Microstructure of Protein−Surfactant Complexes in Gel and SolutionAn

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Microstructure of Protein-Surfactant Complexes in Gel and SolutionsAn NMR Relaxation Study Anna Karin More´n,* Magnus Nyde´n, Olle So¨derman, and Ali Khan Physical Chemistry 1, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Received November 30, 1998. In Final Form: April 29, 1999 The regions formed upon addition of the anionic surfactant sodium dodecyl sulfate (SDS) to the oppositely charged protein, lysozyme, in aqueous solutions are, in succession, solution phase, precipitation region, bluish gel phase, and solution phase, within 20 wt % protein. The dodecyltrimethylammonium chloride (DOTAC)-lysozyme system, where both the protein and surfactant are positively charged, only gives a solution phase. In the lysozyme-SDS-water system, the longitudinal (R1) and the transverse (R2) relaxation rates are determined for the gel and its transformation into the colorless solution via a region of bluish solution (gel dispersed in solution). The results are compared with those of the binary surfactant-water and the lysozyme-DOTAC-water systems. 2H NMR relaxation data of selectively deuterated surfactant, next to its headgroup, show that R2 . R1 in bluish solutions. Larger values of R2, and in most cases of R1, are obtained for bluish solutions compared with colorless solutions that are formed in both proteinsurfactant-water and surfactant-water systems. 2H NMR spectra of the gel and bluish solution, obtained with perdeuterated SDS, show one broad peak, which is resolved into three peaks in the colorless solution. R2 values deduced from line width measurements are in agreement with results from selectively deuterated surfactant. For the gel and bluish solution,1H R2 measurements of lysozyme exhibit a biexponential relaxation, which is not observed in the DOTAC-lysozyme system for corresponding surfactant concentrations. In the oppositely charged system the results obtained from the gel and bluish solution are explained by the presence of large structures, in which the surfactant is present in a more restricted environment than that of a micelle. Upon increasing the surfactant concentration in the colorless solution, the large aggregates dissolve into smaller aggregates and an increasing fraction of surfactants is found in a micellar environment. In the lysozyme-DOTAC-water system only micellar interactions with the protein are detected.

Introduction Ionic surfactants, and particularly the anionic surfactant sodium dodecyl sulfate (SDS), are known to interact strongly with globular proteins. In most cases the protein molecule is denatured as a result of the interaction, especially at high surfactant concentrations. In various protein-surfactant systems the interactions have been investigated through determination of binding isotherms at low protein concentrations.1-12 Depending on the protein-surfactant system, a specific noncooperative binding may occur at low surfactant concentrations, with surfactant binding to specific sites of the protein.13 At higher surfactant concentrations the surfactant binds in * To whom correspondence should be addressed. Fax: 46-46222 44 13. Phone: 46-46-22 8155. E-mail: anna_karin.moren@ fkem1.lu.se. (1) Jones, M. N.; Manley, P. J. Chem. Soc., Faraday Trans. 1 1979, 75, 1736. (2) Fukushima, K.; Murata, Y.; Nishikido, N.; Sugihara, G.; Tanaka, M. Bull. Chem. Soc. Jpn. 1981, 54, 3122. (3) Fukushima, K.; Murata, Y.; Sugihara, G.; Tanaka, M. Bull. Chem. Soc. Jpn. 1982, 55, 1376. (4) Jones, M. N.; Prieto, G.; del Rio, J. M.; Sarmiento, F. J. Chem. Soc., Faraday Trans. 1995, 91, 2805. (5) Subramanian, M.; Sheshadri, B. S.; Venkatappa, M. P. J. Biochem. 1984, 95, 413. (6) Reynolds, J. A.; Herbert, S.; Polet, H.; Steinhardt, J. Biochemistry 1967, 6, 937. (7) Chen, A.; Wu, D.; Johnson, C. S., Jr. J. Phys. Chem. 1995, 99 (9), 828. (8) Oakes, J. J. Chem. Soc., Faraday Trans. 1 1974, 70, 2200. (9) Muller, N.; Mead, R. J., Jr. Biochemistry 1973, 12, 3831. (10) Johnson, T. W.; Muller, N. Biochemistry 1970, 9, 1943. (11) Sarmiento, F.; Prieto, G.; Jones, M. N. J. Chem. Soc., Faraday Trans. 1992, 88, 1003. (12) Sukow, W. W.; Sandberg, H. E.; Lewis, E. A.; Eatough, D. J.; Hansen, L. D. Biochemistry 1980, 19, 912.

a nonspecific and cooperative way to most globular proteins. During this binding micelle-like surfactant aggregates are formed on the protein. At surfactant concentrations beyond the cooperative binding region the number of bound surfactants per protein more or less remains constant.14,15 The different ways of surfactant binding to the protein are, among other techniques, studied and probed by NMR chemical shift as well as line width measurements.8-10,16 Protein-surfactant interactions are also reviewed in a recently published book entitled Polymer-Surfactant Systems.17 The mobility of the complex formed between proteins with reduced disulfide bonds and surfactants at saturation binding is proportional to the molecular weight of the protein measured by polyacrylamide gel electrophoresis (SDS-PAGE technique).18,19 To understand protein-surfactant interactions and also the mechanism of the molecular weight determinations by SDS-PAGE, several structural studies are performed with various techniques, for example NMR,7-10,16 SANS,20-22 and light scatter(13) Dickinson, E. Protein in Solution and at Interfaces. In Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: London, 1993; Chapter 7, p 295. (14) Reynolds, J. A.; Tanford, C. Proc. Natl. Acad. Sci. U.S.A. 1970, 66, 1002. (15) Takagi, T.; Tsujii, K.; Shirahama, K. J. Biochem. 1975, 77, 939. (16) Turro, N. J.; Lei, X. G.; Ananthapadmanabhan, K. P.; Aronson, M. Langmuir 1995, 11, 2525. (17) Polymer-Surfactant Systems; Surfactant Science Series Vol. 77; Marcel Dekker: New York, 1998. (18) Shapiro, A. L.; Vin˜uela, E.; Maizel, J. V., Jr. Biochem. Biophys. Res. Commun. 1967, 28, 815. (19) Shirahama, K.; Tsujii, K.; Takagi, T. J. Biochem. 1974, 75, 309. (20) Guo, X. H.; Zhao, N. M.; Chen, S. H.; Teixeira, J. Biopolymers 1990, 29, 335.

10.1021/la9816611 CCC: $18.00 © 1999 American Chemical Society Published on Web 07/31/1999

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extends to very high water contents (>99 wt %) as a narrow region above and together with the precipitate. From this point on, the bluish solution is referred to as BS. Our aim is to obtain structural information of the gel phase and to study the transition of the gel into the solution phase via the BS region with techniques such as NMR, cryo-transmission electron microscopy (cryo-TEM), DSC, and rheology. In this article a study of the NMR relaxation rate measurements of these regions in the lysozymeSDS-water system is presented and compared with changes appearing as the anionic surfactant is exchanged for the cationic surfactant DOTAC. As a reference, relaxation data of SDS and DOTAC in binary surfactantwater systems are also presented. Theoretical Background Figure 1. Schematic ternary phase diagram of the lysozyme (0-20 wt %)-SDS (0-20 wt %)-water (80-100 wt %) system, showing the phase boundaries at 25 °C. L, solution; G, gel; P, precipitate; P + L, G + L, and P + G + L are two- and threephase regions (from More´n et al.,27 with permission).

ing.23,24 Different structural models have been suggested, for example “rodlike”,25 “necklace and bead”,19,20 “proteindecorated micelle structure”,21,22 and “flexible capped helical cylindrical micelle”.26 The information available is, however, often limited to very dilute protein concentrations. Recently, phase equilibria studies of two proteinsurfactant systems were presented: lysozyme-SDSwater27 and β-lactoglobulin-dodecyltrimethylammonium chloride (DOTAC)-water.28 In these systems the protein and surfactant are oppositely charged, and a wide range of protein and surfactant concentrations is covered. Both systems exhibit similar phase behavior. For reference, the phase diagram of the lysozyme-SDS-water system is reproduced in Figure 1. Three main regions are found: a solution phase that surrounds a precipitation region, and a bluish gel phase. The precipitation region extends to very high water contents, and the gel phase is formed with protein concentrations above 6 wt % lysozyme. Upon increasing the surfactant concentration from the lysozyme-water axis, the succession of regions is as follows: solution, precipitation, gel, solution. The first formation of precipitate occurs at very low surfactant concentrations (≈10-4 wt % for 1 and 3 wt % lysozyme29). Previously we reported a study of the behavior of the precipitation region in combination with the phase equilibria of four lysozyme-sodium alkyl sulfate-water systems of varying surfactant chain length.29 More detailed investigations of the newly discovered gel are, however, needed. The redissolution of the gel into the solution phase upon increased surfactant concentration takes place via a region of bluish solution. The bluish solution, which consists of finely divided gel particles dispersed in solution,27 not only exists above the gel phase but also (21) Ibel, K.; May, R. P.; Kirschner, K.; Szadkowski, H.; Mascher, E.; Lundahl, P. Eur. J. Biochem. 1990, 190, 311. (22) Ibel, K.; May, R. P.; Sandberg, M.; Mascher, E.; Greijer, E.; Lundahl, P. Biophys. Chem. 1994, 53, 77. (23) Tanner, R. E.; Herpigny, B.; Chen, S.-H.; Rha, C. K. J. Chem. Phys. 1982, 76, 3866. (24) Gimel, J. C.; Brown, W. J. Chem. Phys. 1996, 104, 8112. (25) Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1970, 245, 5161. (26) Lundahl, P.; Greijer, E.; Sandberg, M.; Cardell, S.; Eriksson, K.-O. Biochim. Biophys. Acta 1986, 873, 20. (27) More´n, A. K.; Khan, A. Langmuir 1995, 11, 3636. (28) More´n, A. K.; Eskilsson, K.; Khan, A. Colloids Surf., B 1997, 9, 305. (29) More´n, A. K.; Khan, A. Langmuir 1998, 14, 6818.

The dynamics of deuterated surfactant molecules determine their 2H spin relaxation rates.30 The transverse (R2) and the longitudinal (R1) relaxation rates (R1,2 ) 1/T1,2) for a quadrupolar nucleus depend on spectral densities according to eqs 1 and 2.31

3π2 2 χ (2J(ω0) + 8J(2ω0)) 40

(1)

3π2 2 χ (3J(0) + 5J(ω0) + 2J(2ω0)) 40

(2)

R1 ) R2 )

The spectral densities are given by eq 3a-c.

J(0) ) 2τ J(2ω0) )

J(ω0) )

2τ 1 + ω02τ2

2τ 1 + 4ω02τ2

(3a-c)

ω0 is the Larmor frequency of the deuterium nuclei at the given field strength. χ, the quadrupolar coupling constant, is set equal to 167 kHz. τ is the time scale of the motions that induce relaxation in the molecule. The main difference between R2 and R1 lies in the dependence of slow motions, that is, the term J(0) which is included in R2 but not in R1. This fact ensures that R2 always will be faster or equal to R1. R1 is only influenced by the fast dynamics occurring with frequencies ω0 and 2ω0. Their difference ∆R ) R2 - R1 is dominated by changes in the slow motions, and when large aggregates are present ∆R resembles the behavior of R2. For a micelle-forming surfactant system well above the cmc the so-called two-step model describes the dynamics of surfactant molecules well.30 The model relies on the separation of two independent time scales. The first time scale represents the fast local reorientation of the surfactant in the micelle, and the second time scale is given by the slower rotation of the (spherical) micelle and the diffusion of the surfactant along the surface of the micelle. In presence of protein a multisite model will apply. For example, the surfactant molecules not only exist as monomers and micelles but also aggregate onto the protein, and the protein-surfactant complex may, in turn, also aggregate. If the surfactant molecules can be found in i different sites, between which they exchange rapidly, then the observed NMR parameter will be a populationweighted average according to eq 4. (30) So¨derman, O.; Stilbs, P. Prog. NMR Spectrosc. 1994, 26, 445. (31) Abragam, A. The Principles of Nuclear Magnetism; Clarendon Press: Oxford, U.K., 1961.

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R1,2 )

∑i piRi1,2

More´ n et al.

(4)

Here R1,2 is the observed parameter, pi is the fraction of surfactants in site i, and Ri1,2 is the relaxation rate in site i. Accordingly, changes in the observed values of R2 and R1 can then be attributed to variations in the populations of the surfactants at the various sites and/or alterations in the intrinsic relaxation rates. The latter alterations can be brought about by changes in aggregate size (which affects mainly J(0)) or changes in the properties of the surfactant bound to the aggregates, which affect both the local dynamics of the surfactant (which is expected to influence mainly J(ω0) and J(2ω0)) and the fraction of the quadrupolar interaction which is relaxed by the slower motions (in essence the amplitude of the motion the surfactant undergoes). We will also present 1H relaxation data obtained from protons resonating at 7 ppm, originating from the aromatic amino acids in the protein.32 The theoretical analysis discussed above shows that transverse relaxation rates are strongly dependent on the size and shape of surfactant micelles and surfactantprotein complexes; R2 values increase with increasing aggregate size. On the other hand, the longitudinal relaxation rates which measure the fast molecular dynamics are not expected to undergo dramatic changes with the growth of the aggregates. Experimentally determined relaxation rate data will highlight the theoretical analysis. Experimental Section Materials. Lysozyme L-6876 lot no. 111H7010 from chicken egg white, three times crystallized, dialyzed, and lyophilized, was obtained from Sigma. Specifically deuterated SDS and DOTAC at the R-carbon position next to the surfactant headgroup (R-CD2) were synthesized by Synthelec Inc. (Lund, Sweden). For measurements at high surfactant concentrations, mixtures of specifically deuterated SDS and normal SDS (specially pure) obtained from BDH were prepared. Perdeuterated SDS was supplied by Icon Services Inc. (New York). In all 2H NMR measurements deuterium-depleted water was used (Sigma W-1125 lot no. 54H3693). In the 1H spin relaxation measurements of the protein, lysozyme is mixed with either normal SDS (BDH) or DOTAC (Tokyo Kasei (purity > 97%)) in heavy water (>99.8%) from Dr Glaser AG (Basel). All chemicals were used as obtained. Sample Preparation. All samples were prepared by weighing appropriate amounts of substances into screw-capped glass tubes. The total mass of the sample was at least 0.5 g. After intense vortexing, the samples were gently mixed for about 24 h. After the mixing period the samples were left standing in order to equilibrate for another 9 days. The sample (0.4 cm3) was then transferred to an NMR tube, except in the perdeuterated SDS series, where a volume of 0.6 cm3 was used. The concentrations of components in the ternary phase diagram presented in Figure 1 are given in weight percent. In all other figures presented in this work, wt % surfactant is calculated from the amount of surfactant and water in the samples, that is, excluding the protein. Methods. All relaxation experiments were performed on a Bruker DMX 100 NMR spectrometer operating at 15.3 MHz for 2H, at a temperature of 22 °C, using standard experiments.33 The R1 measurements were performed with the standard inversion recovery method, and R2 measurements, with the Hahnecho pulse sequence, both using 18 different delay times in the variable delay list. The delay times were changed with different sample compositions to optimize the experiment. All 2H relaxation rate measurements of the surfactant could be described with a single relaxation rate. R1 and R2 were obtained from three (32) Redfield, C.; Dobson, C. M. Biochemistry 1988, 27, 122. (33) Canet, D. Nuclear Magnetic Resonance, Concepts and Methods; John Wiley & Sons: New York, 1991.

Figure 2. Relaxation rates R2 (circles) and R1 (squares) versus wt % of (a) SDS and (b) DOTAC. parameter fits of the raw NMR data. The 1H R2 measurements on lysozyme displayed both mono- and biexponential relaxation behavior. The series of spectra obtained from perdeuterated SDS were recorded on a Varian 600 MHz spectrometer operating at 92.1 MHz for 2H at 22 °C. Each peak in the spectra was fitted with a Lorentzian function, where the chemical shift was a fixed parameter. R2 was thereafter calculated from the fitted halfheight line widths ∆υ1/2 according to R2 ) π∆ν1/2. The contributions to the measured R2 due to inhomogeneties in the magnetic field are of the order 5 s-1.

Results and Discussion This section is organized as follows: In the subsection treating relaxation rates of specifically R-deuterated surfactant as a function of surfactant concentration, the results of the pure surfactant systems (SDS-water and DOTAC-water) are first presented and compared. Subsequently, the relaxation rates of the surfactants in the presence of lysozyme are introduced and discussed, first with SDS and then with DOTAC. In the following subsection 2H NMR spectra obtained from perdeuterated SDS are discussed in terms of the effect of the protein on different parts of the surfactant. In the last subsection, 1 H R2 values of lysozyme in the presence of both SDS and DOTAC are compared. 2H NMR Relaxation of r-Deuterated Surfactant. SDS-Water. The relaxation rates of the surfactant R2 and R1 measured in the binary SDS-water system as a function of increasing surfactant concentration are presented in Figure 2a. At surfactant concentrations below

Microstructure of Protein-Surfactant Complexes

the cmc at 0.24 wt % SDS,34 the values of R2 and R1 are small and equal (≈10 s-1). For higher surfactant concentrations both relaxation rates increase sharply to about 40 s-1, and thereafter a less pronounced increase in R2 and R1 follows. Above the cmc the value of R2 is always larger and increases faster than R1. DOTAC-Water. R2 and R1 values obtained for the binary DOTAC-water system are presented in Figure 2b as a function of increasing surfactant concentration. The general behavior of both relaxation rates resembles that of the SDS-water system. However, the higher cmc of DOTAC (0.54 wt %34) results in a shift of the curves toward higher surfactant concentrations. Upon comparing the values of ∆R at high surfactant concentrations, a smaller ∆R value is observed in the DOTAC-water system, due to a smaller value of R2. The small and almost identical values of R2 and R1 obtained at surfactant concentrations below the cmc are due to the fast dynamics of surfactant monomers. At higher concentrations the increase in both relaxation rates is induced by self-association of surfactant molecules into micelles, which introduce slow dynamics. The larger value of ∆R in the SDS-water system suggests that the micellar aggregates formed are larger than those in the DOTAC system. This observation is in agreement with results obtained by other independent methods35-37 and can also be rationalized from the phase behavior of the surfactant systems. The first liquid crystalline phase is a cubic phase in the DOTAC-water system38 and a hexagonal phase in the SDS-water system.39 The cubic phase is composed of small spherical micellar aggregates, whereas cylindrical aggregates are packed to form the hexagonal phase. SDS-Lysozyme-Water. The 2H relaxation rates R2 and R1 determined as a function of SDS concentration at constant 4 and 8 wt % lysozyme are presented in Figure 3. As a reference the relaxation rates of the binary SDSwater system from Figure 2a are also included in Figure 3. The fixed protein concentrations of the series are indicated in the ternary phase diagram in Figure 1. The sample prepared with the lowest surfactant concentration in the series with 4 wt % lysozyme is an isotropic and slightly bluish solution, with viscosity close to that of water. Upon first focusing on the behavior of R2, a high value of 500 s-1 is found for the BS. As the surfactant concentration is increased, solutions with a clear colorless appearance are obtained. Initially the value of R2 decreases sharply upon increased surfactant concentrations. Thereafter, a less pronounced decrease in R2 is followed by a shallow minimum at about 8 wt % SDS (140 s-1) and a very slow increase in R2 as the surfactant concentration is further increased. The R1 value at the same protein concentration is around 120 s-1 for the lowest surfactant concentrations. As the surfactant concentration is increased beyond 2 wt % SDS, a decrease and a leveling off in R1 to 65 s-1 at 14 wt % SDS are observed. Compared to R2 the values of R1 are low at all SDS concentrations and exhibit relatively small changes. R2 and R1 of SDS in the presence of the higher protein concentration, that is, 8 wt % lysozyme (Figure 3), exhibit (34) Mukerjee, P.; Mysels, K. J. Critical Micelle Concentrations of Aqueous Surfactant Systems; NSRDS: Washington, DC, 1971. (35) Missel, P. J.; Mazer, N. A.; Carey, M. C.; Benedek, G. B. Solution Behaviour of Surfactants; Plenum Press: New York, 1982; Vol. 1. (36) Lindman, B.; Wennerstro¨m, H. Top. Curr. Chem. 1980, 87, 1. (37) Regev, O.; Khan, A. J. Colloid Interface Sci. 1996, 182, 95. (38) Balmbra, R. R.; Cluney, J. S.; Goodman, J. F. Nature 1969, 222, 1159. (39) Ke´kicheff, P.; Grabielle-Madelmont, C.; Ollivon, M. J. Colloid Interface Sci. 1989, 131, 113.

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Figure 3. Relaxation rates (a) R2 and (b) R1 as a function of SDS concentration in the presence of fixed lysozyme concentrations of 4 wt % (circles) and 8 wt % (triangles). Also included are data for the binary SDS-water system (squares).

a behavior similar to that for the 4 wt % lysozyme case. The sample with the lowest surfactant concentration is a BS prepared just above the gel phase (2.9 wt % SDS). As the surfactant concentration is increased, all samples appear as colorless solutions. The much higher relaxation rates in the presence of protein (R1,2(PS)) compared to the relaxation rates in the binary surfactant-water system (R1,2(S)) indicate strong protein-surfactant interactions for the oppositely charged protein-surfactant system. The very high value of R2(PS) obtained in the BS is due to the relaxation being dominated by surfactant molecules bound to large aggregates with slow rotation and tumbling. The bluish appearance of the gel and solution indeed indicates scattering of light arising from large aggregates, and in transmitted light these samples appear as yellow-orange. This is in agreement with cryo-TEM images of BS’s, where domains of large aggregates and layers are observed together with small dots, with a size corresponding to a surfactant micelle or a protein molecule (results will be published). Upon increased SDS concentrations the value of R2(PS) decreases dramatically and the bluish color disappears. This is attributed to a dissolution of the large aggregates upon further surfactant binding, which may be understood by an increased electrostatic repulsion between the complexes. In cryo-TEM images of colorless solutions only the smaller dots are observed. As the surfactant concentration is increased further, the value of R2(PS) decreases more slowly before it levels out, and R2(PS) does not approach R2(S). In fact, it is noted that R2(PS) at constant 4 wt %

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lysozyme increases slightly at high surfactant concentrations. If R2(PS) was approaching R2(S), this would indicate the formation of an increased number of protein-free surfactant micelles with a lower value of R2(S). Since this is not the case, the added surfactant is likely to contribute to a growth of either protein-free micelles or micelles interacting with the protein. Turning to the behavior of R1, a different functional form is found for R1(PS) compared to that of R1(S) (Figure 3b). The difference is, however, smaller than the difference between R2(PS) and R2(S). As noted above, the maximum value obtained for R1(PS) equals 120 s-1 for both 4 and 8 wt % lysozyme. This is a value considerably higher than that for R1(S) (40 s-1) at the corresponding surfactant concentration. The difference in the R1 values suggests that the initially protein-bound surfactant molecules experience a slightly more restricted local environment than surfactants in a micellar aggregate. The decrease in R1(PS) beyond the maximum value is interpreted as the onset of surfactant self-association, since R1(S) for micelles is lower than R1(PS). The decrease in R1(PS) for both protein concentrations is well accounted for by a two-site model according to eq 4. At the surfactant concentration corresponding to the maximum in R1 all surfactants are assumed to have R11 ) 120 s-1. Further addition of surfactants contributes to a second site with R21 ) 50 s-1, which is a reasonable value for a surfactant micelle. This suggests that the initial sites remain populated also at high surfactant concentrations. Similar observations are reported by Muller and Mead from chemical shift changes in a system with a fluorinated surfactant and bovine serum albumin (BSA).9 There, the protein-bound surfactants experienced two different environments, of which one is micelle-like. A corresponding two-site model is not applicable to the behavior of R2(PS), since R2 is also sensitive to the changing size of the protein-surfactant complexes. The relaxation rate measurements are summarized as follows: In the BS’s very large R2(PS) values indicate the presence of large aggregates, and the protein-bound surfactants experience fast local motions that are more restricted than those of surfactants in a micelle. A drastic decrease in R2(PS) upon increased surfactant concentrations implies dissolution of the large aggregates. Upon further increasing the surfactant concentration, the fraction of surfactant molecules in a micelle-like environment is increased. However, not only are an increased number of surfactant micelles formed, but there is also a growth of either protein-bound or protein-free micelles, since R2(PS) does not approach R2(S) at high surfactant concentrations. DOTAC-Lysozyme-Water. In the lysozyme-DOTACwater system, where both the protein and the surfactant carry a positive net charge, only a single colorless solution phase is observed, with a viscosity close to that of water. The values of R2 and R1 are shown in Figure 4 as a function of DOTAC concentration at constant 8 wt % lysozyme. Also included are the behavior of R2 and R1 in the binary DOTAC-water system. Starting with the values of R2 (Figure 4a), the sample with the lowest surfactant concentration (0.4 wt % DOTAC) in the series exhibits a low R2 value of 40 s-1. As the surfactant concentration is increased to 1 wt % DOTAC, R2 is sharply increased to a maximum value of about 140 s-1. Upon further increasing surfactant concentrations, R2 first decreases and then levels out to a value around 100 s-1 above 6 wt % DOTAC. Turning to R1 (Figure 4b), the value for the lowest surfactant concentration is equal to 30 s-1. R1 is markedly

More´ n et al.

Figure 4. Relaxation rates (a) R2 and (b) R1 as a function of DOTAC concentration in the presence of constant 8 wt % lysozyme (squares) and in the binary DOTAC-water system (circles).

increased to about 60-65 s-1 at 1-2 wt % DOTAC, followed by a small decrease and then a leveling out to about 60 s-1 at high surfactant concentrations. The value of R2(PS) at 0.4 wt % DOTAC (40 s-1) is higher compared to that of R2(S) for the same surfactant concentration (10 s-1), that is, below the cmc of the surfactant. The higher R2(PS) value is explained by surfactant molecules interacting with the protein. The pronounced increase that follows, however, suggests the presence of a significant fraction of surfactant monomers. The presence of a maximum in R2(PS) at about 1 wt % DOTAC (140 s-1) indicates that the protein-surfactant aggregates are larger than the protein-free micellar aggregates formed in the binary system. The decrease that follows may be induced by an increasing fraction of protein-free micelles with a lower R2 value or a decrease in the size of the protein-surfactant aggregates. Since R2(PS) does not approach R2(S) at high surfactant concentrations, it is reasonable that a growth of micellar aggregates occurs also in the similarly charged system. The difference between R1(PS) and R1(S) in the similarly charged system (Figure 4b) is markedly smaller than that in the oppositely charged system. The difference is most pronounced at low surfactant concentrations, and as the surfactant concentration is increased, R1(PS) approaches R1(S). The close values suggest that micellar aggregates of DOTAC are interacting with lysozyme. This is supported by binding isotherm studies of the lysozyme-DOTAB (B stands for bromide) system, where only a nonspecific cooperative surfactant binding to the protein occurs.4 The slightly higher value of R1(PS) at low surfactant concentrations may be due to slightly restricted dynamics of

Microstructure of Protein-Surfactant Complexes

Figure 5. Relaxation rates (a) R2 and (b) R1 as a function of surfactant concentration expressed as number of surfactant molecules per lysozyme molecule in the lysozyme-SDS-water system at constant 4 wt % (squares) and 8 wt % lysozyme (circles) and in the lysozyme-DOTAC-water system at 8 wt % lysozyme (triangles).

surfactant molecules in protein-bound micelles. A shift in the onset of aggregation toward lower surfactant concentrations is expected, since the critical aggregation concentration of a surfactant to a protein or polymer (cac) is lower than the cmc of the binary surfactant-water system.40 Also, a lower cac results in a smaller amount of surfactant monomers at the onset of aggregation and an increase in R1(PS). To facilitate the comparison of the previously shown protein-surfactant series (4 and 8 wt % lysozyme-SDSwater as well as 8 wt % lysozyme-DOTAC-water), R2 and R1 are plotted in Figure 5 as a function of increasing molar ratio between the surfactant and the protein. It can be seen that the R2 values of the lysozyme-SDS-water systems behave similarly upon increased surfactant concentrations. However, larger values of R2 are obtained for 8 wt % lysozyme at all surfactant concentrations. Both systems exhibit an initial high R2 value at about 20 [SDS]/ [lysozyme], which first decreases markedly and then levels out. The value of R2(PS) in the plateau region at high surfactant concentrations depends on the protein concentration and is higher with 8 wt % lysozyme (400 s-1) than that with 4 wt % lysozyme (140 s-1) (Figure 3a). (40) Lindman, B.; Thalberg, K. Polymer-surfactant interactionss Recent developments. In Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: London, 1993; Chapter 5, p 203.

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Upon comparing R2 in the similarly and oppositely charged systems, a much higher value is observed for the latter system, which indicates that the size of the aggregates in that system is markedly larger. Also, in the plateau that follows at high surfactant concentrations the oppositely charged system exhibits a significantly higher R2 value (Figure 5a or 3a and 4a). Hence, the aggregates formed, not only at low surfactant concentrations but also at high surfactant concentrations, are smaller in the similarly charged system. Most likely there is an important contribution to the higher R2 value of the SDS system from the way the surfactant aggregates grow at high surfactant concentrations, a difference that is previously pointed out for the binary surfactant-water systems. At very low surfactant concentrations there is a sharp increase in R2 for the similarly charged system as surfactant aggregates are formed and the monomer fraction is decreased. A corresponding increase is also expected in the oppositely charged system prior to the precipitate formation, that is, when the surfactant concentration is on the order of 10-4 wt % SDS.29 From binding isotherm studies it is also observed that the onset of surfactant binding to lysozyme occurs at much higher surfactant concentrations in the similarly charged proteinsurfactant system compared to the oppositely charged system.2,4,41 The R1 curves of the two protein concentrations in the lysozyme-SDS-water system overlap at low surfactant concentrations (Figure 5b). This indicates that the surfactant more or less binds to the protein in a proportional way. Compared with the case of the similarly charged protein-surfactant system, R1 in the oppositely charged system exhibits a much larger value. The difference is presumably a result of a different additional binding of the surfactant to the oppositely charged protein. As the surfactant concentration is increased and the fraction of surfactants in a micelle-like surrounding is increased, the R1 values of the oppositely charged system approach those of the similarly charged system. 2 H NMR Transverse Relaxation of Perdeuterated SDS. A 2H relaxation study of perdeuterated SDS is performed in order to study the effect of the protein in different parts of the surfactant tail. Three spectra are recorded with an increasing concentration of perdeuterated SDS in aqueous solutions (0.19, 1.8, and 3.7 wt % SDS); see Figure 6a. Four peaks are resolved for the sample with 0.19 wt % SDS (below cmc). The peak that appears to the right in the spectrum originates from deuteriums at the end group (ω-CD3) of the surfactant. Thereafter, several unresolved peaks from deuteriums in the middle methylene groups follow as well as a resolved peak from deuteriums at the β-carbon and finally a peak originating from deuteriums next to the surfactant headgroup (RCD2). As the surfactant concentration is increased to 1.8 and 3.7 wt %, a slight broadening of the peaks is observed due to the presence of micelles. A series of spectra with SDS concentrations ranging from 2.7 to 11.9 wt % at fixed 8 wt % lysozyme are presented in Figure 6b. The spectrum of the gel sample prepared with the lowest surfactant concentration shows a very broad single peak, indicating surfactant bound to very large aggregates. In the following two spectra, obtained within the region of BS, the peak becomes increasingly more narrow. As the surfactant concentration is further increased to 3.9 wt % SDS, that is, going from BS to the colorless solution, a very different spectrum is (41) Jones, M. N.; Manley, P. J. Chem. Soc., Faraday Trans. 1 1980, 76, 654.

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Figure 6. 2H NMR spectra of increasing concentration of perdeuterated SDS in (a) the binary SDS-water system without lysozyme and (b) the lysozyme-SDS-water system at fixed 8 wt % lysozyme. Concentrations in wt % represent SDS.

recorded. The spectra with surfactant concentrations above 3.9 wt % SDS resemble the spectra obtained for the binary SDS-water system, although only three peaks are resolved. A narrow ω-CD3 peak is followed by unresolved peaks from deuteriums of the middle methylene groups (CD2)2-11 and finally a broad peak from R-CD2. It can be seen that the spectra within the colorless solution phase in principle remain unchanged upon increased surfactant concentrations and do not approach the more narrow peaks of the binary SDS-water spectra (Figure 6a). This suggests that in the colorless solution phase there appears to be little difference between the properties of micelles not associated with the protein and those that are associated. Lorentzian band shapes are fitted to the data in Figure 6, and an example of the result for 11.9 wt % SDS at 8 wt % lysozyme is given in Figure 7a. It can be seen that the signals from R-CD2 and ω-CD3 are very well described by single Lorentzian peaks, whereas the peak from (CD2)2-11 is less well fitted. This is explained by the fact that all deuteriums in the (CD2)2-11 peak are not equivalent, and a better description of the peak is given by a sum of Lorentz functions. R2 values are calculated according to R2 ) πυ1/2, where υ1/2 is the fitted half-height line width of the ω-CD3, (CD2)2-11, and R-CD2 peaks. The calculated R2 values are plotted as a function of surfactant concentration in Figure 7b. It is suggested that the broad peak of the bluish samples, with a R2 value of around 1600 s-1, originates from deuteriums at the end position with some contributions from the (CD2)2-11 and the R-CD2 groups. The R2 values of the BS’s obtained with specifically R-deuterated SDS (discussed above) are around 2400 s-1, which makes υ1/2 about 1.5 times larger than that for the peak obtained with perdeuterated SDS. At this slow dynamics, when τ . ω0, a comparison between the two experiments is relevant, since the contribution to R2 is dominated by the

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Figure 7. (a) Example of the fitting (broken line) to the recorded spectrum (solid line). The recorded spectrum at 11.9 wt % SDS and 8 wt % lysozyme is also shown in Figure 6b. (b) Calculated R2 values from the fitted half-height line widths of the peaks in Figure 6: R-CD2 (circles), (CD2)2-11 (squares), and ω-CD3 (diamonds), as a function of SDS concentration. Filled symbols represent the binary SDS-water system, and open symbols, the lysozyme-SDS-water system. Error bars are obtained from the least-squares fitting procedure.

J(0) term according to eqs 2 and 3a, which are independent of the field strength. Also, a less good fitting of the left side of the peak is observed. Upon increasing the surfactant concentration from the bluish to the colorless solution in Figure 7b, the values of R2 of the ω-CD3 group decrease sharply due to dissolution of larger aggregates into smaller ones. Thereafter, R2 of all three peaks within the solution phase remains more or less the same as the surfactant concentration is increased. The R2 value of the ω-CD3 group in the presence of protein (14 s-1) is close to R2 for the pure surfactant-water system (10 s-1). Compared with the R2 values for (CD2)2-11 and R-CD2 (≈200 s-1 for both), its value is very small. Interestingly, the R2 values of the R-CD2 headgroup and the (CD2)2-11 group are much larger in the presence of protein than without it (≈200 s-1 compared to ≈20 s-1). This suggests that the protein interacts with the headgroup of the surfactant in the colorless solution phase and that the environment of the end group has similar properties to those of a surfactant in a micelle. The effect is also observed by Turro et al. for the BSA-SDS-water system.16 In binary surfactantwater systems it is expected that even after considerable micellar growth the value of R2 for ω-CD3 remains small.42 Large R2 values are obtained for different positions in the surfactant tail within the gel and the BS. This indicates that in the gel the surfactant is not present in micelle-like aggregates, and the role of the surfactant in the gel formation remains unclear. (42) So¨derman, O.; Carlstro¨m, G.; Olsson, U.; Wong, T. C. J. Chem. Soc., Faraday Trans. 1988, 84, 4475.

Microstructure of Protein-Surfactant Complexes

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Figure 9. Schematic illustration of the structure of the gel and the colorless solution at higher surfactant concentrations for the protein-surfactant-water systems.

Figure 8. 1H relaxation rate R2 of lysozyme versus SDS concentration at fixed (a) 4 wt % and (b) 8 wt % lysozyme. Monoexponential relaxation only gives small aggregates (filled squares). In the case of biexponential relaxation both large (open squares) and small aggregates (filled squares) are obtained, and the fraction of large aggregates (crosses) is also shown as a function of SDS concentration. 1 H NMR Relaxation of Lysozyme. SDS-LysozymeWater. Previously 2H relaxation of the surfactant in the presence of protein was investigated. As a complement a corresponding 1H NMR transverse relaxation study of the protein is presented. The R2 values of lysozyme as a function of increasing SDS concentration at fixed 4, 6, and 8 wt % lysozyme are determined. In Figure 8 the results from 4 and 8 wt % lysozyme are illustrated. In the BS’s a biexponential echo decay is noted for all three protein concentrations. In this region analysis of the data gives one population that has an R2 value typically of the order 30-50 s-1, whereas the population with the larger size gives R2 > 800 s-1. The fraction of proteins with the higher R2 value is also included in each figure. Upon increasing surfactant concentration in the BS, the fraction of large aggregates is decreased. In the colorless solution phase at 4 and 6 wt % lysozyme the biexponential relaxation turns into a monoexponential relaxation, which again exhibits a biexponential decay at very high surfactant concentrations. The extension of the monoexponential relaxation is dependent on the protein concentration: 4 wt % lysozyme displays a much larger region of monoexponential relaxation than 6 wt %, and for 8 wt % lysozyme a monoexponential decay is not detected. A complete understanding of the 1H data is nontrivial due to the complexity in interpreting relaxation data from coupled spin systems and, in particular, spin systems that relax with the dipolar relaxation mechanism. In addition, the peaks from the protein arise from protons of different

amino acids that may have different R2 values. However, taken together with the 2H relaxation data from the surfactant, we believe that some additional information about the structure in the system can be extracted. In particular 1H spectra of the gel phase show that the protein peaks are broad due to restriction of motion of the gel network. Cryo-TEM has also shown that in the BS’s there appears to exist dispersed gel fragments in solution. This suggests that the presence of the fast relaxation rate and consequently the biexponential echo decay is an indication of the presence of large aggregates. For low surfactant concentrations the appearance of the fast R2 derives from the dispersed gel phase, and when the echo decay again becomes biexponential at much higher surfactant concentrations, we believe that this also is an indication of larger aggregates. It is noted above that R2 of a specifically deuterated surfactant at high surfactant concentrations is faster in the case of 8 wt % than at 4 wt % protein. This result together with the biexponential echo decay for the protein indicates that it is the growth of combined aggregates of protein and surfactant that is responsible for this behavior. DOTAC-Lysozyme-Water. The 1H R2 of lysozyme as a function of increasing DOTAC concentration is also studied at 8 wt % lysozyme (data not shown). Unlike the oppositely charged protein-surfactant system with 8 wt % lysozyme, the similarly charged system exhibits an initial monoexponential relaxation behavior up to a surfactant concentration of 5 wt % DOTAC. As the surfactant concentration is increased further, a biexponential decay is observed, with an increasing fraction of protein present in larger aggregates. This indicates that a growth of the protein-surfactant complexes occurs at high surfactant concentrations also in the similarly charged system. As noted above, the interpretation of 1H relaxation data can be complex, and some comments about the interpretation might be necessary. Although we interpret the deviation from monoexponential behavior as the onset of biexponential behavior, the reason might be even more complex. Specifically we note that the difference between biexponential behavior and multiexponential behavior can be hard to detect. The reason for a multiexponential decay

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can be the denaturation of the protein, thus separating the different amino acids into different R2’s. However, a biexponential form fits the data nicely, and we also note that the errors are random. The 1H relaxation dependence of the protein concentration in the lysozyme-SDS-water system also indicates that protein unfolding would not be the only reason for the biexponential decay. Any denaturing effects in 4 wt % lysozyme would be present at a lower or at least at the same surfactant concentration than that for 8 wt % lysozyme. Thus, the large region of monoexponential decay for 4 wt % lysozyme, which does not occur with 8 wt % lysozyme, further motivates the choice of the “simplified” approach to interpret the 1H relaxation data. Conclusions. A summary and conclusion of the presented and discussed results is given below. The results are also illustrated as a schematic picture in Figure 9. In the oppositely charged lysozyme-SDS-water system, the presence of large aggregates in the BS and gel is probed both in the surfactant (large R2) and in the protein relaxation measurements (biexponential relaxation). As

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the surfactant concentration is increased, the large aggregates dissolve. For surfactant concentrations at which the solution is colorless, the surfactant experiences both a more restricted fast local motion, which is present already in the gel, and a micelle-like environment. At high surfactant concentrations a growth of micelles interacting with the protein is suggested. The similarly charged lysozyme-DOTAC-water system exhibits only the micelle-like interaction between the protein and the surfactant, and the presence of any large aggregates is not detected. Acknowledgment. The Swedish NMR Centre is acknowledged and especially Charlotta Damberg for her assistance with the measurements on the Varian 600 MHz spectrometer. The project is financed by the Swedish Research Council for Engineering Sciences (TFR). LA9816611