Surfactant

ARTICLE pubs.acs.org/Langmuir

Adsorption Behavior of Hydrophobin and Hydrophobin/Surfactant Mixtures at the Air
Water Interface Xiaoli L. Zhang,† Jeffrey Penfold,*,†,‡ Robert K. Thomas,† Ian M. Tucker,§ Jordan T. Petkov,§ Julian Bent,|| Andrew Cox,|| and Richard A. Campbell^ †

Physical and Theoretical Chemistry Laboratory, Oxford University, South Parks Road, Oxford, United Kingdom STFC, Rutherford Appleton Laboratory, Chilton, Didcot, Oxon, United Kingdom § Unilever Research and Development Laboratory, Port Sunlight, Quarry Road East, Bebington, Wirral, United Kingdom Unilever Research Laboratories, Sharnbrook, MK44 1LQ Beds, United Kingdom ^ Institut Laue Langevin, 6 Rue Jules Horowitz, F-38042, Grenoble, Cedex 09, France

)

‡

bS Supporting Information ABSTRACT: The adsorption of the surface-active protein hydrophobin, HFBII, and the competitive adsorption of HFBII with the cationic, anionic, and nonionic surfactants hexadecyltrimethylammonium bromide, CTAB, sodium dodecyl sulfate, SDS, and hexaethylene monododecyl ether, C12E6, has been studied using neutron reflectivity, NR. HFBII adsorbs strongly at the air
water interface to form a dense monolayer ∼30 Å thick, with a mean area per molecule of ∼400 Å2 and a volume fraction of ∼0.7, for concentrations greater than 0.01 g/L, and the adsorption is independent of the solution pH. In competition with the conventional surfactants CTAB, SDS, and C12E6 at pH 7, the HFBII adsorption totally dominates the surface for surfactant concentrations less than the critical micellar concentration, cmc. Above the cmc of the conventional surfactants, HFBII is displaced by the surfactant (CTAB, SDS, or C12E6). For C12E6 this displacement is only partial, and some HFBII remains at the surface for concentrations greater than the C12E6 cmc. At low pH (pH 3) the patterns of adsorption for HFBII/SDS and HFBII/C12E6 are different. At concentrations just below the surfactant cmc there is now mixed HFBII/surfactant adsorption for both SDS and C12E6. For the HFBII/SDS mixture the structure of the adsorbed layer is more complex in the region immediately below the SDS cmc, resulting from the HFBII/SDS complex formation at the interface.

’ INTRODUCTION Biosurfactants are of much current interest1,2 because of their potential applications in areas involving bioremediation3 and biosustainability and biodegradation.4 The glycolipids,1 such as the rhamnolipids, sophorolipids, and mannosylerythritol lipids, and the lipopeptides and proteins,2 such as surfactin and hydrophobin, are some of those most commonly considered and studied. Indeed, the patterns of protein adsorption at interfaces have in particular been extensively studied.5 Much of the attraction and potential of the different classes of biosurfactant lies in their novel surface adsorption and solution self-assembly properties, and yet many of these aspects are poorly characterized or understood. Hydrophobin is a small (7
10 kDa) highly surface active globular protein which is produced by filamentous fungi, and its primary crystal structure has been established.6,7 It is characterized by a conserved pattern of eight cysteine residues which make four intramolecular disulfide bridges and which make the protein r 2011 American Chemical Society

very compact and robust. The structure is nearly globular, with a central β-barrel structure and a small segment of R-helix. Its functionality and hence its surface activity arise from a hydrophobic patch consisting of side chain residues of leucine, valine, and analine, which occupies 20% of the surface area. This structure and the surface segregation into hydrophilic and hydrophobic regions have prompted comparisons with Janus particles.8,9 There are two major classes of hydrophobin which differ in their aqueous solubility, class I being relatively hydrophobic and class II more water-soluble. Examples of class II materials are HFBI and HFBII, which originate from the organism Trichoderma ressei. In this paper we focus entirely on HFBII, produced via fermentation of a modified yeast.

Received: May 9, 2011 Revised: July 17, 2011 Published: July 20, 2011 11316

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The biological function of hydrophobin is diverse, but is related to its strong surface activity and hence usually involves surfaces and interfaces. Hydrophobins are both secreted at and retained within fungal structures. It is their extreme and strong surface activity that is central to all aspects of its functionality. They act as a coating or protective (hydrophobic) surface, aid adhesion, and promote surface modification. Their ability to lower surface tension and aid growth of aerial hyphae and coat spores and promote attachment of fungi to solid surfaces are all examples of their strong surface properties.2 Their unusually strong surface properties have generated much interest in their potential industrial applications, as surface coatings and protective agents, and in adhesion. Their other major attraction is their ability to stabilize dispersions, such as foams and emulsions, and this has considerable potential in aerated foods such as ice cream and other food-based emulsions and foams.2,10 To maximize their potential for applications, a more detailed understanding of their surface and self-assembly properties, and especially of their interaction with conventional surfactants and more flexible proteins, such as β-casein, is essential. Although protein/surfactant interactions11 and protein/surfactant adsorption at interfaces12
14 have been extensively studied, the behavior of HFBII/surfactant mixtures has not been explored. However, HFBII adsorption at interfaces has been studied by surface tension8,15 and X-ray reflectivity and other techniques.16
18 In this paper neutron reflectivity, NR, has been used to probe the adsorption behavior of HFBII and HFBII in combination with the anionic, cationic, and nonionic surfactants of sodium dodecyl sulfate, SDS, hexadecyltrimethylammonium bromide, CTAB, and hexaethylene monododecyl ether, C12E6, at the air
water interface. This is the first paper of a series which will explore some of the fundamental physicochemical properties of HFBII/surfactant and HFBII/protein mixtures at different surfaces and in solution.

’ EXPERIMENTAL DETAILS Neutron Reflectivity. The specular neutron reflectivity measurements were made on the SURF reflectometer19 at the ISIS pulsed neutron source at the Rutherford Appleton Laboratory, U.K, and on the FIGARO reflectometer20 at the Institut Laue Langevin, Grenoble, France. On both reflectometers the measurements were made at a fixed angle of incidence and with the white beam time-of-flight method to cover a wide range of incident wavelengths. On SURF the measurements were made using a single detector at a fixed angle, θ, of 1.5 and neutron wavelengths in the range of 0.5
6.8 Å to cover a Q range of 0.048
0.5 Å
1 and using what are now well-established experimental procedures. On FIGARO, similar measurements were made at a fixed angle of incidence of 3.8 and neutron wavelengths in the range of 2
30 Å to cover a Q range of 0.03
0.4 Å
1. Similar alignment procedures were used, and on FIGARO an area detector was used to subtract the specular signal from the background scattering. The specular reflection of neutrons provides information about inhomogeneities normal to an interface or surface, and the technique is described in detail elsewhere.21 The basis of an NR experiment is that the variation in specular reflection with Q (the wave vector transfer normal to the surface, defined as Q = (4π/λ) sin θ, where λ is the neutron wavelength and θ is the grazing angle of incidence) is simply related to the composition or density profile in a direction normal to the interface. In the kinematic or Born approximation, it is just related to the square of the Fourier transform of the scattering length density profile, F(z): RðQ Þ ¼

16π2 j Q2

Z

FðzÞe
iQz dzj2

ð1Þ

Table 1. Scattering Lengths of Different Components ∑b (10
3 Å)

Component HFBII

16.2

d-SDS

2.76

h-SDS

0.16

d-CTAB

3.29

h-CTAB

0.15

d-C12E6

2.74

h-C12E6

0.13

D2O nrw

0.19 0.0

where F(z) = ∑ini(z)bi, ni(z) is the number density of the ith nucleus, and bi is its scattering length. The key to the use of the technique for the study of the adsorption of surfactants, mixed surfactants, and other mixtures of surface-active materials is the ability to manipulate the scattering length density or neutron refractive index profile (where the neutron refractive index is defined as n = 1
λ2F(z)/2π at the interface using hydrogen (H)/deuterium (D) isotopic substitution (where H and D have vastly different scattering powers for neutrons). This has now, for example, been powerfully demonstrated for the study of surfactant adsorption (for the determination of adsorbed amounts and surface structure) in a wide range of surfactants, surfactant mixtures,22 and polymer/surfactant mixtures23 and in protein/surfactant mixtures.24 For such reasons neutron reflectivity has been applied here for the study of the adsorption of HFBII/surfactant mixtures at the air
water interface. In the specific case of surfactant adsorption, for a deuterated surfactant in null reflecting water, nrw (92 mol % H2O
8 mol % D2O has a scattering length of zero, the same as that of air), the reflectivity arises only from the adsorbed layer at the interface. For a simple monolayer this reflected signal can be analyzed in terms of the adsorbed amount at the interface and the thickness of the adsorbed layer. The most direct procedure for determining the surface concentration of surfactant is to assume that it is in the form of a single layer of homogeneous composition. The measured reflectivity can then be fitted by comparing it with a profile calculated using the optical matrix method for this simple structural model. The parameters obtained for such a model fit are the scattering length density, F, and the thickness, τ, of the layer. The area per molecule is then given by A¼

∑i bi =Fτ

ð2Þ

where ∑bi is the scattering length of the adsorbed surfactant molecule. In the case of the binary HFBII/surfactant mixture, as studied here, eq 2 can be extended as F¼

∑b1 =A1 τ þ ∑b2 =A2 τ

ð3Þ

where bi and Ai are the scattering lengths and area per molecule of each component in the binary mixture. Two different reflectivity measurements are made, with and without the surfactant deuterium labeled. HFBII (in common with many proteins) has a finite contrast, without the need for deuterium labeling. Hence, the adsorption of both the HFBII and surfactant components can be established by solving eq 3 for the complementary measurements. Furthermore, the NR measurements for HFBII alone rely upon the finite contrast of the HFBII. The associated sums of scattering lengths for the different components used in this study are summarized in Table 1. Materials and Measurements Made. The deuterium-labeled surfactants (alkyl chain deuterium labeled) of SDS, CTAB, and C12E6 (abbreviated as d-SDS, d-CTAB, and d-C12E6) were synthesized using established synthetic routes.27
29 The hydrogeneous SDS and CTAB 11317

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Figure 1. (a) Neutron reflectivity versus wave vector transfer, Q, for 0.2 g/L HFBII in nrw (b). The solid line is a model calculation assuming a single layer of uniform composition with d = 30 Å and F = 0.9  10
6 Å
2. (b) Adsorption isotherm (adsorbed amount, Γ, 10
10 mol cm
2) for HFBII. (h-SDS, h-CTAB) were obtained from Sigma. The hydrogeneous C12E6 (h-C12E6) was obtained from Nikkol and used as supplied. SDS and CTAB were recrystallized in ethanol/acetone mixtures, and C12E6 was purified on a chromatography column. The purity of the surfactants was checked using surface tension measurements, and no minima in the surface tension were observed. Class II hydrophobin (HFBII) was produced at an external fermentation company (BAC) using a yeast fermentation route and was subsequently purified by a two-phase extraction at Unilever Research Vlaardingen using the procedures described in more detail elsewhere.10,30 For dissolution into the appropriate aqueous solvent for the NR measurements, the extracted and purified material was freeze-dried. The NR measurements were all made in nrw (unless otherwise stated). High-purity water (Elga Ultrapure) was used, and the D2O was obtained from Fluorochem. The pH was adjusted by the addition of HCl and NaOH. All glassware and the Teflon troughs for the NR measurements were cleaned using alkali detergent (Decon 90) and rinsed thoroughly in highpurity water. All the measurements were made at a fixed temperature of 30 C, chosen to be in excess of the Krafft point of the ionic surfactants.25,26 The cmc values for the different surfactants are 8  10
3 M for SDS, 9  10
4 M for CTAB, and 9  10
5 M for C12E6.25 Care was taken during the neutron reflectivity measurements and during the associated sample preparation to avoid bubble formation, using degassed water, and sonication, because at low concentrations without these precautions lower adsorbed amounts resulted due to depletion of the HFBII in solution from adsorption at the bubble interfaces. The adsorption of HFBII alone was characterized using NR in the concentration range from 10
3 to 0.2 g/L, predominantly at pH 7, but also at pH 3 and 10 over a more limited concentration range. NR measurements were made for mixed solutions of 5  10
2 g/L HFBII in combination with SDS, CTAB, and C12E6 and again predominantly at pH 7. Measurements were made for the isotopic combinations of d-surfactant/HFBII and h-surfactant/HFBII in nrw. The concentration range of the surfactant was 10
5 to 2  10
2 M for SDS, 10
5 to 5  10
3 M for CTAB, and 4  10
6 to 5  10
4 M for C12E6 from below to above the surfactant cmc. For HFBII/SDS and HFBII/C12E6 (at 5  10
2 g/L) some additional measurements were made at pH 3 over a more limited range of surfactant concentrations.

’ RESULTS AND DISCUSSION HFBII Adsorption. The HFBII adsorption at the air
water interface has been measured using NR in the concentration range

from 10
3 to 0.2 g/L. Typical reflectivity data, for 0.2 g/L HFBII in nrw, are shown in Figure 1a, and data for the other HFBII concentrations are shown in Figure S1 in the Supporting Information. The reflectivity data are well described by a single layer of uniform density, as is routinely observed for a wide range of surface-active materials in nrw.21,22 From the variation with HFBII concentration, the corresponding adsorption isotherm is plotted in Figure 1b. Apart from the measurement at the lowest HFBII concentration (10
3 g/L) the adsorbed amount is constant within experimental error. For HFBII concentrations in the range from 2  10
3 to 0.2 g/L the mean value of the adsorbed layer thickness, d, is 31 ( 2 Å, the mean area per molecule, A, is 420 ( 20 Å2, and the adsorbed amount is 0.39 ( 0.02  10
10 mol cm
2. The key model parameters from the analysis of the corresponding NR data are summarized in Table S1 in the Supporting Information. At a concentration of 10
3 g/L the adsorbed amount decreases from ∼0.39  10
10 to 0.29  10
10 mol cm
2. This is consistent with the trends in the surface tension data of Cox et al.8 Due to the unusual surface rheology properties, it is difficult to obtain reliable and reproducible surface tension data. However, the measurements of Cox et al. showed an initial break point at a concentration of ∼0.4 μM (∼3  10
3 g/L), and this was interpreted as the surface saturation concentration (as opposed to a cmc). The break point is consistent with the NR-derived adsorption isotherm presented here. At extremely low HFBII concentrations (eμM) the adsorption shows some marked time dependence. At higher HFBII concentrations, g8  10
3 g/L, the adsorption reached equilibrium on a time scale shorter than that of an individual NR measurement (eminutes). However, at the lowest concentrations (∼μM) it took g60 min for the surface to establish equilibrium adsorption. Hence, for example, at an HFBII concentration of 10
3 g/L (∼1.7  10
7 M) the area per molecule from the initial measurement was ∼980 Å2, and the equilibrium value was ∼580 Å2 (established after a lapse time on the order of 5 h). At an HFBII concentration of 2  10
3 g/L (∼3.4  10
7 M) the area per molecule change from the initial measurements to equilibrium was from 560 to 450 Å2 for a similar lapse time. 11318

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Table 2. Key Model Parameters from Analysis of NR Data for HFBII/CTAB Adsorption at the Air
Water Interface at pH 7a surfactant

HFBII/

concentration

surfactant

(M) 1E
5 1E
4 5E
4 8E
4 1E
3

d

F ((0.05 

A

Γ ((0.04 

combination ((2 Å) 10
6 Å
2) ((20 Å2) 10
10 mol cm
2) hd

35

1.2

1320

0.13

hh

31

1.3

395

0.42

hd

33

1.4

1431

0.11

hh

30

1.3

402

0.41

hd

36

1.3

660

0.25

hh

33

1.2

398

0.42

hd

34

1.6

114 ( 10

1.46

hh

35

0.7

602

0.28

hd

25

3.5

38 ( 2

4.37

24

3.5

39 ( 2

4.21

23

3.5

41 ( 2

4.08

hh 2E
3

hd hh

Figure 2. Neutron reflectivity for 5  10
2 g/L HFBII with (red) 10
4 M d-CTAB, (blue) 10
4 M h-CTAB, (green) 10
3 M d-CTAB, and (black) 10
3 M h-CTAB in nrw. The solid lines are model calculations for a monolayer of uniform composition and for the parameters summarized in Table 2. The data for 10
3 M h-CTAB/5  10
2 g/L HFBII (black) show no reflectivity, and the signal present is purely the background scattering.

Some further NR measurements were made in the saturated adsorption region (g8  10
3 g/L) where the pH was adjusted to 3 and 10, and the NR data are shown in comparison with the data at pH 7 in Figure S2 in the Supporting Information. A summary of the parameters from the analysis of those data are summarized in Table S2 in the Supporting Information. Within the region of saturation adsorption, the increase or decrease in the solution pH had no significant impact upon the adsorbed amount or the thickness of the adsorbed layer of HFBII. The isoelectric point (IEP) of HFBII is ∼3.5, and hence, changing the surface charge in magnitude and in sign and the measurement close to the IEP do not impact the adsorption at the air
water interface. This implies that the surface adsorption of HFBII is dominated by the hydrophobic patches on the surface of the HFBII and not electrostatic interactions, and in particular, measurement close to the IEP does not result in a more highly packed structure. The mean thickness of the adsorbed HFBII layer is ∼30 Å. From the approximate dimensions of the globular protein (24  27  30),6 this corresponds to an orientation with the hydrophobic patches adjacent to the air phase. Similar dimensions are reported from X-ray reflectivity measurements and inferred from other related studies.16
18 The density of the adsorbed layer corresponds to a volume fraction of HFBII at the interface of ∼0.7. HFBII/Surfactant Mixed Adsorption. NR measurements of the competitive adsorption of HFBII with conventional cationic, anionic, and nonionic surfactants, CTAB, SDS, and C12E6, were made at the air
water interface. The measurements were made at a fixed HFBII concentration of 5  10
2 g/L (in the region of saturated adsorption for the HFBII) and at pH 7. The surfactant concentration was varied from well below to in excess of the surfactant cmc. Measurements were made for the isotopic combinations of HFBII/h-surfactant and HFBII/d-surfactant in nrw. Some typical reflectivity data for the HFBII/CTAB mixture are shown in Figure 2, and similar data for the HFBII/SDS

5E
3

hd hh

“hh” refers to the HFBII/h-surfactant isotopic combination and “hd” to HFBII/d-surfactant. a

and HFBII/C12E6 mixtures are shown in Figure S3 in the Supporting Information. Broadly similar data are obtained for the HFBII/SDS and HFBII/C12E6 mixtures, and the key model parameters from the analysis of those data are also summarized in Table 2 and Table S3 in the Supporting Information. In Figure 2 the reflectivities at the lower surfactant concentration of 10
4 M are similar for both “contrasts”, HFBII/h-CTAB and HFBII/d-CTAB. For the combination HFBII/h-CTAB the only contribution to the reflected signal arises from the HFBII component at the interface (h-CTAB is effectively matched to the air and solution phases). Hence, the surface must be dominated by the HBII adsorption at this surfactant concentration. At higher surfactant concentration (10
3 M) the situation is markedly different, and there is no reflected signal above the background for the isotopic combination of HFBII/h-CTAB, but there is a strong reflectivity for the isotopic combination of HFBII/d-CTAB. This implies that in this case the surface adsorption is now dominated by the CTAB adsorption, and there is little or no HFBII adsorption. These general trends in the relative adsorption are also consistent with the variation in the observed thickness of the adsorbed layer. For concentrations < cmc the thickness of the adsorbed layer has a mean thickness of ∼36 Å, whereas for concentrations > cmc the mean thickness is ∼24 Å for CTAB and ∼20 Å for SDS. These variations in the adsorbed layer thickness entirely reflect the change in the surface adsorption, from a surface dominated by HFBII to one dominated by the surfactant. Below the cmc the thickness of the predominantly HFBII layer is systematically thicker than the value obtained for HFBII alone (∼36 Å compared to ∼31 Å), and this reflects a change in the packing to accommodate the small amount of coadsorbing surfactant (see later discussion). Above the cmc the thickness corresponds to that expected for CTAB and SDS monolayers. Using the parameters in Tables 1 and 2 and in Table S3 in the Supporting Information, in eq 3 the simultaneous equations arising from the measurements at the two isotopic combinations can be solved straightforwardly to obtain the area per molecule 11319

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Figure 4. Variation in surface composition (mole fraction of HFBII) for (red) HFBII/CTAB, (blue) HFBII/SDS, and (green) HFBII/C12E6. The vertical tick marks indicate the pure surfactant cmc values.

Figure 3. Adsorbed amount (Γ  10
10 mol cm
2) versus surfactant concentration for 5  10
2 g/L HFBII with (a) CTAB, (b) SDS, and (c) C12E6.

and hence adsorbed amounts of each component, and these values are listed in Table 2 and in Table S3 in the Supporting Information. The relative adsorptions of the HFBII/surfactant mixtures are then plotted in parts a, b, and c of Figure 3 for HFBII/CTAB, HFBII/SDS, and HFBII/C12E6, respectively. For the HFBII/CTAB and HFBII/SDS mixtures the variation in the adsorption of the two components in the mixture follows a similar pattern. For surfactant concentrations < cmc the surface is dominated by the HFBII adsorption, whereas above the cmc it is

dominated by the surfactant adsorption and there is no HFBII adsorption. This transition is relatively abrupt, and there is only a relatively narrow region where there is a substantial adsorption of both components. The adsorption behavior observed implies that the formation of HFBII/surfactant mixed aggregates is energetically more favorable than the mixed adsorption at the air
water interface. This is consistent with the general trend that has been observed in polyelectrolyte/surfactant mixtures23 and in other protein/surfactant mixtures,24,31 where desorption of the polyelectrolyte or protein occurs above the surfactant cmc. Below the surfactant cmc the amount of HFBII adsorbed is close to its saturation value in the absence of surfactant. However, there is evidence that there is a small amount of surfactant coadsorbing in this region of concentrations, and given a relatively large uncertainty in the values obtained for the surfactant, the coadsorption in similar for CTAB and SDS. However, the systematic trends imply that there is a strong coadsorption for the SDS and hence a strong surface interaction. The finite but small coadsorption implies some slight surface synergy and surface interaction between the HFBII and surfactant, and a similar effect was reported for the nonionic polymer/ionic surfactant mixtures of PEO/SDS.32 In other polyelectrolyte/ surfactant and protein/surfactant mixtures a strong synergistic coadsorption is often observed for concentrations below the surfactant cmc.33 The general trends in the coadsorption of the HFBII/C12E6 mixture at the air
water interface are broadly similar to those observed for HFBII/CTAB and HFBII/SDS. However, there is one notable difference. For HFBII/C12E6 at surfactants concentrations > cmc of C12E6 there is a small but finite adsorption of HFBII. This is in marked contrast to the observations for the HFBII/CTAB (SDS) mixtures, where above the surfactant cmc there was no HFBII adsorption evident. This is illustrated in Figure 3c and more clearly demonstrated in comparison with the other mixtures in Figure 4. There is no evidence from the adsorption data that the coadsorption for HFBII/C12E6 at concentrations < cmc is any stronger than for HFBII/CTAB (SDS). Hence, the differences for concentrations > cmc reported here for HFBII/C12E6 must be associated with changes in the solution behavior compared to 11320

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Table 3. Model Parameters from Analysis of Reflectivity Data for SDS/5  10
2 g/L HFBII at pH 3 SDS concentration (M)

contrast

d1 ((2 Å)

F1 ((0.05  10
6 Å
2)

2E
2

hh

38

0.18

hd

20

3.1

hh

52

0.14

hd

26

2.8

hh

50

1.0

hd

16

3.2

hh

56

0.8

hd

19

1.4

5E
3 1E
3 1E
4

Figure 5. Neutron reflectivity for 5  10
2 g/L HFBII/d-SDS/nrw at pH 3, (red) 2  10
2 M SDS, (blue) 5  10
3 M SDS, and (green) 10
3 M SDS. The data are shifted vertically for clarity.

that of HFBII/CTAB (SDS). This would imply that the HFBII/ C12E6 solution complexation is not as pronounced or as energetically favorable as it is for the HFBII/CTAB (SDS) mixtures. HFBII/SDS and HFBII/C12E6 Mixed Adsorption at Low pH. Some NR measurements over a limited range of surfactant concentrations were made for HFBII/SDS and HFBII/C12E6 mixtures at a fixed HFBII concentration of 5 x10
2 g/L at the reduced pH of 3. For the HFBII/SDS mixture at pH 3 NR measurements were made at an HFBII concentration of 5  10
2 g/L and SDS concentrations of 10
4, 10
3, 5  10
3, and 2  10
2 M SDS, in the region where there is a transition in the adsorption behavior at pH 7 from HFBII to SDS dominated adsorption. In this concentration range at pH 7 the adsorption is in the form of a monolayer (see Table 3), and at pH 3 and at an SDS concentration of 2  10
2 M the SDS adsorption is similar (see Figure 5). However, at lower SDS surfactant concentrations, 5  10
3 to 10
4 M, the form of the reflectivity is different, as illustrated in Figure 5 for 5  10
3 and 10
3 M SDS (the data at 10
4 M SDS are shown in Figure S4 in the Supporting Information). The reflectivity has a pronounced interference fringe at high Q and is no longer consistent with a single monolayer. The data shown in Figure 5 are for the isotopic combination HFBII/ d-SDS/nrw, and so are dominated by the SDS adsorption. The data at 5  10
3 M are consistent with a three-layer structure at

d2 ((2 Å)

F2 ((0.05  10
6 Å
2)

d3 ((2 Å)

F3 ((0.05  10
6 Å
2)

10

1.1

19

2.1

36

1.9

37

1.6

the interface, whereas the data at 10
3 and 10
4 M are well described by two layers. The key model parameters are summarized in Table 3. This surface structure is broadly similar to that observed in polyelectrolyte/surfactant mixtures, in situations where a strong surface interaction exists, such as in PEI/SDS mixtures at high pH.24 The complementary measurements of HFBII/h-SDS/nrw (see also Table 3), where the reflectivity is dominated by HFBII, also confirms the presence of HFBII at the interface over this SDS concentration range. At the higher pH 7 there was no HFBII adsorption at the higher SDS concentrations (see Table S3 in the Supporting Information and Figure 3b). The results imply the formation of HFBII/SDS complexes at the surface, which then retain some HFBII at the interface even at the SDS concentrations in excess of the cmc, and in contrast to what is observed at the higher pH 7. Furthermore, the interaction is such that the complexation promotes a more complex surface structure and surfactant distribution at the interface. At pH 3 the HFBII is now below its IEP (the IEP for HFBII is ∼3.5) and is now positively charged. At pH 7 HFBII is negatively charged. At pH 7 the surface behavior of the HFBII/SDS mixture is primarily determined by its competetive adsorption and the relative free energy of the formation of bulk HFBII/SDS aggregates, driven primarily by the hydrophobic interaction. Any surface interaction at that pH will be primarily of hydrophobic origin. At pH 3 the stronger surface interaction implied by the more complex layer structure arising from SDS/HFBII surface complex formation can now have an electrostatic contribution in addition to any hydrophobic contribution. NR measurements were also made for the HFBII/C12E6 mixture at pH 3, for C12E6 concentrations of 4  10
5, 6  10
5, 10
4, and 2  10
4 M. In contrast to the data for HFBII/ SDS at pH 3 the data for HFBII/C12E6 remain as a monolayer. The key model parameters for the two different isotopic combinations measured are summarized in Table S4 in the Supporting Information. In Table 4 the adsorbed amounts of HFBII and C12E6 at pH 7 and 3 are directly compared. The results in Table 4 are consistent with a shift in the competition between the C12E6 and HFBII adsorption in favor of C12E6 to lower C12E6 concentrations. This is not due to an enhanced surface activity of C12E6 at the lower pH34 or due to a shift in the cmc of C12E6. The results imply a shift in the surface synergy or interaction between the HFBII and C12E6 and a less favorable free energy of formation of the bulk HFBII/C12E6 aggregates. General Discussion. There are broad similarities between the adsorption behavior of HFBII/surfactant (CTAB, SDS, and C12E6) mixtures and that reported for polymer/surfactant 11321

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Table 4. Comparison of Adsorbed Amounts of HFBII and C12E6 for 5  10
2 g/L HFBII at pH 3 and 7

C12E6

adsorbed amount

adsorbed amount

((0.05  10
10 mol cm
2) at pH 3

((0.05  10
10 mol cm
2) at pH 7

concentration (M)

HFBII

C12E6

HFBII

C12E6

2E
4

0.07

2.3

0.12

2.7

1E
4

0.14

3.1

0.05

2.7

6E
5

0.19

1.9

0.29

0.29

4E
5

0.25

1.5

0.37

0.15

mixtures23,35 and for other protein/surfactant mixtures.24,31 In common with the HFBII/surfactant mixtures, in polymer/surfactant mixtures and in protein/surfactant mixtures any polymer or protein coadsorbed at the interface at surfactant concentrations < cmc is replaced by surfactant at concentrations > cmc. This is generally assumed to be due to a more energetically favorable formation of bulk solution polymer (protein)/surfactant complexes for surfactant concentrations > cmc. Below the cmc it is usual to encounter some synergistic coadsorption of the polymer (protein) and surfactant in the form of surface complexes, and this has been demonstrated particularly in protein/surfactant31 and in polyelectrolyte/surfactant23 mixtures. In the case of SDS/poly(ethylene oxide), PEO, mixtures,32 there was minimal surface interaction between the SDS and PEO. Hence, below the cmc the adsorption was dominated by PEO, and by SDS for concentrations > cmc. A similar behavior was observed in poly(N-isopropylacrylamide), PNIPAM/octaethylene monodecyl ether, C10E8, mixtures.36 The abrupt change in the pattern of adsorption of HFBII and surfactant at the cmc here is consistent with the competition between surface adsorption and solution complex formation, and not simply due to competitive adsorption associated with different packing criteria. The adsorption patterns for the HFBII/CTAB, HFBII/SDS, and HFBII/ C12E6 mixtures are most closely associated with the trends observed in SDS/PEO mixtures. That is, below the cmc the adsorption is dominated by HFBII, with little coadsorption, and above the cmc HFBII is effectively removed from the surface. In protein/surfactant mixtures other factors need to be taken into account, as was demonstrated by Green et al.31 for the adsorption of lysozyme/SDS mixtures at the air
water interface. Below the cmc there was a strong mixed lysozyme/SDS adsorption, and at much higher SDS concentrations the lysozyme was removed from the surface and only SDS adsorption occurred. At intermediate SDS concentrations, before the lysozyme desorption, the structure of the adsorbed layer changed, and this was attributed to a breakdown of the secondary structures of the lysozyme by the SDS. This is not observed here for the surfactant/HFBII mixtures, where, on the basis of the observed thickness of the adsorbed layer where HFBII dominates the adsorption, the HFBII remains intact. This is not surprising as HFBII is a more compact and tightly bound protein, due to its intramolecular disulfide bridges. It is known that nonionic surfactants interact more weakly with proteins. This was illustrated in a study of the coadsorption between lysozyme and the nonionic surfactant pentaethylene glycol monododecyl ether, C12E5.37 Due to this weak interaction, nonionic surfactant/protein adsorption (for proteins such as β-casein and lysozyme) has been described in terms of competitive

adsorption.38 In that case there was little sign of any associative surface interaction before the desorption of the lysozyme at higher surfactant concentrations. However, some deformation of the protein's globular structure was observed in this more weakly bound protein. Here the pattern of adsorption of the HFBII/ C12E6 mixture is broadly similar to that encountered in the HFBII/SDS and HFBII/CTAB mixtures. However, the major difference is that above the C12E6 cmc there remains a fraction of HFBII at the interface, which is not observed in the other mixtures. As discussed earlier this is attributed to a weaker solution complex formation between HFBII and C12E6. At pH 7 HFBII is nominally anionic, and hence, there is little or no electrostatic interaction between HFBII and SDS or C12E6 and only an opportunity for an electrostatic interaction with CTAB. However, the weak surface synergy and interaction implies that the surface interaction is only weakly hydrophobic, even for CTAB. However, at low pH, pH 3, and below its IEP HFBII is now cationic. Here, there is evidence from the NR data of a more complex surface structure due to the formation of surface HFBII/SDS complexes, driven by the electrostatic interaction between HFBII and SDS. This is similar to what has been observed in polyelectrolyte mixtures, such as poly(styrenesulfonate)/CTAB39 and especially in poly(ethyleneimine), PEI/SDS mixtures,33 and remarkably similar surface structures are observed. For both mixtures studied at low pH, HFBII/SDS and HFBII/C12E6, pH is seen to have a significant impact upon the pattern of adsorption. This contrasts sharply with the situation for the adsorption of HFBII alone, where pH has a minimal impact, and it can be concluded that the surface interaction between neighboring HFBII molecules is dominated by the hydrophobic interaction. The absence of a strong surface interaction between HFBII and CTAB at pH 7 is due to HFBII being only weakly anionic at pH 7 and the intrinsically weaker interaction of the charged groups with the CTAB headgroup than the SDS headgroup.

’ SUMMARY The adsorption of HFBII and HFBII/surfactant mixtures at the air
water interface has been determined using NR. HFBII adsorbs strongly down to low concentrations to form a compact layer, with a mean area per molecule of ∼420 Å2 and a thickness of ∼31 Å. This is consistent with a monolayer of HFBII at the interface, with its hydrophobic patch adjacent to the air phase. The competitive adsorption of HFBII with conventional surfactants CTAB, SDS, and C12E6 at the air
water interface shows that the strong surface activity of HFBII dominates the adsorption for surfactant concentrations < cmc. Above the cmc HFBII is displaced entirely by CTAB or SDS in favor of solution complex formation and is replaced by surfactant at the interface. This is broadly similar to what is observed in other protein/ surfactant mixtures, such as lysozyme/SDS.31,37 The notable difference in the comparison with lysozyme/SDS is that SDS disrupts the secondary protein structure prior to desorption, whereas the more compact tightly bound HFBII remains intact before desorption. The generally weaker interaction observed in nonionic/protein mixtures37 is also observed here, and in the case of HFBII/C12E6 adsorption some HFBII remains at the interface at concentrations greater than the C12E6 cmc. At low pH (pH 3) the impact of the charge reversal of the HFBII, now below its IEP, is evident and a stronger HFBII/SDS interaction is observed. This is manifest in the formation of a 11322

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Langmuir more complex layered structure at the interface for SDS concentrations < cmc, and the surface structure is similar to that previously observed in polyelectrolyte/surfactant mixtures.33,39 These observations provide some important insights into HFBII adsorption, how HFBII adsorption can be manipulated, and how HFBII in combination with conventional surfactants can be used to manipulate and tailor surface properties.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional NR data and key model parameters from the analysis of the NR data for HFBII, HFBII/SDS, and HFBII/C12E6 adsorption. This material is available free of charge via the Internet at http://pubs.acs.org//

’ AUTHOR INFORMATION Corresponding Author

*E-mail: jeff[email protected].

’ ACKNOWLEDGMENT The contribution from John Chapman, Chris Sidebottom, and Tim Litchfield regarding the production and purification of the HFBII from modified yeast is gratefully acknowledged. We acknowledge the provision of neutron beam time on SURF at the ISIS Facility, U.K., by STFC, and on FIGARO at the Instititut Laue Langevin, France. The support and assistance of the instrument scientists on SURF, Arwell Hughes, Phil Taylor, Max Skoda, and John Webster, and of the instrument technician on FIGARO, Simon Wood, is much appreciated. ’ REFERENCES (1) Kitamoto, D.; Morita, T.; Fukuoka, T.; Konishi, M.; Inura, J. Curr. Opin. Colloid Interface Sci. 2009, 14, 315. (2) Linder, M. B. Curr. Opin. Colloid Interface Sci. 2009, 14, 356. (3) Milligan, C. N. Environ. Pollut. 2005, 133, 183. (4) Muthusamy, K.; Gopalakrishnan, S.; Kochupappy, T.; Sivachidambaram, R.; Sivachidambaram, P. S. Curr. Sci. 2008, 94, 736. (5) Dickinson, E. Colloids Surf., B 1999, 15, 161. (6) Hahanpaa, J.; Szilvray, G. R.; Kaljunen, H.; Maksimainen, M.; Linder, M. B.; Rouvinen, J. Protein Sci. 2006, 15, 2120. (7) Kallio, J. M.; Linder, M. B.; Rouvinen, J. J. Biol. Chem. 2007, 282, 28733. (8) Cox, A. R.; Cagnol, F.; Russell, A. B.; Izzard, M. J. Langmuir 2007, 23, 7995. (9) Walther, A.; Muller, A. H. E. Soft Matter 2008, 4, 663. (10) Basheva, E. S.; Kralchevsky, P. A.; Danov, K. D.; Stoyanov, S. D.; Blijdenstein, T. B. J.; Pelan, E. G.; Lips, A. Langmuir 2011, 27, 4481. (11) Goddard, E. D.; Ananthanpadmanabhan, K. P. Interaction of Surfactants with Polymers and Proteins; CRC Press: Boca Raton, FL, 1993. (12) Maldonado-Valderama, J.; Martin-Molina, A.; Martin-Rodriguez, A.; Cabreeizo-Vildez, M. A.; Galvez-Ruiz, M. J.; Langevin, D. J. Phys. Chem. C 2007, 11, 2715. (13) Fainerman, V. B.; Lucassen-Reynders, E.; Miller, R. Colloids Surf., A 1998, 143, 141. (14) Miller, R.; Alehverdjieva, V. S.; Fainerman, V. B. Soft Matter 2008, 4, 1141. (15) Lumsdon, S. O.; Green, J.; Stieglitz, B. Colloids Surf., B 2005, 44, 172. (16) Kisko, K.; Szilvay, G. R.; Vainio, U.; Linder, M. B.; Serimaa, R. Biophys. J. 2008, 94, 198.

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