Langmuir 1995,11, 3636-3643
3636
Articles Phase Equilibria of an Anionic Surfactant (Sodium Dodecyl Sulfate) and an Oppositely Charged Protein (Lysozyme)in Water Anna Karin Moren” and Ali Khan Physical Chemistry 1, Chemical Centre, Lund University, P.O. Box 124, ,9221 00 Lund, Sweden Received February 6, 1995@ The interactions between the positively charged protein, lysozyme and the negatively charged surfactant, sodium dodecyl sulfate (SDS) in water have been investigated by determining the phase equilibria of the ternary system within the concentration range of 20 wt % protein and 20 w t % surfactant. Addition of small amounts of SDS to an aqueous lysozyme solution results in a precipitation, at protein concentrations higher than 0.1 wt %. The formation ofthis precipitate is due to charge neutralization ofthe protein (eight positive charges per protein molecule) by the surfactant. The tie lines converge toward relative concentrations corresponding to charge neutralization and the maximum amount of precipitation occurs where the charge neutralization is complete, On addition of more SDS, a complete redissolution of the precipitate occurs at about 19 SDS molecules per protein, which corresponds to the total number of positive sites along the protein chain. Between 7 and 20 w t % of protein, there is a narrow strip between the isotropic solution phase and the precipitation region, where a bluish-colored,transparent, viscous gel is formed. The molar ratio between SDS and lysozyme within the stability-region of the gel phase is not more than the total number of positive sites of the protein molecule. The shape of the tie lines indicates an associative-type of interaction between the lysozyme and SDS. The phase diagram is discussed in terms of electrostatic and hydrophobic effects.
Introduction Protein-surfactant interactions have been extensively studied in aqueous solution^.^-^ Studies include both oppositely’-s and similarly8-10charged protein-surfactant and protein-nonionic surfactant systems.ll The understanding of the interactions a t a molecular level is, however, complicated since proteins are complex biomacromoleculeswith unique primary structures expressed in terms of their amino acid sequences. These molecular constituents contribute to a wide variety of interactions that lead to the secondary and tertiary structure of a protein.
* To whom correspondence should be addressed: FAX, 46-46222 44 13; phone, 46-46-222 81 88; e-mail, anna-karin.moren@ fkeml.lu.se. Abstract published in Advance A C S Abstracts, September 1, 1995. (1)Ananthapadmanabhan, K. P. In Interactions ofSurfactants with Polymers and Proteins, 1st ed.; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press, Inc.: London, 1993; p 319. (2) Jones, M. N.; Brass, A. In Food Polymers, Gels, and Colloids; Dickinsson, E., Ed.; Cambridge University Press: Cambridge, 1991; p @
65.
(3) Dickinson, E. In Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: London, 1993; p 295. (4)Jones, M. N.; Manley, P. J. Chem. Soc., Faraday Trans. 1 1980, 76, 654. ( 5 )Jones, M. N.; Manley, P. J . Chem. Soc. Faraday Trans. 1 1979, 75, 1736. (6)Fukushima, K.; Murata, Y.; Sugihara, G.; Tanaka, M. Bull. Chem. SOC.Jpn. 1982, 55, 1376. (7) Fukushima, K.; Murata, Y.; Nishikido, N.; Sugihara, G.; Tanaka, M. Bull. Chem. Soc. Jpn. 1981, 54, 3122. (8) Skripkin, A. Y.; Volynskaya, A. V.; Shishkov, A. V.; Gol’danskii, V. I. Molekulyarnaya Biologiya 1989, 23, 365. (9) Subramanian, M.; Sheshadri, B. S.;Venkatappa, M. P. J. Biochem. 1984, 95, 413. (10) Subramanian, M.; Sheshadri, B. S.;Venkatappa, M. P. J . Biosci. 1986, 10, 359. (11)Nishiyama, H.; Maeda, H. Biophys. Chem. 1992, 44, 199.
Ionic surfactants are known to interact very strongly with oppositely charged globular protein^.^-^ For these systems, such as, lysozyme-sodium dodecyl sulfate (SDS), the interactions can be investigated by determining surfactant binding isotherms by different experimental technique^.^-^ These studies are normally carried out by adding increasing amounts of surfactant to a constant very dilute aqueous solution of protein, mostly below 2 wt % protein. The binding isotherms, which are defined as the number of surfactant ions bound per protein molecule, have been explained by Jones and M a n l e ~in ~ ,terms ~ of a specific and noncooperative binding a t low surfactant concentrations followed by the nonspecific and cooperative binding a t higher concentrations. The specific binding involves both electrostatic and hydrophobic interactions, while the nonspecific binding is dominated by the hydrophobic forces. In a few report^,^^^,^ the formation of a precipitate occurs with addition of small quantities of surfactant, but it redissolves on further addition of the surfactant. However, the heterogeneous precipitate region and the single solution phase surrounding the precipitate region have not been characterized in any detail. Probably, this is due to the fact that most studies reported in the literature are limited to a very narrow concentration range of protein. It has become a common practice to examine the phase equilibria of surfactant and surfactant-polymer in binary and multicomponent systems prior to a detailed study of a phase of interest. The phase equilibria studies have contributed significantly to our understanding of surfactant assembly processes in complex systems. It is expected that the characterization, location, and stability ranges of different phases, including the heterogeneous regions, will provide important information toward the understanding of protein-surfactant interactions. An increased knowledge in the bulk phase is also expected to result in
0743-746319512411-3636$09.00/0 0 1995 American Chemical Society
Surfactant-Protein -Water Equilibria
Langmuir, Vol. 11, No. 10, 1995 3637
phase can easily be distinguished when the two phases are in a better understanding of not only these systems but also coexistence. The region containing the precipitate is recognized related systems studied by, for example, adsorption to by its white precipitate sedimentatingat the bottomofthe sample surfaces. In this study, we present a n isothermal ternary tubes while the clear or blue solution forms the supernatant. phase diagram for the system lysozyme (1ys)-SDS-water The border line between the two-phase gel and solution region a t 298 K. Single phases are characterized. The heteroand the three-phase region was determined by obsellring the geneous precipitate region is analyzed in detail and the disappearance of the precipitate as a function of the sample results are discussed in terms of electrostatic and hycomposition. drophobic forces. Determinationof Tie Lines. The samples in the two-phase The anionic surfactant, SDS, forms small, spherical, region were analyzed to determine the tie lines. They were shaken for 20 h and then centrifuged for further 3 h at 25 "C. fairly monodisperse normal micellar aggregates with water up to fairly high surfactant c o n c e n t r a t i o n ~ . ~ ~ J ~First the supernatant liquid was carefully separated from the precipitate with a Pasteur pipet, and then it was added to a Lysozyme is a small, well-defined, compact, globular preweighed glass tube. Both the precipitate and the supernatant protein. It is soluble in water and stable in acidic were lyophilized, and their masses were obtained by standard s01utions.l~Lysozyomeis slightly ellipsoidal with dimengravimetric methods. sions 45 x 30 x 30 A. It has 129 amino acid residues and In order to determine a tie line, one needs to know at least two a molecular mass of about 14 600 Da.I5 Its structure is points in the phase diagram, the original concentration of the stabilized by four disulfide bridges and the interior of the sample, and the composition of the components in either the protein is almost hydrophobic while the surface is mostly supernatant phase or the precipitate. In this case, we used the polar. The solubility of the protein is determined by a supernatant phase, which is also assumed to contain all the water. The unique point of the composition in the supernatant phase delicate balance between different factors and in this study lies somewhere on the axis of the supernatant phases' water it turns out that even if as few as eight amino acid residues concentration. The water concentration in the supernatant phase on the surface are masked by the amphiphile, the solubility is obtained after it has been lyophilized. The determined of the protein falls to near zero. The isoelectric point is boundary line between the single solution and the two-phase 11.0 and the protein is accordingly positively charged in region only leaves one way of drawing the tie lines, since they a n aqueous solution.16 can never cross. The composition of the precipitate phase was Many biological systems of commercial o r technological neither analyzed nor used in determiningthe tie lines. However, importance contain a mixture of proteins and surfactants. knowingthe mass ofthe precipitate provides a method ofchecking the accuracy of the experiment. For example proteins and surfactant mixtures are used An analysis of both SDS and protein in the supernatant liquid in cosmetic products as well as stabilizers of emulsions phase allows the tie lines to be drawn more accurately. However, and foams in the food industry. Surfactants are used in we have not yet been able to develop a method capable of a crystallizing membrane proteins that are employed for simultaneous determination of both the surfactant and protein X-ray diffraction studies. concentrations in a mixed system. The nature of the tie lines, illustrated in Figure 2 will, however, not be affected by any Experimental Section successful quantitative determination of both components. Analysis of the Precipitate. To analyze the behavior of the Materials. Lysozyme No L-6876 obtained from chicken egg precipitate the samples were prepared as in previous section. white, was bought from Sigma and had been crystallized three When the precipitate in the presence of salt was analyzed, a 0.10 times, dialyzed, and lyophilized. SDS (specially pure) was M NaCl and 0.05 M NaCl solution was used as a substitute for supplied by BDH, and NaCl (zur Analyse) was obtained from water. The lyophilized precipitate was obtained after it had been Merck. Millipore filtered water was used. All chemicals were separated from the supernatant, and is expressed in wt % used as obtained. precipitate of the total sample content. The wt % of precipitate Sample Preparation. The samples were prepared by is then plotted against the varying concentration of either weighing appropriate amounts of substances into glass tubes, lysozyme at constant SDS or SDS at constant lysozyme. which were flame sealed. In the dilute region the samples were The separation of the supernatant from the precipitate always prepared in screw-capped glass tubes. After an initial vigorous leaves a small amount of supernatant with the precipitate. This shaking, the samples were put in a shaker for at least 16 h at will have a negligible effect on the total amount of precipitate 25 "C. This time has been shown to be sufficient to achieve an as long as the concentration of SDS and lysozyme is low in the equilibrium in the dilute precipitationregion.8 More concentrated supernatant. But at higher concentrations of SDS and lysozyme gel regions, however, needed to be unperturbed in order to build there might be a non-negligible contribution to the amount of up the network structure. The time needed to obtain a gel is precipitate. In the salt-free system, the separation of the found to be dependent on the sample composition. For example, precipitate from the supernatant is further complicated within close t o the precipitate region a viscous gel was formed imthe three-phase region, which contains the gel. Here, the mediately after shaking. However, these samples were centrisupernatant is blue even after prolong centrifugation (benchfuged to enhance the mixing of local heterogeneous nonequitype centrifuge,6000rpm). The blue solutionis a finely dispersed librium regions,which are embeddedin the veryviscous gel phase. gel in liquid and the gel particles could be detected easily by an If the sample is in the middle of the gel phase region, the gel can optical microscope equipped with a videocamera. However in be formed within a day after proper mixing of the sample. the salt-system the supernatant is a colorless solution, which However, gel samples close to the solution border first form blue indicates that the supernatant does not contain any gel particles. colored solutions, which after a few days turn into the viscous This suggests that in the presence of salt, both the gel and the gel. These gels are normally formed within about 10 days. The precipitate are separated after centrifugation from the superthoroughly mixed samples were kept standing at 25 "C for natant. equilibration. Identification of the Phases and Phase Boundary. The Results and Discussion isotropic solution phase was identified by placing the sample between crossed-polars, as well as by examining the samples The phase behavior of the ternary lysozyme-SDSagainst transmitted light. The gel phase is also isotropic but is water system will be determined by the interactions very viscous and slightly blue-colored. The gel and the solution between the components. As stated in the introduction, the interactions in this case are complex, and therefore (12) Lindman, B.; Wennerstrom, H. Top. Curr. Chem. 1980, 87, 1. it is appropriate to first make fundamental studies of the (13)Fontell, K. Mol. Cryst. Liq. Cryst. 1981, 63, 59. (14)Martindale The extra Pharmacopoeia, 28th ed.; The Pharmaphase equilibria of the system. This is a very simple way ceutical Press: 1982. of gaining knowledge about the general picture of changes (15)Stryer, L. Biochemistry, 3rd ed.; W. H. Freeman and Company: in the phase microstructure that takes place in the system. New York, 1988. To minimize the variables of the studied system, it was (16)Tanford, C.; Wagner, M. L. J. Am. Chem. SOC.1954, 76,3331. ~
~
~~~
Mor& and Khan
3638 Langmuir, Vol. 11, No. 10, 1995
A
/\ /
. lysozyme
+
(1) The Clear Isotropic Solution Phase, L. Within the determined concentration range (Figure 11, both SDS and lysozyme17 are independently soluble in water. In very dilute solutions the protein exists as monomers, but a t pH above 4.5 and a t higher concentrations, it shows an associative behavior and forms dimers and higher oligomers.l8J9 The critical micelle concentration (cmc) of SDS in water is 0.24wt %, and it forms small spherical micelles over the entire concentration range used in this study.13 The addition of very small amounts of SDS M) to a n aqueous solution of 2.5 wt % lysozyme results in a precipitate. It appears that by increasing the lysozyme concentration (10 wt %), the amount of SDS necessary to obtain the precipitation does not increase substantially. The protein and the surfactant interact strongly with each other, and accordingly the SDS concentration required to form independent micelles in the presence of lysozyme is higher than that without the protein.2 The cmc of SDS in water is not reached within the solution region close to the lysozyme-water axis. Therefore, it is likely that the surfactant molecules in this region of the solution exist partly as monomers and partly bound to the protein. To predict the exact behavior of this solution region, further investigations are required. The estimated triangular phase diagram (Figure 1B) is dominated by the upper clear, isotropic single solution phase a t high SDS concentrations. At the surfactantwater axis, the solution region extends to the very dilute water corner and there is eventually a connection to the lower region of the solution phase. The samples in the upper solution region seem to have about the same viscosity, but a t higher surfactant concentrations the viscosity tends to increase. The clear solution phase is visually the most stable phase and can be formed immediately after mixing the components. A n exception is in the border regions close to the heterogeneous region where the samples need longer time to reach equilibrium, (2) The Heterogeneous Regions Containing the Precipitate. The precipitate region, P L, extends to very low surfactant concentrations close to the lysozymewater axis. The extension of the precipitate region is very limited in the water-rich corner with respect to the SDS concentration but increases with increasing concentrations of the protein. At lower SDS conceqtrations, the white precipitate is in equilibrium with the clear solution in a two-phase region. When the number of SDS molecules per lysozyme molecule is increased, a three-phase region P G L appears, and the precipitate is in equilibrium with the clear solution and the blue gel. (3)The Gel Phase, G. The single gel phase begins to form directly above the heterogeneous precipitate region a t around 7 wt % lysozyme and extends with increased protein concentrations. The gel is viscous, isotropic, and has a blue appearance. The stability region of the gel phase is very limited with respect to the SDS concentration, and when the added amount of SDS is increased, the single gel phase is converted first into a two-phase region before it is finally redissolved. It can be difficult to detect the transparent blue gel in the heterogeneous regions, especially when it is present in small amounts. Therefore, the exact border regions of the phase equilibria are difficult to determine. (B)Description of the Different Main Regions. (1) The Precipitation Region. In Figures 1 and 2, there
+
L
L lysozyme
Figure 1. (A) Isothermal three-componentphase diagram for the system lysozyme (0-20 wt %)-SDS (0-20 wt %)-water (80-100 wt %) at 25 "C: solid square, solution; solid circle, precipitation; solid triangle, gel; x in square; blue solution; plus sign, three-phase equilibrium; open circle, gel and precipitate in equilibrium. Solid lines within the triangle indicate the SDSAysozyme molar ratio. (B)The schematic isothermal three-component phase diagram for the system lysozymeSDS-water, showing phase boundaries based on experimental points in part A. L, solution; G, gel; P, precipitate; P + L, G + L, P + G L are two- and three-phase regions.
+
decided not to use any buffer (to avoid screening effects caused by a salt). (A) General Features of the Phase Diagram. At constant temperature (25 "C) and pressure (1 atm) the phase equilibria of the three-component system lysozymesodium dodecyl sulfate (SDS)-water is represented in Figure 1as a triangular phase diagram. The triangular sides correspond to different binary axes: the base line, lysozyme and water; the right side, SDS and lysozyme; the left side, water and SDS. The ternary phase diagram has been mapped out from about 100 samples (Figure 1A) which were prepared within the concentration range of 20 wt % SDS and 20 wt % lysozyme. In the following section we will give a brief introduction to the phase diagram, which contains different phases that were identified in the system. Thereafter the main phases of interest will be discussed in detail.
+ +
(17)Ericsson, B.; Larsson, K.; Fontell, K. Biochzm. Biophys. Acta 1983,729,23. (18)Bruzzesi, M. R.; Chiancone, E.; Antonini, E. Biochemistry 1966, 4 , 1796. (19)Lampreave, F.;Piniero, A.; Brock, 3. H.; Castillo, H.; Sbnchez, L.; Calvo, M. Int. J. Biol. Macromol. 1990,12, 2.
Surfactant-Protein- Water Equilibria
Langmuir, Vol. 11, No. 10, 1995 3639
is a white precipitate produced by the association of SDS and lysozyme a t very low SDS concentrations that is in equilibrium with the isotropic solution phase. The amount of precipitate increases as the SDS concentration is increased a t constant protein concentration; see Figure 3. The precipitate reaches a maximum and becomes saturated after a particular SDS concentration and any further addition of SDS results in its redissolution. When the precipitate is redissolving, a gel phase is formed. This region is accordingly a three-phase region where the gel (GI, the precipitate (P),and the solution phase (L) are in equilibrium (Figure 1). The detection of the gel in the three-phase region is difficult since it is easily dispersed in the heterogeneous mixture. This is especially a problem when the gel is present only in small amounts, as in the dilute region or close to the border region where the precipitate and the solution are in equilibrium. The exact determination of the three-phase region is therefore difficult, but the gel has been observed in the three-phase region down toward 1 wt % of lysozyme, and it is most likely that the gel coexists with the precipitate and the solution phase even at lower protein concentrations. The line representing the complete redissolution of the last trace of precipitate, i.e., the upper border line of the three-phase region, is fairly straight, and the concentrations of the components along this line correspond to about 19 SDS molecules per lysozyme molecule. In a previous work, Jones and M a n l e ~ interpreted ~,~ their binding isotherm in terms of a specific binding until 18 SDS molecules are bound to each lysozyme molecule (pH = 3.2). The origin of the specific binding is a combination of both hydrophobic and electrostatic interaction^.^,^,^^,^^ The hydrophobic contribution to the specific binding is due to interactions between the hydrophobic parts of the protein and the surfactant tail, whereas the electrostatic interaction occurs between the positively charged amino acid residues of the protein and the negatively charged surfactant headgroup. In this system the pH is around 6.5 which corresponds to about 18 positive amino acid and this makes the two systems comparable. Accordingly the specificbinding appears to extend a t least over the whole precipitate region (P L and P G L). As the SDS concentration is increased beyond the specific binding the precipitate is totally redissolved and the protein and surfactant start to interact by the nonspecific binding. The latter binding is mainly due to hydrophobic interactions between the surfactant tails and the nonpolar regions of the protein as well as surfactant-surfactant interactions3 and is of a similar character to the driving force for the micelle formation. Therefore, it is assumed that micellar aggregates on the protein and free micelles in the solution are not present in the system until the nonspecific binding has started.2 Tie Lines. The tie lines have been estimated in the two-phase heterogeneous region where the precipitate and the clear solution are in equilibrium. The tie lines shown in Figure 2 originate from the water corner a t the waterlysozyme binary axis and extend to the lysozyme/SDS side. This means that there is a n association between lysozyme and SDS and not a ~ e g r e g a t i o n .Association ~~ results in one concentrated phase, enriched with the two compo-
+
+ +
(20)Oakes, J. J. Chem. SOC.,Faraday Trans. 1 1974, 70,2200. (21)Yonath, A,; Podjarny, A.; Honig, B.; Sielecki, A.;Traub, W. Biochemistry 1977,16, 1418. (22)Bartik, K.; Redfield, C.; Dobson, C. M. Biophys. J. 1994, 66, 1180. (23)Yang, A,; Honig, B. J . Mol. Biol. 1993,231, 459. (24)Lindman, B.; Carlsson, A.; Gerdes, S.; Karlstrom, G.; Piculell, L.; Thalberg, K.; Zhang, K. In Food Colloids and Polymers: Stability and Mechanical Properties;Walstra, P., Dickinson, E., Eds.; The Royal Society of Chemistry: London, 1993;p 113.
; P+L
100
_.*
3 L
*
4
5
lysozyme
Figure 2. The three-component phase diagram for the system lysozyme (0-5 wt %)-SDS (0-5 wt %)-water (95-100 wt %), showing tie lines of the two-phase region, P + L, at 25 "C.
Notations as in Figure 1.
Table 1. The Original Sample Composition, the Yield of Precipitate after Lyophilizing, and the Amount of Water in the Supernatant at Constant 3.0 wt % of Lysozyme
wt %
wt %
lysozyme
SDS
wt % water
3.0 3.0 3.0 3.0 3.0
0.3 0.4 0.5 0.6 0.7
96.7 96.6 96.5 96.4 96.3
wt % precipitate in sample
wt % water in supernatant
2.2 2.9 3.3 2.8 2.1
98.8 99.5 99.9 99.3 98.3
nents, in this case the precipitate between SDS and lysozyme, and one dilute phase, the supernatant. In the case of segregation, the tie lines would be vertical, with the two phases rich in each of the components. The supernatant in this two-phase region contains very low concentrations of SDS, since the precipitate is in equilibrium with the part of the solution phase close to the protein-water axis. The precipitate which is in equilibrium with the supernatant has a well-defined composition since all the tie lines converge toward about eight SDS molecules per lysozyme molecule a t the SDS/ lysozyme axis. Accordingly this can be described empirically as a n equilibrium between the solid precipitate and a solution of SDS and lysozyme as shown below.
The composition of the precipitate 8 SDSAysozyme indicates a complete charge neutralization of lysozyme by the negative SDS molecules, since a t pH =Z 6.5 a lysozyme molecule is known to have approximately eight net positive charges.16 This is in good agreement with results obtained by Fukushima et al.7 a t pH = 5.8 and a t a much lower lysozyme concentration of 0.06 wt %. Analysis of the Sample. The investigation of the heterogeneous region has been accomplished by estimating the yield of precipitate by replacing water with SDS a t a constant lysozyme concentration (Table 1)as well as by replacing water with lysozyme a t a constant SDS concentration (Table 2). The quantity of lyophilized precipitate is calculated in wt % ofthe total sample and is plotted
Mor& and Khan
3640 Langmuir, Vol. 11, No. 10, 1995 Table 2. The Original Sample Composition, the Yield of Precipitate after Lyophilizing, and the Amount of Water in the Supernatant at Constant 0.6 wt % of SDS
SDS
wt % water
wt % precipitate in sample
wt % water in supernatant
0.5 0.5 0.5 0.5 0.5 0.5
97.0 96.7 96.5 96.3 96.0 92.5
2.3 3.1 3.3 3.5 3.7 4.1
99.3 99.8 99.9 99.9 99.8 96.2
wt % lysozyme
wt %
2.5 2.8 3.0 3.2 3.5 7.0
against the concentration of the varied component in Figures 3 and 4. The figures are based on the assumption that the precipitate does not contain any water aRer it has been freeze-dried. Figure 3 shows the yield of lyophilized precipitate versus the SDS concentration at constant (A) 3 wt % and (B) 1 wt % of lysozyme. At first, there is a n increase in the amount of precipitate when there is a deficit of lysozyme. Above a certain SDS concentration the precipitate starts to redissolve. The maximum yield a t constant 3 wt % of lysozyme takes place a t a point close to 0.5 wt % SDS, corresponding to eight SDS molecules per lysozyme molecule. The maximum amount of precipitate at constant 1wt % of lysozyme is around 0.17 wt % SDS which corresponds to a value slightly less than nine SDS molecules per lysozyme molecule. The results support a previous conclusion namely that the precipitate composition is around eight SDS molecules per lysozyme molecule. The mixture a t the maximum point also gives the most water-rich supernatant, 99.9 wt % ofwater. The decrease in the amount of precipitate in Figure 3 is explained by the increased number of surfactant ions binding specifically to the protein, and thereby the net charge of the SDSAysozyme complex becomes negative, causing a solubilization of the complex. The phase diagram study presented in Figure 1reveals the formation of a gel phase which until now was unknown. The gel, which is formed a t a surfactant concentration above the charge neutralization level, extends to very high water concentrations through multiphase regions. The dissolution of the gel into a clear solution requires more surfactant than the dissolution of the total amount of precipitate a t a SDS/ lysozyme ratio of 18. In previous the authors explained their results, obtained from the analysis of the precipitate, without taking the presence of the gel into account. The blue supernatant is more obvious in the samples containing 3 wt % of lysozyme, but for the 1wt % protein samples a blue gel could be seen above the precipitate after centrifugation. Figure 4 shows the amount of lyophilized precipitate obtained by increasing the lysozyme concentration a t constant 0.5 wt % SDS. At low concentrations oflysozyme, the protein-surfactant complex is in solution, then a t higher concentrations there is a dramatic increase in the amount of precipitate which levels off as the precipitate is saturated. Since precipitation is caused by ion-pair neutralization, the system is expected to attain a n increased yield of precipitate up to 3 wt % of lysozyme, where there is a deficit of lysozyme. At 3 wt % of lysozyme the protein molecules are fully neutralized by the surfactant molecules and accordingly this would give the maximum yield (Figure 3A). On further addition of lysozyme, the amount of precipitate is not expected to increase, since no free SDS molecules are available to neutralize the charged protein molecules, and the excess of lysozyme will remain in the supernatant liquid. However, the amount of lysozyme required to obtain the
SDSIlysozyme
0
5
10 15 SDSllysozyme
20
Figure 3. The w t % of precipitate for samples versus the SDS concentration, expressed as number of SDS molecules per lysozyme molecule, at a constant (A) 3 wt % and (B) 1 wt %
lysozyme.
o ! 0
1
2 4 wt% lysozyme
6
I
8
Figure 4. The wt % ofprecipitate for samples versus lysozyme concentration at a constant SDS concentration (0.5 wt % SDS).
maximum yield of precipitate a t constant SDS concentrations (Figure 4)is slightly higher ( a 3 . 5 wt % lysozyme). The increased precipitate, even above 3 wt % lysozyme a t constant SDS, might have several explanations, and a discussion of this is best left until the precipitate is analyzed. However, one reason might be that the separation of the supernatant and the precipitate always leaves a small amount of supernatant within the precipitate. When the concentration of lysozyme is high, the solution left in the precipitate sample may affect the total amount of lyophilized precipitate. (2) The Gel Phase. When the SDS concentration is increased beyond the precipitation region, containing 7 wt % lysozyme o r more, the heterogeneous region disappears and the sample is turned into a slightly bluecolored, transparent viscous gel phase, G. The blue color ofthe gel indicates the presence oflarge aggregates in the system. The gel phase forms a narrow strip between the upper solution region and the precipitation region (Figure 1). The gel region probably even extends to higher lysozyme concentrations than those studied. The single gel-phase which is formed adjacent to the heterogeneous precipitate region has a very limited capacity to solubilize SDS. When the amount of SDS is increased, the blue gel is redissolved and a less viscous blue solution is formed. If the blue solution is centrifuged for a longer period, the blue gel sediments as a lower phase although the gel is
Surfactant-Protein- Water Equilibria extremely easy to redisperse into the solution. After further increase of the SDS concentration, the viscosity of the solution is lowered and the blue solution is converted into a clear and isotropic one-phase solution. The twophase blue solution region extends to lower protein concentrations as a string between the three-phase region and the clear solution phase. The gel appears to exist as a single phase within the region indicated by G in Figure 1, since no free liquid droplets or precipitate are found to be present in the samples. Moreover, the gel phase coexists with the single solution and the precipitate through the appropriate two- and three-phase regions. However, the extent to which these multiphase regions, containing the gel, reach toward the water corner is difficult to determine. When the gel is heated to 55 "C, it becomes white, and on lowering the temperature to 25 "C, the white color does not disappear. This might be explained by the formation of a heat-set gel, if the protein in the surfactant-induced gel can be further denatured by heat. In absence of surfactant, lysozyme is known to have a denaturation temperature at 78 "C (pH 6.5hZ5 It is observed that the denaturation temperature is lowered in the presence of surfactant, since the surfactant also denatures the protein (private communication with Krister Eskilsson in this lab). Two main kinds of heat-set gels are observed, one transparent and one nontransparent The transparent gel consists of a molecular homogeneous network. The network is proposed to be built up by extended strings of beads as a result of a long-range order, which is induced by the charge of the protein. When the charge of the protein is decreased, the electrostatic long-range forces are reduced. This is suggested to give the nontransparent gel consisting of a randomly aggregated protein network. A further denaturation of the protein in the surfactantinduced gel a t 55 "C would result in a n increased attraction between the protein molecules. The white appearance of the heated surfactant-induced gel could hence be derived from a randomly aggregated protein network. The gel network might also be induced in other ways, e.g. by binding the surfactant polar headgroup to the oppositely charged site of the protein with its alkyl chain connected to the nonpolar part of the second protein. It is also possible that small surfactant aggregates are formed on the protein that bind mainly hydrophobically to protein molecules and form networks reminescent of hydrophobically modified polymer-surfactant gels.27 The surfactant and polymer then form mixed aggregates that induce a cross-linking of the polymer chains. However, further investigations considering the microstructure of the gel are needed before any conclusions can be drawn. Combined techniques of NMR and cryo-TEM (under study) are expected to provide important information regarding the microstructure of the gel. (C) The Effect of Salt. To investigate the salt effect on the phase behavior, 40 samples of different compositions have been prepared and investigated as in the salt-free system. The phase diagram obtained is shown in Figure 5. The pure water in Figure 1has been replaced by a 0.10 M NaCl solution. The investigated concentration range of lysozyme and SDS is 20 wt %, as in the salt-free system. But the interest in this phase diagram is mainly focused on the border regions. The main features of the phase diagram are similar to those observed in the salt-free system. As before, the phase diagram obtained contains ~~
~~
~~
(25) Sochava, I. V.; Belopolskaya, T. V. Food Hydrocollords 1992,6, 97 (26) Dol, E. Trends Food Scr. Technol. 1993, 4 , 1.
(27) Magny, B.; Iliopoulos, I.; Audebert, R.; Piculell, L.; Lindman, B. Prog Colloid Polum. Sci. 1992, 89,118.
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10
20
lysozyme
Figure 6. Isothermal three-component phase diagram for the system lysozyme (0-20 wt %)-SDS (0-20 w t %)-0.10 M NaCl solution at 25 "C: dot in circle, solution; solid circle, threephase equilibrium; x in square, blue solution; solid triangle, white gel. Notations as in Figure 1.
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3642 Langmuir, Vol. 11,No. 10, 1995
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Figure 7. Micrographs for the precipitate and the gel region with (A)and without ( R ) polarizer in the precipitate region. The sample composition is 5.1 wt % lys, 1.4 wt (2 SDS, and 93.5 wt c/r 0.1 M NaCl solution and the magnification x40. Micrographs with (C) polarizer in the gel phase. The sample composition is 9.6 wt % lys, 4.0wt % SDS, and 86.4 wt c/c 0.1 M NaCl solution and without (D) and the magnification x 100.
two-phase region. A corresponding series of samples has also been made where the SDS concentration was kept at 0.5 wt %. All of the samples were mixed and equilibrated in the same way as those in the salt-free system. (1) Analysis of the Precipitate with 0.10 M NaCl and 0.05 M NaCl. The samples with 0.10 M NaCl were prepared a t concentrations corresponding to those of the salt-free system, and the behavior of the precipitate was analyzed in the same way as that of the salt-free system. Under these conditions, it is easy to compare results obtained for the two systems. The extent of precipitate expressed as wt % of precipitate of the total content of the sample is obtained either a t a constant lysozyme content (3 wt %) with a varying SDS concentration or a t a constant SDS content (0.5 wt %I) with a varying lysozyme concentration. The series with varying SDS concentration at a constant 3 wt % lysozyme concentration was complemented by a 0.05 M NaCl solution, to see the effect of lower salt concentrations. The obtained results for 0.10 M NaCl are presented in Tables 3 and 4. At constant concentration of lysozyme (Figure 6, Table 3), there is a sharp initial rise of the amount of precipitate with added SDS. A maximum a t about 0.5-0.6 wt % SDS is attained, as in the salt-free system. The maximum of the precipitation corresponds to the charge neutralization of lysozyme. Within this SDS concentration range the salt-free and the salt systems behave identically. On further addition of SDS, the solubility curves deviate compared to the salt-free system. At concentrations in excess of charge neutralization, the yield of precipitate in
the salt system is higher than that in the salt-free system a t a given SDS concentration. The samples in both systems contain gel in this concentration range. As mentioned earlier, the gel remains as fine particles within the supernatant for the salt-free system. But for the salt system, the dispersed gel particles seem to be of relatively larger size and are hence separated along with the precipitate. The high yield compared with the salt-free system is, therefore, due to the contribution of the gel. In fact, the sample with the highest amount of SDS (1.2 w t 96) contains no precipitate, just the gel in equilibrium with the clear solution. The isotropic gel in the salt-free system and the white gel in the salt-system show very weak textures when viewed through a video-enhanced light-microscope. The microscopic textures of the precipitate are very pronounced and different from the gel as shown in Figure 7. There are certain factors that immediately change the solution properties in the presence of a salt; one of them is the critical micelle concentration of a n ionic surfactant. When salt is added, the repulsive forces between the neighboring charged headgroups of the surfactant are reduced. This inhibits the formation of free micelles in the solution2 and also affects the micellar aggregates formed on the protein. The exact cmc in this study has not yet been measured. Therefore, it will be important to investigate the behavior of the cmc as a function of both protein and salt concentration. Figure 8 shows the yield of precipitate plotted against the lysozyme concentration in the presence of 0.10 M
Langmuir, Vol. 11, No. 10, 1995 3643
Surfactant-Protein- Water Equilibria
Table 4. Amount of Precipitate Obtained by Lyophilizing the Bottom Phase and the Supernatant of the Samples versus the Concentration of Lysozyme for the System Lysozyme-SDS-0.10 M NaCl Solution, at Constant 0.5 wt % SDSa
wt % wt % wt % precipitate water in SDS water in sample supernatant 0.5 97.0 2.8 99.3 2.8 0.5 96.7 3.0 99.3 3.0 0.5 96.5 3.3 99.3 3.2 0.5 96.3 3.4 99.2 3.5 0.5 96.0 3.7 99.3 7.0 0.5 92.5 4.1 95.7 a Note, all water is assumed to be in the supernatant phase. (D)A Comparisonof Phase Diagrams between the Protein and a Polyelectrolyte System. It is interesting to compare the studied system with an oppositely charged polyelectrolyte-surfactant system: sodium hyaluronate (NaHyI-tetradecyltrimethylammonium bromide (TDTAB)-water.28 Hyaluranon (Hy) is a linear anionic polysaccharide. At neutral pH, the carboxylate groups of NaHy are practically fully dissociated. The ternary phase diagram of the polyelectrolyte exhibits many features similar to the protein system. Like the protein system the NaHy-surfactant shows associative behavior in water and has a two-phase region. But the two-phase region is shown to reduce in size with a small addition of salt (70 mM NaBr). This behavior is not observed in the proteinsurfactant system, where salt does not have any significant effect on the size of the heterogeneous region compared to the salt-free system. The reduction in size of the polymer two-phase region is explained in terms of a n electrostatic screening effect as electrolyte is added. The screening results in a reduced binding of surfactant to the polyelectrolyte molecules. In the protein system precipitation of the Lys-DS- complex is more efficient than that of the lys-C1- since the polar head group -OSOS- of the surfactant binds to the cationic amino acid residues and the alkyl chain interacts with the hydrophobic part of the protein. Consequently, the size of the heterogeneous region remains unchanged with the addition of NaC1. wt % lysozyme 2.5
0 1 0
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Figure 8. The wt % of precipitate for samples versus the lysozyme concentration in a system containing (0)0.10 M NaCl and (13) pure water, at a constant concentration of 0.5 wt % SDS. Table 3. Amount of Precipitate Obtained by Lyophilizing the Bottom Phase and the Supernatant of the Samples versus the Concentration of SDS for the System Lysozyme-SDS-0.10 M NaCl Solution, at Constant 3 wt % Lysozymea
wt % wt % water in wt % precipitate wt 9% wt % lysozyme SDS water in sample supernatant 2.2 98.3 3.0 0.3 96.7 96.6 3.1 99.2 3.0 0.4 3.0 0.5 96.5 3.3 99.3 96.4 3.3 99.3 3.0 0.6 3.0 0.7 96.3 3.2 99.2 96.2 3.5 99.3 3.0 0.8 3.0 0.9 96.1 2.9 98.5 a Note, all water is assumed to be in the supernatant phase. NaCl solution a t a constant SDS concentration (0.5wt %). The results are in good agreement with those observed in the salt-free system. The results presented in both Figure 6 and Figure 8 indicate that the phase behavior of the systems with and without salt are in good agreement. The main difference is that while the gel is separated from the liquid in the salt system, it is present with the liquid in the salt-free system. The salt contributes to the total mass ofthe sample and this cannot be ignored. A concentration of 0.10 M NaCl corresponds to an additional 0.6 wt % ofthe samples after lyophilizing. The experimental results are given in Tables 3 and 4and the supernatant in the presence of salt contains around a 0.5 wt % higher value than the corresponding salt-free samples, while the corresponding precipitate values agree very well. It is therefore reasonable to assume that most ofthe salt is dissolved in the supernatant liquid phase.
wt %
Acknowledgment. Originally the project was suggested by Bjorn Lindman. Continuous help, numerous discussions, and critical comments on the manuscript by Bjorn Lindman and Marie Wahlgren are gratefully acknowledged. The project is financed by the Swedish Research Council for Engineering Sciences (TFR). LA950087F (28) Thalberg,K.; Lindman, B.; Karlstrom, G. J. Phys. Chem. 1991, 95, 6004.
