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Langmuir 2002, 18, 3848-3853
Interactions between Mucin and Alkyl Sodium Sulfates in Solution. A Light Scattering Study L. Bastardo,*,†,‡ P. Claesson,†,‡ and W. Brown§ Department of Chemistry, Surface Chemistry, Drottning Kristinas va¨ g 51, Royal Institute of Technology, SE-100 44 Stockholm, Sweden, Institute for Surface Chemistry, P.O. Box 5607, SE-114 86 Stockholm, Sweden, and Department of Physical Chemistry, Box 532, University of Uppsala, SE-751 21 Uppsala, Sweden Received December 7, 2001. In Final Form: February 27, 2002 The properties of negatively charged mucin in aqueous solutions and its interaction with anionic sodium alkyl sulfates with different hydrocarbon chain lengths were studied by means of dynamic light scattering. It was observed that mucin forms aggregates in aqueous solutions with a hydrodynamic radius above 500 nm. These aggregates dissolve when sodium dodecyl sulfate or sodium decyl sulfate is present at sufficiently high concentration, above about 0.2 cmc (critical micellar concentration). On the other hand, sodium octyl sulfate is not very effective in dissolving the mucin aggregates. The hydrodynamic radius of the dissolved mucin, decorated with some associated surfactant, is found to be in the range of 40-90 nm. The observation that the dissolving power of the sodium alkyl sulfates decreases with decreasing surfactant chain length suggests that the association between the surfactant and mucin is hydrophobically driven. The kinetics of the dissolution process depends on the surfactant concentration, a higher surfactant concentration giving rise to a more rapid dissolution of the aggregates. It was also observed that when the ionic strength is increased, the surfactant concentration needed to dissolve the mucin aggregates decreases. This can be explained by reduction of repulsive electrostatic forces by the salt.
Introduction Mucins are glycoproteins of mucous secretions with large molecular weights and extensive polydispersity. The chemical composition of the mucin also depends on the region and species from which it is isolated. The macromolecules contain a large number of oligosaccharide side chains covalently bound to a protein backbone. Most of the glycans are confined to certain regions, which alternate with less densely populated or “naked” stretches of proteins. The terminal sialic acids in these side chains give a strong anionic character to the molecules.1,2 The intact mucin glycoprotein is very large, and molecular weights of 10-50 million g/mol have been reported.3,4 It consists of subunits, typically in the range of 0.1-2 million g/mol, that are connected by disulfide bonds. It has been difficult to establish the exact size of the mucin subunits, because the size of the fragments obtained depends to some extent on the extraction method.1,5 Mucins typically contain 70-90 wt % carbohydrate, and the protein backbone accounts for most of the remaining part. The oligosaccharides may contain from 1 to about 20 monosaccharides in branched as well as linear sequences.1 The typical structure of a mucin molecule is shown in Figure 1. Due to the hydrophilic nature of the glycans and the overall random coil-like conformation, mucins are equipped * To whom correspondence should be addressed. † Royal Institute of Technology. ‡ Institute for Surface Chemistry. § University of Uppsala. (1) Carlstedt, I. From the Department of Physiological Chemistry, University of Lund; Lund University: Lund, Sweden, 1988. (2) Bettelheim, F. A.; Scheinthal, B. M. J. Polym. Sci. 1970, 30, 117. (3) Carlstedt, I.; Sheehan, J. K.; Corfield, A. P.; Gallagher, J. T. Essays Biochem. 1985, 5, 40. (4) Shogren, R.; Jamieson, A. M.; Blackwell, J.; Jentoft, N. Biopolymers 1986, 25, 1505. (5) Varma, B. K.; Demers, A.; Jamieson, A. M.; Blackwell, J. Biopolymers 1989, 28, 785.
Figure 1. The typical structure of a mucin molecule consisting of several subunits connected by disulfide bridges. Note the presence of heavily glycosylated regions separated by naked regions exposing the protein backbone.
to interact strongly with the surrounding water molecules and contribute to gel formation within the mucous layer on the epithelial surfaces, keeping for example the oral tissues moist and lubricated. The mucin-rich film also affects bacterial adhesion and acts as a barrier to carcinogens and viruses, preventing their access to epithelial cell surfaces.6,7 Mucins are major components of saliva, which adsorb readily to the surfaces in the oral cavity, making them a constituent of the pellicle. Mucins are thus in contact with the different components present in oral care products applied in the oral cavity, and it is of importance to understand the interactions between such components and the glycoproteins. Sodium dodecyl sulfate, SDS, has been used as a foaming agent in dentifrices since the mid-30s. It is an anionic strongly protein-denaturing agent with a hydrophobic organic tail, which exhibits high affinity for protein molecules.8 The large size of mucin causes it to adsorb readily to both hydrophilic and hydrophobic surfaces; see for example refs 6, 9, and 10. In a more general sense, mucin can be (6) Shi, L.; Caldwell, K. D. J. Colloid Interface Sci. 2000, 224, 372. (7) Saliva and Oral Health; Edgar, W. M., O’Mullane, D. M., Eds.; British Dental Association: London, 1999. (8) Herlofson, B.; Barkvoll, P. Acta Odontol. Scand. 1993, 51, 39. (9) Malmsten, M.; Claesson, P. M.; Blomberg, E.; Carlstedt, I.; Ljusegren, I. J. Colloid Interface Sci. 1992, 151, 579.
10.1021/la015717u CCC: $22.00 © 2002 American Chemical Society Published on Web 04/10/2002
Interactions of Mucin and Alkyl Sodium Sulfates
viewed as a polyampholyte with an overall anionic character. It is therefore of relevance to recapitulate what is known about interactions between anionic surfactants and polymers, a topic that has been extensively studied during the past years. Some reviews covering various aspects of this vast topic can be found in, for example, refs 11-13. The main interactions between polymers and surfactants are generally regarded to be of electrostatic and hydrophobic nature. The electrostatic interactions between the charged surfactant headgroups as well as between the surfactant and charged groups on the polymer should be considered. The hydrophobic interaction is always of paramount importance for the association between the nonpolar parts of the surfactants in aqueous media, and it may also be present between the surfactant tails and the nonpolar parts of the polymer. SDS interacts strongly with cationic polyelectrolytes, in most cases forming large insoluble aggregates at charge stoichiometry. The internal structure of these aggregates is, in the case of highly charged polyelectrolytes, highly ordered with lamellar or hexagonal organization.14 SDS also interacts strongly with some moderately hydrophobic uncharged polymers, such as poly(ethylene oxide), PEO. In this case, micellar-like SDS aggregates associate with the PEO chain in a bead-and-necklace structure.15,16 When sodium alkyl sulfate surfactants are added to a mucin solution, it is not obvious that any association will occur since both the surfactant and the polymer are negatively charged, which counteracts the association process. However, if the hydrophobic interaction is sufficiently strong, surfactant-mucin association may occur, as is observed for, for example, other negatively charged protein-SDS systems.17 On the basis of the above considerations, one may expect that the ionic strength of the solution as well as the length of the surfactant hydrocarbon chain will affect the association process, if any. To test this hypothesis, bovine submaxillary mucin (BSM) solutions were studied in the absence and presence of sodium alkyl sulfate surfactants by means of dynamic light scattering, since this method is a useful experimental tool for the characterization of polymers and protein systems.2,5,18-20 Both the alkyl chain length and the ionic strength of the medium were varied. To minimize effects due to long-range electrostatic interactions20,21 between separate polymers, very dilute mucin solutions were used (5-100 ppm) in a solution of moderate ionic strength (30 mM NaCl). This NaCl concentration was also chosen because it is similar to the salt concentration in the oral cavity. However, the ionic strength in the oral cavity is influenced by the saliva secretion rate.7 (10) Perez, E.; Proust, J. E. J. Colloid Interface Sci. 1986, 118, 182. (11) Lindman, B.; Kyrre, T. In Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: Boca Raton, FL, 1993; p 427. (12) Claesson, P. M.; Dedinaite, A.; Poptoshev, E. In Physical Chemistry of Polyelectrolytes, 1st ed.; Radeva, T., Ed.; Marcel Dekker: New York, 2001; p 882. (13) Piculell, L.; Lindman, B. Adv. Colloid Interface Sci. 1992, 41, 149. (14) Claesson, P. M.; Bergstro¨m, M.; Dedinaite, A.; Kjellin, M.; Legrand, J.; Grillo, I. J. Phys. Chem. B 2000, 104, 11689. (15) Cabane, B.; Duplessix, R. J. Phys. 1982, 43, 1529. (16) Cabane, B.; Duplessix, R. Colloids Surf. 1985, 13, 19. (17) Ananthapadmanabhan, K. P. In Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC: Boca Raton, FL, 1993. (18) Varma, B. K.; Demers, A.; Jamieson, A. M.; Blackwell, J.; Jentoft, N. Biopolymers 1990, 29, 1990. (19) Griffiths, P. C.; Fallis, I. A.; Teerapornchaisit, P.; Grillo, I. Langmuir 2001, 17, 2594. (20) Sedla´k, M. Langmuir 1999, 15, 4045. (21) Fo¨rster, S.; Schmidt, M. Adv. Polym. Sci. 1995, 120, 51.
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Materials and Methods Samples and Sample Preparation. The BSM preparation and the SDS were purchased from Sigma (catalog number M3895 and L4509, respectively). The BSM, specified by the manufacturer to contain 12% of sialic acid groups, was used as received. The SDS was purified by recrystallization from Milli Q water,22 repeating the procedure twice. Octyl and decylsodium sulfate (purchased from Merck) were used as received. Pro-analysis grade NaCl was obtained from Merck. A stock mucin solution at relatively high concentration was prepared in 30 mM NaCl approximately 12 h prior to experiments. It was diluted to the desired concentration with 30 mM NaCl just prior to use. After the study of the freshly diluted sample, the solutions were allowed to stand at room temperature for a prolonged time and reanalyzed at a later stage. Next, the effects of the SDS on the mucin solution were studied at different SDS concentrations, where all the solutions contained 25 ppm of mucin and 30 mM NaCl. The measurements were performed on freshly prepared solutions and after 3, 13, and 48 h of storage at room temperature. To learn if the ionic strength has any effect on the mucin-SDS solutions, some measurements were done at other NaCl concentrations (1 and 100 mM). The effect of the hydrophobic interaction between mucin and surfactants was investigated by adding sodium alkyl sulfates with other chain lengths to the mucin solution as described above. For this purpose, we used sodium octyl sulfate in one set of experiments and sodium decyl sulfate in another set, both in 30 mM NaCl solution. Dynamic Light Scattering. The dynamic light scattering instrument has been described previously; see for example ref 23. The data were collected using an ALV-5000 digital multiple τ correlator with 288 exponentially spaced channels. The measured intensity autocorrelation function is given by
g(2)(t) ) B(1 + β|g(1)(t)|2)
(1)
where β is a nonideality factor and B is a baseline term. g(1)(t) can be written as the inverse Laplace transform (ILT) of the distribution of relaxation times, τ:
g(1)(t) )
∫
∞
0
A(τ) exp(-t/τ) dτ
(2)
A constrained regularization of the REPES routine was used to perform the ILT analysis. This routine is essentially similar to CONTIN, except that REPES directly minimizes the sum of the squared differences between the experimental and the calculated intensity-intensity autocorrelation function g(2)(t) using a nonlinear programming and allows the selection of the smoothing parameter. The relaxation time distributions were represented in the form of a τ A(τ) versus log τ plots, with τ A(τ) in arbitrary units. This provides an equal area representation. The mean diffusion coefficient (D) was calculated from the second moments of the peaks as D ) Γ/q2, where q ) (4πnD/λ) sin θ/2 is the magnitude of the scattering vector and Γ ) 1/τ is the relaxation rate. Here, θ is the scattering angle, nD is the refractive index of pure solvent, and λ is the wavelength of the incident light. The Stokes-Einstein equation relates the infinite dilution diffusion coefficient, D0, to the hydrodynamic radius (Rh):
D0 )
kT 6πη Rh
(3)
where kT is the thermal energy factor and η is the temperaturedependent viscosity of the solvent. The most reliably determined values for the radii presented in Figures 5-8 are those at high surfactant/NaCl concentrations where the solutions do not contain very large aggregates. Static Light Scattering. Static light scattering measurements were made using a Hamamatsu photon-counting device with a 3 mW He-Ne laser. Toluene was used as the reference (22) Fielden, M. L.; Claesson, P. M. J. Colloid Interface Sci. 1998, 198, 261. (23) Shille´n, K.; Brown, W.; Jonhsen, R. M. Macromolecules 1994, 27, 4825.
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Figure 2. Surface tension isotherm for SDS at different NaCl concentrations, obtained using the Wilhelmy plate method: (a) SDS in NaCl solution (1 mM), (b) SDS in NaCl solution (30 mM), and (c) SDS in NaCl (100 mM).
Figure 3. Surface tension isotherm for sodium decyl sulfate in aqueous 30 mM NaCl solution, obtained using the Wilhelmy plate method.
(Rayleigh ratio, Rtoluene ) 13.59 × 10-4 m-1 at 633 nm). The optical constant is
K ) 4π2n2(dn/dc)2/NAλ4
(4)
with (dn/dc) being the refractive index increment equal to 0.165 mL/g.24 NA is Avogadro,s constant, and λ is the wavelength. R90 is the Rayleigh ratio at angle 90° determined using [(I - I0)/ Itol]Rtol(ns/ntol)2. Here, ns is the solvent refractive index and ntol is that of toluene. I is the measured intensity of the solution, I0 is that of the solvent, and Itol is that of toluene. For molecular weight (Mw) determinations, the reduced scattered intensity (Kc/ R90) was plotted versus concentration and Mw was calculated from the intercept, 1/Mw. This procedure is valid for small particles, for example, the deaggregated mucin, with the product of scattering vector (q) and radius of gyration q Rg , 1, for which there is no angle dependence of the reduced scattered intensity. For large particles, for example, the mucin aggregates, with q Rg < 1 and where the reduced scattered intensity is angle dependent, the radius of gyration, Rg, could be estimated from the ratio of slope to intercept of plots of 1/I sin θ versus sin2(θ/2) in the range where there is a negligible concentration dependence of (Kc/Rθ), where Rθ is the Rayleigh ratio at different angles θ. Surface Tension Measurements. The surface tension of surfactant solutions in the absence of mucin was measured with a Kru¨ss K12 tensiometer, employing the Wilhelmy plate method. The measurements were carried out using a sand-blasted platinum plate to ensure zero contact angle. The plate was cleaned using chromosulfuric acid prior to each isotherm measurement, rinsed with ethanol, and finally dried with nitrogen gas.
Results and Discussion Surface Tension Measurements. Prior to investigating the association between mucin and the alkyl sulfate surfactants, it was regarded as necessary to determine the critical micellar concentration, cmc, of the surfactants at the ionic strengths used in the experiments. This was done by performing surface tension measurements. The results obtained for the SDS solutions at various ionic strengths are shown in Figure 2. We note that the surface tension isotherms do not contain any minimum prior to the cmc, indicating a high purity of the sample. The cmc and the surface tension at the cmc, γcmc, decrease with increasing ionic strength due to the decreased entropy penalty of bringing the counterions close to the micellar surface and the surfactant-loaded air-water interface. The cmc values obtained at 1, 30, and 100 mM NaCl were 8.2, 3.3, and 1.5 mM, respectively. These values are in good agreement with previously reported results.25 (24) Carlstedt, I.; Sheehan, J. K. Biochem. J. 1984, 221, 499. (25) Gunnarsson, G.; Jo¨nsson, B.; Wennerstro¨m, H. J. Phys. Chem. 1980, 84, 3114.
Figure 4. Relaxation time distributions through inverse Laplace transformation of dynamic light scattering data at an angle of 90°. (a) Fresh mucin solution, 25 ppm. (b) Fresh mucin solution with SDS (2 mM). (c) Mucin solution with SDS (2 mM) after 48 h.
The surface tension isotherm for sodium decyl sulfate in 30 mM NaCl is shown in Figure 3. A small minimum in the surface tension isotherm is observed prior to cmc, indicating the presence of some surface-active contaminant, presumably decanol. We note that the cmc (19.3 mM) and the surface tension at cmc are higher for sodium decyl sulfate than for SDS, which is as expected considering the difference in chain length of the hydrophobic tails. Again, good agreement with the literature value was obtained.25 The cmc value for sodium octyl sulfate was taken from the literature (cmc ) 116 mM in 30 mM NaCl).25 Light Scattering: Mucin Solutions without Surfactant. The mucin solutions displayed a rather complex relaxation time distribution, as indicated in Figure 4. The main peak in the freshly prepared 25 ppm mucin solution has a maximum corresponding to a hydrodynamic radius of 720 nm, with a shoulder toward the fast relaxation time. The shoulder corresponds to a hydrodynamic radius of 160 nm. A further small peak at short relaxation times is observed in Figure 4, which seems likely to correspond to internal fluctuations in the macromolecular aggregate, and it will be ignored in the further discussion. The relaxation time distributions found at different mucin concentrations were rather similar (Table 1). We note that the intensity of scattered light is dominated by the larger species, but in fact the mass ratios of the small and large species are roughly the same (Table 1). The mass ratios were calculated by using the relation that the peak area
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Langmuir, Vol. 18, No. 10, 2002 3851
Table 1. Fresh Solutions of Mucina CMucin (ppm)
Rh1 (nm)
Rh2 (nm)
C1
C2
5 10 25 100
77 (0.08) 190 (0.18) 160 (0.17) 73 (0.08)
580 (0.92) 830 (0.82) 720 (0.83) 560 (0.92)
0.39 0.49 0.48 0.41
0.62 0.51 0.52 0.59
a All solutions contained NaCl (30 mM). The relative amounts of molecules (in intensity) for the different hydrodynamic radii are indicated in parentheses. The mass fractions of molecules for the same Rh are indicated as C1 and C2, respectively.
(A) is proportional to the product of concentration (c) and molecular weight (M), that is, 2
C1 A1M2 A1D1 ) ) C2 A2M1 A D 2 2
(5)
2
where D is the diffusion coefficient. The last equality arises from an assumption of a random coil conformation, supported by the data presented below. When mercaptoethanol was added to the pure mucin samples, only a slight reduction in the determined hydrodynamic radius was noted even after prolonged exposure at elevated temperature. However, we did not add any denaturing solvent such as 6 M guanidine hydrochloride, which in some cases has been reported to be needed in order to expose the disulfide bonds and make them accessible to reductive cleavage.4 By comparison of the Rh value, 720 nm, for freshly diluted 25 ppm mucin solutions (Table 1) with the Rh value, 500-600 nm, for aged solutions, it can be seen that the size of the mucin aggregates decreases slightly with time. The sample containing mercaptoethanol (1%) showed a similar small reduction in aggregate size with time, going from Rh ) 500 nm for the fresh solution to 410 nm after more than 48 h. Static light scattering was employed in an attempt to determine the radius of gyration and molecular weight of the large species. The radius of gyration for the aged solution was found to be 820 nm. However, due to a prohibiting large molecular weight, estimated to be above 100 × 106 g/mol, no precise value could be obtained for this quantity. Hence, it seems that the large species detected correspond not to intact mucin molecules but rather to large aggregates. These data are in agreement with previous works made with porcine submaxillary mucin (PSM), where it was reported that in salt solution PSM exists as large, polydisperse aggregates that slowly dissociate.26 The ratio of the radius of gyration and hydrodynamic radius was found to be close to the expected value for a random coil. Hence, this is the overall structure of the aggregates. The second virial coefficient was close to zero; that is, there was no significant interaction between the aggregates. Deaggregation of Mucin by SDS Addition. In the next set of experiments, we investigated the effect of SDS on the mucin solutions containing 30 mM NaCl. At low SDS concentrations, the surfactant had no significant effect on the mucin aggregates. However, as the concentration was increased (above 0.75 mM SDS ≈ 0.2 cmc), the aggregate size started to decrease significantly (fresh solution Rh values are shown in Figure 5 and Table 2). At high SDS concentration, the surfactant-induced change in the mucin aggregates was dramatic. In Figure 4, it can be seen how the relaxation time distribution for pure mucin that initially was monomodal changed into a bimodal curve (26) Shogren, R.; Jamieson, A. M.; Blackwell, J. Biopolymers 1983, 22, 1657.
Figure 5. Hydrodynamic radius of mucin/SDS aggregates. Fresh solutions. The data presented are for the peaks with the largest relative intensity (>50%). All solutions contained NaCl (30 mM). Table 2. Hydrodynamic Radius of Mucin-SDS Aggregatesa CSDS (mM)
Rh1 (nm)
Rh2 (nm)
C1
C2
0.50 0.75 0.85 1.00 2.00 4.00 7.50
120 (0.06) 44 (0.04)
640 (0.94) 560 (0.96) 540 (1.00) 250 (0.52) 280 (0.39) 380 (0.17) 240 (0.55)
0.26 0.35 0.00 0.86 0.91 0.96 0.80
0.74 0.65 1.00 0.14 0.09 0.04 0.20
37 (0.48) 44 (0.61) 77 (0.83) 51 (0.45)
a Fresh solutions. All solutions contained NaCl (30 mM) and 25 ppm of mucin. The relative amounts of molecules (in intensity) for the different hydrodynamic radii are indicated in parentheses. The mass fractions of molecules for the same Rh are indicated as C1 and C2, respectively.
Table 3. Hydrodynamic and Gyration Radii of Mucin and Mucin-SDSa SDS concn (mM)
Rh (nm)
Rg (nm)
Rg/Rh
0.0 2.0 4.0
500 44 44
820 79 80
1.6 1.8 1.8
a All samples contained 25 ppm of mucin and 30 mM NaCl. The samples were allowed to stand at room temperature for more than 48 h.
when 2 mM SDS was present and then, eventually, into a new monomodal distribution curve corresponding to smaller species. In some cases, a weak peak was noted (see e.g. Figure 4) corresponding to small species. That feature was not reproducible and will not be discussed further. Static light scattering measurements were employed to estimate the molecular weight of the smaller species, and a value of 7 × 106 g/mol was obtained. This is within the range normally reported for mucins. Clearly, the presence of SDS results in a breakdown of the mucin aggregates into individual molecules. However, since mucin molecules easily are broken down during preparation and purification3 we do not dare to state that this value corresponds to intact mucin molecules; rather, it should be viewed as a lower limit. The dramatic change in the size of the species present in solution demonstrates that SDS associates with the mucin molecules and that the physical bonds linking the aggregates together are broken. The changes in aggregate size are, of course, also reflected in the measured radius of gyration obtained by static light scattering. The Rg and Rh values for aged solutions are provided in Table 3. The Rg/Rh ratio is in the range of 1.6-1.8 for all solutions, which is typical for
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Figure 6. The mucin/SDS aggregate size as a function of time. All solutions contained NaCl (30 mM). (a) Mucin solution with SDS (0.20 mM). (b) Mucin solution with SDS (0.50 mM). (c) Mucin solution with SDS (0.75 mM). (d) Mucin solution with SDS (1.00 mM). (e) Mucin solution with SDS (2.00 mM).
Figure 7. Hydrodynamic radius for mucin/sodium alkyl sulfates aggregates. Aged solutions (48 h). All of the solutions contained mucin (25 ppm) and NaCl (30 mM). (a) Octylsodium sulfate. (b) Decylsodium sulfate. (c) Dodecylsodium sulfate.
random coil conformations.27 Hence, both the mucin aggregates and the dissolved mucin decorated with associated SDS have this overall conformation. The second virial coefficient remains close to zero also for the mucin molecules associated with SDS. The changes in the aggregate size with SDS concentration and time are summarized in Figure 6 and Table 4. Clearly, an SDS concentration of less than 0.5 mM has a very limited effect even after 48 h. At an SDS concentration of 0.75 mM, the deaggregation time is long, over 13 h. At higher SDS concentrations, the deaggregation time is significantly shorter. The hydrodynamic radius of the mucin subunits with associated SDS obtained after deaggregation is in the range of 40-90 nm. We note that the process of association between SDS molecules and the mucin is expected to be relatively fast. Thus, the slow change in size of the mucin aggregates gives strong support to the hypothesis that the time-limiting step in the deaggregation process is related to the polymer chain relaxation rate, that is, how fast the polymer may disentangle from the other large polymers present in the aggregate.
With these results in mind, we may propose a mechanism for the breakdown of mucin aggregates by SDS addition. The first crucial observation is that we have a rather clear critical SDS concentration (0.75 mM ≈ 0.2 cmc) above which deaggregation occurs. A lower SDS concentration has no effect, and at higher SDS concentrations the deaggregation kinetics is rather fast. These results indicate that the amount of SDS associated with the mucin molecules increases rapidly at the critical SDS concentration, which is indicative of a cooperative association driven by hydrophobic interactions. Both SDS and the glycan side chains that cover the protein core are negatively charged. Hence, electrostatic interactions counteract surfactant binding to the sugar-rich domains. In view of this, it seems likely that the association between mucin and SDS occurs predominantly at the carbohydratedepleted regions and that these regions serve as nucleation sites for the self-association of SDS. Further, the data suggest that it is these regions that are involved in the association process between individual mucin molecules that occurs in the absence of SDS. Addition of mercaptoethanol to the mucin solution deaggregated with SDS did not result in any further reduction in hydrodynamic radius. Thus, disulfide bonds, if present, remain inaccessible to reductive cleavage. Effects of Surfactant Chain Length. Some experiments with sodium alkyl sulfates, having shorter alkyl chains than SDS, were conducted in order to test the hypothesis that hydrophobic interactions drive the association between mucin and these anionic surfactants. The data presented in Figure 7 show that the C12 and the C10 alkyl sulfates effectively dissolve the mucin aggregates, whereas the C8 alkyl sulfate is less effective, even when normalizing the concentration with the cmc of the surfactant. Hence, the alkyl chain has to be sufficiently long to provide the necessary driving force for the association. We note that a lower concentration of SDS, compared to sodium decyl sulfate, is needed in order to induce the deaggregation of the large mucin aggregates. However, on normalizing the surfactant concentration with the cmc, one finds that deaggregation occurs at almost the same fraction of the cmc (about 0.2 cmc) (Table 5). This is a slightly lower value as compared to the critical association concentration, cac, between SDS and poly(ethylene oxide), about 0.5 cmc,15,28 but significantly higher than that between SDS and cationic polyelectrolytes.12 Taking 0.2 cmc as the cac of the surfactant with mucin allows us to estimate the free energy difference for a surfactant unimer
(27) Brown, W. Light Scattering: Principles and Development; Clarendon Press/Oxford Science Publications: Oxford, 1996.
(28) Brown, W.; Fundin, J.; Maria da Grac¸ a, M. Macromolecules 1992, 25, 7192.
Table 4. The Mucin/SDS Aggregate Sizes as a Function of Timea SDS concn (mM)
time (h)
0.20
0 13 48 0 13 48 0 3 13 48 0 3 13 48 0 3 13 48
0.50 0.75
1.00
2.00
Rh1 (nm)
Rh2 (nm)
C1
C2
66 (0.04) 130 (0.13) 66 (0.10) 120 (0.06) 140 (0.13)
740 (0.96) 780 (0.87) 650 (0.90) 640 (0.94) 660 (0.87) 183 (1.00) 560 (0.96) 750 (0.66) 660 (0.73)
0.34 0.48 0.51 0.26 0.42 0.00 0.35 0.83 0.73 1.00 0.86 0.86 0.81 1.00 0.91 1.00 1.00 1.00
0.66 0.52 0.49 0.74 0.58 1.00 0.65 0.17 0.27 0.00 0.14 0.14 0.19 0.00 0.09 0.00 0.00 0.00
44 (0.04) 82 (0.34) 90 (0.27) 83 (1.00) 37 (0.48) 70 (0.38) 77 (0.37) 79 (1.00) 44 (0.61) 98 (1.00) 70 (1.00) 44 (1.00)
250 (0.52) 700 (0.62) 570 (0.63) 280 (0.39)
a All solutions contained NaCl (30 mM). The relative amounts of molecules (in intensity) for the different hydrodynamic radii are indicated in parentheses. The mass fractions of molecules for the same Rh are indicated as C1 and C2, respectively.
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Table 5. Hydrodynamic Radius for Mucin/Sodium Alkyl Sulfate Aggregatesa surfactant
Xcmc
Rh (nm)
surfactant
Xcmc
Rh (nm)
C8 C8 C8 C8 C10 C10 C10 C10 C10
0.19 0.39 0.75 0.89 0.07 0.25 0.43 0.80 0.98
655 565 69 54 388 150 140 91 93
C10 C10 C12 C12 C12 C12 C12 C12
1.16 1.34 0.10 0.14 0.20 0.27 0.54 1.08
52 50 332 183 83 79 90 44
a The samples were allowed to stand at room temperature for 48 h. All solutions contained mucin (25 ppm) and NaCl (30 mM).
Figure 8. Hydrodynamic radius for mucin/SDS aggregates for different salt concentrations. Aged solutions (48 h).
in a micelle associated with the mucin and in a free micelle as
cac (cmc )
0 0 µmic,mucin - µmic,free ) kT Ln
(6)
The value obtained is about -1.6kT. For the association between mucin and sodium octyl sulfate that commences at about 0.5 cmc, the corresponding value is about -0.7kT. Effects of Ionic Strength. The electrostatic repulsion between charge groups can be screened by salt addition. A consequence of this is that the size of the aggregates formed by mucin in the absence of surfactant decreases with increasing salt concentration; see Figure 8. Further, the repulsion between SDS molecules and the glycan side chains of the mucin is also reduced with increasing salt concentration. Hence, the hydrophobic interaction is expected to become more dominant at higher salt concentrations. This hypothesis is supported by experiments that show that the SDS concentration needed to dissolve the mucin aggregates decreases with increasing ionic strength. However, when the surfactant concentration is
scaled with the cmc of the surfactant at the different ionic strengths, the effect is rather minor, as demonstrated by the data displayed in Figure 8, even though for the solutions containing 1 mM NaCl the change in the aggregate size seems to be more gradual than at higher ionic strengths. Hence, the addition of salt decreases the cmc of SDS and the cac between SDS and mucin to a similar extent. It is also clear from the data shown in Figure 8 that the ionic strength of the solution does not significantly affect the size of the deaggregated mucin subunits associated with SDS at high enough surfactant concentration. Conclusions Bovine submaxillary mucin molecules dissolved in surfactant-free 30 mM NaCl contain species with sizes above 500 nm. The addition of mercaptoethanol does not dissolve the aggregates, indicating that the disulfide bonds are not accessible to reductive cleavage in nondenaturating solvents. The large units observed consist of mucin molecules held together by physical bonds. The addition of SDS to these samples results in deaggregation of the large mucin aggregates when the surfactant concentration exceeds about 0.2 cmc. Hence, the negatively charged mucin and the anionic surfactant do associate and this association affects the aggregation state of the mucin. A similar association between mucin and sodium decyl sulfate occurs, and again deaggregation occurs once the surfactant concentration has reached about 0.2 cmc. On the other hand, a smaller effect is observed when sodium octyl sulfate is added to the mucin solution, where deaggregation occurs at concentrations above about 0.5 cmc, indicating that the surfactant chain length influences the association with the mucin. The results demonstrate that the association is primarily driven by hydrophobic interactions. We suggest that it occurs predominantly on the naked protein regions and to a lesser extent on the glycosylated and negatively charged regions. The kinetics of the deaggregation process depends on surfactant concentrations but is generally slow. This suggests that the deaggregation kinetics is controlled by the polymer chain dynamics. The ionic strength of the solution affects the association between mucin and SDS in a manner similar to its effect on the cmc. Acknowledgment. This work was sponsored by the Center for Surfactants Based on Natural Products (SNAP), which is one of the competence centers of VINNOVA (The Swedish Agency for Innovation Systems). The authors also thank Dr. Thomas Arnebrant from the Institute for Surface Chemistry for valuable discussions during this investigation. LA015717U
