Neutron Reflectivity of Adsorbed Protein Films - ACS Symposium

Chapter 22

Neutron Reflectivity of Adsorbed Protein Films 1

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2,4

Peter J. Atkinson , Eric Dickinson , David S. Home , and Robert M. Richardson

Downloaded by HONG KONG UNIV SCIENCE TECHLGY on April 4, 2017 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0602.ch022

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Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, United Kingdom Hannah Research Institute, Ayr KA6 5HL, Scotland School of Chemistry, University of Bristol, Bristol BS8 1TS, United Kingdom 2

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Direct information on the thickness and structure of adsorbed milk protein layers at oil/water and air/water interfaces has been obtained using the technique of specular neutron reflection on the CRISP reflectometer at the Rutherford-Appleton Laboratory. Segment density profiles are presented for β-casein adsorbed at the air/water interface as a function of bulk solution pH and protein concentration. The results are compared with film properties derived using other techniques. Data from a neutron reflectivity study of the competitive adsorption of β-casein and the nonionic surfactant, C E , are also included. 12

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By virtue of their amphiphilic nature, proteins are very surface active and adsorb readily at interfaces. This adsorption behavior is of great practical importance in fields as diverse as the pharmaceutical, biomedical, cosmetic and food industries. Our particular area of interest is the food industry, where proteins, especially dairy proteins, are employed in the stabilization of oil-in-water emulsions. Here the protein layer protects the oil droplets against immediate coalescence and provides long-term stability against flocculation and eventual coalescence. Despite much research on protein adsorption to various interfaces, there is still relatively littie knowledge of the actual conformations adopted by the adsorbed protein molecules. In this paper, we describe the technique of neutron reflection which yields such information directiy as the density distribution normal to the interface. The protein we employ is the milk protein, /3-casein, measuring the conformational profile at the air/water interface as a function of solution p H and protein concentration. Many biocolloidal systems of technical and commercial importance contain small molecule surfactants as well as proteins. The distribution of these molecules between the surface and the bulk phase affects the properties of the systems (stability, rheology etc.)(7). While the competitive displacement of milk proteins by 4

Corresponding author 0097-6156/95/0602-0311$12.00/0 © 1995 American Chemical Society Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

312

PROTEINS AT INTERFACES II

nonionic and anionic surfactants has been demonstrated experimentally at the planar oil/water interface (2) and at the emulsion droplet surface (5) and the behaviour modelled by Monte Carlo (4) and molecular dynamics (5) simulation, the detailed structure of the mixed protein/surfactant layers remains unknown. Preliminary measurements of the competitive displacement of 0-casein by the nonionic surfactant, C^E^ (hexaoxyethylene dodecyl ether) are detailed here, together with a description of a strategy for locating the surfactant in the mixed layer.


Neutron Reflection A general description of the neutron reflection technique has been provided by Richards and Penfold(6). Its application to the study of the conformation of proteins adsorbed at interfaces has been described in detail previously(7). In many of their interactions with matter, the behaviour of slow neutrons can be expressed in theoretical terms based on analogies with light. Thus scattering, interference, diffraction and reflection all have their counterparts in neutron behavior and follow very similar laws to electromagnetic radiation. The specular reflection of neutrons from an interface thus depends quantitatively on the refractive index profile perpendicular to the interface, which is related to the coherent scattering-length density. In turn this scattering-length density profile depends on the chemical composition and number density of scattering species. Hence detailed information regarding the interfacial region is contained in the reflectivity profile. A further advantage of neutrons is their short wavelengths, some two to three orders of magnitude smaller than light, so that interfacial layers are probed on the molecular scale. Neutrons also possess the further distinct advantage that isotopes of the same element may manifest different scattering lengths, hydrogen and deuterium being the most advantageous and most widely used. This is exploited in two ways here, first by contrast-matching the solvent water to air or other liquid, so that only the adsorbed layer is "seen" and the protein profile generates the reflectivity observed and secondly by employing both deuterated and hydrogenated versions of the nonionic surfactant in the competitve adsorption studies. The essence of a neutron reflection experiment is to measure the neutron reflectivity as a function of the momentum transfer vector perpendicular to the reflecting surface. In principle, it should be possible to transform the measured reflectivity data to obtain the adsorbed segment density profile directiy. In practice, as is often the case in attempting to implement a mathematical inversion procedure, the lack of high quality data in ranges of wave vector largely inaccessible due to experimental limitations precludes this approach. Detailed analysis has therefore been accomplished by adopting a two-layer model function for the profiles and seeking a best-fit to the data. Materials and Methods The j8-casein (genetic variant B , obtained from a single cow homozygous for this variant) was prepared from acid casein precipitated from fresh skim milk. Separation from the other caseins was achieved by ion-exchange chromatography on a Sepharose Q column. The fraction corresponding to 0-casein was dialysed

Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

22. ATKINSON ET AL.


exhaustively against distilled water and freeze-dried. Polyacrylamide gel electrophoresis and fast protein liquid chromatography demonstrated its purity to be greater than 99%. Research grade C E was purchased from Sigma Chemicals and used as supplied (stated purity 99%). The deuterated surfactant, denoted d - C E , was a gift from Unilever Research, Port Sunlight, Cheshire, U . K . D 0 was obtained from M S D Isotopes Ltd. A l l solutions were prepared using Milli-Q ultrapure water with Analar grade reagents as buffering agents. The reflectivity profiles were measured using the CRISP instrument at the Rutherford-Appleton Laboratory (Chilton, Oxfordshire, U . K . ) (8). This instrument uses a pulsed polychromatic beam of neutrons and records their time of flight so as to vary wavelength X at the selected fixed grazing incidence angle 0. In a typical experiment a buffered solution (20 mmol/dm imidazole/HCl, p H 7.0) containing 0casein and a variable amount of C ^ all dissolved in air-contrast-matched-water (CMW) (8%D 0) was carefully poured into a clean Teflon trough, approximately 60 cm of solution being required to give a proud meniscus above the edge of the trough. Further details of the apparatus and procedure may be found elsewhere (7,9,70). 12

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2

3

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Results and Discussion We first consider results for 0-casein adsorbed to air-contrast-matched water in the absence of surfactant. Preliminary analysis of the reflectivity profiles is best accomplished by application of the Guinier approximation to the data obtained at low scattering vector, q (q=(4x/X)sin 0)(7,77). For an adsorbed layer on top of a homogeneous subphase the reflectivity can be written as a sum of three terms in the kinematic approximation(72). 2

R(q) « Ro(q)Ap - R, Ap + R (q) 2

(1)

The first term is the Fresnel reflectivity from a clean sharp interface (no adsorbed layer): 2

2

2

Rp(q) = Ro(q)Ap = 16* Ap /q

4

(2)

The quantity Ap is the difference in scattering length density between the incident medium and the subphase. When the subphase is contrast-matched to the air, Ap = 0. Both the first term (R ) and the second term (Ri(q) Ap) disappear and the reflectivity is simply given by F

R(q) = R (q)

(3)

2

In practice, when Ap = 0 the reflectivity is weak and so the kinematic approximation holds very well. Applying a Guinier-type approximation, the behaviour of the function R (q) at low q can be written as 2

2

2

R (q) = (16xVq )m exp(-qV) 2


(4)

314


where a is the second moment of the adsorbate distribution function normal to the interface

o = ((z ) - (z)Y 2

(5)

and m is the scattering length density integrated over the adsorbed layer:


m = J p(z)dz

(6)

2

2

A Guinier plot of In q R(q) against q thus gives a straight line of slope equal to -a and intercept equal to / / i f ^ i ^ m ] . Hence the slope gives a measure of the thickness of the adsorbed layer and the intercept gives the quantity of material adsorbed at the interface. The integrated scattering length density can be related to the adsorbed amount in units of mass per unit area, T, knowing the atomic composition of the adsorbate and using the equation 2

T = (M m/N ) [E bjw

1

(7)

A

where M is the molecular weight of the adsorbate, N is Avogadro's number, and bi is the scattering length of the i * atom in the adsorbate molecule. Table I lists the values calculated for surface coverage and the square root of the second moment of the adsorbed layer profile from profdes measured as a function of buffer p H and bulk protein concentration. w

A

Table I. Derived Guinier plot parameters for pure 0-casein adsorbed at air/contrast-matched-water interface pH

7.0 7.0 7.0 6.0 5.4

Bulk Protein Cone, (wt %)

T (mg/m )

a (nm)

2.7+0.1 2.05 ± 0 . 1 1.91+0.1 2.85±0.1 3.90±0.15

1.80±0.06 1.65+0.07 1.81 ± 0 . 0 7 2.39±0.08 2.57±0.08

2

2

5X105X10" 5X10^ 5X10 5X10"

3

3

3

3

The bulk concentration of 5X10" wt% lies in the expected plateau region of the adsorption isotherm of /J-casein at both the air/water and oil/water interfaces (13). The calculated surface coverage of 2.05 +/- 0.10 mg/m at p H 7.0 is close to that reported by Graham and Phillips (14) (r«2.5 mg/m ). The observed increase in surface coverage inferred from the Guinier plots as bulk protein concentration is increased to 5X10" wt% is also consistent with their findings as is our result that reducing the protein content to 5X10^ wt% gives little change in surface coverage 2

2

2


2


22. ATKINSON ET AL.


(Table I). Layer thickness, calculated from the slopes of the Guinier plots, remains almost constant across the protein concentration range, with possibly a marginal increase at higher surface coverage but a similar slight increase at the lowest concentration employed. The increase at the higher concentration would be expected if secondary layer coverage were being initiated, but the behaviour at low concentrations is not and merits further study to determine i f these variations are significant. We observe a considerable increase in reflectivity as the solution p H is decreased. The layer thickness at pH 5.4 derived from the Guinier plot is some 50% greater than die p H 7.0 value and the adsorbed amount almost 100% larger. This behaviour is consistent with the increased aggregation of 0-casein molecules in solution as the p H approaches the isoelectric point p i (net molecular charge tends to zero), since one expects increased protein surface coverage under condition" where the protein solution is close to phase separation or precipitation. The values calculated here for T and a at p H 5.4 are almost exactly the same as those reported previously (7) for j3-casein adsorbing from unbuffered distilled water at air/water and oil/water interfaces. We conclude from this that the effective p H in that preliminary experiment was close to pi, partially due to the buffering action of the protein itself, and partially to the additional acidity resulting from dissolved carbon dioxide. The preceding observations are completely independent of any model assumed for the structure of the adsorbed protein layer. Further analysis of the reflectivity data was accomplished by model fitting, as described in our earlier publication (7), using the matrix method of Abeles as summarised in ref 15 to calculate the function R (q) from an assumed model for the scattering length density function p(z). Our model divides the interface into a number of uniform layers. The number of layers, their thickness, scattering length density and a roughness parameter may all be adjusted to achieve an optimum fit to the data, using a nonlinear least-squares fitting routine. The scattering length density of the adsorbed layers is closely related to the protein volume fraction, , by the formula 2

p(z) = (z)p + (1 - (z))p ?

®

s

where p is the scattering length density of the pure protein, p is that of the solvent, and (z) is the volume fraction profile of the protein, z being the distance measured normal from the interface. Best fits were achieved with a two layer model exemplified by the plot shown in Figure 1 for the adsorbed /J-casein profile at p H 7.0 at a bulk concentration of 5X10" wt%. The calculated segment density profile indicates a dense inner layer of volume fraction (00 0.9 and thickness (& ) approx. 1 nm immediately adjacent to the interface and beyond that extending into the aqueous phase a more tenuous and extensive outer layer, 4-5 nm thick (d )and of volume fraction (^), 0.14. Such layer thicknesses agree well with hydrodynamic layer thicknesses inferred in studies of j8-casein adsorption to polystyrene latex particles from dynamic light scattering measurements (16,17), bearing in mind the known sensitivity of hydrodynamic measurements for segment density at the periphery of the adsorbed layers. The calculated variations in the fitted parameters with solution p H are plotted in Figures 2a and 2b. The volume fraction of the inner layer (

:

A

1 AIR [

i

1

i

1

1

i

1

i

1

WATER

-

0.4 0.2 0

/

-

1

1

1

1

1

1

1

T» 1

-2

10

Distance from interface

(nm)

Figure 1. Two-layer model fit of segment density distribution calculated for f3casein (5X10 wt%) adsorbed at air/ water interface at pH 7.0. 3

1.0 .2 0.8 o 2 0.6

*-

1

-

1 • *1 •

"(a)

® 0.4 o >

*

0.2 - X * 0.0

I.I

5.0 I

6.0

7.0

PH -r

j

60.0 40.0 20.0 0> —J 0.0 5.0

• .i.

i

• 1

6.0

1

7.0

PH

Figure 2. Results of fitting two-layer model to neutron reflectivity data obtained for /3-casein (5X10 wt%) adsorbed at the air/water interface with subphase buffered to pH values indicated using imidazole (20mM) and HC1. (a) Variation of volume fraction parameters (fa inner and outer) with pH. (b) Variation of derived layer thicknesses (d inner and d outer) with pH. 3

2

x

2



22. ATKINSON ET AL.

Neutron Reflectivity of Adsorbed Protein Films

317

to 0.95 as the solution pH is decreased. It is difficult to see how it could increase further. That of the outer layer (fa) increases by about a third from 0.14 to 0.19 as the p H is decreased from 7.0 to 5.4. The thickness of the inner layer (d^ is almost doubled from just under 10 A at pH 7.0 to 18 A at p H 5.4. The outer layer thickness increases even more from 48 to 68 A over the same p H range. It seems rather likely therefore that a second layer of protein forms on approaching p i , as schematically described in Figure 3. The train-and-tail model for the adsorbed protein molecule at p H 7.0 has been inferred from our earlier studies and fits well the two-layer structure derived from neutron reflectance measurements. Adding a second layer of /J-casein should approximately double dj whilst fa should remain fairly constant. The volume fraction, fa, should increase and the outer layer thickness, d , should also increase as indicated in the simple model in the diagram. The data for pH 6.0 seem to suggest the additional protein packs loosely into the outer diffuse layer. The increase in d is quite large and fa is increased, but d and fa remain about the same. If the extra protein sat neatiy on the first layer, then d would increase by as much as d . That it should be observed to be much greater is an indication that the additional protein is perhaps adopting a more extended or simply different conformation from the molecular layer nearest the interface. When the same model analysis is applied to the reflectivity data obtained as a function of bulk protein concentration at p H 7.0, a similar picture emerges. Again the best-fits are obtained with a two-layer model for the adsorbed protein molecule. Table II lists the parameters determined in these fitting procedures. Increasing the protein concentration by a factor of 10 from the standard bulk concentration of 5X10" wt% increased the surface coverage to 2.7 mg/m (Table I). Fitting indicates that this produces no increase in the volume fraction of the inner layer, but an increase in its thickness from 10.1 A to 13.4 A, almost exactly compensating for the increase in protein loading. No changes are found in either the thickness or volume fraction of the outer layer as a result of this increase in T. ApparenUy the extra protein is accommodated by the molecules compacting closer together in this instance rather than forming a second molecular layer as the behaviour observed on lowering the solution pH suggests. Decreasing the bulk protein concentration to 5X10^ wt% produces littie change in surface coverage, as calculated from the intercept of the Guinier plot. The fitting to a model profile gives an inner layer marginally thinner than the 5X10" wt% profile at 9.5 A and of a lower volume fraction than previously calculated for this protein (fa = 0.85). The outer layer features an unchanged volume fraction of 0.14 but now appears thinner at 42 A. At the lower protein concentration, the adsorbed molecule has apparentiy more space to move around on the surface and the tail extending out into the aqueous phase is more flexible and less constrained by its neighbours. These neutron reflectivity studies of the adsorption of /J-casein at the air/water interface are providing a detailed picture of the behaviour of the adsorbed molecule, largely substantiating the scenario inferred from hydrodynamic measurements of adsorbed layer thickness and changes in this parameter consequent on enzymic digestion of the adsorbed molecule (16-19). 2

2

x

2

t

3

2

3


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Table n . Fitted parameters for segment density profiles derived from reflectivity data for 0-casein adsorbed at air/contrastmatched-water interface (pH 7.0) from different bulk protein concentrations


Bulk Protein Concentration (wt%)

4(A)

4

5X10 5X105X10

4>i

0.85 0.90 0.92

9.5 10.1 13.4

3

2

d (A) 2

42 49 49

0.14 0.14 0.14

1.91 2.05 2.70

The addition of hydrogenated nonioinic surfactant, C E«, to the aqueous phase (pH 7.0) at concentrations of the order of 1X10"* wt% and above leads to a substantial reduction in the reflectivity of the /?-casein layer adsorbed from a bulk protein solution of 5 X 1 0 wt%. Figure 4 shows plots of the logarithm of the measured reflectivity (R) against scattering vector (q) for four different surfactant concentrations. The especially large fall in reflectivity between surfactant concentrations of 2.5X10" wt% and 5X10" wt% is suggestive of substantial displacement of protein from the air/water interface in this surfactant concentration range. Corkhill and co-workers (20) determined the cmc in water to be 5 X 1 0 wt% for this surfactant, so that the observed protein displacement is taking place predominantiy below this critical concentration. The molecular scattering length for hydrogenated C E calculated on the basis of its chemical composition is some 400 times smaller than for /3-casein. Allowing for the 50-fold difference in molecular weight, a complete layer of this surfactant is still calculated to be approximately 8 times less reflective than a monolayer of protein. The low reflectivities observed at surfactant concentrations of 1X10" wt% are consistent with these calculations and indicate total displacement of protein from the air/water interface at these surfactant levels at neutral pH. Because the hydrogenated surfactant is such a low-power reflector of neutrons relative to the 0-casein, it is possible to calculate from the Guinier parameters derived from the reflectivity data a titration curve for the displacement of protein by surfactant (77). Such plots reproduce the behaviour seen in more conventional chemical laboratory studies of the competitive adsortion between jS-casein and nonionic surfactant (27). With the technique of neutron reflection relying heavily on model fitting for data interpretation, it is gratifying it can also reproduce the results of the simpler, less esoteric methods. This imparts greater faith to the reliability and acceptability of parameters derived from the more complicated analytical procedures required for full interpretation of the data. Thus we anticipate that a full analysis of the reflectivity data will provide a detailed picture of the composition and structure of the mixed protein/surfactant layer. To exploit fully the advantage provided in neutron reflection of the varying contrasts of different isotopes, we have recentiy performed experiments involving deuterated surfactant and mixtures of deuterated and hydrogenated surfactant to provide a range 12

3

4

4

3

1 2

6

2


22.

ATKINSON ET AL.


02* >

02

6 d'

pH 5.5

2


~2Xd.

w 01

Neutron Reflectivity of Adsorbed Protein Films - ACS Symposium

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