Molecular Model for Astringency Produced by Polyphenol/Protein Interactions

Molecular Model for Astringency Produced by Polyphenol/Protein Interactions

Elisabeth Jobstl,†,‡ 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 causedby the interaction of polyphenols with basic salivary proline-rich proteins (PRPs). It is widely assumed thatthe molecular origin of astringency is the precipitation of PRPs following polyphenol binding and theconsequent change to the mucous layer in the mouth. Here, we use a variety of biophysical techniques ona simple model system, the binding ofâ-casein to epigallocatechin gallate (EGCG). We show that at lowEGCG ratios, small soluble polydisperse particles are formed, which aggregate to form larger particles asEGCG is added. There is an initial compaction of the protein as it binds to the polyphenol, but the particlesubsequently increases in size as EGCG is added because of the incorporation of EGCG and then toaggregation and precipitation. These results are shown to be compatible with what is known of astringencyin foodstuffs.

Introduction

Polyphenols are widely distributed in the plant kingdomand, therefore, commonly found in plant-based foods andbeverages.1 They are characterized by containing severalphenolic groups (often in the form of galloyl [3,4,5-trihydroxybenzoyl] groups) and have been found to have avariety of effects on animals including humans.2,3 Polyphe-nols of intermediate size have the ability to bind to proteinsand precipitate them and, hence, are also known as tannins.1,2

They have been suggested to reduce the nutritional value ofsome foodstuffs,4-7 but they are also important constituentsof many foods and beverages, such as red wine and tea,because it is the astringency of the tannins in these beveragesthat gives them many of their desirable qualities. It is widelybelieved that salivary proteins may act as a primary defenseagainst harmful (mainly higher molecular weight) tanninsby forming insoluble complexes with them and preventingtheir absorption from the intestinal canal and interaction withother biological compounds.2,3,8 The interaction of polyphe-nols with salivary proteins has long been thought to lead tothe sensation of astringency, which is generally recognizedas a feeling of puckeriness and dryness in the palate.2,5,6,9Itis not confined to a particular region of the mouth but is adiffuse surface phenomenon, characterized by a loss oflubrication,10 which takes a time of the order of 15-20 s todevelop fully.11,12 It is, therefore, quite different from themore well-known taste sensations.

A mucous layer composed of salivary proteins andglycoproteins covers the exposed surface of the mouth tomaintain lubrication. The primary reaction leading to thesensation of astringency is the precipitation of proteins andmucins by polyphenolic compounds. The essential featureis the cross-linking of polypeptides by surface-exposedphenolic groups on the polyphenols, leading to aggregationand precipitation and, therefore, the occurrence of theastringent response.13-15 Saliva is produced by salivary glandsand contains a variety of proteins. The major proteinconstituent of saliva is a group of proteins consisting ofmultiple repeats of an unusual amino acid sequence contain-ing a large amount of proline, commonly referred to asproline-rich proteins (PRPs).16,17Of the three groups of PRPs(acidic, basic, and glycosylated), the main function of thebasic PRPs seems to be the complexation of polyphe-nols.2,14,18

The molecular interaction of polyphenols with PRPs hasbeen studied using a peptide containing a typical repeatsequence of a mouse PRP and the human basic PRP IB-5.15,19,20It was shown that the major requirement is for thepeptide to have an extended conformation and that theprincipal binding sites on these peptides are prolines and thepreceding amide bonds together with the preceding aminoacid (see also ref 18). The pyrrolidine rings of the prolylgroups act as potential binding sites and form “hydrophobicsticky patches” that stack face to face with the galloyl ringsof the phenolic substrate. Other interactions includinghydrogen bonding interactions can further stabilize thecomplex.21

There is, thus, a good deal known about the molecularbasis of polyphenol/protein interactions. However, the eventsafter binding are not as well understood, not least because

* 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.

942 Biomacromolecules 2004,5, 942-949

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

in vivo both the protein and the polyphenol components areheterogeneous. We have, therefore, undertaken a study ofthe interaction using defined molecular species for bothcomponents. The polyphenol used was (-)-epigallocatechin-3-O-gallate (EGCG), a major component of green tea,22 andthe protein component was dephosphorylated bovineâ-casein.â-Casein is an abundant milk protein, representing 36% ofbovine casein. It is a 209-residue protein, with 35 prolinesevenly 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 techniqueshave been used here, because the interaction is complex, andno single technique can provide all the information required.What emerges for the first time is a description of polyphe-nol/protein binding that goes all the way from the initialinteraction to the final insoluble high-molecular-weightcomplex.

Experimental Procedures

Materials. Bovine â-casein was donated by UnileverResearch, Colworth, U.K. The five phosphoserine groupswere removed by enzymatic dephosphorylation. Casein (800mg) was treated with 24 units of acidic potato phosphatase(Sigma) while dialysing against H2O using dialysis tubingwith a molecular cutoff of 10 kDa for at least 48 h at atemperature of 4°C, pH 7.0. EGCG was a donation fromUnilever Research, Colworth, and was>98% pure by NMR.To maintain consistency, the solvent conditions for allexperiments 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 simplifythe spectra.â-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 themeasurement, except for the viscometry, where the sampleswere measured immediately after mixing.

Circular Dichroism (CD) . â-Casein and EGCG weredissolved in H2O and adjusted to pH 7.0 at a concentrationof 20.4× 10-6 mol L-1 (0.48 mg mL-1) and 10× 10-3 molL-1, respectively. A JASCO J-810 spectropolarimeter run-ning the software Jasco J810 CD was used to collect spectraat wavelengths 190-240 nm and 240-300 nm. Quartzcuvettes of path length 1 mm were used for the far-UV region(190-240 nm), and for the higher wavelength region 10-mm quartz cuvettes were used. The scanning speed was 20nm/min, and the response and the bandwidth were 4 s and1.37 nm, respectively. Three different mixtures (EGCG/â-casein ratios of 0.2, 1, and 5:1) were measured, and eachspectrum represents the average of three scans. An EGCGspectrum of the same concentration was subtracted from theEGCG/â-casein spectra.

Transmission Electron Microscopy (TEM). EGCG wasmixed withâ-casein (4.2× 10-6 mol L-1, 0.1 mg mL-1) ina molecular ratio of 4:1 and, immediately after mixing, wasfixed on a carbon grid and stained with uranyl formate. Thesample grids were then examined using a Philips CM100transmission electron microscope at an accelerating voltageof 100 kV.

Analytical Ultracentrifugation (AUC). â-Casein wasdissolved at a concentration of 4.2× 10-5 mol L-1 (1.0 mgmL-1). The measurements were conducted using an OptimaXL-I analytical ultracentrifuge (Beckman Scientific, Inc.,Palo Alto, U.S.A.) equipped with scanning absorption opticsat the National Centre for Macromolecular Hydrodynamicsat the University of Nottingham, U.K. The sedimentationvelocity method was applied with a run speed of 50 000 rpmat 20 °C. Double sector cells of 12-mm path length wereused as sample cells. The data were recorded as concentrationversus radial position, and sedimentation coefficients weredetermined using the time derivative software DCDT+(Biotechnology & Software Consulting, Thousands Oaks,U.S.A.).28,29 All sedimentation coefficients were correctedto standard conditions of 20°C and water as the solvent(s20,w) using eq 1:

whereηT,w andη20,w are the viscosity of water at temperaturesT and 20°C, respectively,ηs/ηw is the calculated relativeviscosities of the solution and water,F20,w and FT,s are thedensities of water at 20°C and the solution at temperatureT, andνj is the partial specific volume of the solution. Toavoid nonideality effects, thes20,w values were then extrapo-lated to infinite dilution. The DCDT+ software fits aGaussian curve to the concentration trace if a single speciesis present or a sum of Gaussian curves if more than onespecies is present. The maximum of the curve yields thesedimentation coefficient and the width of the curve thediffusion coefficient.

Small-Angle X-ray Scattering (SAXS). â-Casein, at aconcentration of 4.2× 10-4 mol L-1 (10.0 mg mL-1), wasmixed in different ratios with EGCG. The measurementswere carried out on beamline 2.1 at the SRS Daresburylaboratories (Cheshire, U.K.). Each sample was measuredfor 60 s at two camera lengths of 2.25 and 8.0 m, andsubsequently the data were merged to obtain the scatteringcurves. Initial data reduction comprised sector integrationof the scattering pattern, normalization, background subtrac-tion, and division by the detector response and was performedusing the programs bsl and otoko.30 Fitting of the scatteringcurves was performed using the software package gift(obtained directly from Prof. O. Glatter, University of Graz)to calculate the radius of gyrationRg, pair distance distribu-tion function, and, hence, the maximum dimension of theâ-casein molecules.31

Dynamic Laser Light Scattering (DLS). â-Casein wasdissolved and filtered through a Minisart sterile filter with apore size of 0.2µm, and the concentration was determinedas 1.8 × 10-4 mol L-1 (4.2 mg mL-1) using a molarextinction coefficient at 280 nm of 10 810 L mol-1 cm-1,calculated on the basis of the amino acid composition.32 Themeasurements were performed using a BI-200SM Goniom-eter version 2.0 from Brookhaven Instruments Corp. withBrookhaven Instruments Particle Sizing Software version3.42. The laser wavelength was 532 nm, and measurementswere conducted at an angle of 90°. A log-normal fitting

s20,w ) sobs

ηT,w

η20,w

ηs

ηw

1 - νjF20,w

1 - νjFT,s(1)

Model for Polyphenol-Induced Astringency Biomacromolecules, Vol. 5, No. 3, 2004 943

procedure was applied to fit the time correlation function toa single particle size.

Viscometry. The protein concentration was determinedspectrophotometrically as 1.7× 10-4 mol L-1 (4.0 mgmL-1). The specific viscosity of the protein solution wascalculated and divided by the absolute concentration ofâ-casein to obtain the reduced viscosity (eq 2), whereηred,ηspec, η, andη0 are respectively the reduced viscosity, specificviscosity, the viscosity of the solution, and the viscosity ofthe solvent, andc is the concentration of the protein.

An Ubbelohde or dilution viscometer with an automaticviscosity timer was used to measure the flow time of theâ-casein solution through a capillary between two definedpoints. The flow time of the pure solvent was determined ina separate experiment. The viscometer was submerged in athermostated water bath at 25 or 35°C, and the solutionswere temperature-equilibrated for 20 min prior to themeasurement. Each measurement was repeated six times, andthe average and the standard deviation for each data pointwere calculated.

NMR PGSE Experiment. The PGSE experiment wasused to determine diffusion coefficients, using a standardpulse sequence33 modified to include water suppression,34

in which magnetization is longitudinal during the diffusiondelay ∆. Gradient pulses were sine shaped. Plotting thesquare of the gradient strength (G) against the logarithm ofthe signal intensity, according to the equation

(whereIG andI0 are the integrals of the intensity at gradientstrengthsG and 0, respectively,γ is the gyromagnetic ratioof the observed nucleus,δ is the length of the gradient pulse,andD is the diffusion coefficient) gives a linear plot. Thediffusion coefficient of the species from which the measuredresonance originated was determined from the fitted gradient.â-Casein was dissolved in D2O:dDMSO (95:5 v/v) at aconcentration of 2.0× 10-4 mol L-1 (4.7 mg mL-1) andmixed with EGCG at different ratios 24 h prior to themeasurements. The experiments were performed on a 500-MHz Bruker DRX-500 NMR spectrometer, and the gradientstrength was varied between 5 and 80% of full power sothat a typical signal reduction of 80% was achieved atmaximum gradient strength. The hydrodynamic radius (Rh)of theâ-casein was calculated according to eq 4 with respectto 1,4-dioxane, which was added as a reference molecule(5.68 mmol L-1) having a knownRh of 0.212 nm.35 TheNMR raw data were processed using FELIX (Accelrys, Inc.,San Diego, CA). The graphs of the logarithm of theintegrated peaks against the square of the gradient strengthswere produced, fitted, and displayed using routines writtenby Jeremy Craven (University of Sheffield):

whereRhProteinandRh

Ref are the hydrodynamic radii of proteinand the reference compound andgProtein and gRef are thegradients of the plots of ln(IG/I0) againstG2.

Results

Preparation and Characterization of DephosphorylatedCasein. The N-terminal portion ofâ-casein contains fivephosphoserine residues and, thus, essentially all of the netnegative charge, while the C-terminal end contains manyapolar residues. This concentration of negative charge onone end and of apolarity on the other results in an amphiphiliccharacter of the protein strand, resulting in soaplike behaviorand micelle formation, especially in the presence of Ca2+.25

To avoid micelle formation, the five phosphoserine groupswere removed enzymatically. The phosphorylated and de-phosphorylated protein were investigated by capillary elec-trophoresis and phosphorus NMR, showing that the proteinhad been dephosphorylated to at least 99% (data not shown).

CD. CD measurements ofâ-casein between 190 and 240nm and 240-300 nm are consistent with the absence of anyR helices,â sheets, or polyproline II helices. Proline residuesprevent the formation ofR helices andâ sheets and onlyform polyproline II helices when there are at least fourconsecutive prolines.36 This lack of evidence for regularsecondary structure is, therefore, expected and was alsoobserved with basic salivary PRPs.26,37On addition of EGCG,there was no change in the far-UV CD spectrum of theprotein (Figure 1A). This demonstrates that there is nochange in the regular secondary structure content as EGCGis added but of course does not preclude changes in theprotein conformation as EGCG is added. The near-UV regionchanges slightly on addition of EGCG (Figure 1B), indicatingsome possible conformational restriction of the aromatic sidechains.

TEM . Transmission electron micrographs (Figure 2) showthe polydisperse nature of the EGCG/â-casein mixtures.Particles looking like monomers, dimers, trimers, and largeraggregates can be distinguished, with approximately theexpected dimensions: the diameter of a monomer in thefigure is approximately 10 nm, compared with an end-to-end distance ofâ-casein of approximately 77 nm and adiameter for a globular protein of this size of 4-5 nm,suggesting that the particles are more compact than anextended chain but not as compact as a globular protein. Themonomer bound to EGCG has a roughly spherical shape. Acomparison ofâ-casein/EGCG aggregates immediately and24 h after mixing reveals a similar nature of the complexes,suggesting that most of the binding and aggregation eventshappen soon after mixing.

AUC. The AUC experiments were run as sedimentationvelocity experiments, in which the species present arefollowed over time as they sediment through the tube. Inthe early stage of the measurement, the different speciescould not be resolved. However, at later times as theseparation became more pronounced, three main speciescould be identified, indicating a polydisperse system. In allmeasurements, there was a very high molecular weightspecies that sedimented very rapidly, which presumably

ηred )ηspec

c)

(η - η0/η0)

c(2)

ln(IG

I0) ) -[γ2δ2G2(∆ - δ

3)]D (3)

RhProtein) gRef

gProteinRh

Ref (4)

944 Biomacromolecules, Vol. 5, No. 3, 2004 Jobstl et al.

corresponds to the very large aggregates seen in the TEMpictures. To fit the data adequately, it was necessary toinclude at least two other species (although it is expectedthat each “species” in fact represents a distribution of particlesizes or shapes). At low EGCG/protein ratios, they hadsedimentation coefficients of approximately 2 and 5 S, whichwould be consistent with their being aâ-casein monomerand a small aggregate, respectively, again in agreement with

the TEM pictures (DCDT+ calculates that, in this solvent,a sedimentation coefficient of 2 S corresponds to a globularprotein of 22 kDa, compared to theâ-casein monomermolecular mass of 24 kDa). Increasing the EGCG/â-caseinratio from 0.125 to 0.25 led to an increase in the sedimenta-tion coefficient from 1.85 to 2.06 S (Table 1). This increaseis rather small and is not large enough to be caused by proteindimerization. On the other hand, it is too large to be causedby binding of additional EGCG molecules. We, therefore,suggest that it arises from a conformational change and thattheâ-casein molecule, which is originally a loose extendedrandom coil, adopts a more spherical shape upon binding toEGCG at low ratios, by binding to the EGCG at severallocations and, therefore, becoming more coiled. This explainsthe increase in the sedimentation coefficient because aspherical particle sediments faster than an elongated particleof the same mass. In contrast, the change in sedimentationcoefficient seen on raising the ratio of EGCG/â-casein from1:1 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 thesedimentation rate on going from monomer to dimer, whichis a factor of 1.4.38

The sedimentation coefficient of the larger species, whichis assumed to consist of several aggregated polyphenol-coatedâ-casein molecules, increased from 5.1 to 8.6 S asEGCG was added, indicating further aggregation. The ratiosof the peak intensities of species 1/species 2 fell significantlyfrom the lower to the higher EGCG/â-casein ratios (Figure3): the ratios changed from 3.9 to 0.05 as the EGCG/â-casein ratio changed from 0.125:1 to 4:1, showing that thelarger aggregates grow at the expense of the smaller ones.

SAXS. SAXS results were analyzed by fitting the decayof the scattering curves (Figure 4A) to give an averageparticle radius of gyration and a maximum moleculardimension (Figure 4B). Very large particles, such as thoseseen in TEM and also in AUC experiments, are too large tobe visible in SAXS experiments. Similarly, EGCG is toosmall to be seen. The results, therefore, are derived fromthe two smaller protein species seen in AUC. However,because the analysis was carried out assuming a singleGaussian-shaped particle distribution, only a single distribu-tion was obtained. The scattering curves cannot be easilyfitted using two distributions of particles.

The radius of gyrationRg of the EGCG/â-casein complexmeasured by SAXS decreased at the ratios EGCG/â-caseinof 0.17:1 and 0.25:1 compared to the original protein. Thisinitial decrease in size is consistent with the AUC resultsand complements them well because the AUC results providemore reliable information on the species present but cannotseparate the effects of size and shape, whereas the scatteringresults report mainly on size but average the two species

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

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

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

EGCG/â-casein s (species 1; S) s (species 2; S)

0.125 1.85 5.100.25 2.06 5.541.0 1.96 5.804 2.99 8.55


together. A further increase in the EGCG concentration ledto aggregation of theâ-casein molecules and, hence, anincrease in the average molecular dimensions. As soon as adimer is formed, the complex is rendered insoluble.15 Oncethis happens, the solution becomes cloudy and the databecome very noisy and unreliable.

A Kratky plot of I(q)q2 againstq (whereq is equal to4π(sin θ)/λ andI(q) is the intensity of scattered radiation atq) can be used to provide information on the shape of theparticles.39,40 For globular particles, the Kratky plot has amarked peak at lowq, approximately 3-4 times more intensethan the high-q tail, whereas for random coil proteins theplot increases monotonically withq. For molten globules(which are typically more expanded than the native state),the peak is much less pronounced, with a more intenseplateau at highq. The Kratky plot for pure casein resemblesthat of a molten globule, becomes more peaked at lowEGCG/casein ratios, and flattens off again at higher ratios,implying that casein itself is extended (as expected for aproline-rich random coil), becomes more spherical onaddition of EGCG, and then becomes again less sphericalonce aggregation starts (data not shown). This agrees withthe TEM images, which suggest that the aggregated particlesare less spherical than the monomers.

The pair distance distribution functionsp(r) at differentEGCG/protein ratios (Figure 4A, inset) also provide informa-tion on particle shape because spherical particles have asymmetrical distribution function whereas ellipsoidal par-ticles have a longer tail at highr. The distribution functionsconfirm the results from Kratky plots, by having a high-rtail at higher EGCG/protein ratios, again implying that theparticle is spherical at low EGCG/protein ratios but becomesless spherical in the aggregated state. At low EGCG/proteinratios, the particles are expected to consist of a singleâ-casein molecule and several EGCG molecules. The results,

therefore, imply that multiple binding of several sites incasein to different phenolic groups in EGCG produces morespherical aggregates.

DLS. Astringency is a sensation that develops over thecourse of approximately 15-20 s11,12 and then is graduallylost, probably by washing off the complexes with freshsaliva.12,41 The relevant time scale to be studying particlesis, therefore, of the order of 1 min after mixing. TEMmicrographs of freshly prepared samples and 24-h sampleswere very similar. However, most of the techniques justdescribed cannot be set up so rapidly, and it is, therefore,important to show whether the particles change in size orshape 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 ofthe â-casein molecules occurs within the first minute aftermixing and the hydrodynamic radius (Rh) then stays constant.They also show the by now expected initial contraction atlow EGCG ratio, followed by an increase in particle size asaggregation occurs.

Figure 3. AUC results. The ratio of species 1 (s ∼ 2 S) to species2 (s ∼ 5 S) decreases dramatically as the EGCG/â-casein ratio isincreased from 0.125:1 to 4:1, demonstrating the progressive ag-gregation caused by the addition of EGCG. The inset is theexperimental data from the mixture EGCG/â-casein 0.125:1 and theirbest fit. The fitted curve is the sum of two species, one (blue)exhibiting a sedimentation coefficient around 1.85 S and the largerspecies (green) around 5.1 S.

Figure 4. SAXS results. (A) Scattering curves and (inset) the pairdistance distribution functions (probability of distance r against r),assuming a single Gaussian particle size distribution, for casein aloneplus three casein/EGCG mixtures (casein, red; EGCG/casein ratios0.25:1, blue; 1:1, purple; and 4:1, green). I(q) is the scatteringintensity. The r value where the pair distance distribution functioncrosses the x axis represents the maximum dimension across aparticle Dmax, and the radius of gyration Rg is approximately equal tothe mean value of r (for a spherical particle, Rg corresponds to thepeak of the p(r) function). (B) Plots of Rg ([, left scale) and Dmax

(3, right scale) as a function of the EGCG/casein ratio.


Figure 5B shows the compaction of theâ-casein moleculeat two different temperatures (20 and 35°C) starting at anEGCG/â-casein ratio of 1:1. TheRh passes through aminimum 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, maybe due to precipitation, which removes large aggregates fromthe solution.

Measurement of DLS requires extensive filtration of thesolutions to remove any dust particles that would have a verylarge effect on the measurements. It is noticeable that theminimum particle size as measured by DLS is found at ahigher ratio (EGCG/casein approximately 10:1) than theSAXS results described previously (0.25:1) or the viscometryresults reported in the following (approximately 0.2:1).Although some of this difference may be explained by theway the different techniques average over polydispersedistributions, it is likely that much of the difference is dueto the fact that there are no casein aggregates present at thestart of the DLS experiments: this statement is justifiedfurther below, in the section on NMR diffusion measure-ments.

Viscometry. One of the most interesting observationsmade here is the apparent reduction in dimensions of thecasein particle at low EGCG concentrations. We wished toconfirm that this was indeed occurring and was due to coilingup of casein around EGCG molecules. A good method forthis is measurement of viscosity because viscosity of apolymeric solute is controlled largely by tangling of thepolymer chains: an extended or random coil molecule hasa high viscosity, whereas a folded molecule has much lowerviscosity.42-44 The viscosity of casein/EGCG solutions wasmeasured and normalized for protein concentration byconversion to a reduced viscosity (eq 2). The reducedviscosity is plotted as a function of the molecular ratio ofEGCG/â-casein in Figure 6. At low ratios (EGCG/â-casein0.2:1), the viscosity exhibits a minimum that may beattributed to coiling up of the protein chain around themultidentate EGCG molecules and, hence, a decrease in themolecular size of theâ-casein chains. On exceeding anEGCG/â-casein ratio of 10:1, the reduced viscosity falls asa result of a loss of material from the solution and, hence, areduction in the concentration caused by precipitation of theâ-casein aggregates. Measurements were conducted at twotemperatures, namely, 25 and 35°C. No appreciable differ-ence in concentration dependence between these temperatureswas noted.

Diffusion Measurement by PGSE Experiment. Toobtain a more quantitative measure of the molecular size,self-diffusion rates were measured by NMR and convertedto absolute numbers by comparison to diffusion rates of aninternal standard (eq 4). The hydrodynamic radius of theâ-casein showed a significant reduction in size up to anEGCG/â-casein ratio of 0.14 (Figure 7A), in agreement withresults using other techniques. At higher EGCG concentra-tions, theRh increased to about 3.0 nm (EGCG/â-casein0.33), which corresponds approximately to a dimer. Dimer-ization and even higher aggregation renders the protein-polyphenol complex insoluble and, therefore, not visible byNMR (Figure 7, inset), as previously observed.21 This mightbe the reason that, at EGCG ratios above 0.33,Rh seems todecrease and the data start to become unreliable.

Figure 7B presents data from aâ-casein solution that wasfiltered prior to the measurements using a 0.2-µm filter.

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

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


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

This matches the DLS results but not the SAXS andviscometry results. It is striking that the two experiments inwhich the solution was prefiltered give minima at a ratio of10, while the other experiments give minima at a ratio ofapproximately 0.25.

Discussion

This study is aimed at deriving a molecular descriptionof the interactions between proteins (particularly salivarybasic PRPs) and dietary polyphenols, which are thought tobe responsible for the astringent sensation of polyphenol-rich foods and beverages such as red wine and tea. Thepurification of single PRPs from saliva is laborious, so as asubstitute for PRPs, bovineâ-casein was used. This is cheapand easily available and has a number of similarities withPRPs: it shares a similar extended conformation, withexposed prolines,25,26 it competes with gelatin to bind topolyphenols,7 and it binds to wine polyphenols in a similarway to the established model polyvinyl-polypyrrolidone,although there is a lower density of polyphenol bindingsites.45 Dephosphorylation removes the hydrophilic phos-phates and, therefore, reduces the tendency of casein to bindcalcium and form micelles.46

Polyphenols are multidentate ligands able to bind simul-taneously, via different phenolic groups, at more than onepoint on the protein strand.1,47 It has previously beensuggested that polyphenol-protein precipitation occurs inthree stages.15 Here, we have confirmed and expanded thethree-stage model (Figure 8):

(1) The free proteins exist in a loose, randomly coiledconformation. Simultaneous binding of the multidentatepolyphenols to several sites on the protein leads to coilingof the protein around the polyphenols. This causes thephysical size to decrease and the structure of the protein tobecome more compact and spherical. Chelated binding atseveral sites increases the overall binding affinity: in supportof this statement, we note that the affinity of a full-lengthPRP (70 residues) for polyphenols is much greater than thatof a 19-residue single PRP sequence.20

(2) As the polyphenol concentration rises, polyphenolscomplexed onto the protein surface cross-link differentprotein molecules and dimerization ensues, causing insolubil-ity.15 This phenomenon is similar to the precipitation of

Figure 7. Hydrodynamic radius of EGCG/casein mixtures, deter-mined by NMR PGSE experiments. (A) Unfiltered solutions. The graphin the inset shows the integrated intensity of the casein NMR signalsrelative to the same concentration of free casein, which should beconstant as long as casein remains soluble. The dashed line indicatesthe ratio at which visible precipitation was first observed. (B) Filteredsolutions.

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


antigens by multidentate antibodies,48 implying that theformation of insoluble complexes is most favorable whenthe ratio of (polyphenol binding site):(protein binding site)is approximately unity.47,49,50It is probably no coincidencethat the onset of aggregation is at a molar ratio of EGCG/casein of about 10:1. EGCG has three free binding groups,while bovine â-casein has 35 prolines and 14 aromaticrings,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 about0.6:1.

(3) As further EGCG is added, dimers aggregate togetherand the resultant large particles precipitate out of solution.Experiments carried out using an ultrafiltered casein solutionshow a much later onset of aggregation, implying thatpreexisting casein aggregates act as nuclei for furtheraggregation.

The AUC measurements show that two main species arepresent during this phase, a monomer and an aggregated statecontaining several protein molecules. As more EGCG isadded, monomers aggregate together so that the main changein average particle size is due to a reduction in the numberof small particles and an increase in the number of largeparticles (Figure 3) rather than simply an increase in theaverage size of the existing particles. Similar conclusionswere reached in a study of gliadin/tannic acid complexation.51

The results presented here show that polyphenol-proteinbinding produces a more cross-linked and hydrophobicprotein. This is suggested to be the basis of the astringentsensation, which is basically a loss of wettability of the thinmucous layer at the palate. The time scale over whichastringency develops is consistent with the observationsreported here. We, therefore, present the three-stage model(Figure 8) as a likely model for how astringency developsin the mouth.

Acknowledgment. Unilever Research Colworth is ac-knowledged for financial funding of the project. We fur-thermore thank Dr. Gunter Grossman (Daresbury laborato-ries) for help with the X-ray experiments, Dr. Matt Conroyfor the TEM micrographs, Dr. Chris Walters for conductingthe AUC experiments, Andrea Hounslow for setting up theNMR experiments, Silva Giannini for interpretation of theCD spectra, and Dr. Ron Young for help with the Ubbelohdeviscometer.

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Molecular Model for Astringency Produced by Polyphenol/Protein Interactions