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Complexation between Dodecyl Sulfate Surfactant and Zein Protein in Solution Juan M. Ruso,‡ Namita Deo,† and P. Somasundaran*,† NSF IUCR Center for Advanced Studies in Novel Surfactants, Langmuir Center for Colloid and Interfaces, Columbia University, New York, New York 10027, and Biophysics and Interfaces, Department of Applied Physics, University of Santiago de Compostela, E-15782 Santiago de Compostela, Spain Received March 30, 2004. In Final Form: July 1, 2004 Interactions between sodium dodecyl sulfate and zein protein, a model system for the understanding of the effect of surfactants on skin, were investigated using a range of techniques involving UV-vis spectroscopy, TOC (total organic carbon analysis), electrophoresis, and static and dynamic light scattering. Zein protein was solubilized by SDS. The adsorption of SDS onto insoluble protein fraction caused the zeta potential of the complex to become more negative. From these values, we calculated the Gibbs energy of absorption, which decreases when the SDS concentration is raised. Finally the structure of the complex, based on the analysis by static and dynamic light scattering, is proposed to be rod like.
1. Introduction Folding and binding are two of the most fundamental aspects of protein behavior. Biological function is generally possible only when a protein is folded into a specific threedimensional conformation, with unfolded proteins being impotent. Biological function also involves interactions with other molecules, as in the case of enzymes binding substrates and products and transport proteins binding the ligands. The interaction of small molecules with macromolecules of biological systems and with specific receptor sites on surfaces of supramolecular organizations is one of the most extensively studied phenomenon in biochemical research. The binding subject includes a vast range of important biochemical phenomena.1 Regardless of whether the interactions are specific or not, they constitute an important part of the protein function. An accurate description of such interactions is of great value in further understanding the behavior of proteins.2 Anionic surfactants, such as sodium dodecyl sulfate (SDS), interact strongly with oppositely charged globular proteins and denature them. At low surfactant concentrations, the number of surfactant molecules bound per protein molecule increases slowly, characterizing a specific and noncooperative binding, followed by a rapid increase in bound surfactant molecules due to nonspecific and cooperative binding. The unfolding of the proteins is believed to occur in the cooperative binding region. Further increase in surfactant concentration leads to a saturation region where additional binding of the surfactant on the protein does not occur and normal micelles form then as excess surfactant is added.3 While many investigations have focused on watersoluble proteins,4,5 there are very few studies with * Author to whom correspondence should be addressed. E-mail:
[email protected]. † Columbia University. ‡ University of Santiago de Compostela. (1) Creighton, T. E. Protein Folding; W. H. Freeman and Company: New York, 1992. (2) Doniach, S. Statistical Mechanics, Protein Structure and Protein Substrate Interactions; NATO ASI Series, Series B: Physics Vol. 325; Plenum Press: New York, 1994. (3) Jones, M. N.; Manley, P. Surfactants in Solution; Mittal, K. L., Lindman, B., Eds; Plenum Press: London, 1984.
insoluble proteins. In this work, we studied a system composed by an insoluble protein (zein) and an anionic surfactant ( SDS) in aqueous solutions. Zein is a group of proline-rich proteins extractable from plant seeds, and it represents an important exception from the conventional notion of insolubility of unmodified proteins in organic solvents. Zein is soluble in ethanol, 2-propanol, acetic acid, phenol, and dimethyl sulfoxide.6 Amino acid composition analysis shows large amounts of hydrophobic residues, such as leucine (20%), proline (9%), alanine (14%), and glutamine (20%). The polypeptide chains form R helices where the polar amino acids are distributed on three symmetric sites. The tertiary structure consists of nine repeated helices.7 The study of surfactant interactions with insoluble proteins (keratin, stratum, and corneum) can yield valuable information about the effects of surfactants on skin and hair. Model experiments made with zein reveal that the ability of the surfactant to solubilize this protein correlates very well with the epidermic alteration power of the surfactant.8 Recent studies have shown that the presence of zein in liposomes makes the liposomes more vulnerable to SDS,9 and the mixing of SDS with nonionic surfactants reduces the denaturation potential of SDS.10 2. Experimental Section Materials. Sodium dodecyl sulfate was obtained from Fluka, Inc. and was used as received. Zein protein was obtained from Fisher Scientific and was also used as received. Absorbance Measurements. The absorbance measurements were made at 25 °C with a Shimadzu UV-240 spectrophotometer (λ ) 277 nm, cell length ) 4 cm). Zeta Potential Measurements. Zeta potential measurements were made using a Pen kem laser zee meter 501 system (4) Goddard, E. D.; Ananthapadmanabhan, K. P. Interactions of surfactants with polymers and proteins; CRC Press: Boca Raton, FL, 1993. (5) Ruso, J. M.; Taboada, P.; Martinez-Landeira, P.; Prieto, G.; Sarmiento, F. J. Phys. Chem. B 2001, 105, 2644. (6) Bromberg, L. J. J. Phys Chem B 1997, 101, 504. (7) Argos, P.; Pedersen, K.; Marks, M. D.; Larkins, B. J. Biol. Chem. 1982, 257, 9948. (8) Pezron, I.; Galet, L.; Clausse, D. J. Colloid Interface Sci. 1986, 180, 285. (9) Deo, N.; Somasundaran, P. Langmuir 2003, 19, 2007. (10) Moore, P. N.; Puvvada, S.; Blankstein, D. Langmuir 2003, 19, 1009.
10.1021/la049182r CCC: $27.50 © 2004 American Chemical Society Published on Web 09/02/2004
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Solubilization of Zein Protein by SDS. Zein protein (1.0%) samples in triple-distilled water were interacted with SDS at different concentrations for 48 h. The undissolved protein was separated by centrifugation followed by membrane filtration (0.2 µm). The protein concentration in the solution was determined using total organic carbon (TOC). The amount of SDS that complexes with zein protein in solution was measured using the two-phases titration method.11 Static Light Scattering Measurements. The light intensity scattered by polymer solutions may be interpreted by the ZimmDebye equation
[
][
]
q2〈R2g〉z 1 Kc2 ) 1+ + 2A2c2 ∆Rθ 3 Mw
(1)
where ∆Rθ is the excess Rayleigh ratio (∆Rθ ) ∆iθr2/I0, where ∆iθ is the excess scattered light intensity per unit volume of the scattering solution, I0 is the incident light intensity, and r is the distance from the sample to the detector), Mw is the weightaverage molecular weight, A2 is the second virial coefficient and R2g is the z-average mean-square radius of gyration. K is the optical constant and, for vertically polarized light, is given by
K)
4π2n2(dn/dc2)2 4
NAλ
(2)
being n the refractive index of the solvent, c2 is the scatter particle concentration, λ is the wavelength of incident light, dn/dc2 is the refractive index increment, q is the scattering vector given by
q)
θ sin ( ) (4πn λ ) 2
(3)
where θ is the scattering angle. Zimm plots have two limiting plots, which, within the experimental error, intersect the ordinate at the same point that represents the molecular weight, 1/Mw. The scattering plots at zero scattering angle, θ ) 0, give quantitative information on the interparticle interactions, with the second virial coefficient, A2, as its initial part at low concentrations. Meanwhile plots at c2 ) 0, give information on the size of the scattered particles, characterized by the radius of gyration. In these experiments, solutions were clarified by ultrafiltration through 0.2 µm filters Dynamic Light Scattering Measurements. In dynamic light scattering, a time correlation function (TCF) of the scattering intensity is calculated from the scattering intensities measured at a certain starting time i(q,0) and at a very short time interval, t, later. In normalized form, the TCF is given by
g2(q,t) )
〈i(q,0)i(q,t)〉 〈i(q,∞)〉2
(4)
where i(q,∞) is the scattering intensity at very low delay times. For theoretical evaluations, mostly the electric field time correlations function is used, g1(q,t), which is related to the intensity TCF g2(q,t) by the Siegert relationship.12
g2(q,t) ) 1 + |g1(q,t)|2
(5)
With increasing t, the intensity of the TCF decays to baseline and can be expressed by a cummulant expansion as given by the equation
ln[g1(q,t)] ) Γ0 - Γ1t +
() ()
Γ2 2 Γ3 3 t t + ... 2! 3!
(6)
where the Γi are the various cummulants. The first cummulant
Figure 1. Absorbance of zein protein at 277 nm as a function of SDS concentration. is Γ1 ) Γ, related to the apparent diffusion coefficient through the relationship
Γ1 ) Γ ) q2Dapp(q,t)
(7)
while the second cummulant is related to the polydispersity of the sample. The first cummulant corresponds to a characteristic relaxation time, τ0, which is related to the ζ-average translational diffusion coefficient, D0. According to Stokes-Einstein equation, the translation diffusion coefficient at zero concentration depends on the hydrodynamic effective sphere radius Rh
D0 )
k BT 6πη0Rh
(8)
where kB is the Boltzmann constant, T is the absolute temperature, and η0 is the solvent viscosity.13 The system used was a BI-9000AT (Brookhaven Instrument Corp.), which allows DLS measurements at various scattering angles. Since the preliminary experiments showed no angular dependence on the particle size, most DLS measurements were performed at an angle of 90° and a temperature of 25 °C. The solutions were clarified as described above.
3. Results and Discussion In Figure 1, the normalized absorbance of solutions at a fixed zein concentration of 0.1 g/10 mL is shown as a function of SDS concentration. As can be observed, the absorbance increases almost linearly until it reaches a plateau at an SDS concentration above 50 mM. This maximum value of absorbance corresponds to the maximum concentration of zein solubilized, which indicates that the total amount of protein has been solubilized. Thus, a minimum concentration of 50 mM of SDS is needed to solubilize 0.1 g of zein protein completely. The number of dodecyl sulfate ions per zein molecule in solution was calculated from TOC measurements. Prior to the analysis of the samples, they were centrifuged and filtered and then the number of SDS molecules was obtained by titration. A plot of the bound dodecyl sulfate molecules is given in Figure 2 against the total concentration of SDS. Each protein molecule has a certain number of binding sites. An exponential binding of SDS on zein protein was observed until most of the free binding (11) Osborn, T. B. Science 1908, 28 417. (12) Berne, B. J.; Pecora, R. Dynamic Light Scattering; WileyInterscience: New York, 1976. (13) Ioan, C. E.; Aberle, T.; Burchard, W. Macromolecules 2001, 34, 326.
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Figure 2. Values obtained for binded SDS as a function of total SDS concentration determined by TOC measurements. Estimated uncertainties (5%
Ruso et al.
Figure 4. Hydrodynamic diameter of the zein-SDS complex as a function of SDS concentration determined by dynamic light scattering.
tion can be used to calculate the number of adsorption sites, N1:14,15
( )
dζ ) d log c
(
(
)
4.606kBT sin h (zeζ1/2kBT) - sin h (zeζ2/2kBT) × ze cos h(zeζ2/2kBT)
)
x8n0kBT[sin h (zeζ1/2kBT) - sin h (zeζ2/2kBT)] - 1 zeN1
(9)
ζ1 and ζ2 are selected zeta potentials on the line, c is the concentration, and n0 is the ionic concentration. Now, the adsorption constant, k2, can be calculated from the equation: Figure 3. Zeta potential of the zein-SDS complex (9) and Gibbs energies of adsorption (O) as a function of SDS concentration.
1 ) c
(
k2 sites of the protein molecules were saturated with SDS, which corresponds to the break point on the plot. On further increasing the surfactant concentration, there is no significant increase in the SDS adsorption. Thus, the break on the adsorption curve corresponds to the maximum number of SDS molecules adsorbed. Once most of the free binding sites of the protein are saturated with surfactant molecules, a small increase in SDS adsorption is expected until all the free sites are fully saturated due to competition among the SDS molecules for the same site. Hence, the increase in the adsorption of SDS is small after the break. It was also found that 1 g of zein protein consumed 1.5 g of SDS, which correlates with the results obtained previously but very different from the result reported by Moore et al.10 In Figure 3, we show the zeta potential of the system SDS plus zein (shown for a zein concentration constant at 0.1%). Zeta potential can be seen to decrease with SDS concentration due to the adsorption of the surfactant onto the protein. Initial binding of SDS on zein is considered to be specific due to electrostatic interactions with the positively charged side chains. However, cooperative binding occurs at very low SDS concentrations and solubilization of zein by SDS occurs,10 and consequently, noncooperative binding occurs. Thus, the following equa-
zeN1
x8n0kBT[sinh (zeζ1/2kBT) - sinh (zeζ2/2kBT)]
)
-1
(10)
Here, c is chosen as the concentration at the ζ-potential midpoint between ζ1 and ζ2. The standard free energy of adsorption, ∆G0ads, can be obtained from the equation
k2 ) exp(-∆G0ads/kBT)
(11)
The standard Gibbs energies of adsorption evaluated from eq 11 are plotted in Figure 3. The results show that Gibbs energies are large and negative at low values of SDS concentration, where binding to the high energy sites takes place, and become less negative as more SDS molecules bind, suggesting a saturation process. Similar behavior was found for the system sodium n-dodecyl sulfate histone HI.16 Dynamic light scattering was employed with the aim of calculating the hydrodynamic radius of the complex. In Figure 4, we plot the calculated diameter as a function of (14) Kayes, J. B. J. Colloid Interface Sci. 1976, 56, 426. (15) Stadilis, G.; Avranas, A.; Jannakoudakis, D. J. Colloid Interface Sci. 1990, 135, 313. (16) Moosavi-Movahedi, A. A.; Housaaindokht, M. R. Physiol. Chem. Phys. Med. NMR 1990, 22 19.
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that calculated from the hydrodynamic radius suggests strong electrostatic interactions among the complexes. This is similar to that for the SDS-lysozyme system.19 It is known that Rg is critically dependent on the radius distribution of the chain density, whereas Rh is relatively more influenced by the short-range distances between continuous atoms along the chain sequence.20 Differences between different structures can be better seen in terms of Rg/Rh because it reflects the conformation of the chain. Thus, if Rg/Rh is of the order of (3/5)1/2 a uniform hardsphere form is predicted. The results given here are far from that for the hard sphere, and this is an indication for the existence of nonspherical objects, such as rods, for the complexes.21 Conclusions Figure 5. Zimm plot for the zein-SDS complex. The lines drawn represent least-squares fits to the data.
the SDS concentration (zein concentration was kept constant). As can be observed, the hydrodynamic diameter remains almost constant in a range of 17.5-19.5 nm, while SDS concentration is increased. This fact correlates very well with the observation that, when SDS is added to the solutions, it tends to interact with the solid zein to dissolve it. Thus, in the concentration range tested, we have more complex particles in solution keeping the same size. The polydispersivity found in all experiments was always less than 0.1. On the basis of our previous observations, we can conclude that, in the range of concentration under study, the addition of SDS results in solubilization of zein keeping the size constant. Thus, in Figure 5, we show a representative Zimm plot for the binary system SDS/zein protein at SDS concentration ranging from 0-0.1 M. The weight-average molecular weight (Mw), radius of gyration (Rg), and second virial coefficient (A2) were obtained from dual extrapolations following eq 1, giving the results: Mw ) 844 000 ( 19 000; Rg) 35 ( 4 nm and A2 ) 9.18095 × 10-10 mL mol g-2. Now, it is interesting to think about these results. First, if we assume, on the basis of the data of titration and absorbance, that when the zein is completely solubilized by SDS and an average number of 147 SDS molecules adsorbed per zein protein, then considering the molecular weight obtained from the Zimm plot, we obtain a number of 11 units of SDS-zein forming the complex. Virial coefficients reflect deviations from ideality, or intermolecular interactions. The second virial coefficient should be related to the interactions between pairs of molecules. The experimental values obtained from the fit of the second virial coefficients is, in this case, positive, suggesting that the effective interaction between the complex is repulsive, which is in contrast to the systems involving attractive interactions.17,18 The hard-sphere potential is the simplest potential used and is the only potential for which the first seven virial coefficients have been calculated. A system of particles with this potential is called a hard-sphere gas or fluid. Assuming a model of hard spheres, it is possible to calculate the radius of the sphere having the same excluded volume from the value of the second virial coefficient:
3A2M2w R3A ) 16πNA
(12)
The value calculated for the hard-sphere approximation is ≈40 nm. Comparison of this value for the radius with
In this work, physicochemical interactions between a protein, zein, and a surfactant has been examined along with the structure of the SDS-zein complex. The data for absorbance and titration yield a value of 1.5 g for binding of SDS to 1 g of zein, suggesting an average number of 147 dodecyl ions per zein molecule. While this values is close to that reported earlier, it is very different from that of Moore et al.10 Such differences are attributed to differences in the protein size, with zein being a polypeptide chain, the length of which can be changed. The thermodynamic parameters for the interactions were calculated assuming that that between the protein and the surfactant is mainly hydrophobic. This is based on the total number of hydrophobic residues of the protein (almost 70%) and on the fact that after an initial electrostatic interaction which unfolds the structure of the protein partially, a noncooperative binding appears. The free energy of adsorption tends to decrease when the ζ-potential of the complex becomes more negative. On the basis of the results obtained for the molecular weight and size of the complex from the light scattering data, it is proposed that rather than individual molecules of zein with bound SDS, complexes formed between 11 protein molecules and their corresponding bound SDS molecules (147) are present under the conditions studied. This is based not only on the values for the size and the molecular weight but also on the existence of the single peak found in dynamic light scattering data and the low value of polydispersivity, suggesting only one kind of structure. Such a structure is proposed to be formed by links between the partially unfolded protein chains and the alkyl chains of SDS forming a network. Furthermore, the ratio between the hydrodynamic radii and the radius of gyration seems to indicate the basic complex to be rod shaped. Acknowledgment. The authors acknowledge the support of the National Science Foundation (Grant Number 9804618) and Industrial/University cooperation research center for adsorption studies in novel surfactants. The financial support of Unilever Research Laboratory, U.S. is gratefully acknowledged. LA049182R (17) McQuarrie, D. A.Statistical Thermodynamics; University Science Books: Sausalito, CA, 1973. (18) Hansen, J. P.; McDonald, I. R. Theory of Simple Liquids; Academic Press: London, 1976. (19) Gimel, J. C.; Brown, W. J. Chem. Phys. 1996, 104, 8112. (20) Wang, X.; Qiu, X.; Wu, C. Macromolecules, 1998, 31, 2972. (21) Young, C. Y.; Missel, P. J.; Mazer, N. A.; Benedek, G. B.; Carey, M. C. J. Phys. Chem. 1978, 82, 1375.
