The interaction of sucrose esters with β-lactoglobulin and β-casein from bovine milk

Food Hydrocolloids Vol.6 no.2 pp.173-186, 1992

The interaction of sucrose esters with from bovine milk

~-lactoglobulin

and

~-casein

David C.Clark, Peter J.Wilde, David R.Wilson and R.Wustneck Food Colloids and Biopolymer Science Department, AFRC Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK Abstract. The interaction of three high hydrophilic-lipophilic balance (HLB), water soluble sucrose monoesters with ~-lactoglobulin and ~-casein from bovine milk was studied by fluorescence titration and equilibrium dialysis techniques. The ~-lactoglobulin bound all forms of the sucrose ester investigated stoichiometrically with a single, comparatively high affinity site. In contrast, ~-casein bound the emulsifier substoichiometrically, possibly by interaction with ~-casein micelles. The interaction of lauryl and stearoyl sucrose esters with ~-lactoglobulin was stronger than with ~-casein. The affinity of binding increased with saturated fatty acid chain length for both proteins, with the stearoyl ester giving a dissociation constant of 2.3 u.M with ~-Iactoglobulin. However, the interaction of the monounsaturated oleic sucrose ester with ~-casein was marginally stronger than with ~-lactoglobulin.

Introduction

Many foods contain mixtures of different types of surface active ingredients. This class of molecules can be divided into two categories: macromolecular surface active species such as proteins, and low molecular weight emulsifiers such as sucrose esters. Often both types of molecules are mixed in the preparation of processed foods to obtain the desired properties in the final product and stability of the ingredients to processing conditions (1). The distribution of these two classes of surface active molecules between the interface and bulk phases is of importance in the control of formation, stability and texture of multiphase foods such as ice-cream and salad dressings (2). Proteins and low molecular weight emulsifiers not only compete for interfacial area (3), but in some cases can interact with each other non-covalently (4). The complexes that result can have completely different functional behaviour compared to that expected from the sum of the individual species. For example, their surface properties may be altered or they may possess novel properties not observed with the individual components. The principal area of exploitation of protein:emulsifier interactions in the food industry is in dough conditioning in bakery products (5). Anionic emulsifiers such as stearoyl-2-lactylates or organic acid derivatives of monoglycerides (i.e. diacetyl tartaric acid esters and succinylated monoglycerides) and hydrophilic non-ionic emulsifiers such as ethoxylated derivatives of monoglycerides or polysorbates are most widely used in this application (6). Inclusion of one of these dough conditioning agents results in improved dough processing characteristics, together with increased volume and a finer texture in the baked product. The mode of action of the emulsifier is primarily through interaction with the gluten proteins during dough mixing. Most other studies of the binding of surfactants by proteins have focused 173

D.C.Clark et al.

on interactions with non-food approved anionic (i.e. sodium dodecyl sulphate, (SDS), cationic (i.e. cetyltrimethylammonium bromide (CTAB) and non-ionic (i.e. Triton X-lOO and l3-octyl glucoside) detergents and has been reviewed recently (7). However, details of the interaction of the polysorbate emulsifier, Tween 20 with 13-lactoglobulin from bovine milk have been reported (4). In this paper we have investigated the interaction between selected milk proteins and the non-ionic emulsifier, sucrose esters. Bovine milk constitutes an important source of protein for the food industry as it contains -3.5% wt protein. Milk proteins may be divided into two fractions, the caseins and whey proteins (8). Casein and whey isolates from bovine milk are widely used in the food industry as a source of functional protein. The casein fraction constitutes nearly 80% of the total protein and may be separated from the whey proteins on an industrial scale by isoelectric precipitation. I3-Casein accounts for -35% of the casein fraction of milk and along with asl-casein is one of the major components. I3-Casein has a molecular weight of -24 000 daltons and an unusual primary structure with a high density of charged amino acids and five phosphorylated serine residues at the N-terminal third of the molecule (8). The remainder of the molecule has no net charge and has a high content of hydrophobic residues making this the most hydrophobic member of the casein family. As a result, isolated l3-casein shows a high propensity to self-aggregate to form micellar structures in solution. This behaviour is distinct from that observed in milk, where the complete family of casein molecules aggregate to form submicelles. These structures in turn interact, along with calcium phosphate, to form super aggregates called casein micelles. Isolated l3-casein forms only small micelle-like structures. I3-Casein is not a globular protein but rather has been shown to exist in a fairly random conformation containing only -10% a-helix and 13% l3-sheet (9). I3-Lactoglobulin is the main component of bovine whey where it accounts for -50% of the total protein-derived nitrogen or 12% of the total protein in milk (8). It has a monomeric molecular weight of 18400 and, in contrast to l3-casein, is a globular protein. Circular dichroism data indicate that it has a secondary structure composed of 16% a-helix, 58% l3-sheet and 25% random coil (10). Between pH 5.5 and 7.0, 13-lactoglobulin monomers interact to form a dimer (11). Between pH 3.5 and 5.2, dimers of 13-lactoglobulin, particularly the genetic variant 13-lactoglobulin A, tetramerize to form an octamer of molecular weight 147000. Of these two milk proteins, the ligand binding properties of 13-lactoglobulin have been most widely studied. With the exception of its cation (particularly Ca 2 +) binding properties, the interaction of l3-casein with ligands has been largely unexplored. There has been some speculation that the physiological role of 13-lactoglobulin involves interaction with (12) and transport of retinol (vitamin A, 13). Retinol is protected from enzymic oxidation in the one-to-one complex that it forms 13-lactoglobulin monomers. However, it is possible that the binding of retinol is non specific since 13-lactoglobulin binds other hydrophobic molecules, including protoporphyrin IX (14) and carbonyl compounds such as 174

Interaction of sucrose esters with milk proteins

alkanones and ketones (15). The affinity for ketones increases with chain length, suggesting that the association is predominantly hydrophobic in nature. The interaction of SDS and 13-lactoglobulin has been studied (16). However, investigation of the interaction of 13-lactoglobulin with low molecular weight food emulsifiers has been limited to studies with the polysorbate emulsifier, Tween 20 (4). It was demonstrated that adsorption of the complex formed by the interaction of these two components at the air-water interfaces of foams was responsible for their destabilisation. In this paper, we report a detailed characterization of the binding of three high hydrophilic-lipophilic balance (HLB), water soluble sucrose ester emulsifiers to l3-caseinand 13-lactoglobulin. An understanding of the interaction between these components was required before the results of a detailed investigation of the influence of complex formation on the processing properties and functional properties of these protein-emulsifier mixtures could be made. Materials and methods I3-Lactoglobulin (L-0130; 3x crystallized) and l3-casein (C-6905; minimum 90%) were obtained from Sigma Chemical Co. (Poole, UK). Sucrose esters (SE) were provided by the Mitsubishi Kasei Corporation (Yokohama, Japan). The three SE investigated were those of lauric (L-1695), stearic (S-1670) and oleic (01570) acids. The first two digits of the code numbers refer to the HLB value of the emulsifier. The second two digits refer to the minimum purity (%) of the fatty acid used in its manufacture. Therefore, the lauryl sample was prepared from at least 95% pure lauric acid, whereas the stearoyl and oleic samples were prepared from 70% pure stearic and oleic acid fractions. The fatty acid composition of the three SE samples was measured by gas chromatography. All the samples were found to be well within the manufacturers' specification. L-1695 and 0-1570 contained only minor levels of contaminating fatty acids. S-1670 contained ~25% of palmitic acid and 0-1570 contained 75% of oleic acid, 10% of linoleic acid and other fatty acids. Anthrone was General Purpose grade from BDH. Ethanol (99.7%) was obtained from J.Burrough (FAD) Ltd (Witham, UK). All other chemicals were 'AnalaR' grade and obtained from BDH Ltd (Poole, UK).

Estimation of SE concentration in solution The solution concentration of SE was determined by an anthrone assay (17,18). All glassware was cleaned by washing with chromic acid. Anthrone reagent was prepared by placing a solution containing 60 ml distilled water, 15 ml of ethanol (99.7%) and 0.4 g suspended anthrone on ice in a conical flask. Gradually 200 ml of concentrated sulphuric acid (sp. gr. 1.84) were added to this reagent with continuous mixing until all the anthrone was dissolved. The SE assay involved addition of 2.5 ml of this anthrone reagent to 0.5 ml of SE solution in a 10 ml sealable glass tube (Soviril tube). The solution was mixed well and incubated at 90°C for 10 min in a heated block containing oil to ensure good conduction of heat. The samples were then cooled on ice and transferred to a 175

D.C.Clark et al.

1 em spectrophotometer cuvette. The absorbance was read at 620 nm in a Perkin-Elmer Lambda 9 UV/visible spectrophotometer. A solution containing all the reagents except the SE was used as a reference.

Bindin g studies using equilibrium dialysis The dialysis cells that were used in this study contained 5 sample chambers per unit (Radleys , Sawbridgeworth, UK). Each cell was composed of two halves which could be bolted together , sandwiching a sheet of dialysis membrane. Acce ss to each half of the solution chamber was possible through a channel that could be sealed with stainless steel screws. Each half of the sample chamber had a volume of 1 ml. Small magnetic stirring bars were introduced into the half chamber containing only SE to accelerate equilibration but minimise the risk of protein denaturation. Regenerated cellulose dialysis membranes were prepared by boiling in distilled wat er for 30 min in the presence of a small quantity of ethylenediaminetetraacetic acid (EDTA) to complex any divalent metal ions. The membranes were then rinsed in cold water and refrigerated until used. The membranes had a nominal molecular weight cut off of 6000 daltons and a thickness of 0.073 mm. In a typical experiment, 4 of the 5 half-sample compartments were filled with solutions containing protein and SE . A calibration solution containing SE alone was placed in both halves of the final compartment , and was one of the solutions used to generate the calibration curve for the anthrone assay . The other 4 chambers were filled with solutions containing a fixed concentration of protein (typically 27.2 umol/dm' ) and variable concentrations of SE in one half chamber and a solution containing an identical concentration of SE alone in the other half. The chambers were sea led with screws and the cells placed on a magnetio stirrer for 20 h to ensure that tru e equilibrium was reached . A 0.5 ml sample was then removed from the half chambers that contained SE alone and assayed. The concentration of SE in this solution was equivalent to the concentration of free (unbound) SE in the chamber containing both SE and protein. The reduction in the concentration of SE observed in this chamber at equilibrium was related to the fraction of sites occupied on the protein .

Binding studies using fluores cence titration Th e interaction of the SE with the 13-lactoglobulin caused an enhancement of tryptophan fluorescence. Therefore, it was possible to measure the extent of association of SE with the protein by fluorescence titration. In a typical experiment, successive aliquots (5-10 ul) of the titrant solution containing SE {l0 urnol/drn') and 13-lactoglobulin (0.054 umol/drrr') in 10 mmol/drrr' sodium phosphate buffer, pH 7.0, were added to 1 ml of 13-lactoglobulin (0.054 umol' dm') in the same buffer contained in a semi-microfluorescence cell. Both titrant and sample solutions contained equivalent concentrations of protein to avoid dilution during the titration. After each addition the sample was gently mixed and the fluorescence emission resulting from excitation at 280 nm (slit 2.5 nm) was mea sured at 337 nm (slit 2 .5 nm) on a Perkin Elmer LS-5 luminescence 176

Interaction of sucrose esters with milk proteins

spectrometer. The data were recorded at room temperature. The titration was stopped when the fluorescence showed no further increase with added SE. The background fluorescence from the SE was small but was measured and subtracted from the data using a reference titration in the absence of protein. The fraction of occupied sites was calculated, as a function of added SE, from the observed fluorescence assuming that the plateau represented total saturation of the available binding sites.

Analysis of binding data In the simplest case, the interaction of SE with a protein may be described by the expression: P + SE

~

P.SE

(1)

where P, SE and P.SE are the protein, the SE and the complex formed between the SE and the protein. This binding is a reversible process and the strength of interaction between the SE and the protein is determined by the dissociation constant (Kd ) for the complex which is defined as: K _ [P].[SE] d [P.SE]

(2)

where the square brackets indicate the molar concentrations of the relative species. It follows that as the magnitude of K d decreases, the strength of the interaction between the SE and protein increases. The key to the determination of the magnitude of K d is the measurement or calculation of the relative concentrations of the three components P, SE and P.SE. It is also true that [P]

=

[P]tot - [P.SE]

(3)

and [SE]

= [SE]tot

- [P.SE]

(4)

where [P]tot is the total protein concentration in the system and [SE]tot is the total SE concentration. Substituting (3) and (4) in (2) gives: [P.sEf - ([SE]tot

+ [P]tot + Kd>.[P.SE] + [Ptot].[SE]tot = 0

(5)

which is a quadratic equation and can be solved for [P.SE]. The binding data can be fitted to this equation provided only a single binding site is active. Alternatively, it is possible to obtain the dissociation constant from equilibrium dialysis or fluorescence titration data using the Scatchard (19) equation:

v/[L]

= (n - v)/K d

(6) 177

D.C.C1ark et al.

A Scatchard plot of v against v/[ L] can be prepared, where v is the fraction of protein with occupied binding sites (i.e. [P.SE]/[P]tot) and [L] is the concentration of free ligand (i.e. 5E). If a straight line is obtained with this plot it indicates that there is only one type of binding site. The gradient of this line is -IIK d , where K d is the dissociation constant. The intercept on the x-axis gives the number of binding sites , n. However, some care is needed in the interpretation of n since in the case of the fluorescence titration the analysis is based on the assumption that only one binding site is present. In the experiments reported here, we followed the analytical pathway shown below. Firstly, a plot of the raw data provided an indication of the type of binding present. Typically , we plotted fractional occupation of the protein binding sites (v) as a function of total ligand concentration . This allowed identification of the binding plateau where saturation of the binding sites occurred. A Scatchard plot was then generated (plotting v against v/[ L]). If a straight line was obtained with this plot it indicated that one class of binding site was present and the gradient of the line gave the K d . In this case, the raw data were fitted to the quadratic equation (5), and agreement was checked between th e result of the Scatchard plot and the value of K d obtained from the quadratic equation. If the Scatchard plot did not give a straight line, the shape of the curve obtained could be used to identify whether the observed binding was positively or negatively cooperative . In the former case , the Hill equation was used to determine the K d and cooperativity coefficients (20) . In contrast, a negatively curved plot signified independent sites.

Results The concentration of SE in a given solution was determined using a colourimetric assay based on the anthrone reaction with sugar. Calibration graphs of absorbance at 620 nm against SE concentration were obtained for L-1695 , S-1670 and 0-1570 SE s. The lower limit of sensitivity was established to be -5-10 umol/drrr' SE. The upper limit using 1 ern pathlength spectrophotometer cells was in the order of 800-1000 umol/drrr' SE. The assay was used to determine the concentration of SE present in samples from equilibrium dialysis experiments investigating the binding of SE to protein, In anticipation of this work , a control experiment was performed to assay samples of SE of known concentration that had been incubated for 10 h at room temperature in the equilibrium dialysis cell. The gradients of the calibration plots from dialysed SE samples were reduced compared with calibration plots from samples that had not been in the equilibrium dialysis cells. This is illustrated in Figure 1 for L-1695. This was consistent with adsorption of SE to the wall s of the dialysis cell or the dialysis membrane. This adsorption increased line arly with the concentration of SE in the concentration range of interest Therefore , in the equilibrium dialysis experiments that follow , the Sf concentration was determined by referring to a calibration graph generated from control samples that had been incubated in the dialysis cells.

178

Interaction of sucrose esters with milk proteins

0.6 0.5 0

0.4

'" CD

-e 0.3 0.2 0.1 0.0 0

100 (L-1695]

200

300

( p M)

Fig. I. Typical calibration lines obt ained for the anthrone assay of the sucrose ester, L-1695. (.), L-1695 that was not incubated in a dialysis cell; (+), L-1695 that had been incubated for 10 h at room temp er ature in the dialysis cell. Th e difference in the gradi ent s of th e lines is due to adsorption of SE onto the.walls of the dialysis cell.

Binding studies with r>-lactoglobulin

Initially the binding of L-1695 to r>-Iactoglobulin was studied by equilibrium dialysis and compared with the fluorescence titration data for the same system . The results were in excellent agreement. Subsequent mea surements were performed using the more reliabl e fluorescence method. The Scatchard plots for the binding of the SEs , L-1695, S-1670 and 0-1570 are shown in Figure 2. The plots gave straight lines and the dissociation const ants calculated from the gradients of the plots are shown in Table I. The slopes of the plots showed that S-1670 was bound with the greatest affinity, followed by L-1695 and 0-1570. The se data indicated that 13-lactoglobulin posse ssed a single class of SE binding site. Therefore, the raw data were reanalysed by fitting to the quadratic equation (5). Plots of the titration data (symbols) and the calculated fits (solid lines) from equation (5) are shown in Figure 3. The average Kds calculated from at least 3 repeat experiments using this method for the different SEs are shown in Table I. Binding studies with l3-casein

Studies of SE binding to l3-casein were performed by equilibrium dialysis. Measurements of binding by fluorimetric titration were unsuccessful. This was probably because binding of the SE did not change the environment of the fluorescent amino acids in this protein. Scatchard plots of the results again produced straight lines (Figure 4). The Kds for the different SEs were again 179

D.C.Clark et al.

0*100l-~~::=~~~~~~~~~~~

0.0

0.25

0.5 v

to

0.75

Fig. 2. Scatchard plots for the bind ing of SE to 13-lactoglobulin. The differ ent SEs are identified by the symbo ls; (+) , S-1670; (0 ). L-1695 and (x ), 0-1570. Table I. Dissociation consta nts fo r prot ein -SE complexes Prot ein

SE

Number of sites

13-1actoglobulin

L-1695 S-1670 0 -1570 L-1695 S-1670 0-1570

1 1 1 0.3 0.7 0.6

l3-casein

Dissociat ion constants (umol/drrr') Fitted" Scatchard'' 6.9 2.3 26.5

1330.0 124.1 17.4

11.6 1.02 24.8 480.0 69.2 27.5

" Fitted K d ave raged from at least 3 det erminations. b K d fro m Scat chard plot s shown in th is paper.

determined from the gradients and are summarised in Table I. As these results indicated that SE binding by this protein was not cooperative the equilibrium dialysis data were reanalysed by fitting to equation (5). Plots of the dialysis data (symbols) and the calculated fits (solid lines) are presented in Figure 5. Average Kd values returned by this analysis are summarised in Table I. There was some difference between the fitted and Scatchard values of the Kds quoted in Table I. It should be stressed that the fitted Kds are averages of several titration or dialysis experiments. In contrast , the Scatchard Kds were those values calculated from the gradients of plots of the data presented in this report. Averaging of the Scatchard Kds provides values that are in better agreement. However , the data shown are in rea sonable agreement and very consistent for binding studies . 180

Interaction of sucrose esters with milk proteins

0.75 >

0.5

0.25 x

5

15

10

20

25

x10-S [SE]

(M)

Fig. 3. A plot of the titration data for the binding of SEs to 13-lactoglobulin along with the calculated fit to equation (5). The data for the different SEs are distinguishable by the symbols where (+) is S-1670, (.) is 0·1570 and ( x) is L-1695. The calculated fits to the data are shown by the solid lines through the data sets.

0.0*10o-h~~~~W""''''''''''''''''r-T'"I-r-r .............-r........~~~ 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 v Fig. 4. Scatchard plots for the binding of SE to l3-casein. The different SEs are identified by the symbols; (
181

D.C.Clark et al.

0.7...r------------------..,

>

0.3

0.2 0.1

2.5

5.0

7.5

10.0

12.5

[SE] x10- S M Fig. 5. A plot of the titration data for the binding of SEs to ~-casein along with the calculated fit to equation (5). The data for the different SEs are distinguishable by the symbols where (+) is S-1670, (0) is 0-1570 and (x ) is L-1695. The calculated fits to the data are shown by the solid lines through the data sets.

Calculation of solution composition from binding data Equation (5) can also be used to calculate the relative concentrations of species (i.e. free protein, free SE and protein:SE complex) present in solution at given total protein and SE concentrations. Examples of the change in composition of solutions containing a constant protein concentration of 0.2 mg/ml (0.02% wt) as a function of increasing SE concentration are shown in Figure 6 for ~-lactoglobulin and Figure 7 for ~-casein. The difference between the binding properties of the two proteins is clear from Figures 6 and 7. The tight binding of S-1670 by ~-lactoglobulin (Figure 6a) results in the complex being the predominant component in solution between 8 umol/drrr' and 17 umol/dnr' SE. It is only at S-1670 concentrations above 17 urnol/drrr' that the free SE becomes the dominant component. At this point the protein was 77% saturated. The weak binding of L-1695 to ~-lactoglobulin means that the complex is never the dominant component in solution (Figure 6b). Under the conditions shown here, there were almost equivalent amounts of the three species at 11 umol/drrr' L-1695. In contrast, the weak interaction between ~-lactoglobulin and 0-1570 means that very little of the added SE complexed with the protein (Figure 6c). A similar picture is seen with ~-casein and 0-1570 (Figure 7c). At the point where there were equivalent amounts of free ~-casein and complex, only -20% of the added 0-1570 had been bound. 182

Interaction of sucrose esters with milk proteins

2.0.10-'

a

c a 1.5'10-'

~

Ca>

1.0.10-'

o

c 0

o

5.0.10'"

0.0.10· 0.10·

2.0'10-'

2.10-' 1.10'" Total [5-1670]

3.10-'

1.10'" 2.10'" Total [L-1695]

3.10'"

b

.sc 1.5'10-' ~c

a>

o

c

1.0.10-'

0

u

5.0.10'"

0.0.10· 0.10·

4.10'"

C

~ 3.10'"

~

~ 2.10-' c 0

u

1'10-' .

0.10· 0.10·

1.10'"

2.10'" 3.10'" Total [0-1570]

4.10-'

5.10'"

Fig. 6. The calculated composition of solutions containing 13-1actoglobulin (0.2 mg/ml) in terms of relative concentrations of free protein (+), free SE (0) and complex (x) as a function of SE concentration. a S-1670, b L-1695 and c 0-1570.

Even weaker binding was shown in the presence of S-1670 (Figure 7a) and negligible binding of L-1695 by l3-casein is observed after addition of 50 umol/ drrr' SE (Figure 7b). 183

D.C.Clark et al.

a

4.10'"

0.10°-+:~;"";:ltj==r:l=T~~~~:;=::;::~~;::;~~ 0.100

2.10'" 3.10'" Total [5-1670]

4.10'"

5.10'"

' -r---------------""JI)

5.1O...

0.100 0.10"

4.10'"

1.10'"

2.10'" 3.10'" Total [L-1695]

4.10'"

5.10'"

1.10'"

2.10'" 3.10'" Total [0-1570]

4.10'"

5.10'"

C

c: 3.10'"

0 :;> 0

1 8

2 •10'" 1.10'" 0.100 0.100

Fig. 7. The calculated composition of solutions containing 13-casein (0.2 rng/ml) in terms of relative concentrations offree protein (+), free SE (0) and complex (x) as a function of SE concentration. a S-1670, b L-1695 and c 0-1570.

Discussion

The results show that in general, l3-lactoglobulin had a greater affinity for SE than the l3-casein. The main exception to this was the unsaturated SE, 0-1570, which bound comparatively tightly to l3-casein. The affinity of both l3-casein and 184

Interaction of sucrose esters with milk proteins

~-lactoglobulin for saturated SE (S-1670 and L-1695) increased with chain length. This is consistent with the binding being of a hydrophobic nature. The binding site on ~-lactoglobulin did not bind the unsaturated 0-1570 as effectively as the saturated SEs. In contrast, ~-casein bound 0-1570 with the highest affinity. A single binding site was identified on each ~-lactoglobulin monomer. This observation agrees with previous work where a single Tween 20 binding site was detected per monomer (4,21). In addition, studies of the interaction of SDS with ~-lactoglobulin resulted in the identification of a single high affinity binding site along with 11 electrostatic binding sites (16). It is possible that the SE binding site is distinct from the retinol binding site. It is likely that the SE binds at the same site as Tween 20 given the similar structures of these molecules. A previous competitive binding study involving investigation of the interaction of Tween 20 with ~-lactoglobulin pre saturated with retinol showed no inhibition of Tween 20 binding (21). Inhibition was expected if both Tween 20 and retinol bound at the same site, since the affinity for retinol is considerably greater than for Tween 20 (12,13). However, it is possible that the site is shared by both molecules. The observed increase in the intrinsic fluorescence upon binding of SE is consistent with either the close proximity of the fatty acyl chain of the SE to a fluorescent tryptophan side chain of the ~-lactoglobulinor a conformational change in the protein resulting in the movement of sidechains to create a more hydrophobic environment around the fluorescent aromatic sidechain. Studies of the 3-dimensional structure of ~-lactoglobulin obtained from X-ray crystallographic studies have lead to the suggestion that the retinol binding site is located in the ~-barrel structure of ~-lactoglobulin. Trp-19 which is located at the base of this structure has been implicated in the binding of the ~-ionone ring of retinol (13). If this is correct it is likely that the change in fluorescence of ~-lactoglobulin upon interaction originates from a change in the environment of this tryptophan. Examination of the published stereoscopic image of the 13-lactoglobulinstructure suggests that it is unlikely that the remaining tryptophan residue (Trp-61) which is located in an exposed loop on the surface of the protein molecule participates in the binding process. In the case of l3-casein, the results showed <1 molecule of SE interacted with each l3-casein monomer. The precise level of interaction was dependent on the type of SE studied. One possible explanation of this observation was that SE interacted only with free l3-casein monomers or ~-casein micelles, but not both species. This is explained in expression (6).

[Monomer.SE] <---> [Monomer] <--> [Micelle] <---> [Micelle.SE] (6) 3 1 2 4 Solutions of l3-casein contain species 111 and 121 in equilibrium. If SE binds to the monomer 111, then addition of SE leads to formation of species 131. This will deplete the concentration of 111 and cause some dissociation of 121 to form more 111. Alternatively, if SE binds to the micelle, then addition of SE will lead to formation of species /41. This will deplete the concentration of micelle 12/ that 185

D.C.Clark et al,

contains no SE and cause some monomer 111 to aggregate to form more micelles 121. It is not possible to determine whether interaction with SE results in production of species 131 or 141 from our existing data. We are currently exploring the possibility of monitoring changes in the amount of micelle present as a function of binding of SE using photon correlation spectroscopy. Knowledge of the K d allows calculation of the solution composition for any given protein and SE mixture. This is extremely useful in determining the mechanism of action of SEs in a variety of roles. For example, there is evidence that interaction of SEs with proteins reduces thermally-induced protein aggregation. This observation is supported by the results of preliminary experiments on the thermal stability of 13-lactoglobulin in the presence and absence of SE, as determined by near-UV circular dichroism. We intend using the binding data presented here to interpret this effect. In addition, the binding data will underpin the elucidation of the mechanisms underlying the observed changes in stability of dispersions containing SE in the presence of l3-casein or 13-lactoglobulin. Acknowledgement The authors gratefully acknowledge that this work was funded by the Mitsubishi Kasei Corporation of Japan. References 1. 2. 3. 4. 5. 6. 7.

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