Food Hydrocolloids Vol. 10 no. 3 pp.323-328, 1996

Association tendency of ~-lactoglobulinAB purified by gelpermeation chromatography as determined by dynamic lightscattering under quiescent conditions*

Mallika Sharma, Zahur U.Haque! and William W. Wilson2

Department of Food Science and Technology, Mississippi Agricultural and Forestry Experimental Station, Box 9805,Mississippi State University, MS 39762 and 2 Department of Chemistry, Mississippi State University

I To whom correspondence should be addressed

AbstractAssociation properties of f3-lactoglobulin AB (f3-Lg) fractionated by gel permeation chromato­graphy (GPC) was studied using dynamic light scattering (DLS) at a concentration of5% w/vandpH 7.0 from 25 to 70°e. f3-Lg fraction with a molecular weight of 18.4 kDa by GPC (monomeric)showed self-association at 25°e. At 25°C ~58% of the protein had an apparent mean diameter<10 nm and the rest between 10and 100 nm. On heating to 35°Call the protein existed as monomersand dimers. With further heating association increased; large particles with an apparent size of100­599 nm were seen >45°C indicating that some degree of denaturation occurs even at 45°e. Theamount of aggregate <10 nm in size decreased sharply >65°e. Data indicate that f3-Lg that wasmonomeric during GPC, where a linear velocity gradient may exist, was associated even at 25°Cunder quiescent conditions of the DLS experiments. Association increased progressively withtemperature and denaturation, as observed by the formation of large aggregation, starting at 45°e.

Introduction

The structural characteristics of a variety of food systemsare related to the physico-chemical phenomena of aggre­gation, coagulation and/or gelation. These phenomena arethe manifestations of protein denaturation processes anddetermine the functional properties of a protein (1).

Conformational changes in proteins, i.e. denaturation,depend not only on temperature, but on the total amount ofenergy (heat) added to the system. Hence, heating time,heating rate, temperature and the amount of proteinpresent will determine the extent of denaturation and thusaffect formation of protein gels (2).

Whey is a nutritively rich by-product that originates fromthe manufacture of cheese. Whey proteins constitute -20%of total milk proteins (3). Of the two gelling proteins inwhey, ~-lactoglobulin (~-Lg) and bovine serum albumin(BSA), ~-Lg is present in much higher concentrations thanBSA, and is considered to be the primary gelling proteinand presumably contributes to the typical behavior of wheyprotein concentrates (WPC) (2,4,5).

Although factors affecting gel formation are well docu­mented (2), there is a paucity of studies tracing changes in

• Approved as MS #18564 of the Miss. Agric. Forest. Exp. Station.

© Oxford University Press

the molecular size (i.e. building blocks) leading to gelformation. Hence, a study of aggregate formation leadingto gel network structure can help elucidate basic 'buildingblocks' leading to network structure. This can help usmanipulate desired characteristics of a gel.

At pH values near its isoelectric point (i.e., pH 5.2) andup to the pH of milk (6.8) and at room temperature, ~-Lg

reportedly exists as a stable dimer with a mol. wt of36.7 kDa (6). Hydrodynamic and small X-ray scatteringsupport a structure of 2 spheres with a diameter of 3.6 nmimpinging by 0.2-0.3 nm with an axial ratio of -2. Thedimer is a prolate ellipsoid with an overall length of6.95 nm and a width of 3.6 nm. There is a dyad axis ofsymmetry at the point of contact of the monomers (6).Formation of dimers, trimers and higher oligomers wasreported to be more extensive at pH 7.5 than at pH 6.5 (7).

Haque et al. studied the association/dissociation behaviorof unheated ~-Lg, k-casein and ~-Lg/k-casein mixtures atpH 6.8 and 25°C using gel permeation HPLC (8). Theyfound that upon addition of glycol ether diamine tetraaceticacid (EGTA), ~-Lg, which normally existed as dimer,existed in equilibrium between dimeric and trimericcomplexes.

324 M. Sharma, Z. U.Haque and W. W. Wilson

Heat aggregation of I3-Lg reported to begin at 70°C, wasfound to be largely influenced by pH and protein concen­tration (9). This was substantiated by Haque and Kinsella(10), who observed using 1-anilinonaphthalene-8-sulfonate(ANS) tagging studies, that I3-Lg denatured at 70°C.However, De Wit and Swinkels reported a denaturationrange of 60-90°C (11).

Thermal aggregation of I3-Lg has been monitoredbetween 25 and 96°C (12-16). Available studies have usedtechniques such as sedimentation equilibrium, differentialscanning calorimetry (DSC) and/or electrophoresis that canintroduce artifacts and tend to dissociate facile interactions,as these are invasive/perturbing techniques.

Dynamic light scattering (DLS) is being increasingly usedto determine molecular size, hydrodynamic radius andkinetics of aggregation in biopolymers (17,18). Lightscattering uses the fact that the amount of light scattered(i.e. scattered intensity) by a single dissolved molecule orsuspended particle is proportional to the vol ' of themolecule/particle. The volume of the particle is related toexcess scattering intensity through concentration. Thus,light scattering is used not only for determination of sizeand shape of single molecules and stable particles but alsofor investigating aggregation and association effects,micelle formation and growth or dissolution of conden­sation nuclei in precipitating systems (19,20). This tech­nique is non-perturbing (non-invasive, in situ) and simple(21). It provides clear 'real time' data under appropriateconditions.

The present study was undertaken to observe changes inassociation behavior on stepwise heating of I3-Lg, fraction­ated by GPC , from 25 to 70°C at pH 7.0 using DLS. Suchobservations can give useful information on the buildingblocks leading to post-denaturation aggregate sizes, andpost-denaturation aggregates ultimately determine themicrostructure of gels that in turn affect rheologicalproperties.

Materials and methods

I3-Lg was isolated from whey protein concentrate, obtainedfrom Mississippi State University dairy plant, by themethod of Mailliart and Ribadeau-Dumas (22). The pre­cipitate containing I3-Lg was dialyzed against deionized,glass distilled water (10 000 fold) and freeze dried. Theprotein was stored in a desiccator at 4°C until use.

The purity of the isolated I3-Lg was established usingvertical slab sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE) according to the method ofHaque and Mozzaffar (23). I3-Lg was shown to be a mixtureof variants A and B.

The presence of self-associated oligomers in the native 13­Lg was ascertained by analytical Gel Permeation Chro­matography (GPC) using a 'Phenomenex' BIOSEP-SEC­S3000 column (300 x 7.8 mm; Phenomenex, Torrance,CA). A Waters 510 pump forced solvent through a

universal injector (U6K) to a differential refractometerdetector (Waters R40 I). The elution buffer used was50 mM sodium phosphate (pH 6.8) with 1% (w/v) sodiumchloride and 0.02% (w/v) sodium azide with a flow rate of1 ml/min.

As GPC showed the presence of a small amount of large­sized oligorners, the protein was fractionated on a prep­arative column 'Phenornenex' BIOSEP-SEC-S3000 (600 x21.2 rnrn: Phenomenex) using the elution buffer as above.One millilitre of 10% (w/v) I3-Lg was injected during eachrun. The fraction corresponding to a molecular weight of18 400 was collected. The fraction was dialysed exhaust­ively against 10 mmol/dm' imidazole buffer (pH 7.0) andthen concentrated in a stirred ultrafiltration AMICON cell(Model 8010; Amicon Corp., Lexington, MA) using aAMICON 'YM 10' membrane. The protein concentrationin the retentate was measured using E"X,.lcm2XO = 9.6 (24).

The concentrated fraction was centrifuged (Eppendorf'.Model 5415C) at 8000 r.p.m. for 10 min. The supernatantwas filtered to exclude dust by circulating for 10 minthrough a closed sample loop directly into the scatter cellusing a peristaltic pump (Rainin Co. Inc., Woburn, MA).The closed loop consisted of an inert tubing, 0.2 IJ.-m lowprotein affinity filter and a water-jacketed scatter cell(Hell rna Model No. 165). The actual protein concentrationin the sample cell was measured as described above.

The DLS assembly consisted of a laser source (Jodon He­Ne laser, Model HN-50) with emission wavelength of632.8 nm. The laser beam was focused onto the scatteringcell, mounted on a goniometer, by means of a mirror andsystem of lenses. Light scattered at an angle of 90° wascollected and focused by means of a 10 cm lens onto aphotomultiplier tube through a fiber optic cable. Thenormalized autocorrelation function was measured by usinga digital correlator (BIC 2030AT Brookhaven InstrumentsCorp., Ronkonkoma, NY) with 72 real time channels and 4delay channels. Details of the method are given elsewhere(25).

The sample, obtained as described above was filteredinto the scattering cell to give a final concentration of50 mg/m!. The sample was heated from 25 to 65°C in stepsof 10° and then to 70°C by maintaining the sample at therespective temperature for 5 min. Water at the desiredtemperature was circulated through the cell for exactly5 min and then water circulation was stopped to ensure thatthe sample was subjected to equal heating time at eachtemperature. Autocorrelation functions were measured ateach temperature immediately after stopping the watercirculation. The data was saved on diskettes and lateranalyzed by Cumulant and CONTIN analysis programs inthe software that accompanied the correlator. The corre­lation function (26) from DLS is given by

C(milT) = B[1 + b exp(-2r milt)] (1)

where m = channel number, ilt = sample time, B =baseline (and is the uncorrelated flat portion of the curve),

Association of fractionated f3-lactoglobulin 325

b = optical constant , r = line width. The diffusioncoefficient (in cm2/sec) is calculated from

where In(gC1l(K ,t)] is the normalized intensity correlationfunction and K 1, K2 and K;I are the first, second and thirdcumulants (or moments).

(37) . The method runs through two cycles, the first anunweighted analysis of the data to select a trial set ofparameters, and the second a weighted analysis to select the'best fit' set of parameters. The CaNTIN analysis programprovides numerical information and distribution plots ofsize distribution.

Results and discussion

The GPC profile of the J3-Lg obtained on the analyticalcolumn showed a major peak corresponding to a molecularweight of 18.4 kD (Fig . 1) which was collected for the DLSexperiment and concentrated to -6% w/v by ultrafiltration.This concentration still gave a monomeric peak with a veryminor oligomeric content (Fig. 2).

The apparent particle/aggregate sizes estimated by theCaNTIN program were arbitrarily divided into fiveaggregate sizes: Aggregate 1 (1-9 nm), Aggregate 2 (10­99 nm), Aggregate 3 (100-599 nm), Aggregate 4 (600­999 nm) Aggregate 5 (>1000 nm). This was done tosimplify the results so that trends could be seen andquantified.

Cumulant analysis showed that GPC fractionated J3-Lghad an apparent mean diameter of 11 nm at 25°C (Fig. 3).J3-Lg reportedly exists as a prolate, ellipsoid dimer with alength of -7.0 nm and a width of 3.6 nm (28). A meandiameter of 11 nm indicates that despite removing theassociated oligomers. the monomeric ~-Lg (by GPC) wasself-associated to some extent in unperturbed solution . Thisfraction was termed the minimally associated state . Onheating to 35°C the mean diameter drops to -6 nm, i.e . theself-associated particles dissociate and the protein existsmostly as dimers. Further heating to 45°C caused theprotein to re-associate, the rate being slow up to 55°C andsharp increasing thereafter . At 70°C the mean diameter was14 nm.

CaNTIN analysis (Table 1 and Fig. 4) confirmed theself-association tendency in a J3-Lg shown by cumulantanalysis. At 25°C, 54% of the fractionated protein had anapparent mean diameter ranging from 3 to 9 nm (Aggre­gate 1), while 40% lay in the range of 10-99 nm (Aggregate2). At 35°C, however, 96.5% existed as Aggregate 1 with amean diameter of 3-9 nm. As the temperature wasincreased, the amount of Aggregate 1 decreased to 90% at65°C. At the denaturation temperature of 70°C the amountof Aggregate 1 decreased sharply to 60% with a concomit­ant increase in Aggregates 2 and 3. Although the decreasein the amount of Aggregate 1 is marginal until 65°C, theincrease in amounts of Aggregates 2 and 3 seen at 65°C andabove account for the sharp increase in mean diameter at65°C and above, seen in the cumulant analysis (Fig. 4) .

Polydispersity values also reflected the trend shown bycumulant and CaNTIN (Fig. 5). The sudden decrease at35°C reflected the dissociation of aggregates at this tem­perature. Polydispersity values increased at 65°C and aboveas a result of the increased association.

The presence of aggregates of size 10-100 nm at 25°C

(3)

(2)

where k = Boltzmann constant, T = absolute temperature,'Tl = viscosity of the solvent and d = hydrodynamicequivalent spherical diameter of the scattering particles.

Cumulant method of data analysis gives the mean/z­average diameter (also termed the hydrodynamic equiv­alent spherical diameter) and polydispersity (27). By thistechnique the correlation function is expanded about anaverage line-width, raw. This method is based on a series ofexponential functions comprising the normalized intensitycorrelation function and is given as a power series in t:

Polydispersity

Polydispersity obtained from cumulant analysis is given byKiK I

2 (second moment/(first moment)" (20). The poly­dispersity is an approximate measure of the degree ofaggregation. A monodisperse sample gives a polydispersityvalue of 0.02 (20).

where DT = translational diffusion coefficient , q = scatter­ing wave vector given by q = (4-rrn/,,-0) . sin 0/2 where, n =index of refraction of the solvent, "-0 = wavelength ofincident light and 6 = scattering angle .

The apparent mean diameter is calculated from theStokes-Einstein equation

CONTIN

The CaNTIN method developed by Provencher (20 ,27), isa regularization method that gives percentile size distri­bution of particles/aggregates. In this method a grid of line­widths is laid out with equal spacing in logff') and apreliminary unsmoothed solution is sought. Thereafter, apenalizing function (regularizer) is added to the model andadditional solutions are sought with increasing weight of theregularizer. For each solution, a statistic (Fisher test), 'aprobability of reject' is calculated by comparing the sum ofsquared residuals for this degree of regularization and thatobtained in the preliminary calculation. The regularizerused is based on the sum of second derivatives of G(r) withrespect to r. The solution for which the probability of rejectis closest to 0.5 is recommended as the 'chosen solution'.This method assumes a relationship between adjacent andneighboring points in the distribution function i.e. acontinuous distribution of amplitudes as described by G(T)

326 M.Sharma, Z. U.Haque and W. W. Wilson

Iact ~-Iactoglobulin

GPC-PRO 3.11

2.00

1.50

N+o~ 1.00

.500>::E

...J<Zl!I

:il -.000

CHROMATOGRAM

60.040.020.0- . 500 +--+~_---<f---+---l--+---t--+-_---<f---+--+---+---t--+-_---If---+-~

.000 80.0

RET VOL (ml)

Figure I GPC profile of ~-Iactoglobulin AB. The column used was 'Phenornenex' BIOSEP-SEC-S3000 (300 x 7.8). Buffer used was 50mmol/drrr' sodium phosphate buffer with 1% (w/v) NaCI and 0.02% (w/v) NaN 3 in a Waters system with RI detector.

CHROMATOGRAM

.000

1.00

lact-2 ~-lactoglobulin after conen:GPC-PRO 3.11

2.00

N+o....x

>::E

...J«zC!l....CIl

1

64.016.0-1. 00 +--+---+---+--+----+---+----1-1--+----1--1---.,If----+----+-l--+---+----+-----.,

4.00 28.0 40.0 52.0

RET VOL (mIl

Figure 2 GPC profile of GPC fractionated ~-Iactoglobulin AB after concentration by ultrafiltration. Conditions were as in Figure I.

Association of fractionated f3-lactoglobulin 327

8535 45 55 65 70TEMPERATURE ('C)

0.10 +--"'T"'--,----,.--,...-.-----,25

0.38

0,14

0.42

0.34>I-iii 0.30a:w3iO.26is>5°·22II.

0.18

85

CUMULANT ANALYSIS

45 55 65 70TEMPERATURE (·C)

355-+-----T---;--""""T--..,....-r-------,

25

20

Table J Change in percentile distribution of aggregates withtemperature in GPC fractionated ~-Iactoglobulin at pH 7.0 1

Figure 4 Percentile size distribution of ~-Iactoglobulin ABaggregates with temperature. Conditions were as in Figure 3. Thesize distribution was estimated by CONTIN analysis.

Figure 3 Association tendency of ~-Iactoglobulin AB fraction­ated by GPC with temperature at pH 7.0 as seen by dynamic lightscattering. Buffer used was imidazole, 10 mmol/dnr' pH 7. Thesample was heated from 25 to 700C. Apparent mean diametersfrom cumulant analysis arc plotted against temperature.

Figure 5 Polydispersity obtained from cumulant analysis isplotted against temperature. Conditions were as in Figure 3.

(-42%), even after removal of oligomers, as seen fromCONTIN analysis shows that ~-Lg undergoes a facileinteraction when undisturbed in solution. The stability of~­

Lg solution at 25°C is partly maintained by entropic forcesas a result of the ordering of water molecules (firsthydration shell) around the hydrophobic patches on itssurface (29-33). In some proteins, even on folding, somehydrophobic clusters remain exposed on the surface (34).Wishnia and Pinder reported the presence of hydrophobicpatch thought to be the binding site for hydrophobic ligands(35).

With increase in temperature, there is less ordering ofwater around these hydrophobic patches. Increase inhydrophobic interactions with temperature favor an in­crease in free energy associated with a decrease inaccessible free surface area (or surface area that isaccessible to water) when proteins associate (29). This isperhaps the driving force resulting in progressive associ­ation which was seen when ~-Lg is heated up to thedenaturation temperature of 70°C.

It is thought that the stability of a protein-proteincomplex results from balance of large free energy contri­butions of different signs. The free energy term favoringassociation is derived from a decrease in accessible freesurface area (or surface area that is accessible to water)when proteins associate (29). Association confers greaterthermodynamic stability with aggregate size. This con­ceivably explains the progressive association that is seenwith temperature as seen from cumulant, CONTIN andpolydispersity values.

The appearance of Aggregate 3 (100-599 nm) at 45°Csuggests that some denaturation (with subsequent aggre­gation) begins to occur at this temperature. Around 70°C ~­

Lg undergoes irreversible and rapid denaturation (36)causing exposure of its hydrophobic interior. This is seen inthe sudden decrease at 70°C of the small aggregate size (I­to nm) with concomitant increase in aggregates of highersizes, especially Aggregate 3 (100-500 nm). The persist­ence of aggregate size (to-l00nm) throughout the tempera-

CONTIN ANALYSIS

e Agg.l (1-9 nm)* Agg.2 110-99 nm)• Agg.3 1100-599nm)• Agg.4(600-999 nm)• Agg.51 >1000 nm)

35 45 55 65 70 75TEMPERATURE I'C)

20 25-10

100

10

80

90

zQ 70...JCD 60a:...UI 50o~ 40...ffi 30offi 20n,

Temp Agg. I Agg.2 Agg.3 Agg.4 Agg.5eq

25 57.9 42.1 0 0 035 98.2 1.8 0 0 045 89.8 7.3 3.0 0 055 94.0 4.4 1.1 0 065 90.7 3.9 5.3 0 070 62.1 22.3 15.6 0 0

I Contin analysis.

328 M.Sharma, Z. U.Haque and W. W. Wilson

ture range studied seems to suggest it is an intermediate inaggregation to larger sizes.

In conclusion, the data indicated that under unperturbedconditions 13-Lg, which was monomeric by GPC, showsself-association even at 25°C and this progressively in­creased with temperature until the reported denaturationtemperature of 70°C.

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Received on October 13, 1994; accepted on August 16,1995