1274
Langmuir 1994,10, 1274-1280
Forces between Proteoheparan Sulfate Layers Adsorbed at Hydrophobic Surfaces Martin Malmsten,’??Per Claesson,fJ and Gunter Siege15 Institute for Surface Chemistry, P.O. Box 5607, S-114 86 Stockholm, Sweden, Laboratory for Chemical Surface Science, Department of Chemistry, The Royal Institute of Technology, S-100 44 Stockholm, Sweden, and Biophysical Research Group, Institute of Physiology, Free University of Berlin, 0-1000 Berlin 33, Germany Received August 31,1993. I n Final Form: January 18, 1994” The forces between proteoheparan sulfate layers adsorbed at hydrophobic surfaces were investigated by direct surface force measurements. In 0.2 mg/mL proteoheparan sulfate and 0.1 mM NaCl the forces were monotonically repulsive, with one distant regime having a decay length of about 180 A, and one steeply repulsive proximal part at distances smaller than about 100 A. Only small effects were observed on dilution with 0.1 mM NaC1, indicating “irreversible”adsorption. The decay length of the distant force component was 280 A after dilution, whereas the proximal repulsive component remained unchanged. Furthermore, after dilution, a weak adhesion (FIR = 200 rNlm) was observed. On addition of CaCl2, the decay length of the distant repulsion decreased from 280 A in 0.1 mM NaCl to 86 and 77 A in 1.25 and 2.5 mM CaC12, respectively. The decay lengths in CaCl2 solutions are significantly larger than the expected Debye lengths, demonstrating the predominance of steric forces. Furthermore, on addition of CaCl2 the magnitude of the adhesion increased from 200 to loo0 rNlm, and the proximal repulsion was observed at slightly smaller distances. It was found that both steric and electrostatic forces contribute to the interactions between proteoheparan sulfate layers.
Introduction Proteoglycans are anionic biomacromoleculesconsisting of highly carboxylated and sulfated glycosaminoglycan chains covalently attached to a protein core (Figure 1).ll2 As such, they are strong polyelectrolytes with a high linear charge density (typically corresponding to about -120 mV in pure ~ a t e r ) .The ~ heparan sulfate proteoglycan is incorporated into endothelial cell membranes via their protein core, while the glycosaminoglycanchains protrude into the extracellular solution. Among other things, proteoglycanshave been suggested to control the contractioddilatation of smooth muscle tissue. In particular, the heparan sulfate proteoglycan has been suggested to act as a sensor for blood flow, where the signal transduction to the cell interior is likely to involve changes in the pattern of cation binding to the biomacromolecule.112 Proteoheparan sulfate is also an important component in basement membranes. The proteoheparan sulfate, which is primarily located adjacent to cell surfaces, to a large extent determines the charge properties of the basement membrane.4 These molecules are thus important for the interactions between the basement membrane and adjacent cells. In particular, proteoheparan sulfate and related molecules have been reported to interact with specific sites on some adhesion molecules like laminin and fibronectin.5 In a previous investigation,we examined the adsorption of heparan sulfate and proteoheparan sulfate at hydro+ Institute for Surface Chemistry. 8 The Royal Institute of Technology.
I Free University of Berlin.
Abstract published in Advance ACS Abstracts, March 1, 1994. (1)Siegel, G.;Walter, A.; Bostanjoglo, M.; Jans, A. W. H.; Kinne, R.; Piculell, L.; Lindman, B. J. Membr. Sci. 1989,41, 353. (2) Siegel, G.; Walter, A.; Rockborn, K.; Buddecke, E.; Schmidt, A.; Gustavsson, H.; Lindman, B. Polym. J. 1991,23, 697. (3)Malmsten, M.;Siegel,G.; Buddecke, E.; Schmidt, A. Colloids Surf. B Biointerfaces 1993, 1, 43. (4)Johnson, L. D. In Structure and Properties of Cell Membranes; Benga, G., Ed.; CRC Press: Boca Raton, FL, 1985. (5)Klebe,R. J.;Bentley,K. L.;Hanson,D.P. InProteinsatInterfacesPhysiochemical and Biochemical Studies; Brash, J. L., Horbett, T. A., Eds.; ACS Symposium Series No. 343; American Chemical Society: Washington DC, 1987.
0743-7463/94/2410-1274$04.50/0
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Proteid Linkage region Peptide Figure 1. Schematic structure of
Polysaccharide chain
proteoheparan sulfate. The N-sulfateand 0-sulfateester groupsof the heparan sulfate chains are arranged in highly sulfated domains along the molecule, separated by sulfate-poor and sulfate-free sequences. The iduronic acid content of heparan sulfate constitutes about 30% of that of total hexuronic acid. philic and hydrophobized silica surface^.^ It was found that proteoheparan sulfate adsorbs at hydrophobic silica surfaces, the adsorbed amount increasing strongly on addition of CaC12. Heparan sulfate, on the other hand, does not adsorb at either hydrophobic or hydrophilic silica surfaces, even in the presence of excess salt. Thus, the adsorption of proteoheparan sulfate at hydrophobic surfaces occursvia ita protein moiety. The effects of excess salt are complex, and electrostatic considerations alone do not provide a satisfactory explanation of the experimental observations. The adsorption and desorption processes of proteoheparan sulfate are slow, and the desorption on dilution is far from complete (Figure 2). In another study, we investigated the forces between mucin layers adsorbed at hydrophobic surfaces.6 The forces between mucin (rat gastric mucin) coated surfaces are purely repulsive at equilibrium, extending to separations of about 2000 A. The layers could be squeezed into a thickness of only 100-200 A by applying a high compressive force. Hence, considering ita large radius of (6) Malmsten, M.; Blomberg, E.; Claesson,P.; Carlstedt, I.; Ljusegren, I. J. Colloid Interface Sci. 1992, 151, 579.
0 1994 American Chemical Society
Langmuir, Vol. 10, No. 4,1994 1276
Forces between Proteoheparan Sulfate Layers
4 rL
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Time (s) Figure 2. Amount of proteoheparan sulfate adsorbed at hydrophobized silica as a function of time (crossed circles). At zerotime, proteoheparan sulfate (0.1mg/mL in water) wasadded. At point a, 1.25mM CaClz was added, whereasat point b, rinsing with water (200 mL over 10min) was initiated. Illustrated also is the (null) adsorption of heparan sulfateat hydrophobizedsilica (open circles). Data are taken from ref 3. gyration, about 2000 A, rat gastric mucin adsorbs in a “flat” conformation a t hydrophobic surfaces. The interaction forces were independent of the excess electrolyte concentration. Thus, in this case the repulsion is of steric, rather than electrostatic,origin. Rat and pig gastric mucins do not behave the same, and in particular, an attraction and more pronounced relaxation effects were found for the latter, more highly charged, mucin. This points to the difficulty in comparing results obtained with different mucins and emphasizes the importance of thoroughly specifying the mucins studied. It also indicates that nonequilibrium forces should be taken into account when considering interactions between mucus layers and between mucus and foreign substances,and that these effects could be significant for the barrier properties of mucus. From these previous studies, it is clear that there is a need for studies of the conformation of proteoheparan sulfate adsorbed at hydrophobic surfaces and, in particular, the effects of excess salt on the conformation of the adsorbed proteoheparan sulfate. It is also feasible that the surface force technique might be quite successful in providing information on the detailed conformational effects in proteoheparan sulfate systems.
Experimental Section Materials. Water was first purified by a Milli-RO lOPLUS pretreatment unit, includingdepthfiltration,carbon adsorption, and decalcination preceding reverse osmosis. Subsequently, it was led through a Milli-Q PLUS185 unit, which treats the feed water with UV light (185 and 254 nm) before leading it into a Q-PAK unit consisting of an active carbon unit followed by a mixed bed ion exchanger,an Organexcartridge, and a final 0.22pm Millipak 40 filter. Before use, the water was degassed under vacuum. Proteoheparan sulfate (pure Na+ salt, average disaccharide molecularweight520 Da) was prepared as described previously.’ This biomolecule has an average molecular weight of 175 kDa and is a highly negatively charged macromolecule. Ninety-five percent of all molecules lie within the range 165-185 kDa. It contains a protein core (M, = 38 kDa) to which a few heparan sulfate side chains (M, = 35 kDa) are covalently linked (Figure 1).The heparan sulfateside chains, which may be obtained from proteoheparan sulfate by exhaustive proteolytic digestion or by a &elimination reaction, consist of repeating uronic acid (1-4) glucosamine disaccharides, but exhibit a broad chemical and (7) Schmidt,A.; Schaer, E.;Buddecke, E. Eur. J.Biochem. 1988,173, 661.
configurationalvariability with respect to the ratio of glucuronic acid/iduronic acid, the number and position of 0-sulfate ester groups, and the ratio of N-sulfatelo-sulfate, One disaccharide unit contains 1 carboxyl and 0.5 sulfate groups on average. SurfacePreparations. All surfacepreparations were carried out in laminary flowcabinets. The deposition of the hydrophobic monolayerwas performed with a computerized Langmuir trough system (KSV Chemicals, Helsinki). The mica surfaces were immersed in a water-fiied all-Teflon trough. Recrystallized dimethyldioctadecylamonium bromide (DDOA;Eastman) was dissolved in a mixture of ethanol and hexane (15 mg of DDOA in 5 mL of ethanol and 95 mL of hexane). This solution was added dropwiseonto the air/water interface. After allowingthe solution to evaporate, the monolayerwas compressedto a surface pressure of 25 mN/m, corresponding to an area per molecule of about 65 &. The mica surfaces were then pulled out of the solution at a speed of 5 mm/min (surfaces tilted 45O) while the surface pressure was kept constant. After deposition (transfer ratio 1.3;detailsregarding the high transfer ratio, as well as other aspects of this film, have been discussed in detail previouslF1O), the surfaces were immediately mounted in the surface force apparatus. The advancingand recedingcontact angleon DDOAcoated mica have previously been determined to be about 9396O and 6O0, respecti~ely.~*~ Despite this rather large contact angle hysteresis, these surfaces have been found to be stable and reproducible in a large number of experiments. Surface Force Measurements. The forces between proteoheparan sulfate layerswere investigatedwith a Mark I1surface forceapparatus.” Two thin sheets of mica (silveredon one side) were glued with an epoxy resin, Epon 1004 (silvered side down), onto cylindricalsilicadisks. Thesurfaceswere made hydrophobic by a Langmuir-Blodgett deposition as described above. Directly after the deposition,the disks were mounted in a crossed-cylinder geometry inside the surface force apparatus. The dietance between the surfaces was determined interferometrically, with an accuracy of about 0.2 nm, by using fringes of equal chromatic order. Forces down to 10-7 N were determined by the deflection of a spring supporting one of the surfaces. The force measured betweencrossed cylinders (F,) normalized by the localgeometric mean radius ( R ) is related to the free energy of interaction per unit area between flat surfaces (Gf) according t o I 2
provided that the radius of the cylinders (about 2 cm) is much larger than the surface separation ( D ) . This is the case in our experimentalsetup. Another requirementthat has to be fulfiied in order to apply eq 1 is that the surfaces do not deform when pushed together.I3 The surfaceforceexperimentswere performed in the following way: First, the contact position of the hydrophobized surfaces was determined in air, defining the distance of zero separation between the surfaces (Le., the thicknesses stated are due to the proteoglycanlayer). This procedure does not allow a determination of the thickness of the Langmuir-Blodgett f i i . However, these have been found to be reliable in numerous previous experiments. Thereafter, a droplet of the proteoheparan sulfate solution (about 0.2 mg/mL in 0.1 mM NaCl) was placed between the surfaces. (Due to the proteoglycan adsorption, a complete wetting of the surfaces was observed.) Consecutively,the forces were determined at adsorption equilibrium. (Adsorption was performed from 0.1 mM NaCl rather than from pure water in order to achieve a reasonably high adsorbed amount (cf. Figure 2).8 Furthermore, assuming 1.5 charges per disaccharide, the Na+concentration due to the proteoheparan sulfate is about 0.5 mM, Le., [Na+]tow. = 0.6 mM. Moreover, the prohglycan concentration was chosen such as to correspond to plateau (8) Herder, P. C.; Claesson, P. M.; Herder, C. E. J. Colloid Interface Sci. 1987, 119, 155. (9) Claeseon,P. M.; Christeneon, H. K. J. Phye. Chem. 1988,92,1660. (10) Claeeson,P.;Carmona-Ribeiiro,A. M.; Kurihara,K. J.Phye. Chem. 1989, 93,917. (11) Israelachvili, J. N.: Adams, G. E. J. Chem. Soc., Faraday Tram. 1 1978, 74, 975. (12) Derjaguin, B. V. Kolloid-Z. 1934, 69, 155. (13) Parker, J. L.; Attard, P. J. Phys. Chem. 1992,96,10398.
Malmsten et al.
1276 Langmuir, Vol. 10, No. 4,1994 10000
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DISTANCE (A) Figure 3. Forces normalized with the local radius as a function of separation after adsorption from 0.2 mg/mL proteoheparan sulfatein 0.1mM NaCl. Filled and open symbolsrepresent forces measured on approach and separation, respectively. Two different force runs are shown in order to illustrate the degree of
reproducibility.
adsorption, and to be low enough to avoid viscous forces.) Subsequently,the measuring chamber was filled with a 0.1 mM NaCl solution,resultingin a dilution of the proteoheparansulfate solution by roughly a factor of 3000, effectively “removing” nonadsorbedproteoheparan sulfate molecules,whereasadsorbed molecules remained at the interface to a large extent (Figure 2). After equilibration, the forces were measured at this salt concentration. Finally,the forceswere determinedafter addition of 1.25 and 2.5 mM CaC12. The temperature was 20 O C , while the pH was 5.6-5.8 throughout the surface force measurements. Results a n d Discussion Proteoglycans are biological amphiphilic macromolecules consisting of a hydrophobic polypeptide backbone and highly charged polysaccharide (glycosaminoglycan) side chains. While the polypeptide moiety is anchored in the hydrophobic membrane interior, the polysaccharide chains protrude into the cell exterior.’p2 Sincethe modeling of such a membrane-bound system in a biological sense is extremely difficult, we have chosen to develop simpler model systems. From previous ellipsometry work, we found that hydrophobic surfaces provide a good anchoring of the polypeptide backbone, whereas the glycosaminoglycan chains are nonadsorbing (Figure 2).3 Therefore, the use of hydrophobic surfaces allows a firm anchoring and a proper orientation, thus fulfilling two vital requirements of a model of the biological system. We therefore chose to work with a similar system in the present investigation, aiming at elucidating the interactions between proteoheparan sulfate layers. As can be seen in Figure 3,the forces between proteoheparan sulfate layers adsorbed from a0.2 mg/mL solution in 0.1 mM NaCl are monotonically repulsive on compression. (Note that the hydrophobic surfaces provided good anchoring of the proteoheparan sulfate macromolecules, as discussed previously, and that no dynamic effects were observed.) The repulsion consists of a distant regime,with a decay length of about 180 A, and a proximal “hard wall” regime at distances of separation smaller than about 100 A. In comparison, the Debye length of the electrostatic double-layer interaction at this electrolyte concentration is less than 130-190 A, and depends, e.g., on the degree
of protonation of the heparan sulfate carboxyl g r 0 ~ p s . l ~ Assuming a purely electrostatic interaction, and that nonadsorbed polyelectrolytes are expelled from the region between the interacting surfaces (as has been shown to occur for both polyelectr01yte~~J~ and micellar17J8systems), and fitting the force curve to nonlinear Poisson-Bolt” (PB) theory, gives an apparent interfacial potential of -90 mV, with the plane of charge located at the hard wall separation. We note in passing that this is comparable to the electrostaticpotential of the glycosaminoglycan chains obtained from model calculations with the (cylindrical) Poisson-Boltzmann cell model (-120 mV in a salt-free s ~ l u t i o n ) . ~Nevertheless, we refer to the interfacial potential as an apparent value due to the neglect of, e.g., ion-ion correlation effects in the nonlinear P B modells21 and to the difficulties associated with an exact determination of the plane of charge for noninteracting surfaces. However, presumably, the repulsion is of both electrostatic and steric origin. At about 100 A, a significant deviation from the exponentially repulsive behavior is observed, and at 90 A, a hard wall is encountered. Presumably, this is due to a pronounced overlap of the proteoglycanmoieties, involving close promixity between polypeptide domains at the two surfaces. This is further supported by the finding of an adhesion on separation from this close contact. It is interesting to note that the adhesion strength increased somewhat with increasing time at close contact, indicating that slow structural rearrangements occur within the adsorbed layer at close contact. In contrast, the forces were reversible at distances of separation larger than about 150 A (cf. Figure 8). The finding that we were able to reach such short distances of separation is somewhat unexpected, since one would expect the glycosaminoglycan chains to be excerting a strong electrostericrepulsion. The present resultstherefore seem to indicate that the adsorbed amount is rather low, which is consistent with previous ellipsometry results (Figure Hence, due to the low surface coverage, the heparan sulfate side chains in the interaction region can adopt an orientation more or less parallel to the surface in order to minimize the unfavorable electrosteric repulsion. We note in passing that for the same reason charged gangliosides in a deposited phospholipid monolayer diffuse away from the contact zone when two surfaces are brought close together.22 On dilution with 0.1mM NaCl by a factor of 3000,there were only minor changes in the force curves (Figures 4 and 5 ) . The major effect is that the decay length of the distant part of the interaction curve becomes larger. Hence, before and after dilution, the decay lengths are 180 and 280 A, respectively. This finding is consistent with a slightly lower ionic strength (the proteoheparan sulfate concentration between the surfaces is strongly reduced). In fact, the decay length after dilution agrees closely with that of a simple double-layer interaction (K-1 = 300 A) a t this ionic strength. After dilution, the adhesion reaches 200 pN/m, and the surfaces jump apart at a separation of about 130 A. The (14) Hiemenz, P. C. Principles of Colloid and Surface Chemistry; Marcel Dekker: New York, 1986. (15) Claeseon, P. M.; Ninham, B. W. Longmuir 1992,8,1408. (16) Berg, J. M.; Neuman, R. D.; Claesson, P. M. J. Colloid Interface Sci., in preb. (17) Pashley, R. M.; Ninham, B. W. J. Phys. Chem. 1987,91, 2902. (18)Marra, J.; Hair, M. L. J. Colloid Interface Sci. 1989, 128, 511. (19) Guldbrand, L.; Jbwon, B.; Wennerstrbm.. H.;. Lime, P.J. Chem. Phys. 1984,80, 2021. (20) Kjellander, R.; Marcelja, S. J. Phys. Chem. 1986,90,1230. (21) Attard, P.; Mitchell, J.; Ninham, B. W. J. Chem. Phys. 1988,89, A95R
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(22) Parker, J. L. J. Colloid Interface Sci. 1990,137, 571.
Langmuir, Vol. 10, No. 4, 1994 1277
Forces between Proteoheparan Sulfate Layers 10000
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of separation after adsorption from 0.2 mg/mL proteoheparan sulfate in 0.1 mM NaC1, followed by dilution by a factor of 3000 with 0.1 mM NaC1. Filled and open symbols represent forces measured on approach and separation, respectively. T w o different force runs are shown in order to illustrate the degree of reproducibility. Theforcesare illustrated on (a,top) alogarithmic scale and (b, bottom) a linear scale. attractive force is weaker than the van der Waals force present between nonpolymer-coated colloidal particles in the primary minimum. However, it is stronger than the van der. Waals force originating from the bare mica surfaces. At the distance of the minimum (about 100 A), the latter force contribution is only 30-40 pN/m, whereas the magnitude of the attraction measured in the present system is 200-1000 mN/m. Clearly, there is an attractive force component between the adsorbed proteoheparan sulfate layers. The attraction certainly has a contribution from van der Waals interactions between the adsorbed macromolecules, but hydrophobic and contact forces, including hydrogen bonding and ion bridges, also contribute strongly to the measured adhesion force. The fact that we observe a more pronounced adhesion after dilution is probablydue toa reduction in the adsorbed amount (Figure 2). Even though the range of the doublelayer force increases on dilution (due to a reduced ionic strength), the magnitude of the apparent interfacial potential decreases somewhat from -90 to -80 mV. This
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of separation after adsorption from 0.2 mg/mL proteoheparan sulfate in 0.1 mM NaC1. Filled and open symbolsrepresent the forces measured on approach after and before dilution of the proteoheparan sulfate solution with 0.1 mM NaCl by a factor of 3000, respectively. T w o force runs are shown for each case in order to illus'trate the degree of reproducibility. The forces are illustrated on (a, top) a logarithmic scale and (b, bottom) a linear scale. results in areduction of the electrostatic double-layer force at short separations. The reduction is slightly less than 1000 pN/m (from about 2000 to about 1000 pN/m) just outaide the hard wall (Figure 5), which is close to the change in depth of the force minimum observed on separation (cf. Figures 3 and 4). Hence, the increase in adhesion can largely be explained as resulting from a lowering of the double-layer force. However, a reduction of the adsorbed amount is also expected to facilitate close proximity between, e.g., the polypeptide moieties at the different surfaces, which, in turn, could increase the adhesion as commonly observed for polymers.2s On addition of CaC12, the range of the distant regime interaction is reduced (Figures 6,7, and 9). Hence, at 1.25 and 2.5 mM CaC12, the decay lengths are 86 and 77 A, respectively. At these electrolyte concentrations, the (23) Napper, D. H. Polymeric Stabilization of Colloidal Dieperaions; Academic Press: London, 1983.
Malmsten et al.
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Debye lengths are 50and 35 A, respectively. Clearly, steric forces23 rather than simple double-layer forces dominate the observed interaction. Of course, electrostatic interactions are still important since they strongly influence the conformation of the heparan sulfate side chains and thus the range of the interlayer forces. Furthermore, the distance of separation at the onset of the proximal repulsive regime is reduced on addition of CaC12. For example, a force of 1000 pN/m is encountered a t about 160 A in 0.1 mM NaC1, while the corresponding values a t 1.25 and 2.5 mM CaC12 are both about 80 A. The data at 1.25 and 2.5 m M CaC12 show that there is a contraction of the glycosaminoglycanchains (Figures 6, 7, and 9). This is not unexpected, since at low excess electrolyte concentrations, these are expected to be highly stretched normal to the surface, due to repulsive interand intrachain electrostatic interactions within the adsorbed layer, as well as to an image charge repulsion between the chain charges and the low dielectric constant
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On reduction of the linear charge density of the glycosaminoglycan chains, or on screeningof the double layer, these effects become less pronounced, allowing an entropically favored contraction of the The influence of electrostatic interactions on the conformation of adsorbed and grafted polyelectrolytes has been studied previously. Thus, for adsorbed (homo)polyelectrolytes, low adsorbed amounts and thin adsorbed layers are generally found. Increasing the excess electrolyte concentration generally results in an increased adsorbed amount and thicker adsorbed layers.26 For grafted polyelectrolytes on nonadsorbing surfaces, on the (24) Israelachvili, J. N. Intermolecularand SurfaceForces;Academic
Press: London, 1986.
(25)Cohen Stuart, M. A.; Fleer, G. J.; Lyklema, J.; Norde, W.: Scheutjens, J. M. H. M. Adv. Colloid Interface Sci. 1991, 34, 477. (26) Fleer,G.J.;CohenStuart,M.A.;Scheutjem, J.M.H.M.;Coagrove, T.;Vincent,B. Polymers at Interfaces; Chapman & Hak London, 1993.
Forces between Roteoheparan Sulfate Layers
Langmuir, Vol. 10, No. 4, 1994 1279
other hand, a strong swelling normal to the surface is expected. On addition of excess electrolyte, a contraction of the polyelectrolyte brushes is expected.27 150 Despite the large interest in polymers and polyelectro0 lytes at interfaces, there have been surprisingly few studies on the interfacial behavior of both adsorbed and grafted polyelectrolytes with the surface force apparatus. How100 z=l ever, Kurihara et al. studied the surface forces between end-anchored poly(methacry1icacid) and poly@-glutamic acid) layers, and found that an increasing degree of ionization resulted in an extension of the polyelectrolyte chains normal to the surface, and that addition of a divalent counterion resulted in a polyelectrolyte c o n t r a ~ t i o n . ~ ~ ~ ~ Analogous findings on the effects of the degree of ionization were found by Claesson et al. for the (co)polyelectrolyte chitosan.30 Finally, a contraction of proteins resulting from -50 a change in ionic strength has previously been reported by Belfort and Lee.31 0 100 200 300 400 500 600 On increasing the CaCl2 concentration, the adhesion DISTANCE (A) strength increases (Figures 4,6,and 7). Hence, in 0.1 mM Figure 8. Forces normalized with the local radius as a function NaCl it is about 200 pN/m, while in the presence of 1.25 of separation after adsorption from 0.2 mg/mL proteoheparan and 2.5 mM CaCl2, it is about 1OOOpNlm. This is expected, sulfate in 0.1 mM NaC1, followed by dilution by a factor of 3000 since in the presence of CaCl2, the attractive interactions with 0.1 mM NaCl and addition of 2.5 mM CaCla. Filled and may dominate to a higher extent, due to the screening of open symbols represent forces measured on approach and the electrostatic repulsion between the surfaces. Similarly, separation, respectively. the addition of Ca2+ supports the aggregation of proteoglycan chains in solution, Le., enhances network forloooo mation, and increases the viscosity of proteoglycan solutions.2 Note, however, that the adhesion is much weaker than that observed in the absence of steric interactions (cf., e.g., DLVO t h e ~ r y l ~ Clearly, ~ ~ ~ ) . steric interactions are important a t small distances of separation. 1000 -: Furthermore, the addition of CaCl2 results in the E proximal hard wall being displaced from about 90 to about v 80 A. Although the screening of the electrostatic repulsion contributes to this reduction, it cannot account for the O whole effect. Instead, as was the case with the distant 1 1 . 0 100 force regime,a contraction of the glycosaminoglycan chains in the presence of CaC12 is likely to cause this effect. One important aspect in surface force investigations is that of the reversibility of the interaction on approach and separation. At large separations (D > 150-200 A), 10 the forces are reversible, indicating quasi-equilibrium conditions (Figure 8). Considering also the magnitude of 0 100 200 300 400 500 the interaction at these separations, this indicates that DISTANCE (A) possible conformational rearrangements of the glycosaminoglycan chains occur fast compared to the experimental Figure 9. Comparison of the forces obtained between proteotime frame (typically a few seconds per data goint). heparan sulfate layers adsorbed at hydrophobic surfaces as a function of the distance of separation at different excess Furthermore, quasi-equilibrium conditions are observed electrolyte concentrations. The forces were measured on apat the hard wall position. However, in the regime between proach in 0.1 mM NaCl (squares),1.25 mM CaCla (diamonds), 150-200 A and the position of the hard wall repulsion, the and 2.5 mM CaCh (triangles). forces measured are hysteretic, with a purely repulsive force measured on approach and a partly attractive force the structurally related mucus glycoproteins on hydroobtained on separation. This shows that a rearrangement phobic surfaces.6 For example, dynamic effects are of the layer occurs on compression. It is not likely that pronounced in the latter systems, while they are virtually a slight rearrangement of the layer will result in any larger absent for proteoheparan sulfate. One explanation for change in the van der Waals interaction. Instead, this this difference is the fact that the molecular weight of finding strongly indicates that attractive contact forces of mucins (M, 10 X 106) is much larger than that of the type discussed above are formed. proteoheparan sulfate (M,= 0.175 X 109,which is likely It is interesting to note that the forces acting between to result in higher viscosity and slower dynamic^.^^^^^ proteoheparan sulfate layers adsorbed at hydrophobic Furthermore, in the mucus glycoproteins, charged and surfaces are strikingly different from those acting between hydrophobic regions alternate along the macromolecule chain.6 In proteoheparan sulfate, on the other hand, the (27) Miclavic, S. J.; Marcelja, S. J. Phys. Chem. 1988, 92, 6718. hydrophobic region is more clearly separated from the (28) Kurihara, K.; Kunitake,T.; Higashi, N.; Niwa,M.Langmuir 1992, charged regions of the macromolecule. One could therefore 8, 2087.
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(29) Kurihara, K.; Abe, T.; Kunitake, T.; Higashi, N.; Niwa, M. h o c . IUMRS-ICAM 1993, in press. (30) Claesson, P. M.; Ninham, B. W. Langmuir 1992,8, 1406. (31) Belfort, G.;Lee, C. S. Proc.Natl. Acad. Sci. U S A . 1991,88,9146.
~
(32) Horn, R. G.; Him,S.J.; Hadziioannou, G.; Frank,C. W.; Catala, J. M. J. Chem. Phys. 1989,90,6767. (33) Klein, J. Contemp. Phys. 1979,20,611.
M a l m t e n et al.
1280 Langmuir, Vol. 10, No. 4, 1994 expect the latter to be able to orient more effectively than mucins at hydrophobic surfaces, resulting in fewer intraand interlayer hydrophobic associations on compression. Finally, the amount of mucin adsorbed in the previous study is likely to be significantly higher than that in the present investigation,3*6once more contributing to slow structural relaxation effects. From the present investigation, no conclusion can be reached whether the only effect of CaCl2 is to screen the electrostatic interactions or if the previously observed nonelectrostaticbinding of Ca2+to proteoheparan sul.fak18 occurred simultaneously. The interactions between divalent ions and carboxylic acid groups have been addressed in other studies. For instance, a dramatic lowering of the electrostatic double-layer force and a large increase in adhesion force between long-chainedcarboxylic acid layers in the presence of divalent ions have been demonstrated.3436 In this respect, Cd2+ions were found to be more efficient than Ca2+, showing that not only nonspecific electrostatic forces are a t play. Calcium ions also promote adsorption of negatively charged poly(acry1ic acid) at negatively charged surfaces, and the interaction between the poly(acry1icacid) layers are rather strongly attractive at short separations.36 Hence, there is no doubt that divalent ions strongly influence the interactions between negatively charged groups and between anionic polymers. To distinguish between specific ion binding effects and nonspecific screening of electrostatic forces in the case of proteoheparan sulfate layers would require studies with different cations, e.g., Mg2+,K+, and Na+, a topic which is outside the scope of the present investigation. Although the present study suggests that Ca2+indeed influences the conformation of the membrane-bound proteoheparan sulfate,the effectsseem to be rather limited. It is not clear at present how the screening of the
electrostatic interaction and the concomitant contraction of the glycosaminoglycan chains may account for the physiological effects of Ca2+on, e.g., smooth muscle tissue. Again, further studies, inter alia a t higher interfacial amounts of proteoheparan sulfate (accomplished by adsorption from a higher excess electrolyte concentration), are required. Finally, we note that the calculations made by Parker and Attard show that a possible deformation of the glue supporting the surfaces is of no importance for the longrange forces measured.l8 Furthermore, our conclusions based on the range of proximal repulsion and the compressed layer thickness are not affected by the possible deformation, since this would affect the local radius and thus the normalized force,FfR, whereas the interferometric measurement of the surface separation is not influenced.
(34)Claesson, P. M.; Berg, J. M. Thin Solid Films 1989,176, 167. (35) Berg, J. M.; Claesson, P. M. Thin SoZid Films 1989,178, 261. (36) Berg, J. M.; Claeeeon,P. M.; Neuman, R. D.J. Colloid Interface Sci. 1993, 161, 182.
Acknowledgment. Professor E. Buddecke is gratefully acknowledged for providing the proteoheparan sulfate sample.
Summary The forces between proteoheparan sulfate layers adsorbed at hydrophobic surfaces have been investigated with the surface force technique. This biomacromolecule adsorbs in a highly oriented conformation a t hydrophobic surfaces, the adsorption being largely irreversible with respect to dilution. The force curves observed are composed of one distant slowly decaying regime and one proximal hard wall re ime at distances of separation smaller than about 100% The forces in the distant regime were reversible on approach and separation, while an adhesion was observed at smaller separations. On addition of CaC12, there was a reduction in the range of the interaction, while the adhesion strength increased. It is concluded that both electrostatic and steric effects contribute to the observed force behavior. In particular, on addition of CaC12, there was a contraction of the adsorbed proteoheparan sulfate molecules.
