protein-protein interactions, as well as transient and long term interactions between neighboring polypeptide side chains [7].
It is known that UHP can induce structural unfolding, precipitation, protein denaturation, and even dissociation of some proteins into subunits. Studies have demonstrated that protein flexibility may play an important role in antigen-antibody recognition and in the biological functions of proteins [22]. The high pressure and moderate temperature treatment might induce stiffness which is thought to be unfavorable for antigenantibody interactions. In addition, the decreased allergenicity of BSA might be attributed to changes in protein conformation caused by high pressure and moderate temperature. Conformational changes such as protein refolding or re-aggregation may actually hide some conformational epitopes or IgG and IgE binding sites so that some allergen activities could be minimized.

CONCLUSION
The findings of this study highlight the relevance of detailed thermodynamic and kinetic analysis of the BSA-IgG interactions using SPR. A reduction in affinity was induced by pressure levels of 100 -600 MPa in the temperature range of 25 -55 °C. Structural analysis showed that UHP caused protein unfolding and/or aggregation. The treatments led to intense protein-protein interactions via the formation of complex aggregates caused by formation of new disulfide bonds. The results also show the potential of UHP for inducing changes in BSA structure and allergenicity. The combination of UHP and moderate heat treatment will not only prevent beef and milk allergies, but could also be applied to produce hypoallergenic beef and milk.