Langmuir 1993,9, 242-248
242
A Neutron Reflectivity Study of the Adsorption of &Casein at Fluid Interfaces E. Dickinson,+D. S. Horne,*J J. S. Phipps,g and R. M. Richardson1 Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, U.K., Hannah Research Institute, Ayr KA6 5HL, U.K., and School of Chemistry, University of Bristol, Bristol BS8 ITS, U.K. Received June 3,1992. In Final Form: September 16,1992 Direct information on the thickness and structure of adsorbed protein layers at owwater and &/water interfacea is necBB881y for a full understanding of the role of proteins in the stabilization of emuleione'and foams. Thie paper shows that the technique of specular neutron reflectivity can reliably give such information. By UBB of the CRISP instrument at the Rutherford-Appleton Laboratory (Oxford, U.K.), measurements of neutron reflectivity have been made for monolayers of the disordered milk protein &casein at the n-hexan-ter and airwater interfaces. For a bulk protein concentration of 6 X 10-8 w t 9% ,the surface concentration at the air/water interface is estimated as 3.8 mg m-2 and the firet moment of the segment deneity distribution is calculated to be 3.1 nm with the origin at the water surface. Based on modeling techniques to obtain best-fit segment deneity profiles, we find that the distribution of protein normal to both oil/water and air/water interfaces is well deecribed by a dense inner layer of ca. 2 nm thickness directly at the interface and a more tenoue secondary layer of thickness 6 7 nm extending into the aqueoua phase. This structural description of the adsorbed &casein layer is broadly coneistent with recent measurements of film properties derived from adsorption isotherms, dynamic light scattering, and proteolytic digestion of the admrbed protein. nantly nonpolar and very hydrophobic, making the @-caseinmolecule as a whole distinctly amphiphilic.' A considerable body of information on the adsorption behavior of &casein has accumulated. The first and original measurements of proteins at interfaces related only to the adsorption isotherms. Recent isotherm measurements on &casein by Hunter et aL2 largely confirm and extend the earlier studies of Graham and Phillip~.~*~ From their calculations of the average area per molecule at plateau adsorption, Hunter et aL2infer that perhaps one-third of the protein molecule is oriented out from the surface of the interface. This behavior is consistent with values of 10-15 nm for the hydrodynamic layer thickness measured using photon correlationspectroscopy when the @-caseinis bound to polystyrenelatex particles? implying that portions of the adsorbed molecule project into the solution for a considerable distance. Somewhat smaller values, namely 5'3 or 9 11131,' are obtained when layer thickness is measured by ellipsometry. In studies of polymer adsorption,informationregarding the orientation of the molecule at the interface, in particular the fraction of segments entrained close to the surface, is deduced from measurements of reducedsegment mobility as manifested in spectral changes, either infrared, electron spin resonance, or pulsed magnetic resonance.* With proteins an additional approach may be employed using proteolytic enzymes to probe the acceeeibility of hydrolysis-sensitive bonds in the surface layer of the adsorbed molecules.*ll In recent studies of @-casein adsorption, Dalgleish and LeaverloJ1combined observa-
Introduction Despite much reeearch on the adsorption of proteins at varioue interfaces (aidwater, oil/water, and solid/water), there is still relatively little knowledge of the actual conformationsadopted by the adsorbed protein molecules. Thie knowledge will be eeeential if a detailed quantitative understanding of the relationship between molecular structure and the surface and emulsifying properties of proteins ia to be attained. Though statistical theorieshave yet to be extended to proteins, their application to the behavior of polymers at interfaces has delineated some key data provisions to be determined by experiment. Thus, the adsorption isotherm, the effective layer thickness of the adsorbed molecule, the segment density distribution normal to the surface,the fraction of eegmenta directly in contact with the surface, and the fraction of surface sites to which the protein is adsorbed, are all generally thought to be essential pieces of knowledge. Our interest here is in the adsorption behavior of the milk protein, 8-casein. Many of the oil-in-wateremulsions currently produced by the food industry are stabilized by sodium caseinate. The excellent emulsifying behavior of commercial caseinate is associated in large part with the high surface-activity of the two major individual components of the blend, aBl-caseinand &casein. The disordered 8-caseinrepresenta a direct contrast to the behavior of globular proteins such as lysozyme and /3-lactoglobulin, which have also been the subject of many adsorption studies. The globular proteinsare generally compact,their tertiary etructme stabilized by a number of disulfide bonds. On the other hand, &casein is a single-chained protein of molecular weight 24000 with little ordered secondary structure and no disulfide linkages. The N-terminal21amino-acid eequence contains one-third of the charged residues poseeeeed by the &casein molecule at pH 7. The remainder of the polypeptide side chains are predomi-
(1) Swaiegood, H.E. In Deuelopmentr in Dairy Chemistry 1.; Fox, P. F., Ed.; Elaevier Applied Science: Barking, h x , 1982; p 1. (2) Hunter,J. R.; Kilpntrick,P.K.;Carbonell,R. G. J.Collordlnterface Sci. 1991, 142, 429. (3) Graham,D. E.; Phillip, M.C. J. Colloid Interface Sci. 1979, 70,
427. (4) Graham,D. E.; Phillip, M. C. In Theory and Practice of Emulsion Technology; Smith,A. L., Ed.; Academic P w : London, 1976; p 75. (6)Dalglehh, D. C . Colloids Surf. 1990,46, 141. (6) Benjamins, J.; de Feijter, J. A.; E v m , M. J. A.; Graham,D. E.; Phillips, M.C. Faraday Discuss. Chem. SOC.1976,68, 218. (7) M o r r h y , B. W.; Han, C. C. J.Colloidlnterface Sci. 1978,66,423. D. , 5.;Cohen (8) Barnett, K. G.; Congrove, T.; Vincent, B.; S ~ O M Stuart, M.A. MOCr01n0kCUk8 1981,14, 1018.
* To whom correspondence should be addreesed. of Leeds. c h Institute. University of Brietol.
+ University
1 Hannah m
0743-7463/93/2409-0242$04.00/0
(a
1993 American Chemical Society
Langmuir, Vol. 9, No. 1, 1993 243
Adsorption of @-Caseinat Fluid Interfaces
tions of peptide releasewith measurementsof the decrease in hydrodynamic thickness of the protein layer when subjected to enzymic digestion, allowing a possible configurational structure for the protein at the interface to be postulatsd. Direct information on the conformationadopted by the molecule is also available,in principle,through smallangle neutron scattering. In this paper, however, we report the use of an alternative neutron technique, reflectometry,to probe the structure of @-caseinadsorbed at the air/water and oil/water interfaces. This techniques gives information on the neutron refractive index profile normal to the interface.12 Since refractive index is simplyrelated to the scatteringlength density, specular reflection of neutrons may be employed to make useful measurements of the adsorbed amount, the film thickness, and the density profile perpendicular to the surface. Results obtained to date by this technique include adsorption data for surfactants at the air/solution and liquid/solid interface, and polymers at the air/liquid interface.12 More recently, the reflectometrytechnique has been extended to the study of adsorption of surfactants and polymers at the liquid/ liquid interface.13 This work is further developed here with our study of 6-casein adsorption. Experimental Section Neutron reflectivity measurements were carried out on the CRISP instrument14 at the Rutherford-Appleton Laboratory, near Didcot, England. This is a dedicated reflectometer, which uses a fixed angle of incidence and a pulsed, polychromatic neutron beam. Specularly reflected neutrons are received by a single detector, and their wavelengths are analyzed by time-offlight. Perpendicular wave-vector transfer is thus scanned by measuring reflectivity as a function of wavelength. In order to study the liquid/liquidinterface,a specialapparatus has been designed, which has been fully described el~ewhere.~J~ The large incoherent scattering cross section of hydrogen (and, to a lesser extent, deuterium) prevents the transmission of a neutron beam through more than a few millimeters of oil or water. The problem of penetration of the neutron beam through a bulk liquid phase is overcome by using a thin layer of oil, typically 10-20 pm thick, spread on a water surface. For a 10 cm long sample at the grazing angles of incidence used (8 I1.5'), this reduces the pathlength through the oil phase to a few millimeters and gives acceptable transmission. In all the experiments the oil used was n-hexane, for which the attenuation coefficient is approximately constant over the wavelength range used. A pool of water is placed in a PTFE trough sealed in an enclosure and presents a proud meniscus to the incoming neutron beam. The film is maintained in contact with a small reservoir of hexane around the water. Its thickness is maintained by cooling the water (using an electrical Peltier device), such that the rate of condensation of hexane onto the surface just balances its rate of drainage. Since the drainage rate is very slow, a temperature difference of 1K between the water surface and the surroundings is usually sufficient. Layer thickness can be controlled by adjustment of this temperature difference. A schematic diagram of the experiment is given in Figure 1. Theoretical Background. In a reflection experiment, reflectivity is measured as a function of wave-vector transfer, Q, perpendicular to the surfaceor interface. The value of Q is related (9) Shimizu,M.; Ametani, A.; Kaminogawa,S.;Yamauchi, K. Biochim. Biophys. Acta 1986,869,259. (10) Dalgleiah, D. G.; Leaver, J. J. Colloid Interface Sci. 1991, 141, 288. (11) Leaver, J.; Dalgleish, D. G. Biochim. Biophys. Acta 1990,1041, 217. (12) Penfold, J.; Thomas,R. K. J. Phys.: Condens. Matter 1990,2, 1369. (13) Cosgrove,T.;Phipps,J. S.;Richardson,R. M. Colloids Surf. 1992, 62,199. (14) Penfold,J.; Ward, R. C.; Williams, W. G.J.Phys. E: Sci. Instrum. 1987,20,1411.
4 I I
I
'i
w//mum
\
s//b
PeMwcaaAers
-PI&
Figure 1. Schematic diagram of neutron beam passing into reflectometer cell for studies at the aidliquid and liquid/liquid interfaces.
to the grazing incident angle, 8, and the wavelength, A, by Q=-4r sin 8
x
One can thus scan either 8 or A. CRISP scans A at constant 8, with a useful wavelength range of 0.5-6.5 h In order to measure the maximum possible range of Q,several experimenta must be performed, using different angles of incidence, and the data then combined into a single reflectivity profile. The minimum measurable reflectivity is normally limited to about lo4 by the isotropic, incoherent background scattering from the sample. In a typical experiment, on CRISP, reflectivity can be measured over a Q range of 0.01-0.25 A-1. The reflectivity of an interface can be calculated from ita refractive index profile (perpendicular to the surface) using the same equations as for perpendicularly polarized light. The refractive index, n, is simply related to the scattering length density, p, by
and p is related to the local density of scatterers by p
=xnibi
(3)
1
where ni is the number density of the ith type of nucleus and bi is its scattering length. Typically, refractive indices differ from unity only by a few parts per million, and hence grazing angles of incidence must be used. Much of the versatility of the technique arises from the fact that the scattering lengths of hydrogen and deuterium have opposite signs. This enables the scattering length density of compounds containing hydrogen to be adjusted over asubstantial range using isotopic substitution. In the experimenta described in this paper we use isotopic substitution in hexane to obtain a scattering length of approximately zero, giving the hexane the same scattering length as air, and thus rendering it effectively 'invisible" to the incoming neutron beam. This is known aa "contrast-matching". Reflectivity data are analyzed by model-fitting, using the matrix method of AMlh which is s u m m a n d inref 15tocalc~late the reflectivity from the perpendicular refractive index profile. The interface is divided into a number of uniform layers. A smoothly changing refractive index profile may be modeled by introducing an error function roughness parameter between the layers, as shown by Nbvot and Croc6.16 The thicknees, scattering length density, and roughness of the layers may be adjusted to achieve an optimum fit to the data, usinga nonlinear least-squares fitting routine. We have used the protein volume fraction, 4, as a parameter in the fits. It is closely related to the scattering length density of the adsorbed layers by the formula (15) Heavens, 0. S. Optical Properties of Thin Films; Butterwo& London, 1955. (16)Nbvot, R.; C r d , P. Rev. Phys. Appl. 1980, 16, 761.
Dickirwon et
244 Lmgmuir, Vol. 9, No. 1, 1993 P ( Z ) = 9 ( Z ) P P + (1- b ( Z ) ) P S (4) where pp is the scattering length density of the pure protein, ps is that of the solvent, and #(z) is the volume fraction profiie of the protein. Our aspiration was to find a single volume fraction profile that would be consistent with the reflectivities measured with two values for the scattering length density of the aqueous subphase. With thia limited number of contrasts, it is unfortunately not poeeible to gunrantee that a model which fib the data is unique. Errors are assessed by f i i g each parameter at several values around the optimum and allowing the other parameters to adjust accordingly. The error is then the region in parameter space around the optimum for which the fit remains acceptable. The quality of each fit is assessed visually and also by the calculated x2 parameter from the least-squares fitting routine. The latter approach is not entirely satisfactory, since the error bare are calculatedonly for neutron counting statistics, where systematic errore may outweigh purely statistical errors. The relative values of x2 for different fits to the same data set should, however, remain a good measure of their relative quality. The matrix method provides an exact calculation of the reflectivity but gives little insight into the relationship between reflectivity and structure. For reflectivities lower than 10-2, the reflectivity is given to an extremely good approximation by the formula''
(5)
We make use of this formula to give a simple Guinier-type analysis of the reflectivity, using a method f i t suggested by Crowley.18 The interfacial region is represented by a ' f i i " on top of a homogeneous subphase. Representing the subphase by a step function, we have
~ ( =4ApW-2) + pr(z) (6) where Ap ia the difference in scattering length density between the incident medium and the subphase, H(z) is the Heavyside function which steps from 0 to 1at the origin, and p&) is the densityfunction for the film. Using the known Fourier transform of the step function, this gives
R(Q) R,(Q)App2- Rl(Q)Ap+ R,(Q)
(7)
where
-
R, = 1 6 ~ ' R , = 3 2 * 1 1 p f ( z ) sin Qz dz, 4 '
e"
R, = q l p r ( z ) e i e 2dzI2 (8)
e'
RoL\pZis the reflectivity of a clean, sharp interface, which we call
the Fresnel reflectivity. By expanding the R1and RZterms in eq 7 as Taylor series, we obtain
Rp,
16~' R, = r2exp(*c')
e"
(10) where us is the mean square deviation of the adsorbed layer thickness, (2") is the nth moment of the distribution about E = 0, and (11)
For the 'Guinier" analysiswe make the further approximation that (z3)/6(2)= uz (12) This holds exactlyfor a uniform layer and isa good approximation for most other types of distribution. The fmal Guinier approx(17) Ab-Nieben, J. 2.Phycr. B Condem. Matter 1986,61, 414. (18) Crowley, T.L.D.Phil. The&, University of Oxford, 1984.
Pf%J
/
41.
4 M
Figure 2. Scattering length density profide of hydrogenous polymer adsorbed at the surface of a deuterated solvent. See theoretical background section. imation is therefore
III [Q?R - R,)I = III [ i 6 2 ( r - 2 r ( ~ ) ~-pgZuZ )i (13) The approximation will break down with the Taylor expansion when Qz > 1. When the subphase is contrast-matched with air (Ap = 01, the RI and ROterms in eq 7 dieappear, and we may use the intercept of the Guinier plot to obtain the scattering density integrated over the adsorbed layer, r. This may be converted into an adsorbed amount, rm(in mass per unit area), using the formula (14)
where NAis the Avogadro number, M is the molecular weight of the adsorbed molecule, and &bi is the scattering length of one molecule. When the subphase is DzO and the adsorbing compound is not deuterated, the majority of the scattering in the adsorbed layer comes from the DzOitself. However, provided that the adsorbate does not protrude significantly out of the water, the Guinier plot should yield the same value for u as when the subphaseiscontrastmatched with air. Consider the situation shown in Figure 2. With an air-contrast-matched subphase, the scattering length density profiie will be as shown in Figure 2(ii), and with a &O subphase it will be as in Figure 2(iii). By rotating and inverting Figure 2(iii), we obtain Figure 2(iv). Because reflectivity is an example of a phaseleea Fourier transform (at least within the kinematic approximation that we are employing),profiiea 2(iii) and 2(iv) will give the same reflectivity. Figure 2(v) can be conveniently separated into the step function and the (laterally inverted) macromolecule profile (Figure 2(v)), and hence the Guinier analysis will yield the same u value for both contraeta. Sample Materiala. The DzO was used as supplied by MSD Isotopes Ltd. n-Hexyl deuteride (hexane-dl)was prepared from 1-bromohexanevia a Grignard reagent and Ds0 and is approximately contrast matched with air. Ultrapure water was obtained from a Millipore 'Milli-BI system. The &casein was prepared in the laboratory by acid precip itation of whole casein from skim milk obtained by centrif'ugation of whole bulk milk from the Hannah Research Institute herd. Solutions of the casein mixture in 6 M urea, 60 mM imidazole/ HCI pH 7.0 buffer were chromatographed on columna of Sepharose-Q fast-flow ion-exchange material with a gradient of NaCl. The material in the peak corresponding to @-caseinwas collected, dialyzed exhaustivelyagainatdistilledwater,andfreezedried. SDS-PAGEand FPLC on reversed-phasecolumna showed the protein to be better than 95% pure, the major impurities being other casein fractions.
Results Adsorption Experiments at the Aidwater Interface. I n order to meximize the sensitivity of the neutron experiments, we chose a protein concentration of 6 X 10-3
Langmuir, Vol. 9, No. 1, 1993 246
Adsorption of 8-Casein at Fluid Interfaces 1 ,O O r
[I
-12 -lo
I
t
t
-le -20
.
A
-----L
I I 0.00’
0. 00
0. 25 ‘ 0
0.50
0.75
1.00
50
100
150
Distance / Angstrom
x 10‘ / Angstrom-‘
Figure 3. Guinier plota of observed reflectivity (R) of @-casein (6 X 10-3 wt %) at the &/water interface at ambient temperature: (a) DzO; (b) air-contrast-matched water.
\ ,.
0
Figure5. Volume fraction profiles of adsorbed@-caseinobtained by fitting neutron reflectivity data measured at the &/water interface to a two-layer model: reflection from (a) DzO and (b) air-contrastmatched water.
-:I /
0.75-
4
-2
-6
0 / Angstrom-’
Figure 4. Observed reflectivity ratios (RIRf)for @-casein(5 X
10-3 wt % ) adsorbed onto (a) DzO and (b) air-contrastmatched water.
w t % in the plateau region of the adsorption isotherm at
both the &/water and oiVwater interfacmS Two contrasts were uaed, with the protein dissolved in (a) D20 and (b) air-contrast-matched water. In a preliminary analysis, the two seta of reflectivity data were plotted according to the Guinier formulation (Figure 3). From the extrapolated intercept of the air-contrast-matched plot, a value of 3.8 mg m-2 is obtained for the surface concentration of adsorbed &cawin. The slopes of the plots are very similar, giving a value for the second moment about the center of the distribution (a) of 2.0 nm. Since the values of u from the two plots are the same, it appeare. that the protein is entirely submerged in the aqueous phase. By use of the two Guinier plota simultaneously, the f i t moment, (z ), of the aegment density is calculated to be 3.1 nm with the origin at the water surface. The reflectivity ratios (RIRF)for the two contrasts are shown in Figure 4. T h e interference fringe, visible in the DzO data set, is a sensitive indicator of the ‘average” layer thickness. For the air-contrast-matched data, the initial slope is clear but the position of the maximum is very sensitive to the level of background subtracted, and so the layer thicknea is not so well determined from this set of data. However, the fringe does seem rather broader than for the D# case,suggesting a thinner layer. The solid lines in Figure 4 are two-layer model fita, calculaM using the techniques described earlier. Attempta to find a structure for the adsorbed &casein film which is compatiblewith both data seta were unsuccessful, and 80 it seem that, despite the promieii nature of the Guinier plota, the protein distributions in the two aqueous
0. 00
0.05
0. 10
0. 15
0 / Angetrom-1
Figure 6. Semilog plot of reflectivity (R)of @-casein (6 X 10-3 w t %) (a) on DzO and (b) on air-contrast-matched water. Solid
lines are calculated using volume fraction profile (a) of Figure 5.
subphasesare really slightly different. The protein volume fraction profiles corresponding to the solid fita of Figure 4 are shown in Figure 5 for comparison. Figure 6 show both seta of reflectivity data with calculated reflectivities using only the volume fraction profiles of Figure 5a. Agreement with the air-contrast-matched data is quite poor, even though the volume fraction profile that does fit this air-contrast-matched water data is not too dissimilar. This illustratesthe sensitivity to structure of the reflectivity technique. T h e fit parameters are listed in Table I. The scattering length density of each layer has been calculated using eq 4. The scattering length density of the polymer, pp, has been estimated by summing the scatteringlengths of allthe atoms in the &caseinmolem.de, assuming it contained no water of hydration, and dividing by the partial specific volume, V, reported by McMeekin et al.1Q
Pp
=
VM
There is a possibility that the value obtained from eq The most likely source of inaccuracy is bound water of hydration, either in the B-casein used for the original specific volume 16 by this method may not be accurate.
(19) McMeekin, T.L.;Grovea, M. L.;Hipp, N.J. J. Am. Chem. Soc. 1949, 71,3298.
246 Langmuir, Vol. 9, No. 1, 1993
Dickinson et 41.
Table I. Fitted Punmeten for Adsorbed &Casein Segment h n r i t y Profiler. subphase Dit= 62 61 Ddudnm uJ0.93 0.05 7.2 t 0.3 0.14 f 0.02 2.5 2.0 Dz0 1.8 0.2 0.21 f 0.03 0.95 0.02 5.0 f 0.3 1.9 2.0 CM water 2.0 0.2 0.15 f 0.03 0.96 0.03 5.4 f 0.3 1.9 1.7 oiVwater Dz0 2.0 f 0.2 0.15 0.03 1.9 1.7 CM water 2.0 0.2 0.96 f 0.03 5.4 f 0.3 0 D1and Dz are the thicknesses of the inner and outer layers; 61 and 6 2 are the correspondingprotein volume fractions; u denoten the quare root of the second moment of the adsorbate profile. ufis calculated from the model fit; uE is obtained from the Guinier analysis. interface &/water
** *
*
**
experiments or for this neutron reflection experiment. Though initiallyhydrogenous, bound water would rapidly exchange with deuterium when in DzO solutionsand thus change the effective pp. We believe an aasumption of an uncertaintyoffO.10X 10-5A-2inourestimateofpp=O.l8 x W A-2 to be pessimistic, but even this only introduces an error of kO.06 in the highest values of volume fraction, 9, determined by the model fit. There are several possible reasons why the profiies in air-contrast-matched water and D2O appear different. Firstly, since the DzO subphase gives much higher reflectivity than the air-contrast-matched water, the time scales of the D2O experimentswere very short (4h each), and thus the systems may not have reached equilibrium. Graham and Phillips20 observed that, at a bulk concentration of lo-' wt %, the adsorbed amount took some 5 wt % took h to reach a constant value, but that at only 30 min and appeared to go through a weak maximum in this period. At our chosen concentration of 5 X wt %,asteady-state adsorbed amount should therefore have been rapidly reached, though some rearrangement in the conformation of the layer over a more extended period cannot be ruled out. If this were indeed to be the case, then our two experimentswith air-contrast-matchedwater and D2O could be looking at two slightly different situations. A second poseible reason for a differencein protein film structure on D20 and H20 could arise from a change in the ionization behavior on isotopicsubstitutionproducing a difference in the protein conformation. Many of the aminoacid residuesin &casein have ionizable side chains: these are distributed in clusters along the chain. The overall net charge at pH 7.0 is around -13. While the generalobservation can be made that groups in D2O ionize lese readily than in H20,2I it is difficult to predict from thia whether the protein ought to be more or less readily extended in D2O. The net charge, resulting from the partial ionization of both acidic and basic side chainsalong the protein backbone, is determined by many complex equilibria, and the different influences of D20 and H2O on these equilibria cannot be reliably estimated. Adamption Experiments at the n-Hexane/Water Interface. T h e same two subphase contrasts were used in the measurements of ,3-caseinat the n-hexanelwater interface,inbothcaseswithair-contrasta"ahed n-hexane as the upper phase. The low-boiling-point hydrocarbon solvent is W i g the role of the "oil" phase here. Unfortunately, the available Q range for the measurement on air-contrast-matched water wa8 rather limited, since the attenuating upper n-hexane phase reduced the maximum measurable reflectivity by a factor of about 10. In both cases,because of the n-hexane attenuation, it is not possible to assign a unique value to the adsorbed amount, since the abeolute scale for the air-contrast-matched data set is not known. However, Graham and Phillipsmhave (20) Graham, D. E.; Phillip, M.C. J. Colloid Interjace Sci. 1979, 70, 403. (21) hankr,l.Woter,A CompreheMioe Treatise;Plenum: New York, 1973; p 167.
-I0 -12
Ic .
I
a)
',+**=
*
- . .
.
-14
b)
-20
'
0 . 00
1.00
2. 00 0 2 x 10
3. 00
4.00
5.00
/ Angstrom-2
Figure 7. Guinier plota of observed reflectivity of @-wein(6 X 10-3 wt %) at the n-hexane/water interface. Reepective subphases are (a) DzO and (b) air-contrast-matchedwater. 1. ooy
,
0.75.
&
0.50-
LL
I
0.25t
12,
0. 00 0. 00
0.05
0. 10
0. 15
13 / Angctrom-l
Figure 8. Observed reflectivity ratios (RIRd for @-casein(6 X 1O-a wt ?6) adsorbed onto (a) DzO/hexane and (b)air-contrast matched water/hexane.
shown that valuesof the adsorbed amount at the oiUwater and air/water interfacea are very similar,and so for fitting purposesthescale factor wasfixedtoreflectthisassumed similarity. Guinier plots are shown in Figure 7. Again, there is good agreement between the second moment values (u) from the two sets of data, suggesting that the /?-casein does not protrude very far into the n-hexane phase. Despite the fact that one end of the molecule is subtantially hydrophobic, the measurements show that the @-caseinis not solubilized by the oil phase. Experiencein attempting to dissolve the protein &owe that @-caseinis very water-soluble but apparently completely insoluble in n-hexane. The u values from these Guinier plots are similar to those found in the analysisof the adsorbed structure at the &/water interface, albeit a few tenthe of a nanometer smaller. T h e reflectidty ratios derived from the raw experimental data are shown in Figure a The frinee present in the data at the aidwater interface is noticeably absent, but otherwise the data are quite similar. Model fitting again demands a two-layer structure for the adearbed protein
Langmuir, Vol. 9, No.1, 1993 247
Adsorption of 6-Casein at Fluid Interfaces 1. OOr
0.75
0.
in
oov
0
d
50
100
150
O i s t o n c a / Angstrom
Figure 9. Volume fraction profiie satisfying both seta of
reflectivity data obtained for @casein at the it-hexanelwater interface.
on the water side of the interface. On this occasion the same parameters (Table I) provide a fit satisfying both data seta. The layer adjacent to the interface has a thickness of 2.0 f 0.2 nm and a protein volume fraction of 0.96 0.03,with a further layer of 5.4 f 0.3nm thickness and protein volume fraction 0.15 f 0.03 extending into the aqueous phase, as shown in Figure 9. This fitted segment density profile is very similar to the structures envisaged at the &/water interface, although the value of u is slightly smaller, and hence the protein conformation would appear to be slightly less expanded at the oi4water interface than at the &/water interface.
*
Discussion Agreement between the resulta of these neutron reflectivity experiments and existing studies on @-caseinadsorption is generally very good. The value of 3.8 mg m-2 for the adsorbed amount at the &/water interface is slightly larger than the value obtained by Graham and Phillips,3 but slightly less than the more recent value of Hunter et ale2for the same bulk solution concentrations. Both groups used radiotracer techniques, modifying the &caseineither by acetylation3or by reductive methylation2 to introduce the radiolabel, carbon-14. Methylation retains the same net charge in the protein and is the chemical modification which gives adsorption resulta closest to ours using the unmodified &casein. Ellipsometry, which also uses unmodified protein, gives values of the order of 4 mg m-2 for the surface loading in our concentration range.6 The overall thickness of the adsorbed protein layers, determined here by neutron reflectivity, agree well with previous measurementa by ellipsometry and photon correlation specttoecopy (PCS). Our valuea for the totallayer thickness of between 7 and 9 nm are almost identical to the ellipsometry values of one group7 and a little larger than those of another! who quote a value of 5 nm. The values obtained by PCS for the layer thickness of @-casein adsorbed onto polystyrene latex spheres are somewhat larger at about 15 nm than our neutron reflectivity estimates, but it is well appreciated that PCS measures a hydrodynamic radius whose value is sensitive to the presence of the molecular segmenta at the very periphery of the adsorbed layers, whereas reflectivity is directly a measure of average segment density. Taking this into account, we believe that reasonably consistent values for the adsorbed layer thickness are obtained by all these techniques for this approximately random-coil protein. This thick &casein layer contrasts with the much thinner adsorbed layers of globular proteins such as @-lactoglo-
bulin, where values of the order of 2 nm are meaeured both by PCS21J2 and small-angle X-ray scattering.2g "he most important resulta from these neutron reflectivity meaeurementa of adsorbed @asein f h are the estimates of the eegment density profile. It has to be admitted that the splitting of the adsorbed film into discrete layers is somewhat arbitrary, although it is in keeping with the well-known separation of polymer segments on surfaces into "trains"d i r d y a t t t h e surface and'loops" and "Ws"further away. In a study of polymer adsorbed at the &/water interface Lee et al.u have shown how both a very asymmetric two-layer model (similar to that used here for @-casein)and a %elf-similar"(z-'Is) scaling profile will fit the same data set. However, their profile data are all at very low wave vectors and have no interference features. The interference fringe in the &casein data, combined with the high atbrbed amount, does require a sharp boundary betwen the dense and the diffuse layer, and so in this case a smooth profile would not fit the data. The use of subphasesof M e r i n g contraata to produce dissimiIar experimental resulta,but which are well fitted by identical or largely s i m i i profilea, lends further confidence to the predicted profiles. Such predictions of a dense, thin, inner layer and a diffuse, thicker, outer layer are consistent with the speculated structures of the adsorbed layer suggested by Leaver and Dalgleish.loJ1 Using trypsin proteolysis, these authors compared the rate of release of specific peptides from /3-casein adsorbed onto polystyrene latices and emulsion dropleta with the formation of the same peptides free in aqueous solution. Whereas dissolved B-casein was initially hydrolyzed seemingly randomly at a number of trypsin-sensitive bonds involving lysine or arginine residues, the hydrolysis of the protein at the interface was a more orderly event.11 There the crucial initiating step was found to be the cleavage of the N-terminal peptides 1-25 and 1-28 from the molecule. No hydrolysis at other trypsin-sensitivesites of the adsorbed molecule occurred until after this had taken place. This suggests that, with the exception of the large hydrophilic moiety in the N-terminal region, moat of the &casein molecule is inaccessible to the proteinase. These enzyme digestion studies were found to be correlated with the changes in measured hydrodynamic radii of the latadcasein complex and the casein-coatad emulsion droplets in the presence of trypsin. Trypsin attack causes the radius of the complex to decrease rapidly by about 10 nm. The final value obtainedll was larger than that measured for the original latex particle, indicating that an adsorbed layer of thickness 3-5 nm still remained on the latex particle. Combining the reaulta from these observations,Dalgleieh and LeaverlO suggested that the conformation adopted by the adsorbed 8-caseinallows the N-terminal portion of the molecule (residues 1-48) to form either a loop or a tail, rendering the susceptible residues 25 or 28 readily acceesible to trypsin in solution. Conversely, none of the other potential sites is attached,either because the relevant part of the @-casein molecule is in an unsuitable conformation (lying flat at the interface), or the trypsin is prevented from approaching by the protruding N-terminalpeptidea. Once the phosphopeptides have been removed by the initial proteolytic attack, the residue of the molecule is (22) Dalgleieh, D. G.; haver, J. In Food Polymer, G$s and Colhidr, Dickineon, E., Ed.;Royal Society of Chemistry: Cmbrldp, 1991; p 113. (23) Meckie, A. R.;Miqina, J.; Dunn, R.;North, A. N. In Food Polymers, Gels and colloids, D i c h n , E., Ed.; Royal Society of Chemistry: Cambridge, 1991; p 98, (24) Lee, L.T.;Guinelin, 0.;Farnoux, B.; Lapp, A. ~aCromkCUk8 1991,24,2618.
248 Langmuir, Vol. 9, No.1, 1993
open to reaction,or alternatively,a conformational change
Dickineon et ai.
that the largely hydrophobic remainder of the molecule adsorbsin a conformation close in density to that adopted by the molecule in bulk solution. The results of a neutron reflectivity study of the globular protein, bovine serum albumin (BSA) adsorbedat the air/ water interface have recently been rep0rted.a Compared with the disordered @-casein investigated here, the globular mema that the decreaw in radius must represent the length BSA was found to adsorb in a much more compact of the protrusion. configuration,though again a two-layer model was able to The estimates of the segment density profile provided by the neutron reflectivity studies reported here mirror provide the best fit-a dense inner layer of thicknessjust theinferredstruct~~suggeatedfortheadaorbed@-caaein 1.1nm and an outer layer extending only a further 2 nm.a molecule based on the enzyme attack studies. Neutron It is noteworthy,pe!haps, that the inner layer of BSA was inferred to have a smilarly high volume fraction (0.93) as reflection also suggesta a two-layer model. The diffuae estimated here for @-caseinat both oil/waterand &/water outer layer, of some 6-7 nm thicknew,has a volume fraction interfaces. in the range 0.15 to 0.2. This would be equivalent to approximately 20% of the molecule (4U-50 residues) Acknowledgments. The authors thank Dr. Jeff Penoccupying the region beyond the inner layer. The dense fold and the instrument scientista at the W for their inner layer predicted by neutron reflectance also has a encouragement and aseistance and SERC for financial thickneasof the same order of magnitude as suggested by support. This research was partly funded by the Scottish PCS;we recall that PCS is more sensitive to peripheral Office Agriculture and Fisheries Department. structures, which allows the poesibility of rearrangement of the problyzed macropeptide. The high volume (25) Eagleeham, A.; Herrington,T.M.; Penfold,J. Colloids Surf. 1992, fraction of the inner layer simply reflecta the observation 65,9. may OCCUT to facilitate the attack. Dalgleish and haver10 explain the rapid decrease in radius as the loss of the N-terminal peptide, the change of 10 nm reflecting the extent to which this peptide protrudes from the surface. No other proteolytic attack occurs as rapidly, and 80 it
