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pH Sensitive Adsorption of Polypeptide/Sodium Dodecyl Sulfate Mixtures J. Penfold,*,† I. Tucker,‡ R. K. Thomas,§ D. J. F. Taylor,§ and X. L. Zhang§ ISIS, Rutherford Appleton Laboratory, Chilton, Didcot, OXON, OX11 0QX, UnileVer Research and DeVelopment Laboratory, Port Sunlight, Quarry Road East, Bebington, Wirral, CH63 3JW, and Physical and Theoretical Chemistry Laboratory, Oxford UniVersity, South Parks Road, Oxford, OXON, OX1 2JB ReceiVed April 20, 2006. In Final Form: June 28, 2006 Neutron reflectivity and surface tension have been used to investigate the pH sensitivity of the adsorption of poly-L-lysine hydrobromide and sodium dodecyl sulfate mixtures at the air-solution interface. The surface tension variation with surfactant concentration is complex, and between the critical aggregation concentration and critical micellar concentration there is a marked increase in the surface tension. The neutron reflectivity results show that this is associated with a depletion of the surface of polypeptide/surfactant complexes. The variations in the adsorption and surface tension with pH are attributed to changes in the polypeptide conformation at the interface and in solution.
Introduction Recent studies have shown that the strongly interacting polyelectrolyte-ionic surfactant mixtures give rise to a complex solution and surface behavior.1 In particular, the strong surface interaction and the formation of surface polymer-surfactant complexes gives rise to a complex pattern of surface tension and adsorption behavior.2-6 Surface complex formation gives rise to a strong synergism in surface activity, and significantly enhanced surfactant adsorption is observed to very low surfactant concentrations.7 The different patterns of behavior observed in mixtures such as poly(styrene sulfonate)/alkyl trimethylammonium bromide, PSS/CnTAB,2-4 and poly(dimethyldiallylammonium chloride)/sodium dodecyl sulfate, polyDMDAAC/SDS,5,6 have been rationalized in terms of the competition between the formation of bulk polymer-surfactant micelle and surface polymer-surfactant complexes. More recent studies on polyethyleneimine, PEI/SDS,7 and poly(acrylic acid), PAA/CnTAB,8 mixtures have highlighted the role of both electrostatic and hydrophobic interactions in the transition from weak to strong polyelectrolyte/surfactant complexation, which is driven by changing pH. Furthermore, measurements on linear and branched forms of PEI with SDS7 have demonstrated the importance of polymer conformation on the surface adsorption properties of polyelectrolyte/surfactant mixtures. In related studies biological polyelectrolytes, such as DNA,9 and polypeptides such as poly-L-lysine10 have been used as model systems for polyelectrolyte/surfactant complexation and adsorp* To whom correspondence should be addressed. E-mail: j.penfold@ rl.ac.uk. † ISIS. ‡ Unilever Research and Development Laboratory. § Oxford University. (1) Goddard, E. D. J. Coll. Int. Sci. 2002, 256, 228. (2) Taylor, D. J. F.; Thomas, R. K.; Penfold, J. Langmuir 2002, 18, 4748. (3) Taylor, D. J. F.; Thomas, R. K.; Li, P. X.; Penfold, J. Langmuir 2003, 19, 3712. (4) Taylor, D. J. F.; Thomas, R. K.; Hines, J. D.; Humphreys, K.; Penfold, J. Langmuir 2002, 18, 9783. (5) Staples, E.; Tucker, I.; Penfold, J.; Warren, N.; Thomas, R. K. Langmuir 2002, 18, 9783. (6) Staples, E.; Tucker, I.; Penfold, J.; Warren, N.; Thomas, R. K.; Taylor, D. J. F. Langmuir 2002, 18, 5147. (7) Penfold, J.; Tucker, I.; Thomas, R. K.; Zhang, J. Langmuir 2005, 21, 10061. (8) Zhang, J.; Thomas, R. K.; Penfold, J. Soft Matter. 2005, 1, 310.
tion studies. Furthermore the interaction between polypeptides and small surface active molecules is of direct interest in the study of the effects of surfactants on natural membranes and in relationship to gene therapy and drug delivery.11,12 Because of their relatively simple structure, the poly-R-amino acids are suitable polypeptide systems to study, and we have studied here the effect of pH on the surface adsorption of polyL-lysine/SDS mixtures. Here there is an intimate link between polymer conformation, pH, and the strength of the polyelectrolyte/ surfactant interaction. The random coil/β-sheet to R-helix transition in polypeptides, such as poly-L-lysine, in solution is well established from circular dichroism, CD, measurements.13 At high pH polylysine is in the form of an R-helix. As the pH decreases the amino groups become more protonated and the repulsion between the highly charged groups results in a more extended configuration, so that at lower pH it is in the form of a β-sheet or random coil. CD measurements14 show that in solution complexation with SDS causes the polylysine to undergo a conformational change at high SDS concentrations (>20 mM). Babin et al.15 have exploited this pH-driven conformational change in the micellization of block copolymers of polylysine and polyisoprene to manipulate micelle size with pH. Ponomarenko et al.16 have investigated the lamellar nature of the insoluble complexes formed by polylysine and mixtures of alkyl sulfates. Kurawaki and Kusumoto17 investigated the conformational changes in co-polypeptides containing L-lysine in aqueous solution, induced by the cooperative binding of SDS, and observed random coil to β-sheet or R-helix transformations depending upon the nature of the other peptides group. The effects of (9) Stubenrauch, C.; Albouy, P. A.; Klitzing, R. v.; Langevin, D. Langmuir 2000, 16, 3206. (10) Buckingham, J. H.; Lucassen, J.; Hollway, F. J. Coll. Int. Sci. 1978, 67, 423. (11) Sjogren, H.; Ericsson, C. A.; Evenas, J.; Ulvenlund, S. Biophys J. 2005, 89, 4219. (12) Marie, M.; Champeil, P.; Moller, J. V. Biochim. Biophys. Acta 2000, 1508, 86. (13) Lahan, J.; Mitragotri, S.; Tran, T. N.; Kaido, H.; Sundaram, J.; Choi, I. S.; Hoffer, S.; Somorjai, G. A.; Langer, R. Science 2003, 299, 371. (14) Satake, I.; Yang, J. T. Biochem. Biophys. ReV. Comm. 1963, 54, 981. (15) Babin, J.; Hernandez, J. R.; Lecommandoux, S.; Klok, H. A.; Achard, M. F. Faraday Disc. 2005, 128, 179. (16) Ponomarenko, E. A.; Tirrell, D. A.; MacKnight, W. J. Macomolecules 1998, 31, 1584. (17) Kurawaki, J.; Kusumoto, Y. J. Coll. Int. Sci. 2000, 225, 265.
10.1021/la061072s CCC: $33.50 © 2006 American Chemical Society Published on Web 08/04/2006
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surfactant adsorption on the conformation of different polypeptides and copolypeptides have been reported.11,18-20 The surface tension studies of Buckingham et al.10 were used to imply that this SDS-induced transition occurs at lower surfactant concentrations at the interface, although no direct evidence was available. They interpreted the invariance of the surface tension with polypeptide concentration as associated with the polylysine being adsorbed at the interface in the β-sheet conformation. Eckenrode et al.21 attributed the low adsorption at low pH of polylysine onto polystyrene as due to the extended configuration of the polypeptide at low pH. To clarify the role of conformation on the surface adsorption in polypeptide/surfactant mixtures, a direct measure of the adsorption and of the structure of the adsorbed layer is required, and neutron reflectivity measurements can provide directly that crucial information. To that end neutron reflectivity and surface tension measurements were made for a 58 000 MW poly-Llysine hydrobromide/SDS mixtures at pH 3 and 10 and over a wide range of SDS concentrations. Experimental Details The specular neutron reflectivity measurements were made on the SURF reflectometer22 at the ISIS pulsed-neutron source at the Rutherford Appleton Laboratory, U.K. The measurements were made using a single detector at a fixed angle, θ, of 1.5°, using neutron wavelengths, λ, in the range 0.5-6.8 Å to provide a Q range of 0.048-0.5 Å-1, using now well-established experimental procedures. The specular reflection of neutrons provides information about inhomogeneities normal to an interface or surface, and the technique is described in detail elsewhere.23 The basis of a neutron-reflectivity 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) R(Q) )
16π2 | F(z)e-iQz dz|2 Q2
∫
(1)
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 surfactant adsorption 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 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, and polymersurfactant mixtures.24 For a deuterated surfactant in null reflecting water, nrw (92 mol % H2O/8 mol % D2O has a scattering length of zero, the same as air), the reflectivity arises only from the adsorbed layer at the interface. This reflected signal can be analyzed in terms of the adsorbed amount at the interface and the thickness of the adsorbed layer. The (18) Satake, I.; Yang, J. T. Biochem. Biophys. Res. Comm. 1973, 54, 930. (19) Kubota, S.; Ikeda, K.; Yang, J. T. Biopolymers 1983, 22, 2737. (20) Hayakawa, K.; Murata, H.; Satake, I. Coll. Polym. Sci. 1990, 268, 1044. (21) Eckenrode, H. M.; Dai, H. L. Langmuir 2004, 20, 9202. (22) Penfold, J.; Richardson, R. M.; Zarbakhsh, A.; Webster, J. R. P.; Bucknall, D. G.; Rennie, A. R.; Jones, R. A. L.; Cosgrove, T.; Thomas, R. K.; Higgins, J. S.; Fletcher, P. D. I.; Dickinson, E.; Roser, S.; McLure, I. A.; Hillman, A. R.; Richards, R. W.; Burgess, A. N.; Simister, E. A.; White, J. W. J. Chem. Soc,. Faraday Trans. 1997, 93, 3899. (23) Thomas, R. K.; Penfold, J. J. Phys: Condens. Matt. 1990, 2, 1369. (24) Lu, J. R.; Thomas, R. K.; Penfold, J. AdV. Coll. Int. Sci. 2000, 84, 143.
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.24 The parameters obtained for such a fit are the scattering length density, F, and the thickness, τ, of the layer. The area per molecule is then given by A)
∑b /Fτ i
(2)
i
where ∑bi is the scattering length of the adsorbed surfactant molecule. This has been shown to be an appropriate method for surfactants, surfactant mixtures, and polymer/surfactant mixtures.24 In the evaluation of the adsorbed amounts of the SDS at the interface, it is assumed that, for the polymer/surfactant mixtures discussed here, the polymer has a scattering length sufficiently close to zero and that its contribution is negligible. We have not synthesized the deuterium-labeled polymer and do not obtain a direct estimate of the amount of polymer at the interface by a direct extension of eq 2. However, the amount of polymer at the interface can be estimated indirectly by making the measurements in a different way. For measurements with both surfactants deuterated in a D2O subphase, any deviation from the reflectivity of pure D2O (the deuterated surfactants are here closely matched to the D2O) arises from the polymer at the interface, and simple modeling can be used to estimate its amount and its spatial extent at the interface. Analyzing such reflectivity data as a single layer of uniform composition will give a layer thickness τ and a scattering length density F. The difference between the measured scattering length density, F, and that of D2O, FD2O, is assumed to be due to the presence of polymer such that F ) (1 - φp)FD2O
(3)
where φp is the polymer volume fraction in the layer. The surface tension measurements were made on a Kruss K10T maximum pull digital tensiometer using the du Nouy ring method with a Pt/Ir ring. Before each measurement the ring was rinsed in UHQ water and flamed with a Bunsen burner, and repeated measurements were made until equilibrium was established. The protonated surfactant, sodium dodecyl sulfate, h-SDS, and chain-deuterated SDS, d-SDS, were synthesized as described elsewhere25 and purified before use by recrystallization from an ethanol/acetone mixture. The poly-L-lysine hydrobromide was obtained from Sigma-Aldrich with a molecular weight of 58 000 and was used as supplied. Deuterium oxide was supplied by SigmaAldrich, and high-purity water (Elga Ultrapure) was used throughout. The glassware and Teflon troughs used for the measurements, and sample preparations were cleaned in alkali detergent (Decon 90), followed by copious washing in high-purity water. The solution pH was adjusted by the addition of HCl of NaOH and measured using a Accumet Model 50 pH meter. The surface tension and neutron reflectivity measurements were made at 25 °C and at pH 3.0 and 10.0. The polymer concentration was 20 ppm, and the SDS concentration was varied in the range 10-6 to 0.01 M. Measurements were made predominantly for the isotopic combination d-SDS/polyL-lysine hydrobromide/nrw, but at surfactant concentrations of 10-5, 10-4, and 10-3 M and at pH 3.0 and 10.0 the isotopic combination of d-SDS/poly-L-lysine/D2O was also measured.
Results and Discussion Figure 1 shows the variation in surface tension with SDS concentration for 20 ppm poly-L-lysine hydrobromide/SDS mixtures at pH 3 and 10. The surface tension behavior shows a strong dependence on SDS concentration and solution pH. At pH 3 and 10 the surface tension variations show the same overall trends with SDS (25) Lu, J. R.; Morrocco, A.; Su, T. J.; Thomas, R. K.; Penfold, J. J. Coll. Int. Sci. 1993, 158, 303.
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Figure 1. Surface tension for SDS/20 ppm poly-L-lysine hydrobromide as a function of SDS concentration: (O) pH 3; (2) pH 10.
concentration, except at the highest SDS concentration (∼10-2 M) the surface tension is systematically higher at pH 10 than at pH 3. With increasing SDS concentration there is an initial decrease in the surface tension, followed by a narrow plateau region, an abrupt increase in surface tension at ∼10-4 M SDS, and finally a decrease to an eventual plateau at the SDS cmc, ∼10-2 M. Broadly similar behavior has been observed in other polyelectrolyte/ionic surfactant mixtures for SDS/polyDMDAAC,6 PEI/SDS,7 C16TAB/PSS,3 and cationically modified starch/SDS mixtures.26 For SDS/polyDMDAAC,6 the initial decrease in the surface tension was associated with the adsorption of polyelectrolyte/surfactant complexes at the interface. The increase in surface tension at higher SDS concentrations was attributed to a partial depletion of the surface due to competition with bulk polyelectrolyte/surfactant micelle complex formation. The surface tension data for 25 000 linear PEI/SDS mixtures at pH 3 and 7 closely resemble that observed here for the polyL-lysine hydrobromide/SDS mixtures.7 If the surface tension is taken as a measure of the surface activity of the poly-L-lysine/SDS complexes, then the data in Figure 1 would imply that the complexes formed at low pH are more surface active. However, the difficulties in interpreting surface tension data for polyelectrolyte/ionic surfactant mixtures, in terms of adsorption and in the presence of strong surface complexation, have been extensively discussed and highlighted.1 In their pioneering work on polylysine/SDS, Buckingham et al.10 obtained broadly similar surface tension data for polylysine/ SDS as that reported here. To quantify the adsorption they applied the Gibbs equation to the SDS/polylysine mixtures at low SDS concentrations to obtain the surface excess of SDS. At low concentrations, ignoring activity corrections and assuming complete ionization with all the bromide ions replaced by dodecyl sulfate ions, and negligible surface excess of sodium ions, then
-dγ ) RTΓs dln[CNaS(CPBrn)1/n]
(4)
where Γs is the surface excess of dodecyl sulfate ions, γ is the surface tension, CNaS and CPBrn are the concentrations of SDS and polylysine, and n is the degree of polymerization of the polylysine. For large n then eq 4 approximates to
dγ ≈ -RTΓs dlnCNaS (26) Merta, J.; Stenius, P. Coll. Surf. A 1999, 149, 367.
(5)
That is, the slope at low concentrations should be a measure of the SDS adsorption. A similar approach was applied by Langevin et al.27 to the polyanionic/cationic surfactant mixtures of polyacrylamide sulfonate/dodecyl trimethylammonium bromide, PAMPS/DTAB, where an independence of the surface tension with polymer concentration, similar to that observed for SDS/ polylysine by Buckingham et al.,10 was observed. However, here neutron reflectivity data provide a direct measure of the SDS adsorption, and we will return to this later in the discussion. The neutron reflectivity measurements for the isotopic combination of d-SDS/polylysine/nrw provide a direct measure of the adsorption of the SDS at the interface in the presence of the polylysine. The results of the analysis of the data, using eq 2, for 20 ppm polylysine at pH 3 and 10, and a range of SDS concentrations are summarized in Table 1. The results, plotted as adsorbed amount of SDS, and the thickness of the adsorbed layer, as a function of SDS concentration are plotted in Figures 2 and 3, respectively. In addition, in Figure 2 the adsorption of SDS in the absence of polylysine is also plotted for comparison. Measurements for the isotopic combination of d-SDS/polylysine/D2O, where the SDS is effectively index matched to the D2O, provides a measure of the amount of polylysine at the interface, as discussed earlier. By use of eq 3 the amount of polymer at the interface can be estimated, and this is shown as a volume fraction in Figure 4. The variation in the amount of SDS at the interface shows a complex behavior with SDS concentration and solution pH (see Figure 2). At high SDS concentrations (>10-3 M) the amount of SDS at the interface is coincident with that measured for pure SDS. Whereas at low SDS concentrations (10-2 M) than is relevant here and involve transition to the R-helix form. Hence we attribute the changes in adsorption with pH in the region of surface depletion (10-4 to 10-3 M) as due to different polylysine conformations in solution at pH 3 and 10. As polyelectrolyte/ionic surfactant
(in monomeric form) complexes are not generally soluble and are more surface active,1-7 the surface depletion is assumed to arise from the competition between the formation of surface polyelectrolyte/surfactant and solution polyelectrolyte/surfactant micellar complexes.6,7 From the same “packing” arguments of Buckingham et al.10 it is likely that the polylysine/SDS micelle complexes are more favorable when the polylysine is in its R-helix conformation, and hence the greater depletion is observed at pH 10. Indeed, evidence from other studies11,18-20 are consistent with cooperative surfactant adsorption inducing/stabilizing the R-helix conformation of polylysine and polylysine-based copolypeptides.
Summary A combination of neutron reflectivity and surface tension has been used to investigate the pH dependence of the adsorption of SDS/poly-L-lysine mixtures at the air-solution interface. At low SDS concentrations (,cmc) strong adsorption of SDS/polyL-lysine complexes is observed, giving rise to a marked enhancement of SDS at the interface. At these low SDS concentrations the adsorption is independent of pH, and it is concluded that the poly-L-lysine is in a “near-extended” or β-sheet conformation at the interface. The marked increase in the surface tension at higher SDS concentrations, before the eventual decrease as the SDS concentration approaches its cmc, is associated with a depletion of SDS/polylysine complexes from the surface in favor of the formation of more soluble polypeptide/SDS micelle complexes. Here there is a marked pH dependence in the adsorption and the surface tension which is attributed to a more favorable formation of polypeptide/SDS micelle complexes when the polylysine is in its R-helix conformation at high pH. LA061072S
