Interaction of β-Lactoglobulin with Resveratrol and its Biological

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Interaction of β-Lactoglobulin with Resveratrol and its Biological Implications Li Liang,† H. A. Tajmir-Riahi,‡ and Muriel Subirade*,† Chaire de recherche du Canada sur les protéines, les bio-systèmes et les aliments fonctionnels, Institut de recherche sur les nutraceutiques et les aliments fonctionnels (INAF/STELA), Université Laval, Sainte-Foy, Canada, and Département de Chimie-biologie, Université du Québec à Trois-Rivières, C. P. 500, Trois-Rivières (Québec), G9A 5H7, Canada Received June 30, 2007; Revised Manuscript Received September 28, 2007

β-Lactoglobulin (β-LG), the major whey protein in the milk of ruminants, has a high affinity for a wide range of compounds. Resveratrol (3,5,4′-trihydroxystilbene), a natural polyphenolic compound found in grapes and red wine, exhibits many physiological effects associated with health benefits. In this study, the interaction of resveratrol with β-LG was investigated using circular dichroism, fluorescence and UV–vis absorbance. Self-association of resveratrol possibly occurs at high concentrations. Resveratrol interacts with β-LG to form 1:1 complexes. Resveratrol is bound to the surface of the protein because β-LG-bound polyphenol is in a weaker hydrophobic environment relative to 75% ethanol. The binding constant for the resveratrol-β-LG interaction is between 104 and 106 M-1, as determined by protein or polyphenol fluorescence. The β-LG-resveratrol interaction may compete with self-association of both the polyphenol and the protein. It has no apparent influence on β-LG secondary structure but partially disrupts tertiary structure. Complexing with β-LG provides a slight increase in the photostability of resveratrol and a significant increase in its hydrosolubility.

Introduction Resveratrol (3,5,4′-trihydroxystilbene, Figure 1) is a natural polyphenolic compound produced in plants (e.g., grapes, peanuts, mulberries) in response to injury and fungal attack. Resveratrol can also be found in food products and beverages such as peanut butter, red wine, and grape juice.1,2 In 2006, it was found for the first time in dark chocolate and cocoa liquor.3 This compound has aroused widespread interest due to biological effects associated with health benefits. Due to its polyphenolic structure, resveratrol possesses antioxidant activity and may decrease reactive oxygen species generation in blood platelets, oxidant-induced apoptosis, and low-density lipoprotein oxidation.4,5 Resveratrol can inhibit platelet aggregation induced by thrombin, collagen, and adenosine diphosphate6 and reduce inflammation via inhibition of prostaglandin production, cyclooxygenase-2 activity, and nuclear factor-B activity.5 The antiproliferative activity of resveratrol is associated with the inhibition of D-type cyclins and cyclin-dependent kinase (Cdk) 4 expression as well as the induction of tumor suppressor p53 and Cdk inhibitor p21.7 These biological effects may account for its cardioprotective benefit and chemopreventive activity against the initiation, promotion, and progression stages of carcinogenesis.8–10 Resveratrol exists as trans- and cis-isomers. Most of its biological activities are attributed to the trans-isomer. Although a few biological activities of cis-resveratrol have been reported, such as inhibition of collagen-induced platelet aggregation and kinase activity related to cancer,11,12 it is not clear whether cisresveratrol might extensively exhibit biological activities comparable to those of the trans-isomer. In solution, trans-resveratrol converts to its cis-isomers under exposure to light.13–15 The * To whom correspondence should be addressed. Tel.: +1 418 656 2131 × 4278. Fax: +1 418 656 3353. E-mail: [email protected]. † Université Laval. ‡ Université du Québec à Trois-Rivières.

Figure 1. Structure of trans-resveratrol.

stability of trans-resverarol, which ranges from hours to several days, depends on the pH of solution.13 It may form complexes with natural and modified cyclodextrins, increasing both its stability and water solubility.14 The biological activities of resveratrol depend on its absorption, bioavailability, and metabolism.5 Drugs that are poorly soluble in water often exhibit low bioavailability because their absorption may be kinetically limited by low rates of dissolution and capacity-limited by poor solubility.16 Due to the low water solubility of resveratrol, it must be bound to protein or conjugated to remain at a high concentration in serum.17 Previous studies have showed that resveratrol may interact with proteins such as breast cancer resistance protein, human cyclindependent kinase, lipoproteins, and serum albumin.17–20 β-Lactoglobulin (β-LG), the most abundant protein of whey, is of major interest in the food industry because of its nutritional and functional properties. The structure of this protein is wellknown. At neutral pH, β-LG exists as a mixture of monomers and dimers,21 of which the equilibrium ratio depends on the association constant of the dimer and on the protein concentration. Each monomer consists of 162 amino acid residues and has a molecular mass of 18 kDa. As a member of the lipocalycin family, β-LG is a small globubar protein folded into a calyx formed by eight antiparallel β-strands and an R-helix located at the outer surface of the β-barrel.22,23 β-LG exhibits an affinity for a variety of hydrophobic and amphiphilic compounds, including retinol,24 fatty acids,25 phospholipids,26 and aromatic compounds.27 Binding to β-LG pro-

10.1021/bm700728k CCC: $40.75  2008 American Chemical Society Published on Web 12/08/2007

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vides protection to retinol and β-carotene from degradation due to heat, oxidation and irradiation.28 It has therefore been proposed that β-LG could be used as a versatile carrier of hydrophobic molecules in controlled delivery applications.29 The binding constants for the different compounds with β-LG vary widely, from as little as 1.5 × 102 M-1 for 2-heptanone to 6.8 × 105 M-1 for palmitate and 5 × 107 M-1 for retinol.30 Three potential binding sites have been reported for ligand binding to β-LG: the internal cavity of the β-barrel, the surface hydrophobic pocket in a groove between the R-helix and the β-barrel, and the outer surface near Trp19-Arg124.31 Polar aromatic compounds, such as p-nitrophenyl phosphate, 5-fluorocytosine, ellipticine, and protoporphyrin, bind to this outer surface site.32,33 On the other hand, a dynamic fluorescence study on the interaction of β-LG with 1-anilinonaphthalene-8-sulfonate has indicated two binding sites including the internal and external binding sites.27,34 Various interactions, such as hydrophobic interaction, hydrogen bonding, and electrostatic interaction may occur between β-LG and ligands.24,27 These interactions are also the driving force for the structural transition of proteins.35 Hydrophobic and amphiphilic ligands could therefore affect the native structure of β-LG. Cationic surfactants such as dodecyltrimethylammonium chloride and didodecyldimethylammonium bromide and anionic phospholipids such as dimyristoylphosphatidylglycerol have been reported to loosen the tertiary structure and bring about the transition from β-sheet to R-helix in the secondary structure of β-LG.36,37 In the present study, the interaction between β-LG and resveratrol was investigated using circular dichroism, fluorescence, and UV–vis absorbance to characterize the potential of β-LG as carrier of resveratrol. The binding constant and binding site of resveratrol on β-LG were determined, and the effects of the resveratrol-protein interaction on β-LG structure and on resveratrol stability and solubility were discussed.

Experimental Section Materials. β-LG (B variant, purity g 90%) and resveratrol (trans-isomer, purity > 99%) were purchased from the SigmaAldrich Chemical Co. and used without further purification. Sample Preparation. β-LG stock solution was made by dissolving in 10 mM phosphate buffer at pH 7.4 to obtain concentrations of 100 µM and 120 µM, measured by spectrophotometer using a molar extinction coefficient of 17600 M-1 cm-1 at 278 nm.27 Resveratrol stock solution was prepared daily by dissolving at a concentration of 1.2 mM in 75% ethanol and then diluting with 10 mM phosphate buffer at pH 7.4 to a concentration of 120 µM. Samples were prepared by mixing β-LG and resveratrol stock solutions in varying proportions. The highest resulting ethanol concentration was about 7%, which had no appreciable effect on protein structure. The samples were incubated for about two hours at room temperature prior to analysis. Circular Dichroism (CD) Measurement. CD spectra of β-LG were recorded at resveratrol concentrations of 0, 5, 10, 20, 40, and 80 µM on a Jasco J-710 spectropolarimeter. The concentrations of β-LG were 10 µM for far-UV region (190–250 nm) and 20 µM for near-UV region (250–320 nm). Path lengths were 0.1 cm for far-UV region and 1 cm for near-UV region. Ellipticity was recorded at a speed of 100 nm/min, 0.2 nm resolution, 11 accumulations, 1.0 nm bandwidth. Buffer background was subtracted from the raw spectra.

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Figure 2. Fluorescence emission spectra of resveratrol at (a-g): 2.5, 5, 10, 20, 40, 80, and 108 µM in 10 mM phosphate buffer at pH 7.4. Inset: Evolution of fluorescence intensity as a function of resveratrol concentration.

Steady-State Fluorescence Measurement. Protein intrinsic fluorescence was measured at constant β-LG concentration (10 µM) and resveratrol concentrations of 0, 2.5, 5, 10, 20, 40, 80, and 108 µM using a Cary Eclipse Fluorescence spectrophotometer (Varian Inc.). Emission spectra were recorded from 300 to 550 nm with an excitation wavelength of 295 nm and from 290 to 500 nm with an excitation wavelength of 280 nm. The fluorescence of resveratrol was measured at each of its concentrations and at 10 µM or 40 µM in the presence of 0, 2.5, 5, 7.5, 10, 20, 40, and 80 µM β-LG. These spectra were recorded from 330 to 600 nm with an excitation wavelength of 320 nm. Spectral resolution was 5 nm for both excitation and emission. UV–Vis Absorbance Measurement. Absorbance spectra were recorded on a HP 8453 UV–visible spectrophotometer (Palo alto, CA). The path length was 1 cm. To study the stability of resveratrol exposed to light, samples prepared within 30 min were stored under tubular lamps that emit at all visible wavelengths. Absorbance spectra from 240 to 400 nm were measured at specified time intervals for 40 µM resveratrol, 80 µM β-LG, and the mixture of the two. The β-LG spectra were subtracted from the spectra of the resveratrol-βLG mixtures. Resveratrol solubility was studied using resveratrol solution in 75% ethanol diluted 20-fold in 10 mM phosphate buffer at pH 7.4, with or without β-LG, at a final concentration of 80 µM. The initial resveratrol concentrations in 75% ethanol were 6, 12, 24, 36, and 48 mM. Diluted solutions were shielded from ambient light for two hours then centrifuged at 9600 RPM for 12 min (Biofuge 22R, Heraeus Instruments) and 150 µL of supernatant were diluted in 1050 µL of 10 mM phosphate buffer at pH 7.4 for UV–vis absorbance spectrum measurement.

Results and Discussion Structure of Resveratrol in Aqueous Solution. The influence of resveratrol concentration on its fluorescence emission spectra is shown in Figure 2. The emission maxima (λmax) are around 404 nm, which is consistent with the value previously reported.19 Fluorescence intensity increases as concentration increases up to 40 µM, beyond which fluorescence intensity begins to decrease, indicating that self-quenching of resveratrol fluorescence occurs at higher concentrations. Previous studies have shown that polyphenols tend toward self-association in solution as a result of hydrophobic stacking of aromatic phenolic

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Figure 3. (A) Far-UV and (B) near-UV circular dichroic spectra of β-LG in the presence of various resveratrol concentrations in 10 mM phosphate buffer at pH 7.4. The concentrations of β-LG are 10 µM for the far-UV region and 20 µM for the near-UV region, respectively.

38,39

rings. Resveratrol possibly causes self-association at a higher concentration than 40 µM, which results in polyphenol fluorescence self-quenching.40 Influence of Resveratrol-β-LG Interaction on the Structure of β-LG. Circular dichroic spectroscopy is a valuable technique for studying structural transitions of proteins in solution because it can reveal very small alterations in protein structure.41 Far-UV and near-UV CD spectra can be used to characterize, respectively, the secondary structure and side-chain environments of proteins. Figure 3 shows far-UV and near-UV CD spectra of β-LG in the absence and presence of resveratrol at the different concentrations. The CD spectra of native β-LG are consistent with those previously reported.37,42 The far-UV CD spectrum of β-LG shows a typical β-sheet structure with a broad negative minimum around 216 nm (Figure 3A). After protein complexing with resveratrol, far-UV CD spectra remains similar to that of native β-LG, suggesting that the interaction of resveratrol with β-LG has no significant effect on protein secondary structure at resveratrol concentrations lower than 80 µM. The near-UV CD spectrum of native β-LG features the two negative minima at 285 and 292 nm, attributable to Trp residues at positions 19 and 61. Trp61 is partly exposed to the aqueous solvent and makes only a minor contribution to the CD Trp signal, while Trp19 is in an apolar environment within the cavity of β-LG, making it easily detectable in the spectrum.43 The two negative bands at 285 and 292 nm gradually decrease with increasing of resveratrol concentration, suggesting that the interaction of resveratrol with β-LG partially disrupts the anisotropic environment of Trp residues.44

Figure 4. Fluorescence emission spectra of 10 µM (A) and 40 µM (B) resveratrol in the presence of β-LG. (a) Resveratrol alone in 10 mM phosphate buffer at pH 7.4; (b-h) resveratrol with β-LG at 2.5, 5, 7.5, 10, 20, 40, and 80 µM; (i) resveratrol alone in 75% ethanol. Inset: Double reciprocal linear plot of 1/∆F as a function of 1/[β-LG] according to eq 3.

Binding Constant and Site of Resveratrol Binding to βLG. Fluorescence is a useful approach to investigating intermolecular interactions because the photophysical character of the fluorophore is sensitive to the polarity of its surrounding environment. Generally, λmax shifts to a shorter wavelength and fluorescence intensity increases as this polarity decreases. As shown in Figure 4, λmax of pure resveratrol is around 404 nm in phosphate buffer and shifts to 383 nm in 75% ethanol with a conspicuous increase in fluorescence emission intensity. When interacting with β-LG, the fluorescence emission spectra of 10 µM and 40 µM revesratrol show similar changes. The gradual blue shift of λmax and increase in resveratrol fluorescence intensity are observed as β-LG concentration increases, suggesting that resveratrol transfers from the hydrophilic environment of the aqueous solution to a more hydrophobic environment. However, even at the highest β-LG concentration (80 µM), λmax shifts only to 394 nm and the increase in emission intensity is less than that brought about in 75% ethanol, suggesting that the β-LG environment of the bound polyphenol is not very hydrophobic, in other words, not the internal cavity. We therefore suggest that the binding site of resveratrol is at the surface of β-LG. Intrinsic fluorescence of protein has been widely used to investigate the structural transition and binding properties of

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Figure 5. Fluorescence emission spectra of β-LG at excitation wavelengths of 295 nm (A) and 280 nm (B) in 10 mM phosphate buffer at pH 7.4. (a) 10 µM β-LG alone; (b-h) 10 µM β-LG with 2.5, 5, 10, 20, 40, 80, and 108 µM resveratrol; (i) 108 µM resveratrol alone. Insets: Plot of F0/F versus [resveratrol] as per the Stern-Volmer equation (top); log[(F0 - F)/F] vs log[resveratrol] as per eq 2 (bottom).

proteins in solution.19,45 Figure 5 shows the intrinsic fluorescence emission spectra of β-LG in the presence of different concentrations of resveratrol. β-LG contains two Trp residues and four Tyr residues per monomer.46 When the excitation wavelength is 295 nm, only Trp produces a fluorescent emission. The fluorescence of the partly exposed Trp61 is probably quenched by the proximity of the Cys66-Cys160 disulfide bond, while Trp19 in its apolar environment contributes about 80% of the total fluorescence.47 In its native state, β-LG has a λmax of 338 nm (Figure 5A). Interaction with resveratrol gradually shifts this to a longer wavelength and decreases Trp fluorescence intensity as the concentration of the polyphenol increases. At 20 µM resveratrol, λmax is at 352 nm and the Trp fluorescence intensity is only 53% that of pure β-LG. These results indicate that the interaction of resveratrol with β-LG makes the environment of Trp residues more hydrophilic, which is consistent with the near-UV CD data. The interaction of resveratrol with β-LG, thus, partly disrupts the tertiary structure of the protein. For resveratrol concentrations below 20 µM, an isoemissive point at 368 nm occurs, suggesting that the polyphenol-induced structural transition of β-LG is a two-state mechanism between native and partly unfolded states. At resveratrol concentrations higher than 40 µM, the emission maximum around 352 nm disappears and a new maximum appears around 394 nm, corresponding to the spectrum of the polyphenol, suggesting that Trp fluorescence of β-LG is mostly quenched by resveratrol.

Figure 5B shows the fluorescence emission spectra of β-LG in the presence of resveratrol with an excitation wavelength of 280 nm, at which both Trp and Tyr emit fluorescence and the quantum yield of Trp is higher than that excited at 295 nm. For pure β-LG, the fluorescence emission intensity is about 1.8 times that obtained at 295 nm, while λmax is still about 338 nm. The influence of resveratrol is basically similar at both excitation wavelengths, although the stronger emission at 280 nm produces some differences: (1) the isoemissive point shifts to a longer wavelength of 383 nm;48 (2) the higher concentration of resveratrol is needed for the similar change (i.e., the occurrence of resveratrol fluorescence emission around 400 nm at the resveratrol concentration of 40 µM and nondisappearance of protein intrinsic fluorescence around 350 nm even at the highest resveratrol concentration (108 µM)). The intrinsic fluorescence was analyzed according to the Stern-Volmer equation49

F0/F ) 1 + kq × τ0 × [resveratrol] ) 1 + K × [resveratrol] (1) F0 and F are the fluorescence emission intensities without and with resveratrol; [resveratrol] is resveratrol concentration; kq is the fluorescence quenching rate constant; τ0 is the fluorophore fluorescence lifetime without quencher; and K is a constant, equal to the reciprocal of the quencher concentration

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when the fluorescence intensity decreases by half. When the excitation wavelength is 295 nm (Figure 5A), only the fluorescence data at 40 µM resveratrol or less can be analyzed according to the Stern-Volmer equation because the β-LG fluorescence emission around 350 nm disappears at higher resveratrol concentrations. The linear plot of F0/F as a function of [resveratrol] according to Stern-Volmer equation is given in the upper inset of Figure 5. From the slope of the straight line, K values are (4.6 ( 0.2) × 104 M-1 and (3.4 ( 0.2) × 104 M-1 at the excitation wavelengths of 295 and 280 nm, respectively. According to a previous study, τ0 is 1.28 ns for the Trp residues of β-LG at neutral pH,50 giving kq of about 3.6 × 1013 M-1s-1 and 2.6 × 1013 M-1s-1 at 295 and 280 nm, respectively, which are much higher than maximal dynamic quenching constant (2.0 × 1010 M-1s-1).49,51 For the static quenching, the binding constant Ks and binding number n can be calculated according the following equation49,52,53

log[(F0-F)/F] ) logKs + n log[resveratrol]

(2)

The linear plot of log[(F0 - F)/F] as a function of log[resveratrol] is given in the lower inset of Figure 5. From the slope of the straight line, n values are 1.2 and 0.9 at 295 and 280 nm, respectively, indicate that β-LG interacts with resveratrol to form 1:1 complexes. From the intercept, Ks values are 1.9 ((1) × 105 M-1 and 1.8 ((0.1) × 104 M-1 at excitation wavelength of 295 and 280 nm, respectively. At neutral pH, β-LG exists as a mixture of monomers and dimers and the proportion of dimer is 29% when the protein concentration is 10 µM.25 The formation of protein oligomers is not taken into account when analyzing protein intrinsic fluorescence according to eqs 1 and 2, which possibly leads to an overestimation of the binding constant.54 Generally, complexes involving noncovalent bonds are reversible. For example, the binding of retinol to retinol binding protein is involved in an equilibrium between retinol and retinol-protein complexes.55 Likewise, there may be equilibrium between free and β-LG-bound resveratrol. For 1:1 complexes, the resveratrol fluorescence data in Figure 4 can be analyzed by the following equation56

1/∆F ) 1/∆Fmax + 1/(Ka × ∆Fmax × [β-LG])

(3)

∆F is the change of resveratrol fluorescence intensity in the presence and absence of β-LG; ∆Fmax is the maximal change of fluorescence intensity; Ka is the binding constant; and [β-LG] is the concentration of β-LG. As shown above, self-association of resveratrol possibly occurs at concentrations above 40 µM. This is not considered in the analysis of resveratrol fluorescence by eq 3, with polyphenol concentrations of 10 or 40 µM. The double reciprocal linear plot of 1/∆F as a function of 1/[β-LG] according to eq 3 is given in the inset in Figure 4. From the intercept and slope of the straight line, ∆Fmax and Ka can be calculated. At resveratrol concentrations of 10 µM and 40 µM, Ka values are 3.0 ((0.7) × 104 M-1 and 1.4 ((0.3) × 104 M-1, respectively. The similarity of the binding constants suggests that the binding mode of resveratrol to β-LG is the same at different resveratrol concentrations, with an average binding constant of about 2.2 × 104 M-1. At polyphenol concentrations of 10 µM and 40 µM, ∆Fmax values are 46 and 84, respectively, which are only about 58% of the change for pure resveratrol in 10 mM phosphate buffer at pH 7.4 relative to 75% ethanol, indicating that β-LG-bound resveratrol is not in a very hydrophobic environment and the binding site of resveratrol is possibly at the surface of β-LG. Combined with the protein

Figure 6. (A) Absorbance spectra of resveratrol solution for 5 min (a1), 21 h (a2), and 30 h (a3) after preparation and difference absorption spectra and between resveratrol/β-LG mixture and pure β-LG for 5 min (b1), 21 h (b2), and 30 h (b3) after preparation in 10 mM phosphate buffer at pH 7.4; (B) change of absorbance at 305 nm over time, resveratrol (square) and resveratrol/β-LG mixture subtracted pure β-LG (circle). Resveratrol and β-LG concentrations are 40 and 80 µM, respectively.

intrinsic fluorescence results, the binding constants are between 104 M-1 and 106 M-1, which is less than that for retinol binding to β-LG at the internal cavity site.24 At neutral pH, β-LG exists as a mixture of monomers and dimers and the equilibrium shifts toward the monomeric form with decreasing β-LG concentration.21,25 The equilibrium between monomeric and associated resveratrol may exist in solution due its self-association at high concentrations. This phenomenon has already been observed for retinol, which associates in water even at a concentration as low as 2 µM. When interacting with retinol binding protein, equilibrium exists between monomeric and associated retinol, between monomeric retinol and retinol-protein complexes, and possibly between associated retinol and retinol-protein complexes.55 Similarly, the interaction of resveratrol with β-LG possibly competes with the self-association of polyphenol and protein. Influence of Resveratrol-β-LG Interaction on Resveratrol Stability. Under exposure to light, trans-resveratrol in solution converts to its cis-isomer.13,14 To investigate the influence of β-LG on the photostability of trans-resveratrol, the absorbance spectrum of resveratrol and the difference absorbance spectrum between the resveratrol-β-LG mixture and pure protein were recorded (Figure 6A). Five minutes after sample preparation, resveratrol is in the trans-form and the absorbance spectrum of pure polyphenol and difference spectrum in the presence of β-LG are the same with a broad absorbance maximum between 304 and 318 nm.13 Resveratrol maintains the transconfiguration both in the absence and presence of β-LG for 21 h. After 30 h of exposure to light, the absorbance maximum shifts

Interaction of β-Lactoglobulin with Resveratrol

Figure 7. Absorbance spectra of resveratrol (solid line, a-d) and difference absorbance spectra for the resveratrol-β-LG mixture and pure β-LG (dashed line, e-h) in 10 mM phosphate buffer at pH 7.4. The concentration of β-LG is 80 µM. The initial concentrations of resveratrol in 75% ethanol are 12 (black, a and e), 24 (red, b and f), 36 (blue, c and g), and 48 mM (magenta, d and h).

to 285 and 289 nm in the absence and presence of β-LG, respectively, indicating conversion from the trans- to the cisform.13 Figure 6B shows that over a period of 30 h, absorbance at 305 nm decreases both in the absence and in the presence of β-LG. However, the presence of protein slows the decrease slightly, absorbance decreasing to about 80% of the initial value in the absence of β-LG and to about 90% in its presence after 21 h of exposure to light. These results suggest that β-LG cannot prevent the isomerization of trans-resveratrol but can improve its photostability. Influence of Resveratrol-β-LG Interaction on Resveratrol Solubility. The low solubility of resveratrol in aqueous solution significantly limits its oral administration in animal studies. Recently, it has been shown that complexes of resveratrol with cyclodextrins are hydrosoluble.14 Because resveratrol could form complexes with β-LG, we speculate that this would increase the solubility of the polyphenol. When 6 mM resveratrol in 75% ethanol is diluted in 10 mM phosphate buffer at pH 7.4 to a final concentration of 300 µM, no precipitation is observed. Increasing the initial resveratrol concentration in ethanol to 12 mM causes precipitation to occur (A500 ) 0.5) upon dilution, but not in the presence of 80 µM β-LG (A500 ) 0). These results indicate that complexing with β-LG can inhibit the aggregation of resveratrol. When resveratrol concentrations in ethanol are 36 and 48 mM, precipitation occurs even in the presence of β-LG. Figure 7 shows the absorbance spectra of resveratrol in phosphate buffer and the difference spectra of the resveratrol-βLG mixture and pure β-LG (after centrifuging and 8-fold dilution of supernatant) for the various initial resveratrol concentrations in ethanol. When the initial resveratrol concentration in ethanol is 12 mM, its maximal absorbance in the presence of protein is about 2.5 times that in its absence. That is to say, the resveratrol concentration increases up to 600 µM in the presence of β-LG but is only 240 µM for pure polyphenol, indicating that complexing with β-LG increases the hydrosolubility of resveratrol. With further increasing the initial resveratrol concentration in ethanol, the maximal absorbance in the presence of β-LG decreases. At 48 mM in ethanol, the maximal absorbances indicate a hydrosolubility of 330 and 220 µM in the presence and absence of protein, respectively. These results suggest that the ability of protein to enhance resveratrol hydrosolubility is inversely related to the initial concentration of resveratrol in ethanol. The solubility of resveratrol in the

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absence of β-LG also decreases as its concentration in ethanol increases, which may affect the ability of the protein to increase the polyphenol solubility in water. At high resveratrol concentrations, protein-polyphenol complexes might be coverage or cross-linking via excessive resveratrol, leading to the formation of large, insoluble complexes.57,58 It is known that the interaction of β-LG with ligands depends not only on the binding constants, but also on the solubility of the pure compounds.24 For instance, the solubilities of retinol and palmitic acid are, respectively, 0.06 µM and 28 µM in water. Although the binding constant to β-LG is about 2 orders of magnitude higher for retinol than for palmitic acid, the higher solubility of palmitic acid leads to a 5-fold preferred binding compared to retinol. Compared with retinol and palmitic acid, the higher solubility of resveratrol should mean high affinity to β-LG, even though the binding constants for the interaction of resveratrol with β-LG are lower.

Conclusions Resveratrol can interact with β-LG to form 1:1 complexes, without apparent effects on the secondary structure of the protein but with partial disruption of the tertiary structure. Resveratrol molecules appear to bind to the surface of β-LG with a binding constant between 104 and 106 M-1. The interaction of resveratrol with β-LG may compete with both polyphenol and protein selfassociations. It does not prevent the isomerization of transresveratrol, but can partially improve its photostability. Complexing with β-LG can increase the solubility of resveratrol in aqueous solution, which might be useful for improving the bioavailability of resveratrol in vivo. Thus, β-LG can be considered as a good carrier of resveratrol and resveratrol-βLG complexation a useful model of drug–protein interaction. Acknowledgment. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) discovery program and the NSERC Canada Research Chair in Proteins, Biosystems, and Functional Foods. The authors wish to thank Dr. Voyer (CREFSIP, Université Laval) for the access to CD instruments.

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