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Molecular Model for Astringency Produced by Polyphenol/ Protein Interactions Elisabeth Jo¨bstl,†,‡ John O’Connell,§ J. Patrick A. Fairclough,† and Mike P. Williamson*,‡ Department of Molecular Biology and Biotechnology and Department of Chemistry, University of Sheffield, Sheffield S10 2UH, United Kingdom, and Unilever Research, Colworth House, Sharnbrook, Bedford MK44 1LQ, United Kingdom Received December 8, 2003; Revised Manuscript Received February 6, 2004

Polyphenols are responsible for the astringency of many beverages and foods. This is thought to be caused by the interaction of polyphenols with basic salivary proline-rich proteins (PRPs). It is widely assumed that the molecular origin of astringency is the precipitation of PRPs following polyphenol binding and the consequent change to the mucous layer in the mouth. Here, we use a variety of biophysical techniques on a simple model system, the binding of β-casein to epigallocatechin gallate (EGCG). We show that at low EGCG ratios, small soluble polydisperse particles are formed, which aggregate to form larger particles as EGCG is added. There is an initial compaction of the protein as it binds to the polyphenol, but the particle subsequently increases in size as EGCG is added because of the incorporation of EGCG and then to aggregation and precipitation. These results are shown to be compatible with what is known of astringency in foodstuffs. Introduction Polyphenols are widely distributed in the plant kingdom and, therefore, commonly found in plant-based foods and beverages.1 They are characterized by containing several phenolic groups (often in the form of galloyl [3,4,5trihydroxybenzoyl] groups) and have been found to have a variety of effects on animals including humans.2,3 Polyphenols of intermediate size have the ability to bind to proteins and precipitate them and, hence, are also known as tannins.1,2 They have been suggested to reduce the nutritional value of some foodstuffs,4-7 but they are also important constituents of many foods and beverages, such as red wine and tea, because it is the astringency of the tannins in these beverages that gives them many of their desirable qualities. It is widely believed that salivary proteins may act as a primary defense against harmful (mainly higher molecular weight) tannins by forming insoluble complexes with them and preventing their absorption from the intestinal canal and interaction with other biological compounds.2,3,8 The interaction of polyphenols with salivary proteins has long been thought to lead to the sensation of astringency, which is generally recognized as a feeling of puckeriness and dryness in the palate.2,5,6,9 It is not confined to a particular region of the mouth but is a diffuse surface phenomenon, characterized by a loss of lubrication,10 which takes a time of the order of 15-20 s to develop fully.11,12 It is, therefore, quite different from the more well-known taste sensations. * To whom correspondence should be addressed. Fax +44 114 272 8697. E-mail [email protected]. † Department of Chemistry, University of Sheffield. ‡ Department of Molecular Biology and Biotechnology, University of Sheffield. § Unilever Research.

A mucous layer composed of salivary proteins and glycoproteins covers the exposed surface of the mouth to maintain lubrication. The primary reaction leading to the sensation of astringency is the precipitation of proteins and mucins by polyphenolic compounds. The essential feature is the cross-linking of polypeptides by surface-exposed phenolic groups on the polyphenols, leading to aggregation and precipitation and, therefore, the occurrence of the astringent response.13-15 Saliva is produced by salivary glands and contains a variety of proteins. The major protein constituent of saliva is a group of proteins consisting of multiple repeats of an unusual amino acid sequence containing a large amount of proline, commonly referred to as proline-rich proteins (PRPs).16,17 Of the three groups of PRPs (acidic, basic, and glycosylated), the main function of the basic PRPs seems to be the complexation of polyphenols.2,14,18 The molecular interaction of polyphenols with PRPs has been studied using a peptide containing a typical repeat sequence of a mouse PRP and the human basic PRP IB5.15,19,20 It was shown that the major requirement is for the peptide to have an extended conformation and that the principal binding sites on these peptides are prolines and the preceding amide bonds together with the preceding amino acid (see also ref 18). The pyrrolidine rings of the prolyl groups act as potential binding sites and form “hydrophobic sticky patches” that stack face to face with the galloyl rings of the phenolic substrate. Other interactions including hydrogen bonding interactions can further stabilize the complex.21 There is, thus, a good deal known about the molecular basis of polyphenol/protein interactions. However, the events after binding are not as well understood, not least because

10.1021/bm0345110 CCC: $27.50 © 2004 American Chemical Society Published on Web 03/13/2004

Model for Polyphenol-Induced Astringency

in vivo both the protein and the polyphenol components are heterogeneous. We have, therefore, undertaken a study of the interaction using defined molecular species for both components. The polyphenol used was (-)-epigallocatechin3-O-gallate (EGCG), a major component of green tea,22 and the protein component was dephosphorylated bovine β-casein. β-Casein is an abundant milk protein, representing 36% of bovine casein. It is a 209-residue protein, with 35 prolines evenly distributed throughout the amino acid sequence.23,24 The dephosphorylated form has an extended conformation,25 similar to that of salivary PRPs.26,27 A range of techniques have been used here, because the interaction is complex, and no single technique can provide all the information required. What emerges for the first time is a description of polyphenol/protein binding that goes all the way from the initial interaction to the final insoluble high-molecular-weight complex.

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Analytical Ultracentrifugation (AUC). β-Casein was dissolved at a concentration of 4.2 × 10-5 mol L-1 (1.0 mg mL-1). The measurements were conducted using an Optima XL-I analytical ultracentrifuge (Beckman Scientific, Inc., Palo Alto, U.S.A.) equipped with scanning absorption optics at the National Centre for Macromolecular Hydrodynamics at the University of Nottingham, U.K. The sedimentation velocity method was applied with a run speed of 50 000 rpm at 20 °C. Double sector cells of 12-mm path length were used as sample cells. The data were recorded as concentration versus radial position, and sedimentation coefficients were determined using the time derivative software DCDT+ (Biotechnology & Software Consulting, Thousands Oaks, U.S.A.).28,29 All sedimentation coefficients were corrected to standard conditions of 20 °C and water as the solvent (s20,w) using eq 1: s20,w ) sobs

Experimental Procedures Materials. Bovine β-casein was donated by Unilever Research, Colworth, U.K. The five phosphoserine groups were removed by enzymatic dephosphorylation. Casein (800 mg) was treated with 24 units of acidic potato phosphatase (Sigma) while dialysing against H2O using dialysis tubing with a molecular cutoff of 10 kDa for at least 48 h at a temperature of 4 °C, pH 7.0. EGCG was a donation from Unilever Research, Colworth, and was >98% pure by NMR. To maintain consistency, the solvent conditions for all experiments were H2O/DMSO (95:5 v/v), pH 7.0 ( 0.2, except that for the NMR pulsed gradient spin-echo (PGSE) experiments D2O:dDMSO (95:5 v/v) was used to simplify the spectra. β-Casein and EGCG were dissolved at 2.1 × 10-4 mol L-l (5 mg mL-1) and 10 × 10-3 mol L-1, respectively, and mixed at varying ratios 24 h prior to the measurement, except for the viscometry, where the samples were measured immediately after mixing. Circular Dichroism (CD). β-Casein and EGCG were dissolved in H2O and adjusted to pH 7.0 at a concentration of 20.4 × 10-6 mol L-1 (0.48 mg mL-1) and 10 × 10-3 mol L-1, respectively. A JASCO J-810 spectropolarimeter running the software Jasco J810 CD was used to collect spectra at wavelengths 190-240 nm and 240-300 nm. Quartz cuvettes of path length 1 mm were used for the far-UV region (190-240 nm), and for the higher wavelength region 10mm quartz cuvettes were used. The scanning speed was 20 nm/min, and the response and the bandwidth were 4 s and 1.37 nm, respectively. Three different mixtures (EGCG/βcasein ratios of 0.2, 1, and 5:1) were measured, and each spectrum represents the average of three scans. An EGCG spectrum of the same concentration was subtracted from the EGCG/β-casein spectra. Transmission Electron Microscopy (TEM). EGCG was mixed with β-casein (4.2 × 10-6 mol L-1, 0.1 mg mL-1) in a molecular ratio of 4:1 and, immediately after mixing, was fixed on a carbon grid and stained with uranyl formate. The sample grids were then examined using a Philips CM100 transmission electron microscope at an accelerating voltage of 100 kV.

ηT,w ηs 1 - νjF20,w η20,w ηw 1 - νjFT,s

(1)

where ηT,w and η20,w are the viscosity of water at temperatures T and 20 °C, respectively, ηs/ηw is the calculated relative viscosities of the solution and water, F20,w and FT,s are the densities of water at 20 °C and the solution at temperature T, and νj is the partial specific volume of the solution. To avoid nonideality effects, the s20,w values were then extrapolated to infinite dilution. The DCDT+ software fits a Gaussian curve to the concentration trace if a single species is present or a sum of Gaussian curves if more than one species is present. The maximum of the curve yields the sedimentation coefficient and the width of the curve the diffusion coefficient. Small-Angle X-ray Scattering (SAXS). β-Casein, at a concentration of 4.2 × 10-4 mol L-1 (10.0 mg mL-1), was mixed in different ratios with EGCG. The measurements were carried out on beamline 2.1 at the SRS Daresbury laboratories (Cheshire, U.K.). Each sample was measured for 60 s at two camera lengths of 2.25 and 8.0 m, and subsequently the data were merged to obtain the scattering curves. Initial data reduction comprised sector integration of the scattering pattern, normalization, background subtraction, and division by the detector response and was performed using the programs bsl and otoko.30 Fitting of the scattering curves was performed using the software package gift (obtained directly from Prof. O. Glatter, University of Graz) to calculate the radius of gyration Rg, pair distance distribution function, and, hence, the maximum dimension of the β-casein molecules.31 Dynamic Laser Light Scattering (DLS). β-Casein was dissolved and filtered through a Minisart sterile filter with a pore size of 0.2 µm, and the concentration was determined as 1.8 × 10-4 mol L-1 (4.2 mg mL-1) using a molar extinction coefficient at 280 nm of 10 810 L mol-1 cm-1, calculated on the basis of the amino acid composition.32 The measurements were performed using a BI-200SM Goniometer version 2.0 from Brookhaven Instruments Corp. with Brookhaven Instruments Particle Sizing Software version 3.42. The laser wavelength was 532 nm, and measurements were conducted at an angle of 90°. A log-normal fitting

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procedure was applied to fit the time correlation function to a single particle size. Viscometry. The protein concentration was determined spectrophotometrically as 1.7 × 10-4 mol L-1 (4.0 mg mL-1). The specific viscosity of the protein solution was calculated and divided by the absolute concentration of β-casein to obtain the reduced viscosity (eq 2), where ηred, ηspec, η, and η0 are respectively the reduced viscosity, specific viscosity, the viscosity of the solution, and the viscosity of the solvent, and c is the concentration of the protein. ηred )

ηspec (η - η0/η0) ) c c

(2)

An Ubbelohde or dilution viscometer with an automatic viscosity timer was used to measure the flow time of the β-casein solution through a capillary between two defined points. The flow time of the pure solvent was determined in a separate experiment. The viscometer was submerged in a thermostated water bath at 25 or 35 °C, and the solutions were temperature-equilibrated for 20 min prior to the measurement. Each measurement was repeated six times, and the average and the standard deviation for each data point were calculated. NMR PGSE Experiment. The PGSE experiment was used to determine diffusion coefficients, using a standard pulse sequence33 modified to include water suppression,34 in which magnetization is longitudinal during the diffusion delay ∆. Gradient pulses were sine shaped. Plotting the square of the gradient strength (G) against the logarithm of the signal intensity, according to the equation ln

() [

)]

IG δ ) - γ2δ2G2 ∆ D I0 3

(

(3)

(where IG and I0 are the integrals of the intensity at gradient strengths G and 0, respectively, γ is the gyromagnetic ratio of the observed nucleus, δ is the length of the gradient pulse, and D is the diffusion coefficient) gives a linear plot. The diffusion coefficient of the species from which the measured resonance originated was determined from the fitted gradient. β-Casein was dissolved in D2O:dDMSO (95:5 v/v) at a concentration of 2.0 × 10-4 mol L-1 (4.7 mg mL-1) and mixed with EGCG at different ratios 24 h prior to the measurements. The experiments were performed on a 500MHz Bruker DRX-500 NMR spectrometer, and the gradient strength was varied between 5 and 80% of full power so that a typical signal reduction of 80% was achieved at maximum gradient strength. The hydrodynamic radius (Rh) of the β-casein was calculated according to eq 4 with respect to 1,4-dioxane, which was added as a reference molecule (5.68 mmol L-1) having a known Rh of 0.212 nm.35 The NMR raw data were processed using FELIX (Accelrys, Inc., San Diego, CA). The graphs of the logarithm of the integrated peaks against the square of the gradient strengths were produced, fitted, and displayed using routines written by Jeremy Craven (University of Sheffield): ) RProtein h

gRef Ref Rh gProtein

(4)

where RProtein and RRef h h are the hydrodynamic radii of protein and the reference compound and gProtein and gRef are the gradients of the plots of ln(IG/I0) against G2. Results Preparation and Characterization of Dephosphorylated Casein. The N-terminal portion of β-casein contains five phosphoserine residues and, thus, essentially all of the net negative charge, while the C-terminal end contains many apolar residues. This concentration of negative charge on one end and of apolarity on the other results in an amphiphilic character of the protein strand, resulting in soaplike behavior and micelle formation, especially in the presence of Ca2+.25 To avoid micelle formation, the five phosphoserine groups were removed enzymatically. The phosphorylated and dephosphorylated protein were investigated by capillary electrophoresis and phosphorus NMR, showing that the protein had been dephosphorylated to at least 99% (data not shown). CD. CD measurements of β-casein between 190 and 240 nm and 240-300 nm are consistent with the absence of any R helices, β sheets, or polyproline II helices. Proline residues prevent the formation of R helices and β sheets and only form polyproline II helices when there are at least four consecutive prolines.36 This lack of evidence for regular secondary structure is, therefore, expected and was also observed with basic salivary PRPs.26,37 On addition of EGCG, there was no change in the far-UV CD spectrum of the protein (Figure 1A). This demonstrates that there is no change in the regular secondary structure content as EGCG is added but of course does not preclude changes in the protein conformation as EGCG is added. The near-UV region changes slightly on addition of EGCG (Figure 1B), indicating some possible conformational restriction of the aromatic side chains. TEM. Transmission electron micrographs (Figure 2) show the polydisperse nature of the EGCG/β-casein mixtures. Particles looking like monomers, dimers, trimers, and larger aggregates can be distinguished, with approximately the expected dimensions: the diameter of a monomer in the figure is approximately 10 nm, compared with an end-toend distance of β-casein of approximately 77 nm and a diameter for a globular protein of this size of 4-5 nm, suggesting that the particles are more compact than an extended chain but not as compact as a globular protein. The monomer bound to EGCG has a roughly spherical shape. A comparison of β-casein/EGCG aggregates immediately and 24 h after mixing reveals a similar nature of the complexes, suggesting that most of the binding and aggregation events happen soon after mixing. AUC. The AUC experiments were run as sedimentation velocity experiments, in which the species present are followed over time as they sediment through the tube. In the early stage of the measurement, the different species could not be resolved. However, at later times as the separation became more pronounced, three main species could be identified, indicating a polydisperse system. In all measurements, there was a very high molecular weight species that sedimented very rapidly, which presumably

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Model for Polyphenol-Induced Astringency

Table 1. Sedimentation Coefficients of the Two Species, Fitted to the Experimental AUC Data

Figure 1. CD spectra for dephosphorylated β-casein and the 1:1 EGCG/β-casein complex. (A) 190-240 nm. (B) 240-300 nm. ([, β-casein, 2, complex).

Figure 2. Transmission electron micrograph of EGCG mixed with β-casein in the ratio 4:1.

corresponds to the very large aggregates seen in the TEM pictures. To fit the data adequately, it was necessary to include at least two other species (although it is expected that each “species” in fact represents a distribution of particle sizes or shapes). At low EGCG/protein ratios, they had sedimentation coefficients of approximately 2 and 5 S, which would be consistent with their being a β-casein monomer and a small aggregate, respectively, again in agreement with

EGCG/β-casein

s (species 1; S)

s (species 2; S)

0.125 0.25 1.0 4

1.85 2.06 1.96 2.99

5.10 5.54 5.80 8.55

the TEM pictures (DCDT+ calculates that, in this solvent, a sedimentation coefficient of 2 S corresponds to a globular protein of 22 kDa, compared to the β-casein monomer molecular mass of 24 kDa). Increasing the EGCG/β-casein ratio from 0.125 to 0.25 led to an increase in the sedimentation coefficient from 1.85 to 2.06 S (Table 1). This increase is rather small and is not large enough to be caused by protein dimerization. On the other hand, it is too large to be caused by binding of additional EGCG molecules. We, therefore, suggest that it arises from a conformational change and that the β-casein molecule, which is originally a loose extended random coil, adopts a more spherical shape upon binding to EGCG at low ratios, by binding to the EGCG at several locations and, therefore, becoming more coiled. This explains the increase in the sedimentation coefficient because a spherical particle sediments faster than an elongated particle of the same mass. In contrast, the change in sedimentation coefficient seen on raising the ratio of EGCG/β-casein from 1:1 to 4:1 is from 1.96 to 2.99 S, an increase of 1.5 times. This compares well with the expected increase in the sedimentation rate on going from monomer to dimer, which is a factor of 1.4.38 The sedimentation coefficient of the larger species, which is assumed to consist of several aggregated polyphenolcoated β-casein molecules, increased from 5.1 to 8.6 S as EGCG was added, indicating further aggregation. The ratios of the peak intensities of species 1/species 2 fell significantly from the lower to the higher EGCG/β-casein ratios (Figure 3): the ratios changed from 3.9 to 0.05 as the EGCG/βcasein ratio changed from 0.125:1 to 4:1, showing that the larger aggregates grow at the expense of the smaller ones. SAXS. SAXS results were analyzed by fitting the decay of the scattering curves (Figure 4A) to give an average particle radius of gyration and a maximum molecular dimension (Figure 4B). Very large particles, such as those seen in TEM and also in AUC experiments, are too large to be visible in SAXS experiments. Similarly, EGCG is too small to be seen. The results, therefore, are derived from the two smaller protein species seen in AUC. However, because the analysis was carried out assuming a single Gaussian-shaped particle distribution, only a single distribution was obtained. The scattering curves cannot be easily fitted using two distributions of particles. The radius of gyration Rg of the EGCG/β-casein complex measured by SAXS decreased at the ratios EGCG/β-casein of 0.17:1 and 0.25:1 compared to the original protein. This initial decrease in size is consistent with the AUC results and complements them well because the AUC results provide more reliable information on the species present but cannot separate the effects of size and shape, whereas the scattering results report mainly on size but average the two species

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Figure 3. AUC results. The ratio of species 1 (s ∼ 2 S) to species 2 (s ∼ 5 S) decreases dramatically as the EGCG/β-casein ratio is increased from 0.125:1 to 4:1, demonstrating the progressive aggregation caused by the addition of EGCG. The inset is the experimental data from the mixture EGCG/β-casein 0.125:1 and their best fit. The fitted curve is the sum of two species, one (blue) exhibiting a sedimentation coefficient around 1.85 S and the larger species (green) around 5.1 S.

together. A further increase in the EGCG concentration led to aggregation of the β-casein molecules and, hence, an increase in the average molecular dimensions. As soon as a dimer is formed, the complex is rendered insoluble.15 Once this happens, the solution becomes cloudy and the data become very noisy and unreliable. A Kratky plot of I(q)q2 against q (where q is equal to 4π(sin θ)/λ and I(q) is the intensity of scattered radiation at q) can be used to provide information on the shape of the particles.39,40 For globular particles, the Kratky plot has a marked peak at low q, approximately 3-4 times more intense than the high-q tail, whereas for random coil proteins the plot increases monotonically with q. For molten globules (which are typically more expanded than the native state), the peak is much less pronounced, with a more intense plateau at high q. The Kratky plot for pure casein resembles that of a molten globule, becomes more peaked at low EGCG/casein ratios, and flattens off again at higher ratios, implying that casein itself is extended (as expected for a proline-rich random coil), becomes more spherical on addition of EGCG, and then becomes again less spherical once aggregation starts (data not shown). This agrees with the TEM images, which suggest that the aggregated particles are less spherical than the monomers. The pair distance distribution functions p(r) at different EGCG/protein ratios (Figure 4A, inset) also provide information on particle shape because spherical particles have a symmetrical distribution function whereas ellipsoidal particles have a longer tail at high r. The distribution functions confirm the results from Kratky plots, by having a high-r tail at higher EGCG/protein ratios, again implying that the particle is spherical at low EGCG/protein ratios but becomes less spherical in the aggregated state. At low EGCG/protein ratios, the particles are expected to consist of a single β-casein molecule and several EGCG molecules. The results,

Figure 4. SAXS results. (A) Scattering curves and (inset) the pair distance distribution functions (probability of distance r against r), assuming a single Gaussian particle size distribution, for casein alone plus three casein/EGCG mixtures (casein, red; EGCG/casein ratios 0.25:1, blue; 1:1, purple; and 4:1, green). I(q) is the scattering intensity. The r value where the pair distance distribution function crosses the x axis represents the maximum dimension across a particle Dmax, and the radius of gyration Rg is approximately equal to the mean value of r (for a spherical particle, Rg corresponds to the peak of the p(r) function). (B) Plots of Rg ([, left scale) and Dmax (3, right scale) as a function of the EGCG/casein ratio.

therefore, imply that multiple binding of several sites in casein to different phenolic groups in EGCG produces more spherical aggregates. DLS. Astringency is a sensation that develops over the course of approximately 15-20 s11,12 and then is gradually lost, probably by washing off the complexes with fresh saliva.12,41 The relevant time scale to be studying particles is, therefore, of the order of 1 min after mixing. TEM micrographs of freshly prepared samples and 24-h samples were very similar. However, most of the techniques just described cannot be set up so rapidly, and it is, therefore, important to show whether the particles change in size or shape over the course of minutes or hours. DLS is, therefore, useful because it can be measured over a rapid time scale. The DLS results (Figure 5A) show that the compaction of the β-casein molecules occurs within the first minute after mixing and the hydrodynamic radius (Rh) then stays constant. They also show the by now expected initial contraction at low EGCG ratio, followed by an increase in particle size as aggregation occurs.

Model for Polyphenol-Induced Astringency

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Figure 6. Reduced viscosity of EGCG/casein mixtures at 25 °C ([) and 35 °C (b).

Figure 5. DLS results, showing the dependence of the hydrodynamic radius Rh on time and EGCG/casein ratio. (A) The time-dependence of the DLS measurements (EGCG/β-casein ratios ×, 0.1:1; b, 1:1; 2, 10:1; and 9, 20:1). Data are normalized by dividing by the Rh of β-casein such that casein alone has a normalized Rh of 1. (B) Dependence of particle size on EGCG/casein ratio, at two different temperatures (b, 20, and 9, 35 °C), averaged over the first 5 h.

Figure 5B shows the compaction of the β-casein molecule at two different temperatures (20 and 35 °C) starting at an EGCG/β-casein ratio of 1:1. The Rh passes through a minimum at a ratio of 10:1 and then increases up to 33.3:1. The apparent decrease in particle size at 40:1, 35 °C, may be due to precipitation, which removes large aggregates from the solution. Measurement of DLS requires extensive filtration of the solutions to remove any dust particles that would have a very large effect on the measurements. It is noticeable that the minimum particle size as measured by DLS is found at a higher ratio (EGCG/casein approximately 10:1) than the SAXS results described previously (0.25:1) or the viscometry results reported in the following (approximately 0.2:1). Although some of this difference may be explained by the way the different techniques average over polydisperse distributions, it is likely that much of the difference is due to the fact that there are no casein aggregates present at the start of the DLS experiments: this statement is justified further below, in the section on NMR diffusion measurements.

Viscometry. One of the most interesting observations made here is the apparent reduction in dimensions of the casein particle at low EGCG concentrations. We wished to confirm that this was indeed occurring and was due to coiling up of casein around EGCG molecules. A good method for this is measurement of viscosity because viscosity of a polymeric solute is controlled largely by tangling of the polymer chains: an extended or random coil molecule has a high viscosity, whereas a folded molecule has much lower viscosity.42-44 The viscosity of casein/EGCG solutions was measured and normalized for protein concentration by conversion to a reduced viscosity (eq 2). The reduced viscosity is plotted as a function of the molecular ratio of EGCG/β-casein in Figure 6. At low ratios (EGCG/β-casein 0.2:1), the viscosity exhibits a minimum that may be attributed to coiling up of the protein chain around the multidentate EGCG molecules and, hence, a decrease in the molecular size of the β-casein chains. On exceeding an EGCG/β-casein ratio of 10:1, the reduced viscosity falls as a result of a loss of material from the solution and, hence, a reduction in the concentration caused by precipitation of the β-casein aggregates. Measurements were conducted at two temperatures, namely, 25 and 35 °C. No appreciable difference in concentration dependence between these temperatures was noted. Diffusion Measurement by PGSE Experiment. To obtain a more quantitative measure of the molecular size, self-diffusion rates were measured by NMR and converted to absolute numbers by comparison to diffusion rates of an internal standard (eq 4). The hydrodynamic radius of the β-casein showed a significant reduction in size up to an EGCG/β-casein ratio of 0.14 (Figure 7A), in agreement with results using other techniques. At higher EGCG concentrations, the Rh increased to about 3.0 nm (EGCG/β-casein 0.33), which corresponds approximately to a dimer. Dimerization and even higher aggregation renders the proteinpolyphenol complex insoluble and, therefore, not visible by NMR (Figure 7, inset), as previously observed.21 This might be the reason that, at EGCG ratios above 0.33, Rh seems to decrease and the data start to become unreliable. Figure 7B presents data from a β-casein solution that was filtered prior to the measurements using a 0.2-µm filter.

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This matches the DLS results but not the SAXS and viscometry results. It is striking that the two experiments in which the solution was prefiltered give minima at a ratio of 10, while the other experiments give minima at a ratio of approximately 0.25. Discussion

Figure 7. Hydrodynamic radius of EGCG/casein mixtures, determined by NMR PGSE experiments. (A) Unfiltered solutions. The graph in the inset shows the integrated intensity of the casein NMR signals relative to the same concentration of free casein, which should be constant as long as casein remains soluble. The dashed line indicates the ratio at which visible precipitation was first observed. (B) Filtered solutions.

Similar behavior is seen, except that the minimum apparent size is slightly smaller and the EGCG/casein ratio at which the minimum occurs is much higher, at approximately 10.

This study is aimed at deriving a molecular description of the interactions between proteins (particularly salivary basic PRPs) and dietary polyphenols, which are thought to be responsible for the astringent sensation of polyphenolrich foods and beverages such as red wine and tea. The purification of single PRPs from saliva is laborious, so as a substitute for PRPs, bovine β-casein was used. This is cheap and easily available and has a number of similarities with PRPs: it shares a similar extended conformation, with exposed prolines,25,26 it competes with gelatin to bind to polyphenols,7 and it binds to wine polyphenols in a similar way to the established model polyvinyl-polypyrrolidone, although there is a lower density of polyphenol binding sites.45 Dephosphorylation removes the hydrophilic phosphates and, therefore, reduces the tendency of casein to bind calcium and form micelles.46 Polyphenols are multidentate ligands able to bind simultaneously, via different phenolic groups, at more than one point on the protein strand.1,47 It has previously been suggested that polyphenol-protein precipitation occurs in three stages.15 Here, we have confirmed and expanded the three-stage model (Figure 8): (1) The free proteins exist in a loose, randomly coiled conformation. Simultaneous binding of the multidentate polyphenols to several sites on the protein leads to coiling of the protein around the polyphenols. This causes the physical size to decrease and the structure of the protein to become more compact and spherical. Chelated binding at several sites increases the overall binding affinity: in support of this statement, we note that the affinity of a full-length PRP (70 residues) for polyphenols is much greater than that of a 19-residue single PRP sequence.20 (2) As the polyphenol concentration rises, polyphenols complexed onto the protein surface cross-link different protein molecules and dimerization ensues, causing insolubility.15 This phenomenon is similar to the precipitation of

Figure 8. Proposed binding model: The original random coiled PRP binds to multidentate polyphenols on more than one site because each proline and each aromatic ring represents a possible binding site. At a low polyphenol concentration, the protein binds in several places to the polyphenol molecules leading to a contraction of the loose random coil and decrease in the molecular size of the protein. Upon addition of more polyphenols, intermolecular cross-linking takes place and aggregates are formed that finally precipitate.

Model for Polyphenol-Induced Astringency

antigens by multidentate antibodies,48 implying that the formation of insoluble complexes is most favorable when the ratio of (polyphenol binding site):(protein binding site) is approximately unity.47,49,50 It is probably no coincidence that the onset of aggregation is at a molar ratio of EGCG/ casein of about 10:1. EGCG has three free binding groups, while bovine β-casein has 35 prolines and 14 aromatic rings,23 the other major binding site for polyphenols.15 Aggregation, therefore, starts to become obvious at a (polyphenol binding site):(protein binding site) ratio of about 0.6:1. (3) As further EGCG is added, dimers aggregate together and the resultant large particles precipitate out of solution. Experiments carried out using an ultrafiltered casein solution show a much later onset of aggregation, implying that preexisting casein aggregates act as nuclei for further aggregation. The AUC measurements show that two main species are present during this phase, a monomer and an aggregated state containing several protein molecules. As more EGCG is added, monomers aggregate together so that the main change in average particle size is due to a reduction in the number of small particles and an increase in the number of large particles (Figure 3) rather than simply an increase in the average size of the existing particles. Similar conclusions were reached in a study of gliadin/tannic acid complexation.51 The results presented here show that polyphenol-protein binding produces a more cross-linked and hydrophobic protein. This is suggested to be the basis of the astringent sensation, which is basically a loss of wettability of the thin mucous layer at the palate. The time scale over which astringency develops is consistent with the observations reported here. We, therefore, present the three-stage model (Figure 8) as a likely model for how astringency develops in the mouth. Acknowledgment. Unilever Research Colworth is acknowledged for financial funding of the project. We furthermore thank Dr. Gunter Grossman (Daresbury laboratories) for help with the X-ray experiments, Dr. Matt Conroy for the TEM micrographs, Dr. Chris Walters for conducting the AUC experiments, Andrea Hounslow for setting up the NMR experiments, Silva Giannini for interpretation of the CD spectra, and Dr. Ron Young for help with the Ubbelohde viscometer. References and Notes (1) Haslam, E. Plant polyphenols: Vegetable tannins reVisited; Cambridge University Press: Cambridge, U.K., 1989. (2) Bennick, A. Crit. ReV. Oral Biol. Med. 2002, 13, 184-196. (3) Mehansho, H.; Butler, L. G.; Carlson, D. M. Annu. ReV. Nutr. 1987, 7, 423-440. (4) Joslyn, M. A.; Goldstein, J. L. Wallerstein Lab. Commun. 1965, 28, 143-160. (5) Joslyn, M. A.; Goldstein, J. L. AdV. Food Res. 1964, 13, 179-217. (6) Bate-Smith, E. C. Phytochemistry 1973, 12, 907-912. (7) Luck, G.; Liao, H.; Murray, N. J.; Grimmer, H. R.; Warminski, E. E.; Williamson, M. P.; Lilley, T. H.; Haslam, E. Phytochemistry 1994, 37, 357-371. (8) Mole, S.; Butler, L. G.; Iason, G. Biochem. Syst. Ecol. 1990, 18, 287-293.

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