Thermal stability of the complex formed between carotenoids from sea buckthorn (Hippophae rhamnoides L.) and bovine β-lactoglobulin

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 173 (2017) 562–571

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa

Thermal stability of the complex formed between carotenoids from sea buckthorn (Hippophae rhamnoides L.) and bovine β-lactoglobulin Iuliana Aprodu 1, Florentina-Mihaela Ursache 1, Mihaela Turturică 1, Gabriela Râpeanu 1, Nicoleta Stănciuc ⁎,1 Dunarea de Jos University of Galati, Faculty of Food Science and Engineering, Domneasca Street 111, 800201 Galati, Romania

a r t i c l e

i n f o

Article history: Received 30 May 2016 Received in revised form 28 September 2016 Accepted 15 October 2016 Available online 17 October 2016 Keywords: β-Lactoglobulin Carotenoids Sea buckthorn Fluorescence Molecular modeling

a b s t r a c t Sea buckthorn has gained importance as a versatile nutraceutical, due to its high nutritive value in terms of carotenoids content. β-Lactoglobulin (β-LG) is a natural carrier for various bioactive compounds. In this study, the effect of thermal treatment in the temperature range of 25 to 100 °C for 15 min on the complex formed by β-LG and carotenoids from sea buckthorn was reported, based on fluorescence spectroscopy, molecular docking and molecular dynamics simulation results. Also, the berries extracts were analyzed for their carotenoids content. The chromatographic profile of the sea buckthorn extracts revealed the presence of zeaxanthin and β-carotene, as major compounds. The Stern-Volmer constants and binding parameters between β-LG and β-carotene were estimated based on quenching experiments. When thermally treating the β-LG–carotenoids mixtures, an increase in intrinsic and extrinsic fluorescence intensity up to 90 °C was observed, together with blue-shifts in maximum emission in the lower temperature range and red-shifts at higher temperature. Based on fluorescence spectroscopy results, the unfolding of the protein molecules at high temperature was suggested. Detailed information obtained at atomic level revealed that events taking place in the complex heated at high temperature caused important changes in the β-carotene binding site, therefore leading to a more thermodynamically stable assembly. This study can be used to understand the changes occurring at molecular level that could help food operators to design new ingredients and functional foods, and to optimize the processing methods in order to obtain healthier food products. © 2016 Elsevier B.V. All rights reserved.

1. Introduction β-Lactoglobulin (β-LG), the major whey protein in bovine milk, is a small globular protein with 162 amino acid residues, having a molecular mass of 18,400 Da. The protein is classified as a member of the lipocalinprotein family because of its high affinity to small hydrophobic ligands [28]. Thus, β-LG can bind various hydrophobic compounds and drugs such as fatty acids, lipids, aromatic compounds, vitamins and polyamines [29,31,34]. Burova et al. [8] suggested that β-LG has two intramolecular disulfide bonds (Cys66-Cys160, Cys106-Cys119) and one free thiol group (Cys121), which is buried between the β-barrel and the major α-helix. The protein contains 16 free amino groups that can act as binding site for potential covalent ligands as well [37]. The tertiary structure is dominated by the β-barrel and consists of nine anti-parallel β-sheets and a major α-helix at the C-terminal end of the polypeptide chain [53]. The β-barrel is formed by two β-sheets, where strands A to D form one sheet, and strands E to H form the other (with some participation from strand A, facilitated by a 90° bend at Ser [21]). The loop EF ⁎ Corresponding author at: Dunărea de Jos University of Galati, Faculty of Food Science and Engineering, Domnească Street 111, Building E, Room 304, 800201 Galati, Romania. E-mail address: [email protected] (N. Stănciuc). 1 www.funfood.ugal.ro, www.bioaliment.ugal.ro, www.sia.ugal.ro.

http://dx.doi.org/10.1016/j.saa.2016.10.010 1386-1425/© 2016 Elsevier B.V. All rights reserved.

that connects strands E and F at the open end of the β-barrel acts as a gate ([50], chap. 6, [28]). At the quaternary structure level, the protein is mostly present in monomeric or dimeric form, this equilibrium being significantly influenced by the environmental conditions. β-LG is frequently used as ingredient in food industry, because of the good techno-functional properties, high nutritional value, solubility over a wide pH range and GRAS (generally recognized as safe) status. Due to the abovementioned structural particularities, β-LG is considered as a natural carrier for various bioactive compounds to improve their bioavailability. The binding ability depends on the pH value. At pH higher than 7.0, the EF loop is open, allowing ligands to enter into the hydrophobic core [28]. Harvey et al. [19] suggested that β-LG contains three binding sites for different hydrophobic molecules: the first one is in the central cavity, known as calyx, the second is the surface cleft which lies between the α-helix and the surface of the barrel, and the third one is located at the monomer-monomer interface. Carotenoids are tetraterpenoid pigments found in plants, bacteria, and fungi that are among the most widely distributed colored compounds in nature [42]. They are lipophilic biomolecules and are classified as carotenes if their sole constituents are hydrocarbons or xanthophylls, and if they also contain one or more oxygen atoms [5]. β-Carotenes (BC) are naturally occurring and the most abundant lipophilic carotenoid precursors of vitamin A. BC is widely used as a colorant

I. Aprodu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 173 (2017) 562–571

in foods and beverages. However, the utilization of BC as a functional food in the food industry is currently limited because of its poor water solubility, high chemical instability, and low in vivo bioavailability. Encapsulation of BC can be used to improve the aqueous solubility, physicochemical stability, and bioavailability [33]. Provitamin A activity is the ability of carotenoids to form vitamin A (retinol and retinal) by the action of carotene dioxygenase [52]. Although provitamin A activity is the major function of carotenoids, potent antioxidant activity of carotenoids through singlet oxygen quenching and deactivation of free radicals plays important roles in the prevention of certain types of cancer, cardiovascular diseases, and macular degeneration [38]. Apart from this, carotenoids play important roles in cellular and organelle function [40]. Sea buckthorn berries are among the most nutritious of all fruits and have important medicinal properties [23]. The composition of sea buckthorn has been extensively studied, revealing that numerous positive effects are associated to the high nutritive value in terms of carotenoids, vitamins, organic acids, flavonoids, macro- and micronutrient elements. Sea buckthorn has gained much importance as a versatile nutraceutical crop with diverse uses, from controlling soil erosion to being a source of horse fodder, nutritious foods, drugs, and skin-care products ingredient [15]. Due to its natural antioxidant activity, different parts of the sea buckthorn can be used for the treatment of diseases, such as flu, cardiovascular diseases, mucosal injuries and skin disorders. Regarding the content in carotenoids, Anderson et al. [3] identified mainly the following compounds: zeaxanthin, β-carotene, β-cryptoxanthin, lutein, lycopene and γ-carotene. The aim of the present study was to deepen the understanding of thermal treatment effects on the β-LG-sea buckthorn extract complex (further called as β-LG-CSB), in relation with protein structural changes as followed mainly by in situ fluorescence spectroscopy. The fluorescence spectroscopy methods involved the use of intrinsic and extrinsic intensity fluorescence, phase diagram, synchronous spectra, three-dimensional fluorescence spectroscopy and quenching experiments. The BC binding site on β-LG and the effect of complexation on the conformational stability and the secondary structure of β-LG are reported here, based on the quenching experiments, molecular docking and molecular dynamics simulation results. 2. Materials and Methods 2.1. Materials β-LG (purity N 90%, genetic variants A and B) from bovine milk, βcarotene, 1-anilino-8-naphtalenesulphonic acid (ANS), acrylamide and potassium iodide (KI) were purchased from Sigma (Sigma-Aldrich Co., St. Louis, MO). Sea buckthorn (Hippophae rhamnoides L.) berries were purchased from the local market (Galati, Romania) in the month of October of the year 2015, and immediately stored at −70 °C until use. Unless otherwise stated, all other reagents were of analytical grade. 2.2. Carotenoids Extraction Five grams of sea buckthorn berries were extracted in 35 mL of ethanol:hexane solutions (4:3, v/v) containing 0.05 g magnesium carbonate on an orbital shaker for 1 h at room temperature. After extraction, the supernatant was separated and the residue was re-extracted with 70 mL ethanol:hexane solutions (4:3, v/v). The resulted residue was washed with 25 mL ethanol and afterwards with 12.5 mL hexane. The residue was washed again with 100 mL NaCl of 10% concentration and 150 mL of water. The carotenoids sea buckthorn (CSB) extract was concentrated at 40 °C to dryness, dissolved in 10 mL ethanol (70%) and filtered through 0.45 μm membranes. CSB were quantified using a colorimetric method. In brief, 1 mL of CSB ethanolic extract was added to 0.5 mL of 0.05 g/L NaCl, vortexed for 30 s, and centrifuged at 1500g for 10 min. The supernatant was diluted, and the absorbance at

563

460 nm was measured. The amount of CSB was calculated by plotting a calibration curve with β-carotene as standard (0–0.5 mg/mL). 2.3. Determination of Total Carotenoids Content Total carotenoids content (TCC) in sea buckthorn extracts was analyzed using a colorimetric method. Briefly, 1 mL of aliquot of extract in ethanol was added to 0.5 mL of 0.05 g/L NaCl, vortexed for 30 s, and centrifuged at 1500g for 10 min. The supernatant was diluted, and the absorbance at 460 nm was measured. The amount of TCC was calculated by plotting a calibration curve with β-carotene as standard (0–0.5 mg/ mL). 2.4. Quenching Experiments With β-Carotene The (un)-heat treated protein samples (0.04 mL of 3 mg/mL β-LG in 0.01 M Tris-HCl buffer solution at pH 7.7) were diluted in 3 mL of appropriate buffer and titrated by successive addition of BC (1 mg of standard solution prepared in 2 mL of hexane). The excitation wavelength was set at 292 nm, while the emission spectra were collected from 310 nm to 400 nm with increments of 0.5 nm. Both the excitation and emission slit widths were set at 10 nm. The Stern-Volmer constants, binding constants and number of binding sites were calculated as previously reported [13]. 2.5. Preparation of β-LG-CSB Complex To obtain the protein solution, β-LG was weighed and dissolved in 0.01 M Tris-HCl buffer solution (pH 7.7) at a concentration of 3 mg/ mL. Fresh CSB solution was prepared by dissolving the CSB in ethanol to give 4 μM concentrations. The β-LG-CSB complex was prepared by simple mixing of the two components. The CSB extract was added to the protein solution to reach a final protein/CSB molar ratio of 1:1. The resulting ethanol concentration never exceeded 5% (v/v), which had no appreciable effect on protein structure [31]. 2.6. Heat Treatment Plastic tubes (1 cm diameter) were filled with 0.04 mL of β-LG-CSB complex. The samples were heated at different temperatures ranging from 25 to 100 °C for 15 min, using a thermostatic water bath (Digibath-2 BAD 4, Raypa Trade, Barcelona, Spain). Then, the tubes were cooled in ice water to avoid any further thermal denaturation. 2.7. Fluorescence Spectroscopy 2.7.1. Intrinsic Fluorescence All fluorescence spectra were performed on a LS-55 luminescence spectrometer (Perkin Elmer Life Sciences, Shelton, CT, USA), equipped with the software Perkin Elmer FL Winlab. The excitation wavelength was set at 274 nm, 280 nm and 292 nm, while the emission spectra were collected from 310 nm to 420 nm, with increments of 0.5 nm. Both the excitation and emission slit widths were set at 10 nm. 2.7.2. Extrinsic Fluorescence In order to evaluate the binding of hydrophobic ANS, the thermally (un)-treated β-LG-CSB complex was incubated for 15 min in the dark with 10 μL of 8 mM ANS solutions. Then, the samples were excited at 365 nm and emission was collected between 400 and 600 nm. The excitation and emission slits were both 10 nm, and the scan speed was 500 nm/min. 2.7.3. Phase Diagram Measurements of the fluorescence intensity were performed at excitation wavelength of 292 nm, whereas the intensity was collected at

564

I. Aprodu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 173 (2017) 562–571

320 nm and 365 nm. The slits of the excitation and the emission were set at 10 nm value, and the scan speed was 500 nm/min.

3. Results and Discussions 3.1. Carotenoid Content

2.7.6. Fluorescence Quenching Experiments Fluorescence quenching experiments were performed with acrylamide and KI. 8 M acrylamide and 5 M KI were prepared freshly in 10 mM Tris-HCl buffer at pH 7.7. Quenching titrations were performed by sequentially adding aliquots of the quencher stock solutions to the (un)-heat-treated samples and gently stirring. The excitation wavelength was set at 292 nm, and the fluorescence emission spectra were scanned from 310 to 420 nm. The fluorescence quenching data were analyzed by fitting to the Stern-Volmer equation, as described by Dumitraşcu et al. [14]. 2.8. Molecular Dynamics, Docking and Refinement Procedures The atomic model of β-LG (PDB ID: 4DQ3, [32]) was taken from Brookhaven Protein Data Bank and was preliminary refined by removing all non-protein compounds. In order to understand how β-LG and BC molecules interact in native conditions, the equilibrated protein model was used as receptor for the BC ligand in the docking procedure using the PatchDock algorithm [47]. The top ten models of the β-LG-BC complex, obtained on molecules shape complementarity principles and ranked on atomic contact energy values criteria, were further refined by the use of FireDock tool [4], which adjusts the relative orientation of the molecules within the rigid body docking complexes. The values of the softened attractive and repulsive van der Waals energy terms were used for the final ranking of the resulting β-LG-BC complexes. The model characterized by the lowest value of the binding energy, indicating high affinity of the compounds within complex, was further heated to 25 and 90 °C by weak coupling each component of the optimized solvated complex to a Berendsen thermostat to control the temperature. Subsequent molecular dynamics steps were meant to attenuate eventual temperature and energy oscillations. All energy minimization and molecular dynamics calculations were run in parallelization conditions on an Intel® Core™ 2 CPU 6300 1.86 GHz processor-based machine, using GROMACS 4.6 package [20] and gromos43a1 force field to define the topology. The final equilibrated models were used to check the atomic level details on the interaction between β-LG and BC molecules using PDBSum, PDBePISA and LigPlot+ tools [24,26,27]. 2.9. Statistical Analysis All experiments were performed in triplicates with duplicate samples. The results were expressed in terms of average values. Statistical analysis of data was performed using the data analysis tool pack of the Microsoft Excel software.

3.2. Fluorescence Quenching Mechanism of Heat Treated β-LG by BC The interactions between β-LG and BC were examined by investigating the influence of increasing the concentration of BC on the fluorescence intensity spectra of β-LG preliminary heated at temperatures ranging from 25 °C to 100 °C for 15 min. BC quenched the Trp fluorescence (Fig. 1), due to energy transfer between the excited indole (Trp) ring and the ligands, changes of polarity in the neighborhood of the tryptophanyl residues, or both effects [39]. It has been reported [34] that Trp residues are buried in a non-polar environment if maximum fluorescence emission (λmax) is lower than 330 nm. If λmax is higher than 330 nm, the Trp is assigned a polar environment, which in most cases implies solvent exposure. When heating the β-LG solutions at temperatures higher than 70 °C significant red-shifts (from 331 nm at 25 °C to 337 nm at 80 °C, to 343 nm at 90 °C and to 347 nm at 100 °C, respectively) were observed, suggesting structural changes associated with the exposure of the Trp residues. With increasing BC concentration, the maximum emission wavelength of β-LG was red shifted from 331 nm to 333 nm at 25 °C and 60 °C, whereas 7 nm and 3–4 nm red

a) Fluorescence intensity (a.u.)

2.7.5. Excitation-Emission Matrix Spectroscopy (EEMS) For EEMS or 3-dimensional (3D) spectral measurements, the emission wavelength range was selected from 200 to 500 nm, and the initial excitation wavelength was set to 200 nm with increment of 10 nm, and the number of scanned spectra was 20. All other parameters were adjusted as mentioned for the intrinsic fluorescence studies.

A TCC content of 7.45 ± 1.11 mg β-caroten/g FW was determined in sea buckthorn extracts. Korekar et al. [23] reported a variation in TCC from 0.1 to 14.4 mg/100 g FW in seventeen natural population of sea buckthorn from trans-Himalaya, whereas Pop et al. [43] suggested a variation in TCC between 0.53 and 0.97 mg/g DW in berries. The HPLC profile suggested the presence of the zeaxanthin and β-carotene, as major compounds (data not shown).

350 300

a

250 200

f

150 100 50

0 310

330

350

370

390

410

Wavelength (nm)

b) 500

Fluorescence intensity (a.u.)

2.7.4. Synchronous Spectra The synchronous fluorescence spectra were recorded from 240 nm to 340 nm by scanning simultaneously the excitation and emission monochromator. The wavelength interval (Δλ) was fixed individually at 15 and 60 nm, at which the spectrum only shows the spectroscopic behavior of Tyr and Trp residues, respectively. Appropriate blanks corresponding to the buffer were subtracted to correct the fluorescence background.

a

450 400 350 300

f

250 200 150 100 50 0 310

330

350

370

390

410

Wavelength (nm) Fig. 1. The fluorescence spectra of the interaction between thermally treated β-LG and BC at 25 °C (a) and 90 °C (b). The BC concentration (from a–f) varied from 0 to 0.093 μM.

I. Aprodu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 173 (2017) 562–571

Fluorescence intensity (a.u.)

a)

Table 1 The binding parameters of the heat treated β-LG by BC at different temperatures.

25 50 60 70 80 90 100 a b c

3.48 3.99 4.17 4.36 4.99 6.83 5.70

± ± ± ± ± ± ±

0.55 0.27 0.19 0.20 0.06 0.03 0.24

a

Rb

Kb (10−8 L·mol−1)

n

0.996 0.989 0.996 0.996 0.993 0.994 0.995

1.11 0.92 0.87 1.07 1.06 0.87 0.78

1.56 1.31 1.25 1.47 1.34 0.96 1.04

± ± ± ± ± ± ±

600 500 400 300 200 100

0.05 0.04 0.02 0.02 0.07 0.05 0.03

Standard deviation. R is the correlation coefficient for the KSV values. R is the correlation coefficient for the Kb values.

Rc ± ± ± ± ± ± ±

0.04 0.06 0.05 0.03 0.07 0.04 0.02

0.992 0.999 0.991 0.993 0.990 0.998 0.998

330

50°C

350

370

390

Wavelength (nm) 60°C 70°C 80°C

410

90°C

100°C

b) 700 600 500 400 300 200 100 0 310

25°C

330

50°C

350 370 390 Wavelength (nm) 60°C 70°C 80°C 90°C

410

100°C

c) Fluorescence intensity (a.u.)

Fluorescence spectroscopy may serve as a universal tool for the study of protein-ligand interactions. The method delivers important insights into understanding the structural features that influences the binding mechanism between any target compound and proteins, and is successfully applied to study the interaction between them. Intrinsic fluorescence of proteins arises from Trp, Tyr, and Phe residues. However, Trp dominates over the other two aromatic amino acids as a relatively strong fluorescence emitter due to its higher molar absorption coefficient, higher quantum yield, and the possibility of efficient energy transfer mechanisms from Tyr and Phe toward Trp [36]. However, these authors suggested that, under special circumstances, Tyr fluorescence can be unmasked by quenching the Trp residues. In general though, the intrinsic fluorescence of Tyr and Trp containing proteins is complex, and strongly depends on the environment and photophysics of these residues, especially of Trp. Therefore, in our study, in order to get a complete view of the intrinsic fluorescence of the β-LG-CSB complex, three excitation wavelengths were used to evaluate the complex stability in terms of fluorescence intensity and maximum emission wavelengths, as follow: 274 nm, 280 nm and 292 nm. Fig. 2 shows the heat induced structural changes of β-LG-CSB complex monitored by emission spectrum. When exciting at 274 nm, 280 and 292 nm the protein equilibrated at 25 °C, the emission peaks corresponding to the hydrophobic residue of β-LG were located at 336 nm,

KSV (10−10 L·mol−1)

700

25°C

3.3. Intrinsic Fluorescence of the Complex

T(°C)

800

0 310

Fluorescence intensity (a.u.)

shifts were observed at 80 °C and 90–100 °C, respectively, indicating that BC addition caused the loss of the protein compact structure, exposing the hydrophobic subdomain where Trp is placed. In order to elucidate if the binding mechanism between β-LG and BC is either static or dynamic, the data were analyzed using the SternVolmer equation (Table 1). A high linearity was obtained in the whole temperature range studied, indicating that quenching of β-LG fluorescence emission by BC is static. In the temperature range of 25 °C to 90 °C, the KSV values increased from 3.48 ± 0.55 × 10− 10 mol/L to 6.83 ± 0.24 × 10−10 mol/L, followed by a decrease at higher temperature. The apparent binding constants (Kb) of β-LG-BC complex and the number of binding sites (n) are given in Table 1. The Kb values decreased with increasing temperature from 1.11 ± 0.05 × 10−8 mol/L at 25 °C to 0.87 ± 0.02 × 10−8 mol/L at 60 °C, suggesting that some alterations occurred around the binding sites and changed the binding ability between β-LG and BC. Increasing the temperature to 70 °C lead to the increase of the binding constant, followed by a decrease at even higher temperatures. The Kb values were in line with those reported by Mensi et al. [34], who suggested higher affinity of BC for β-LG variant A (2.07 ± 0.40 × 10−8 mol/L) when compared with variant B (1.23 ± 0.10 × 10−8 mol/L). The n values for the whole temperature range varied between 1.56 ± 0.04 at 25 °C to 0.96 ± 0.04 at 90 °C, leading to the assumption that, regardless of the thermal treatment applied, the β-LG molecule has at least one binding site with high affinity for BC.

565

450 400 350 300 250 200 150 100 50 0 310

25°C

330

50°C

350

370

Wavelength (nm) 60°C 70°C 80°C

390

410

90°C

100°C

Fig. 2. Heat-induced structural changes of β-LG-CSB complex monitored by emission spectrum. The excitation wavelength was 274 nm (a), 280 nm (b) and 292 nm (c) and the spectrums were collected between 310 and 420 nm. Three independent tests were carried out in each case and SD was lower than 3.5%.

340 nm and 327 nm, and spectrum bandwidth was equal to 61 nm, 65 nm and 68 nm, respectively. These values correspond to an emission spectrum occurring from a hydrophobic residue located in a hydrophilic area of the protein. The thermal treatment led to a significant increase in fluorescence intensity up to 90 °C, followed by a decrease at 100 °C. When exciting at 274 nm, the maximum increase in fluorescence intensity (by 148%) was found at 90 °C (Fig. 2 a). In the whole temperature range studied, the λmax was red shifted from 334 nm at 25 °C to 341 nm at 90 °C and 345 nm at 100 °C, suggesting an increase of hydrophilicity in the vicinity of hydrophobic residues. The fluorescence intensity increased with 118% (Fig. 2 b) and 147% (Fig. 2 c) when exciting the complex at 280 nm and 292 nm, respectively. When excited at 280 nm, blue-shifts of 2–5 nm were registered in the temperature range of 50° to 80 °C, followed by 3–5 nm red-shifts at higher temperatures. The decrease of fluorescence intensity at 100 °C could be explained by the effect of heating on the protein unfolding

I. Aprodu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 173 (2017) 562–571

properties [6], which could possibly enable protein aggregation by inducing the formation of intermolecular bonds [46]. Significant red-shifts were found in the whole temperature range, varying from 7 nm at 60 °C to 17 nm at 100 °C, when excited at 292 nm. From our results, it can be concluded that in the un-heated complex, the Trp and Tyr residues are assigned to be buried in the protein core when maximum fluorescence emission is equal or lower than 330 nm, and exposed to solvent when λmax is higher than 330 nm. Each monomer of β-LG contains two Trp and four Tyr residues [30]. These authors suggested that fluorescence properties of β-LG are mainly due to the Trp residues distributed in different parts of the molecule: Trp61, which is completely exposed and Trp [19], which is buried in the hydrophobic core of the protein. Albani et al. [2] suggested that only Trp [19] residue buried in the hydrophobic pocket of β-LG emits, whereas the Trp61 residue present at the protein surface near the aperture of the pocket does not emit. The weak or absence of Trp61 fluorescence might be the result of the presence of a disulfide bridge (Cys66-Cys160) in proximity to this residue, inducing complete fluorescence quenching [41] and/or the self-quenching of Trp61 by the nearby Trp61 of the other monomer, in the β-LG dimeric form [45]. Tyr42 and Tyr102 residues are buried, while Tyr20 and Tyr99 are exposed [30]. The observed changes in emissive properties of β-LG-CSB complex are associated with structural changes occurring in the tertiary structure of β-LG. Changes in λmax of β-LG-oleic acid complex due to thermal treatments were also observed by Simion-Ciuciu et al. [48], who suggested red-shifts of 2 nm at 75 °C, and 4 nm at 80 °C and 85 °C when excited at 292 nm, and 8 nm red-shift at 85 °C when excited at 274 nm. 3.4. Extrinsic Fluorescence Due to its both hydrophilic and hydrophobic properties, ANS is an excellent tool for investigating changes in fluorescence due to binding of ligands to proteins. Changes in ANS fluorescence indicate alterations in the protein conformations due to ligand-protein interaction [12]. β-LG possesses two different binding sites for ANS: an external site, close to a hydrophobic patch on the protein surface, and an internal site, located in the hydrophobic core of the protein [49,51]. The external site is responsible for a nonspecific interaction with ANS, unlike the internal one which contains one disulfide bridge (Cys106-Cys119) [11]. In the present study, the ANS fluorescence intensity of (un)-treated β-LGCSB complex was measured by exciting the samples at 365 nm and measuring the emission between 400 and 600 nm. An increase in ANS fluorescence intensity up to 90 °C, as well as a blue-shift from 507 nm at 25 °C to 495 nm at 70 °C as a result of ANS binding to β-LG-CSB complex are shown in Fig. 3. Further increase in temperature up to 100 °C resulted in a blue-shift of ANS-fluorescence of 10 nm, indicating a change of fluorophore environment from hydrophilic to hydrophobic.

ANSfluorescence intensity (a.u.)

566

300 250 200 150 100

0 420

440

25°C

460

480

500

520

540

Wavelength (nm) 60°C 70°C 80°C

50°C

90°C

560

100°C

Fig. 3. Heat-induced structural changes of β-LG-CSB complex monitored ANS fluorescence intensity. The excitation wavelength was 365 nm and the spectrums were collected between 400 and 600 nm. Three independent tests were carried out in each case and SD was lower than 3.5%.

heating. Busti et al. [9] suggested that β-LG should be considered as a mixture of monomers and dimers when the concentration of the protein in solution is lower than 4 mg/mL. From Fig. 4 it can be concluded that the dissociation of the dimers predominates in the temperatures range of 25 °C to 70 °C, followed by the unfolding at higher temperature. Our results are in good agreement with those reported earlier by Stănciuc et al. [49]. In our previous study, we showed that the unfolding of β-LG is a two-stage process, involving reversible disruption of unique tertiary structure in the first stage, followed by irreversible disruption of persistent secondary structure in the second stage. 3.6. Synchronous Spectra Synchronous spectra of β-LG-CSB complex were monitored by scanning simultaneously the excitation and emission monochromators, while maintaining a fixed wavelength difference (Δλ) between them, with the aim of studying the microenvironment of the fluorophore groups within the complex at thermal denaturation. The characteristic features of the Tyr residues and Trp residues were obtained by setting the Δλ at 15 nm and 60 nm, respectively. The synchronous spectra of the complex at different temperatures at Δλ of 15 and 60 nm are shown in Fig. 5 a and b. The shifts in the position of λmax correspond to changes of polarity around the chromophore molecules. The addition of CSB caused a blue-shift in λmax from 300 to 293 nm in case of Tyr, and from 280 nm to 276 nm in case of Trp. However, our molecular modeling results indicated that, regardless of the simulated temperature, no burial of the Trp or Tyr residues was observed because of interfacing the protein and BC molecules. From Fig. 5 a, one can see that the 120

3.5. Phase Diagram

90°C

100

I365(a.u.)

This method employs the construction of a diagram by plotting Iλ1 versus Iλ2 (where I1 and I2 are the spectral intensity values measured at wavelengths λ1 and λ2); in order to describe the protein unfolding pathway by detecting partially folded species and hidden intermediates [25]. Changes in the protein environment might lead to linear or nonlinear correlation. A linear plot involves an all-or-none transition between the two conformations, while a non-linear plot reflects the sequential character of structural transformations [22]. This method was used in the present study to analyze the unfolding/refolding mechanism of β-LG in complex with CSB, by identifying intermediate states upon heating. In Fig. 4 is presented the phase diagram of heat induced structural changes of β-LG-CSB complex, resulted by plotting the fluorescence intensity of the complex at 320 nm vs 365 nm for different temperature values. The linearity of the correlation suggests an all-or-none transition between the two structurally distinct conformations induced by

50

100°C

80 60

80°C

70°C 25°C

40

50°C

60°C

20 0 40

50

60

70

80

90

100

110

I320(a.u.) Fig. 4. Phase diagram analysis of heat-induced conformational changes of β-LG-CSB complex based on intrinsic fluorescence intensity values measured at wavelengths 320 and 365 nm. The temperature values are indicated in the vicinity of the corresponding symbol. Three independent tests were carried out in each case and SD was lower than 2.5%.

I. Aprodu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 173 (2017) 562–571

Fluorescence intensity (a.u.)

a) 140 120 100 80 60 40 20 0 260

25°C

270

50°C

280

290 300 Wavelength (nm) 60°C 70°C 80°C

310

90°C

320

100°C

Fluorescence intensity (nm)

b) 700 600 500 400 300

567

conformational variation of the new complex [14]. The 3D fluorescence spectra of β-LG-CSB complex at 25 °C and 90 °C are given in Fig. 6. The changes of three dimensional fluorescence spectra parameters of βLG-CSB complex are listed in Table 2. In addition, the contour map provides bird's eye view of 3D fluorescence spectra. Peak 1 denotes the Rayleigh scattering peak (λex = λem), the strong peak 2 mainly reveals the spectral characteristics of Trp and Tyr residues, peak 3 reflects the fluorescence characteristic of polypeptide backbone structures, whereas peak 4 is the second-order scattering peak (λem = 2 λex) (Fig. 6). The data given in Table 2 illustrates that the intensity of peak 1 changes more significantly than peak 2, especially at higher temperatures. The presented results indicated that the interaction between CSB and β-LG, followed by thermal treatment induced significant conformational changes. The increase in peak 1 intensity at temperatures higher than 70 °C reveals a decrease in polarity surrounding the Trp and Tyr residues, with increasing the exposure of some hydrophobic regions. The fluorescence intensity of peak 2 decreased with the temperature increase up to 70 °C, followed by an increase at even higher temperatures. The red-shifts in λmax at higher temperatures revealed that heat treatment induced the unfolding of the polypeptide chains. The shift in the emission is clearly visualized in the 3D measurements (Fig. 6). 3.8. Quenching Experiments

200 100 0 240

25°C

250

50°C

260

270 280 290 300 Wavelength (nm) 60°C 70°C 80°C 90°C

310

100°C

Fig. 5. Synchronous fluorescence spectra of β-LG-CSB complex at Δλ = 15 nm (a) and Δλ = 60 (b) nm after heat-treatment at different temperature. Three independent tests were carried out in each case and SD was lower than 2.5%.

spectrum had a maximum at 293 nm at 25 °C, whereas heating caused 2–3 nm blue-shifts at 90 °C–100 °C. In Fig. 5 b, a red-shift of 2 nm for Trp residues was observed. Therefore, it can be concluded that the heat treatment induced conformational changes that led to burial of Tyr residues and exposure of Trp residues. A detailed analysis of the βLG-BC models used for simulating the molecular behavior at thermal treatment indicated that the overall exposure of the Trp residues to the solvent decreases with the temperature increase from 25 °C to 90 °C. Due to the side chains rearrangements at high temperature the Trp [19] residue, which was reported to be completely buried in native state, became partially exposed (solvent accessible surface area of 1.23 Å2 at 90 °C), whereas the exposed surface of Trp61 residue decreased from 70.20 Å2 at 25 °C to 53.15 Å2 at 90 °C. Concerning the Tyr residues, an increase of the exposed surface by 27.11 Å2, 19.94 Å2 and 0.97 Å2 of Tyr20, Tyr102 and Tyr42 was observed at 90 °C with respect to the native protein. On the other hand, the solvent accessible surface area of the Tyr99 residue decreased from 64.70 Å2 at 25 °C to 28.84 Å2 at 90 °C. It is also indicated that the polarity around the Tyr residues was decreased and the hydrophobicity was increased, but the polarity around the Trp residues was increased and the hydrophobicity was decreased [21]. 3.7. Three-Dimensional Fluorescence Spectroscopy It has been suggested that 3D fluorescence spectra can provide more detailed information about the conformational changes of proteins [35]. The maximum emission wavelength and the fluorescence intensity of the residues have a close relation to the polarity of their micro-environment. Excitation and emission wavelengths along with fluorescence intensity are used on the axes, in order to obtain useful insights regarding the obtained complex at different temperatures. Moreover, the contour spectra can provide information to understand the structure and

Quenching studies exploiting the intrinsic Trp fluorescence of proteins can give information regarding the location of the fluorophore within macromolecules, thus providing useful data regarding the protein structure [18,54]. Changes in the emission spectra of Trp are usually employed to evaluate structural changes of proteins, as they affect the local environment that surrounds the indole ring, causing shifts in λmax and fluorescence intensity. In particular, the maximum fluorescence intensity can be quenched by the added molecules, as the excited state of the indole ring can donate electrons to the neighboring molecules [44]. Quenching experiments were performed for monitoring the temperature induced conformational changes of β-LG-CSB complex. The different mechanisms of quenching can be classified as dynamic quenching (collisional encounters) or static quenching (ground-state complex formation) between fluorophores and quenchers [17]. Acrylamide and KI are external quenchers (charged and non-charged) used to analyze the solvent accessibility and the polarity of the Trp residues microenvironment. Acrylamide quenches exposed and partially exposed Trp residues, while KI quenches only the fluorescence of exposed Trp, located at or near to the surface of the molecules. The Stern Volmer constants (KSV) of β-LG-CSB complex with acrylamide and KI at different temperature values are shown in Table 3. Acrylamide quenching yielded a linear Stern-Volmer plot (data not shown), which implies that the fluorescence quenching takes place on a simple collisional basis [10]. For quenching experiments with acrylamide, the KSV values showed a sharp increase in the temperature range of 25 to 70 °C, followed by a significant increase at higher temperature (Table 3), which can be attributed to the fact that Trp residues become more accessible to the acrylamide. Therefore, the maximum KSV value (5.88 ± 0.007 · 10−3 mol−1 L) was obtained at 100 °C and minimum (2.29 ± 0.25 · 10−3 mol−1 L) at 60 °C, suggesting that Trp residues had the highest exposure at 100 °C, being least accessible to quencher at 60 °C. More pronounced slopes of the Stern-Volmer plot indicate more flexible protein structure and/or higher concentration of unfolded molecules [9]. These authors suggested that the marked increase in KSV values at higher temperatures can be ascribed to the presence of non-native monomers and aggregates of low molecular weight of denatured molecules, irreversibly formed during the heat treatment. Chadborn et al. [10] explained that quenching by KI may be more complicated, as may yield a nonlinear quenching relation, which is inconsistent with the simple collision based Stern-Volmer equation. The iodide may quench in a collisional fashion the fluorescence of Trp

568

I. Aprodu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 173 (2017) 562–571

Fig. 6. The EEMS spectra of β-LG-CSB complex at 25 °C (a) and 90 °C (b). Three independent tests were carried out in each case and SD was lower than 3.5%.

residue upon binding, by fast moving within the protein. In our study, a linear Stern-Volmer plot was obtained (data not shown). The lowest KSV value was calculated at 25 °C (2.27 ± 0.26 · 10− 3 mol− 1 L) and the highest at 50 °C (4.18 ± 0.44 · 10−3 mol−1 L) (Table 3). It can be concluded that the accessibility of Trp residues to the quencher was significantly modified by heating up to 50 °C. Thus, the irreversible lowtemperature transition affects the domain where Trp residues are located in β-LG structure, or the relative position of the CSB with respect to the protein. Concerning the thermal stability of the microenvironment of Trp residues within β-LG molecule, Fessas et al. [16] suggested that, in a lower temperature range, the most stable domain is that containing the buried Trp [19]. Brownlow et al. [7] described this region as laying on the bottom of the putative substrate-binding cavity, and is made up of a cluster of highly hydrophobic residues (including Tyr99, Tyr102, and Val [15]), located on turn regions of the molecule. Fessas et al. [16] suggested that this hydrophobic region is unaffected by dimer dissociation induced by physical treatments, such as thermal treatment.

3.9. In Silico Investigation on the Interaction Between β-LG and BC at Single Molecule Level The atomic events responsible for the thermal dependent behavior of the complex formed by β-LG and BC, which is the most representative compound of the carotenoids extract, were checked after performing molecular dynamics simulations at 25 and 90 °C, temperature indicated as relevant by the fluorescence spectroscopy measurements. A detailed check of the molecular models showed that only 77% of the secondary structure of the native β-LG was conserved at 90 °C. Heating the β-LGBC complex led to changes in the hydrogen bonding pattern, and consequently in the involvement of the amino acids residues in defining different types of secondary structure motifs. The protein treatment at high temperature caused the increase of the amino acids organized in strands (from 37.3% at 25 °C to 44.3% at 90 °C), the most important changes being noticed in the beta and gamma turs motifs. With respect to the initial molecular model, the thermal treatment up to 90 °C

Table 2 Characteristic parameters of the three-dimensional fluorescence spectra of β-LG-CSB complex. Temperature, °C

Peak 2 Peak position (λex/nm, λem/nm)

Stokes shift Δλ (nm)

Fluorescence intensity (a.u.)

Peak 1 Peak position (λex/nm, λem/nm)

Stokes shift Δλ (nm)

Fluorescence intensity (a.u.)

25 50 60 70 80 90 100

230/335 230/335 230/335 230/332 230/340 230/341 230/341

105 105 105 102 110 111 111

456.9 335.95 201.23 233.88 348.12 535.90 694.14

280/335 280/337 280/336 280/335 280/340 280/341 280/347

55 57 56 55 60 61 67

282.96 295.07 214.97 312.42 477.70 596.26 581.72

I. Aprodu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 173 (2017) 562–571 Table 3 The Stern Volmer quenching constant (KSV) of the heat treated β-LG-CSB complex at different temperatures. Temperature, °C

KSV (10−3 L·mol−1) Acrylamide

25 50 60 70 80 90 100 a

2.48 2.68 2.29 2.74 3.77 5.52 5.88

± ± ± ± ± ± ±

KI a

0.25 0.17 0.25 0.10 0.08 0.06 0.007

2.27 4.18 3.58 2.99 2.32 1.48 1.44

± ± ± ± ± ± ±

0.26 0.44 0.55 0.26 0.21 0.36 0.37

Standard deviation.

favored molecular rearrangements that caused the formation of new non-interacting native-like α-helical motifs, altering the conformation of the 3–10 helices. As a consequence of protein unfolding at high temperatures, an increase of the total solvent accessible protein surface from 7239.8 to 7976.1 Å2 was observed. The rearrangements of the amino acids side chains and the exposure of new residues to the surface coupled with the partial burial of the initially exposed ones, induced significant changes in the interaction between β-LG and BC molecules. At 25 °C the BC molecule directly interfaces the following amino acids of β-LG: Ile [2], Val [3], Thr4, Thr6, Lys [8], Ile78 and Glu89 (Fig. 7, a); weak contacts are also established with Gln [6], Ala80, Val81, Lys91, Leu93, Ser110 and Gln115. The most important hydrophobic effect contribution to the total solvation energy gain of the interface corresponded to Lys [8] (0.81 kcal/mol), Ile78 (0.39 kcal/mol), and Lys91 (0.50 kcal/mol). When

569

heating the protein up to 90 °C, slight changes in the BC binding site was observed and the total surface of the protein, which gets buried upon formation of the assembly with BC, increased from 304.7 to 386.1 Å2. When comparing the models equilibrated at 25 °C and 90 °C, a significant increase with the temperature in the solvent exposure of the Ala80 and Val81 residues (from 17.71 Å2 at 25 °C to 79.30 Å2 at 90 °C) was observed (Fig. 7, b), as well as an increase of their hydrophobic interactions with the BC molecule. Due to molecular rearrangements, the Lys [8] residue gets buried within protein core and no interaction with the ligand could be observed. On the other hand, although an additional exposure to the solvent molecules was observed in case of the Glu89 and Lys91, due to the side chain reorientation no involvement in the interaction with BC was obtained at 90 °C. The migration toward protein surface of Leu95 residue, which was completely buried in the native protein, favored the extensive interaction over the entire exposed surface of 28.98Å2 with one of the two cyclohexene rings of BC molecule (Fig. 7, b). The change of the ligand binding surface induced burial of the Ala142, Leu143 and Pro144 residues when in complex with BC molecule, therefore limiting the potential interaction with other β-LG molecules [1]. In agreement with the observation of Loch et al. [32], the formation of β-LG-BC assembly is the result of complex enthalpy and entropy driven events consisting on important conformational changes of the molecules when binding. The small decrease of rigid-body entropy change (TΔSdiss) at β-LG-BC complex dissociation (from 6 kcal/mol at 25 °C to 5.4 kcal/mol at 90 °C) indicated that the stability of the assembly is not significantly affected by the temperature increase. On the other hand, the increase of the free energy of assembly dissociation (ΔGdiss) from − 3.6 to − 2.5 kcal/mol indicates that the β-LG-BC complex is more thermodynamically stable, and the external driving forces needed to dissociate the complex are higher at 90 °C. Moreover, the solvation

Fig. 7. Contact mapping for the β-LG-BC complex equilibrated at 25 °C (a) and 90 °C (b). The amino acids from the β-LG binding surface which are involved in hydrophobic contacts with BC molecule (named Bcr163) are represented by an arc with spokes radiating toward the ligand atoms they contact. The figures were drawn using LigPlot+.

570

I. Aprodu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 173 (2017) 562–571

energy of β-LG folding (ΔGf) decreased from −127 to −154.4 kcal/mol, indicating that the structure of the thermally treated protein within complex is more stable. 4. Conclusions Detailed information regarding thermal stability of β-LG-CSB complex based on fluorescence spectroscopy investigation and molecular modeling were reported in this study. Our fluorescence spectroscopy results suggested partial unfolding of the protein within complex, especially at temperatures higher than 80 °C. In the whole temperature range studied, the λmax was red shifted, suggesting an increase of hydrophilicity in the vicinity of hydrophobic residues. Based on the linearity of the phase diagram, an all or none transition process was suggested, involving the dissociation of the dimers in the temperatures range of 25 °C to 70 °C, followed by the unfolding at higher temperature. Binding of carotenoids and thermal treatment led to a decrease in the polarity around the Tyr residues and an increase in the hydrophobicity, whereas the polarity around the Trp residues was increased and the hydrophobicity was decreased. The higher flexibility of the protein molecule in the complex was evidenced by the Stern-Volmer quenching constants, which had significantly higher values at higher temperature for quenching experiments with acrylamide, and significantly lower for quenching experiments with KI. Finally, the results of the molecular dynamics simulations indicated that, regardless of the temperature, the complex between the β-LG and BC molecules is stabilized by hydrophobic contacts. The atomic level events taking place in the protein model heated at high temperature caused important changes in the BC binding site, therefore leading to a more thermodynamically stable assembly. Data obtained from this study is highly applicable in the food and pharmaceutical industries from the perspective of obtaining new nutraceuticals and/or functional products. Acknowledgements This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS-UEFISCDI, project number PN-II-RU-TE-2014-4-0115. References [1] J.J. Adams, B.F. Anderson, G.E. Norris, L.K. Creamer, G.B. Jameson, Structure of bovine β-lactoglobulin (variant A) at very low ionic strength, J. Struct. Biol. 154 (2006) 246–254. [2] J.R. Albani, J. Vogelaer, L. Bretesche, D. Kmiecik, Tryptophan 19 residue is the origin of bovine β-lactoglobulin fluorescence, J. Pharm. Biomed. Anal. 91 (2014) 144–150. [3] S.C. Anderson, M.E. Olsson, E. Johansson, K. Rumpunen, Carotenoids in sea buckthorn (Hippophae rhamnoides L.) berries during ripening and use of pheophytin a as a maturity marker, J. Agric. Food Chem. 57 (2009) 250–258. [4] N. Andrusier, R. Nussinov, H.J. Wolfson, FireDock: fast interaction refinement in molecular docking, Proteins: Structure, Function, and Bioinformatics 69 (2007) 139–159. [5] P. Bhosale, P.S. Bernstein, Vertebrate and invertebrate carotenoid-binding proteins, Arch. Biochem. Biophys. 458 (2007) 121–127. [6] A. Borkar, M.K. Rout, V. Hosur, Denaturation of HIV-1 protease (PR) monomer by acetic acid: mechanistic and trajectory insights from molecular dynamics simulations and NMR, J. Biomol. Struct. Dyn. 29 (2012) 893–903. [7] S. Brownlow, Morais, J.H. Cabral, R. Cooper, D.R. Flower, S.J. Yewdall, I. Polikarpov, A.C.T. North, L. Sawyer, Bovine β-lactoglobulin at 1.8 A° resolution – still an enigmatic lipocalin, Structure 5 (1997) 481–495. [8] T.V. Burova, Y. Choiset, V. Tran, T. Haertle, Role of free Cys121 in stabilization of bovine beta-lactoglobulin B, Protein Eng. 11 (1998) 1065–1073. [9] P. Busti, C.A. Gatti, N.J. Delorenzi, Thermal unfolding of bovine β-lactoglobulin studied by UV spectroscopy and fluorescence quenching, Food Res. Int. 38 (2005) 543–550. [10] N. Chadborn, J. Bryant, A.J. Bain, P. O'Shea, Ligand-dependent conformational equilibria of serum albumin revealed by tryptophan fluorescence quenching, Biophys. J. 76 (1999) 2198–2207. [11] M. Collini, L. D'Alfonso, G. Baldini, New insight on β-lactoglobulin binding sites by 1anilinonaphtalene-8-sulfonate fluorescence decay, Protein Sci. 9 (2000) 1968–1974. [12] A. Divsalar, A.A. Saboury, A.A. Moosavi-Movahedi, Conformational and structural analysis of bovine β-lactoglobulin-A upon interaction with Cr+3, Protein J. 25 (2006) 157–165.

[13] L. Dumitraşcu, N. Stănciuc, E. Bahrim, I. Aprodu, Insights into the binding of ferulic acid to the thermally treated xanthine oxidase, Luminescence 31 (2016) 1259–1266. [14] L. Dumitraşcu, N. Stănciuc, A. Ciumac, G. Bahrim, I. Aprodu, pH and heat-dependent behaviour of glucose oxidase down to single molecule level by combined fluorescence spectroscopy and molecular modeling, J. Sci. Food Agric. 52 (2015) 8095–8103. [15] J. Fan, X. Ding, W. Gu, Radical-scavenging proanthocyanidins from sea buckthorn seed, Food Chem. 102 (2007) 168–177. [16] D. Fessas, S. Iametti, A. Schiraldi, F. Bonomi, Thermal unfolding of monomeric and dimeric β-lactoglobulins, Eur. J. Biochem. 268 (2001) 5439–5448. [17] A.B.T. Ghisaidoobe, S.J. Chung, Intrinsic tryptophan fluorescence in the detection and analysis of proteins: a focus on Förster resonance energy transfer techniques, Int. J. Mol. Sci. 15 (2014) 22518–22538. [18] X. Guo, X. Li, Y. Jiang, Q. Wu, H. Chang, X. Diao, Y. Sun, X. Pan, N. Zhou, A spectroscopic study on the interaction between p-nitrophenol and bovine serum albumin, J. Lumin. 149 (2014) 353–360. [19] B.J. Harvey, E. Bell, L. Brancaleon, A tryptophan rotamer located in a polar environment probes pH-dependent conformational changes in bovine β-lactoglobulin a, J. Phys. Chem. B 111 (2007) 2610–2620. [20] B. Hess, C. Kutzner, D. Van Der Spoel, E. Lindahl, GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation, J. Chem. Theory Comput. 4 (2008) 435–447. [21] Z.J. Hu, Y. Liu, W. Jiang, R.M. Zhao, S.S. Qu, Fluorometric investigation of the interaction of bovine serum albumin with surfactants and 6-mercaptopurine, J. Photochem. Photobiol. B Biol. 80 (2005) 235–242. [22] I.A. Kataeva, V.N. Uversky, J.M. Brewer, F. Schubot, J.P. Rose, B.C. Wang, L.G. Ljungdahl, Interactions between immunoglobulin-like and catalytic modules in Clostridium thermocellum cellulosomal cellobiohydrolase CbhA, Protein Eng. Des. Sel. 17 (2004) 759–769. [23] G. Korekar, P. Dolkar, H. Singh, R.B. Srivastava, T. Stobdan, Variability and the genotypic effect on antioxidant activity, total phenolics, carotenoids and ascorbic acid content in seventeen natural population of sea buckthorn (Hippophae rhamnoides L.) from trans-Himalaya, LWT Food Sci. Technol. 55 (2014) 157–162. [24] E. Krissinel, Crystal contacts as nature's docking solutions, J. Comput. Chem. 31 (2010) 133–143. [25] I. Kuznetsova, K. Turoverov, V. Uversky, Use of the phase diagram method to analyze the protein unfolding-refolding reactions: fishing out the “invisible” intermediates, J. Proteome Res. 3 (2004) 485–494. [26] R.A. Laskowski, PDBsum new things, Nucleic Acids Res. 37 (2009) D355–D359. [27] R.A. Laskowski, M.B. Swindells, LigPlot+: multiple ligand–protein interaction diagrams for drug discovery, J. Chem. Inf. Model. 10 (2011) 2778–2786. [28] M. Li, Y. Maa, M.O. Ngadi, Binding of curcumin to β-lactoglobulin and its effect on antioxidant characteristics of curcumin, Food Chem. 141 (2013) 1504–1511. [29] L. Liang, M. Subirade, β-Lactoglobulin/folic acid complexes: formation, characterization, and biological implication, J. Phys. Chem. B 114 (2010) 6707–6712. [30] L. Liang, M. Subirade, Study of the acid and thermal stability of β-lactoglobulinligand complexes using fluorescence quenching, Food Chem. 132 (2012) 2023–2029. [31] L. Liang, H. Tajmir-Riahi, M. Subirade, Interaction of β-lactoglobulin with resveratrol and its biological implications, Biomacromolecules 9 (2007) 50–56. [32] J.I. Loch, P. Bonarek, A. Polit, D. Riès, M. Dziedzicka-Wasylewska, K. Lewiński, Binding of 18-carbon unsaturated fatty acids to bovine β-lactoglobulin-structural and thermodynamic studies, Int. J. Biol. Macromol. 57 (2013) 226–231. [33] D.J. McClements, L. Zou, R. Zhang, L.S. Trujillo, T. Kumosani, H. Xiao, Enhancing nutraceutical performance using excipient foods: designing food structures and compositions to increase bioavailability, Compr. Rev. Food Sci. Food Saf. 14 (2015) 824–847. [34] A. Mensi, Y. Choiset, H. Rabesona, T. Haertlé, P. Borel, J.-M. Chobert, Interactions of βlactoglobulin variants A and B with vitamin A. Competitive binding of retinoids and carotenoids, J. Agric. Food Chem. 61 (2013) 4114–4119. [35] M.D. Meti, S.T. Nandibewoor, S.D. Joshi, U.A. More, S.A. Chimatadar, Multi-spectroscopic investigation of the binding interaction of fosfomycin with bovine serum albumin, Journal of Pharmaceutical Analysis 5 (2015) 249–255. [36] G. Mocz, J.A. Ross, Fluorescence techniques in analysis of protein–ligand interactions, in: M.A. Williams, T. Daviter (Eds.), Protein-Ligand Interactions, Methods and Applications, Second EditionHumana Press, Springer, New York Heidelberg Dordrecht London, 2015. [37] F. Morgan, D. Molle, G. Henry, A. Venien, J. Leonil, G. Peltre, S. Bouhallab, Glycation of bovine beta-lactoglobulin: effect on the protein structure, Int. J. Food Sci. Technol. 34 (1999) 429–435. [38] L. Müller, C. Caris-veyrat, G. Lowe, V. Böhm, Lycopene and its antioxidant role in the prevention of cardiovascular diseases — a critical review, Crit. Rev. Food Sci. Nutr. (2015), http://dx.doi.org/10.1080/10408398.2013.801827. [39] S. Muresan, A. van der Bent, F.A. de Wolf, Interaction of β-lactoglobulin with small hydrophobic ligands as monitored by fluorometry and equilibrium dialysis: nonlinear quenching effects related to protein-protein association, J. Agric. Food Chem. 49 (2001) 2609–2618. [40] R.K. Saini, S.H. Nile, S.W. Park, Carotenoids from fruits and vegetables: chemistry, analysis, occurrence, bioavailability and biological activities, Food Res. Int. 76 (2015) 735–750. [41] G. Palazolo, F. Rodriguez, B. Farruggia, G. Picó, N. Delorenzi, Heat treatment of β-lactoglobulin: structural changes studied by partitioning and fluorescence, J. Agric. Food Chem. 48 (2000) 3817–3822. [42] H. Pfander, P. Lester, Carotenoids, Methods in Enzymology, Academic Press 1992, pp. 3–13.

I. Aprodu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 173 (2017) 562–571 [43] R.M. Pop, Y. Weesepoel, C. Socaciu, A. Pintea, J.P. Vincken, H. Gruppen, Carotenoid composition of berries and leaves from six Romanian sea buckthorn (Hippophae rhamnoides L.) varieties, Food Chem. 147 (2014) 1–9. [44] S. Rahimi Yazdi, M. Corredig, Heating of milk alters the binding of curcumin to casein micelles. A fluorescence spectroscopy study, Food Chem. 132 (2012) 1143–1149. [45] D. Renard, J. Lefebvre, M.C. Griffin, W.G. Griffin, Effects of pH and salt environment on the association of β-lactoglobulin revealed by intrinsic fluorescence studies, Int. J. Biol. Macromol. 22 (1998) 41–49. [46] R.M. Rodrigues, A.J. Martins, O.L. Ramos, F.X. Malcata, J.A. Teixeira, A.A. Vicente, et al., Influence of moderate electric fields on gelation of whey protein isolate, Food Hydrocoll. 43 (2015) 329–339. [47] D. Schneidman-Duhovny, Y. Inbar, R. Nussinov, H.J. Wolfson, PatchDock and SymmDock: servers for rigid and symmetric docking, Nucleic Acids Res. 33 (2005) W363–W367. [48] A.M. Simion-Ciuciu, I. Aprodu, L. Dumitrașcu, G.E. Bahrim, P. Alexe, N. Stănciuc, Probing thermal stability of the β-lactoglobulin-oleic acid complex by fluorescence spectroscopy and molecular modeling, J. Mol. Struct. 1095 (2015) 26–33.

571

[49] N. Stănciuc, I. Aprodu, G. Râpeanu, G. Bahrim, Fluorescence spectroscopy and molecular modeling investigations on the thermally induced structural changes of bovine β-lactoglobulin, Innovative Food Sci. Emerg. Technol. 15 (2012) 50–56. [50] A. Thompson, M. Boland, H. Singh, Milk Proteins — From Expression to Food, Elsevier, 2009 165–166. [51] V. Vetri, V. Militello, Thermal induced conformational changes involved in the aggregation pathways of β-lactoglobulin, Biophys. Chem. 113 (2005) 83–91. [52] J. Von Lintig, K. Vogt, Filling the gap in vitamin a research molecular identification of an enzyme cleaving β-carotene to retinal, J. Biol. Chem. 275 (2000) 11915–11920. [53] S.C. Wilde, C. Treitz, J.K. Keppler, T. Koudelka, K. Palani, A. Tholey, H.M. Rawel, K. Schwarz, β-Lactoglobulin as nanotransporter – part II: characterization of the covalent protein modification by allicin and diallyl disulfide, Food Chem. 197 (2016) 1022–1029. [54] C. Xu, J. Gu, X. Ma, T. Dong, X. Meng, Investigation on the interaction of pyrene with bovine serum albumin using spectroscopic methods, Spectrochim. Acta A Mol. Biomol. Spectrosc. 125 (2014) 391–395.