Normal and Shear Forces between Surfaces Bearing Porcine Gastric

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Normal and Shear Forces between Surfaces Bearing Porcine Gastric Mucin, a High-Molecular-Weight Glycoprotein Neale M. Harvey,† Gleb E. Yakubov,‡ Jason R. Stokes,||,‡ and Jacob Klein†,§,* †

The Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, United Kingdom Unilever R&D, Colworth Science Park, Bedford MK44 1LQ, United Kingdom § Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel ‡

ABSTRACT: A surface force balance was used to measure the normal and shear forces between two mica surfaces each bearing an adsorbed layer of porcine gastric mucin (“Orthana” mucin), genetically similar to human MUC6. This mucin is a highly purified, 546 kDa, weakly negative, polyampholytic molecule with a “dumbbell” structure. Both bare (HP) and hydrophobized (HB) mica substrates were used, and forces were measured under 1 and 30 mg/mL mucin solutions, under pure (no-added-salt) water, and under 0.1 M aqueous Naþ solution. Normal surface forces were monotonically repulsive in all cases, with onset of repulsion occurring at smaller surface separations, D, in the 0.1 M salt solutions (∼20 nm, compared with ∼40 nm for no added salt). Repulsion on HP mica was greater on surface compression than decompression, an effect, attributed to bridging and slow-relaxing additional adsorption on compression, not seen on HB mica, a difference attributed to the denser coverage of mucin hydrophobic moieties on the HB surface. Friction forces increased with compression in all cases, showing hysteretic behavior on HP but not on HB mica, commensurate with the hysteresis observed in the normal measurements. Low friction coefficients μ (= ∂Fs/∂Fn < 0.05) were seen up to mean pressures ÆPæ ≈ 0.5 to 1.0 MPa, attributed to low interpenetration of the opposed layers together with hydration lubrication effects, with higher μ (up to 0.4) at higher ÆPæ attributed to interlayer entanglements and to bridging (for the case of HP mica). Shear forces increased only weakly with sliding speed over the range investigated (80-820 nm s-1). The lower friction with HB relative to HP mica suggests a selectivity of the HB surface to the hydrophobic moieties of the mucin that in consequence exposes relatively more of the better-lubricating hydrophilic groups. This surface-selectivity effect on lubrication may have a generality extending to other biological macromolecules that contain both hydrophilic and hydrophobic groups.

’ INTRODUCTION Mucins are a family of large (0.1-50 MDa) glycoproteins, found throughout Nature. Secreted mucins are the main proteinaceous component of mucus1 and are ubiquitous in metazoans2 (including mammals), for example, in the eye,3,4 saliva,5,6 the gastric tract,1,7 the cervix,8 and the bronchial tract.9 In the situation of an adsorbed mucin layer between two rubbing surfaces, boundary lubrication by mucin may occur. Distinct from thick film lubrication, which is dominated by rheology, boundary lubrication occurs when a lubricant layer is molecularly thin and contacts between asperities support the normal load.10 Boundary lubrication is common in both technology10,11 as well as in biology12-15 and has been observed with molecules such as proteins,16,17 surfactants,18,19 and polymers.20,21 The interest in mucin as an in vivo boundary lubricant arises from the widespread presence of mucin in living systems and the molecule’s known association with surfaces. Mucin adsorbs to the hard and soft surfaces of the human mouth,22-24 the surface of the eye25 and, in the case of cell-bound mucins, protrudes from cell membranes.26 Artificial surfaces such as dentures and contact lenses may have their surface properties altered by mucin adsorption.27 Mucins thus interact with many surfaces of different surface chemr 2011 American Chemical Society

istries, in both man-made and natural applications, and have been studied adsorbed to both hydrophobic16,28-31 and hydrophilic32-37 surfaces in previous work. Mucins vary in size and structure, but have certain common features (reviews38-40), including regions of heavy O-glycosylation with oligosaccharides that can comprise up to ∼80% of the molecular mass. The oligosaccharides contain many highly hydrated -OH groups,41-43 and previous studies have shown that well-solvated molecular moieties can lead to good boundary lubrication by polymers, both brushes20,44-46 and adsorbed.17,21,47-49 The effect was partially attributed to strong solute-solvent interactions that were preferred over solutesolute interactions, with such “good solvent” conditions giving rise to osmotic repulsion between polymer-bearing surfaces and the formation of a thin, polymer-depleted interfacial layer that sheared readily under applied lateral motion. Hydrated charges have also been shown to be highly lubricious in previous surface force balance (“SFB”, also known as the surface forces apparatus) Received: November 16, 2010 Revised: January 18, 2011 Published: February 22, 2011 1041

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Biomacromolecules work,18,20,50 behavior that the hydrated polar -OH groups in mucin may mimic. Such “hydration lubrication” has been attributed to the strongly bound but rapidly exchanging hydration sheaths that exist around charges or polar groups, leading to a ball-bearing-like lubrication at shear rates lower than the frequency of exchange.50-52 The study of mucin’s physical behavior at an interface is important for insight into the role mucin may play in biological boundary lubrication (which typically involves pressures of up to ∼5 MPa and shear rates up to ∼106 s-1 12 and a variety of surfaces). Relevant previous work on interfacial physical behavior includes atomic force microscopy,32,34,53,54 SFB measurements,29,35,36,55 and classical tribometry,16,28,56 although not all of these studies investigated friction. The SFB, which we use in the present study, is capable of providing information that complements and extends these previous studies. This capability arises from a number of features: first, the ability of the SFB to measure absolute surface separations; second, the molecular smoothness of the interacting surfaces; third, the low lateral pressure gradient associated with compression of the surfaceattached layers, which could avoid “ploughing” effects (this gradient varies as 1/R 57 and so is much weaker for the SFB compared with scanning probe methods); finally, the high sensitivity of the apparatus to both normal and shear stresses, that is, forces per unit area, which are in turn related via the Derjaguin approximation to the interaction energy per unit area, E(D), of two parallel flat surfaces obeying the same force law (where D is the surface separation) and thus to the intrinsic molecular properties of the adsorbed species. In the Derjaguin approximation, Fn(D)/R  E(D), where Fn(D) is the force between surfaces of mean curvature R a closest distance D apart. The sensitivity is thus δFn/R, which for the SFB is ∼5  10-5 N/m and for a tipped AFM ∼10-2 N/m (although for a colloidalprobe AFM, the stress sensitivity is similar to the SFB). The present study is the first to use an SFB to investigate the friction properties of adsorbed mucin and is also the first SFB study to use a particular mucin from a batch for which the amino acid analysis, and the compositions of the oligosaccharide residues are known. Investigations were performed using both bare (hydrophilic, “HP”) mica and hydrophobized (“HB”) mica, in view of the polyampholytic nature of mucin and the wide variety of surfaces, both natural and artificial, to which mucin may adsorb in vivo.

’ MATERIALS AND METHODS Materials. Pharmaceutical-grade porcine gastric “Orthana” mucin was purchased from A/S Orthana Kemisk Fabrik (Kastrup, Denmark) and extensively dialyzed using a 16 kDa cellulose membrane against ultrapure water (Elga, Marlow, U.K.). The dialyzed mucin was lyophilized and stored at e-10 C until use. All other “pure” water used for cleaning and experiments was mains water filtered through activated charcoal, a RiOs 5 reverse-osmosis device and finally a Milli-Q Gradient A10 (both Millipore, Watford, U.K.) to yield water of resistivity 18.2 MΩ cm and TOC e 4 ppb. Mucin solutions were produced by dissolving weighed portions of lyophilized mucin in pure water and are expected to have been of pH similar to the water (pH ∼5.8,58 the slight acidity arising from atmospheric CO2 dissolving in the water). Filters used were single-use 0.2 μm MiniSart hydrophilic cellulose acetate (Sartorius). A short summary of previous work on this mucin31,34,41,59-61 is as follows: Processed and used as described, Orthana mucin is a 91-97%

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Figure 1. “Orthana” mucin is monomeric and its structure is dumbbelllike, with typically two globular sections linked by a heavily glycosylated spacer.41,61,63 Cartoon reprinted with permission from ref 41. Copyright 2007 American Chemical Society. pure porcine gastric mucin genetically similar to human MUC6.41 Chemical hydrolysis followed by chromatography established that sialic acid is only present in trace quantities. The main sample impurity is most likely bound water that remained despite extensive freeze-drying. Raman spectroscopy, UV analysis of gel filtration samples, and conductivity measurements revealed traces of lipids, free peptides, and free sugars at trace concentrations (too low to quantify) and salt impurities ∼0.01 mM for a 1 mg/mL mucin solution. PAGE analysis showed that small molecules (>1 kDa) were absent, for example, serum albumins, which have been suspected to be contaminants in other mucin samples.62 The Orthana molecular weight is 546.4 kDa, of which 71-76% is carbohydrate.41 The molecular structure is a monomeric dumbbell41,61,63 (Figure 1), and the radius of gyration, Rg, ∼55 nm at ionic strength ∼10-5 M, falls to ∼33 nm at 10-2 M (measured using static light scattering;41 small-angle X-ray scattering61 gave somewhat smaller values). Other dimensions are given in Figure 1. The spacer that connects the two “bells” is heavily O-glycosylated with predominantly neutral di- and trisaccharides. The mucin has no isoelectric point59 but remains weakly net negative throughout the pH range 2-11. Positive, negative, and hydrophobic amino acids are present in the molecule. Muscovite mica used in this work was from a mixed batch of grade I and grade II Special (S&J Trading, NY); silver used for evaporation was from Aldrich (99.9999% pure); the glue used was Shell Epon Resin 1004F. Acids and H2O2 used for cleaning were reagent grade; ethanol was analytical grade (Riedel-de Ha€en, absolut puriss p.a.). Salts (at least 99.995% pure) were from Sigma Aldrich and used as received. Stearyl triammonium iodide (STAI) (synthesized as in ref 64) was a gift from Dr. Nir Kampf and Dr. Gilad Silbert (Weizmann Institute of Science, Israel). Surface Force Balance. The SFB (Figure 2) has been previously described in detail.52,65 The surface separation, D, is controlled via a three-stage mechanism, with an externally sectored piezotube (PZT) providing the finest motion and the lateral (shear) motion of the upper surface. The absolute value of D is tracked using interferometric fringes of equal chromatic order (FECO),66,67 whereas shear motion is measured (with resolution (0.5 nm) using a capacitance probe (Accumeasure AS5000, MTI Instruments). Forces are measured by spring deflection (spring constant ∼150 N m-1 for Fn and ∼250 N m-1 for Fs). In the present experiments, Fn was measured to around (1 μN, limited by nonlinearity in the drive mechanisms and thermal drift over the large range of Fn investigated. Fs, limited by background mechanical noise, was measured to better than (1 μN and D with a precision of around (0.5 nm (values that are lower than the capabilities of the SFB when optimized,68 but adequate for the study at hand). Experimental Procedure. Mica was hand- cleaved into 3 to 4 μm uniformly thick facets. Facets were “downstream” melt-cut69 with a hot platinum wire and half-silvered on one side (to thickness ∼55 nm). Two pieces of mica, cut from the same facet, were glued silver side down onto 1042

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by draining and filling the boat with pure water four times; 0.1 M NaNO3/ NaCl conditions were established by partial replacement of pure water with salt solution. The system was left for 1 to 2 h for equilibration after rinsing or addition of salt. A liquid meniscus was always maintained between the surfaces to avoid surface contamination. FECO positions were measured once the fringes either stopped moving (suggesting layer equilibrium) or appeared to have slowed down to an apparent steady rate (suggesting layer equilibrium with overlaid thermal drift). The mean pressure ÆPæ over the contact region area A, was calculated using Hertzian contact theory,73 with an effective bulk modulus K for the mica/glue combination measured from the FECO deformation under large loads applied after all other data had been recorded. From this ÆPæ = Fn/A = (Fn1/3 K2/3)/(π R2/3) for all Fn. (R is the measured radius of curvature of the mica sheets.) Fn/R = 10 mN m-1 corresponds to ÆPæ ≈ 1.0 MPa. All results presented were reproduced on at least two contact spots within each experiment and, except where stated, in at least two separate, independent SFB experiments. Figure 2. (a) Schematic of the surface force balance.52,65 Two symmetric semisilvered mica pieces are glued (silver side down) onto planocylindrical fused-quartz lenses and mounted in a cross-cylinder geometry. They are submerged under the liquid of interest for force measurements. (b) An alternating voltage applied across the sectors of the piezotube causes a lateral sweeping motion for the application of shear. A feedback loop to a common central sector keeps the surface separation constant during shear. plano-cylindrical fused-quartz lenses and mounted in the SFB in a crossed-cylinder geometry. Glassware was cleaned for at least 4 h in a 2:1 98% H2SO4/30% H2O2 “piranha” solution (piranha solution reacts violently with organic molecules and should be handled with extreme care in a fume hood while wearing personal protective equipment); metalware and Teflon pieces were cleaned in 50 C 20% HNO3 for at least 1 h. Glassware cleaning was followed by copious water rinses with tap water and then pure water; metalware and Teflon pieces were rinsed with pure water then boiled in pure water. Water rinses were followed in a laminar flow hood by a filtered ethanol rinse and blow-dried with filtered nitrogen. Experiments were performed in a temperature-stabilized room at temperatures in the range 21-25 C. The bare surfaces were brought into contact in air for calibration of the PZT and separated; then, pure water was injected into the boat and contact was re-established to calibrate D = 0.69 Cleanliness was verified by observing a jump in and out of adhesive contact under water and (when tested) an absence of measurable friction during the jump-in.70 For experiments on HB mica, the surfaces were removed following calibration of contact under water and hydrophobized by immersion in a 68 C 0.4 mg/mL aqueous STAI solution for 10 s, followed by 10 s in 68 C pure water to remove unadsorbed STAI (a modification of Tadmor et al.’s method64) and immediately remounted in the SFB. We repeated the cleanliness checks to verify that the system was still clean. Previous work71 has established a 102 and 62 respective advancing and receding contact angle for pure water on STAI-hydrophobized mica. Half of the water in the boat was replaced with water injected through a 0.2 μm MiniSart filter that had been previously rinsed through with >80 mL of water, and a jump in/out of contact was confirmed to ensure that the filter did not introduce contamination to the injected liquid.72 In a preliminary investigation (using a 30 mg/mL mucin sample and comparing solution mass densities before and after filtering), the filter model used removed 40 nm) is not visible in Figure 3 because only a selection of runs is shown but can be seen in the “range” bands drawn in Figure 6. It arises, at the lower compressions, from finite relaxation times (which were noticeable from D ≈ 150 nm and were observed by eye to be up to ∼5 s in the range D ≈ 30-80 nm). The higher scatter in the added-salt data may have arisen from slow desorption of mucin from the surfaces. A log ordinate plot (Figure 3b) showed a straight-line relationship for Fn(D)/R for 40 nm > D > 10 nm, with exponential decay lengths κ -1 of 8.6, 6.2, and 10.5 nm for the 1 mg/mL mucin, 30 mg/mL mucin, and pure water rinsed systems, respectively. The addition of 0.1 M NaNO3 reduced κ -1 to 4.9 nm. The measured decay lengths suggest that forces in the region 40 > D > 10 nm are predominantly steric and not pure electrostatic double-layer effects. The range of the steric forces suggests that the adsorbed protein under 0.1 M salt was contracted compared with its size in no-added-salt water. For 1043

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Figure 3. Fn(D)/R on HP mica, prior to application of shear. The legend is the same for both graphs. (a) Main: all no-added-salt data appear approximately similar, but under 0.1 M salt, the onset of repulsion decreases. No net attraction is seen for any system. The small difference between the 1 mg/mL mucin and 30 mg/mL mucin may arise from trace salt impurities and counterions in the mucin solution, both of which are more plentiful in the more concentrated solution and so would lead to greater screening of electrostatic charge. Inset: a single compression/decompression pair of runs to highlight run direction hysteresis. (b) Main Figure data from part (a) using a log ordinate axis.

rinsed surfaces under pure water in the range 40 > D > 70 nm, the repulsion had a decay length of 22.3 nm, which implicates electrostatic double-layer forces prior to an onset of steric forces at D ≈ 40 nm, although it is not impossible that some steric component is contributing to the decay of forces in this regime. The 40 > D > 70 nm data for rinsed surfaces under pure water imply a far-field surface potential of -74 mV and a surface charge of -3.2 mC/m2 (calculated via the Grahame equation74). For D < 10 nm, the Fn(D)/R behavior of all systems converged, indicating a dominant contribution to Fn(D) independent of solution conditions (which we attribute to steric effects of excluded volume75). Under the highest compressions applied (Fn/R > 30 mN m-1) the “hard-wall” D was ∼4 nm for no-added-salt and ∼3.5 nm for added-salt systems. Normal Force, HB Mica. Control measurements between two HB mica surfaces across pure water (Figure 4, diamond markers) showed |Fn/R| < 0.25 mN/m until a “jump-in” occurred from a separation DJI to an equilibrium position at D = DHB, the measured thickness of two adsorbed STAI layers in adhesive contact. Measurements of DJI, DHB, and FPO/R, the pull-off force required to separate the adhered surfaces, were made in a number of independent experiments, including those from which Fn(D)/R data are presented. Average values were ÆDJIæ = 8.4 ( 1.7 nm (15 jump-ins, 7 experiments), ÆDHBæ = 2.1 ( 1.2 nm (20 measurements, 8 experiments), and ÆFPO/Ræ = 505 ( 78 mN m-1 (22 measurements, 6 experiments). The variation in DHB is attributed to alignment issues on remounting. These values, broadly consistent with those of previous studies on STAI or similar surfactant layers,18,64,76-78 indicate a JKR79 adhesive surface energy γ = 54 ( 8 mN/m and an adsorbed STAI layer of thickness 1.1 ( 0.6 nm on each surface. The results in Figure 4 are plotted as Fn(D0 )/R, where D0 = D - DHB evaluated for each experiment. Discounting the thickness of the STAI layers in this way, the results for mucin on HB mica (Figure 4 main) were similar to those on HP mica. No net attraction was observed at any D. Monotonic repulsion, both prior to and after

Figure 4. Fn(D)/R on HB mica. Main: Typical HB mica preshear results for mucin along with control measurements taken across pure water in the absence of mucin. The abscissa, D0 , is D less the ∼2 nm thickness of the STAI layers in contact. The normal force behavior is very similar to that on HP mica (Figure 3). Inset: the same data (control measurements excluded) on a log ordinate to highlight their exponential nature.

the application of shear, onset above the inter-run scatter at D0 ≈ 40 nm under 1 mg/mL mucin and D0 ≈ 100 nm for rinsed surfaces under pure water. The addition of 0.1 M salt (NaNO3 for the data shown in Figure 4; NaCl in a separate experiment) reduced the 1044

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Figure 5. Friction is measured by means of “shear traces” (traces B-E). Lateral motion (trace A) is applied to the top surface using the sectored piezotube. Any friction makes the top surface “couple” to the bottom surface, and they move in registry (near-vertical portions of traces D and E) against a spring. When the restoring force of the spring, Fs, just exceeds the static friction, Fs*, the surfaces slide (plateaux in traces), and this force is recorded as the friction as a function of D, Fn and ÆPæ.

repulsion onset to D0 ≈ 30-40 nm prior to the application of shear. Postshear measurements under 0.1 M salt were either of the same range as preshear (NaCl) or somewhat shorter ranged (NaNO3, repulsion onset D0 ≈ 20 nm) and may be attributed to desorption during the longer NaNO3 experiment. A log ordinate plot of the data (Figure 4 inset) showed that the mucin data in the range 40 nm > D > 15 nm lay in straight lines with κ-1 = 9.5, 13.1, and 4.0-8.4 nm for 1 mg/mL mucin rinsed under pure water and 0.1 M salt, respectively. For the rinsed system under pure water, the repulsion in the region D0 ≈ 40100 nm was of considerably longer decay length (around κ-1 ≈ 55 nm on average) than that at smaller D0 , as similarly observed on HP mica and indicative of an electrostatic double-layer with a range greater than that of steric effects. The effective surface potential in this far-field region was around -57 mV (corresponding to a relatively low surface charge of -0.9mC/m2). The reduction in the onset of repulsion from the no-added-salt systems in the presence of 0.1 M salt (in which any electrostatic double-layer effects would have a range of only a few nanometers) suggests, as also on HP mica, that the adsorbed protein contracted in the high salt environment relative to its size under salt-free water. The behavior of all solution systems converged at D0 < 15 nm, similar to the convergence observed on HP mica. Under high loads, the “hard wall” D0 was ∼5 nm for all systems on HB mica, close to the “hard wall” D = 3.5 to 4 nm on HP mica. Relaxation effects at intermediate D were also similar to those on HP mica. The most notable Fn(D)/R difference between HP and HB mica was the almost complete absence of compression/decompression hysteresis on HB mica, visible by comparing Figure 4 (HB), where Fn(D)/R is the same on decompression as on compression, with Figure 3 (HP), where Fn(D)/R is longerranged on compression.

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Figure 6. Fn(D)/R on HP mica, effect of shear. Measurements of Fn(D)/R after the application of shear to the surfaces (points) are largely unchanged from Fn(D)/R measured prior to application of shear (bands, drawn using data in Figure 3 and other representative data). This shows that the surfaces are not permanently altered by the application of shear within the range of loads and sliding speeds used.

Shear Measurements. Figure 5 shows typical “shear traces” indicative of how shear data are recorded. A constant-speed “back and forth” motion is imposed on the top surface using the sectored piezotube (trace A). At large D (trace B), only noise due to ambient vibrations is measurable in the traces. At smaller D, frictional coupling between the surfaces causes bending of the shear springs to give rise to a shear force Fs. When Fs equals the static friction, the surfaces slide past each other, manifested as a plateau in the trace, providing a direct measure of the sliding shear force Fs*. Figure 6 shows postshear Fn(D)/R data for mucin systems on HP mica and compares them with the range of preshear data. Essentially, shear did not alter the Fn(D)/R profiles, an observation also made on HB mica (data not shown), excluding the special case of NaNO3 previously discussed. These observations suggest that little removal of the mucin occurred during the shear process and that the shear traces can be taken as characteristic of the mucin layers revealed by the normal force profiles. Figure 7 shows a plot of Fs*(D) for rinsed mucin-coated HP mica surfaces with, for reference, a summary of postshear Fn(D) profiles using data from Figure 6. Fs*(D) did not vary much with either the salt concentration or whether the surfaces were being compressed or decompressed, in contrast with Fn(D) (Figures 3, 4 and 6). The closer-in onset of Fs* than Fn is reflected in the lowfriction-coefficient regime at low loads in Figures 8 and 9. Friction Variation with Load. Plotting the sliding shear force Fs* against the simultaneously measured Fn gives a direct measure of the friction coefficient μ, defined as μ = ∂Fs*/∂Fn. Figure 8 shows Fs*(Fn) for rinsed mucin-coated HP mica surfaces; Figure 9 shows data from all systems tested on HB mica. On HP mica, there was a clear difference between Fs* measured on compression and decompression, the latter being significantly larger in any given cycle. On HB mica, this difference 1045

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Figure 7. Fs*(D), HP mica, for rinsed systems. Both pure water and 0.1 M salt systems demonstrate nearly identical Fs*(D) to each other. (The data shown are also representative of 1 and 30 mg/mL mucin systems.) Friction onsets around D = 5 nm (forces at greater separations are noise (∼1 μN) that sets a limit for the resolution of low Fs*). This onset is much smaller than even the smallest onset of Fn, as shown by the Fn guides drawn using postshear data from Figure 6. The small D values measured (slightly below the “hard-wall” mentioned in the main text) may arise from transient shear-induced effects that cause the adsorbed layers to thin somewhat.

was absent. This “hysteresis” effect was probably caused by time effects and is consistent with the hysteresis observations in Fn(D)/R. Fs*(Fn) for mucin solutions on HP mica prior to rinsing (not shown) was similar to the results shown in Figure 8 for rinsed surfaces under pure water. All of these no-added-salt systems on HP mica demonstrated a low-friction zone at loads up to Fn ≈ 40 μN (ÆPæ ≈ 0.8 MPa), with μ ≈ 0.02 to 0.03. At higher loads, μ increased to a limiting value of 0.15 to 0.30. The addition of salt increased μ compared with the no-added-salt cases, which may be understood with reference to the Fn(D)/R profiles (Figure 3) that show that for a given Fn the layers approach more closely in the high salt environment. A low-friction zone existed for the added-salt system on HP mica but only up to Fn ≈ 10 μN (ÆPæ ≈ 0.5 MPa). Figure 9 shows representative Fs*(Fn) results on HB mica. Qualitatively, the results are similar to those on HP mica (a lowfriction coefficient regime at low loads, followed by a sharp nonlinear increase in friction with load at higher loads; the addedsalt system demonstrated higher friction than the no-addedsalt), except for the absence of hysteresis in the HB mica data. The low-friction regime at low loads on HB mica, however, extended to greater Fn than on HP mica: μ ≈ 0.01 to 0.02 was observed at up to Fn ≈ 60 μN (ÆPæ ≈ 1.0 MPa) for no-added-salt systems and at up to Fn ≈ 20 μN (ÆPæ ≈ 0.7 MPa) for the addedsalt system. Friction Variation with Shear Speed, vs. Figure 10 demonstrates Fs*(vs) on HP mica. A fixed applied Fn was used for each data set and ∼2 min elapsed between each vs investigated. The data thus also demonstrate the effect of time in contact. Fs* increased

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Figure 8. Fs*(Fn), HP mica, rinsed systems. Lines drawn are point-topoint and join data from a particular run. Both systems show a marked difference between loading and unloading behavior commensurate with the hysteresis observed in normal behavior as well as a large degree of scatter between runs. § = 11 min interval in contact with no increase in load, showing that time in contact alone increased Fs* on HP surfaces. A general increase in the slope (∂Fs*/∂Fn) is visible when salt is introduced. The shear speed was 400 ( 35 nm s-1.

Figure 9. Fs*(Fn) for mucin systems on HB mica. A nonlinear relationship is found, and added salt increases Fs* at a given Fn, both as observed on HP mica (Figure 8). On HB mica, however, run direction hysteresis is absent, and the low friction region persists to larger loads than on HP mica. The shear speed was 400 ( 35 nm s-1.

with time in contact and weakly with vs. Fs*(vs) appeared to rise more sharply at the lowest sliding speeds, although the effect of time in contact somewhat complicates the Fs*(vs) data. The increase in 1046

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Figure 10. Fs*(vs), HP mica. Several data sets are shown, all compression, each at a fixed D and applied Fn. Lines connect the points in each data set in the order they were recorded. “x” marks the first datum in each set. At a given Fn, Fs* increases with vs, and the effect is greater at larger Fn. A greater effect, however, is the increase in Fs* with time in contact. (Typically 1 to 2 min elapsed between points in each data set.) The nonmonotonic correlation between ÆPæ and Fn is because R and K, the surface radius of curvature and effective bulk modulus, respectively, varied between contact spots, thus affecting the calculated ÆPæ.

Fs* with vs suggests a viscous component to energy dissipation between the rubbing surfaces. Fs*(vs) on HB mica is shown in Figure 11 and when compared with the data shown in Figure 10 shows the very small effect of time in contact on Fs* for HB mica surfaces. (The data were collected using the same protocols as those on HP mica.) A weak increase in Fs* with vs was observed.

’ DISCUSSION The main findings of this study are that Orthana mucin, on both HP and HB substrates, formed similar adsorbed layers that were purely net repulsive and had a longer range of interaction in the absence of salt than in the presence of 0.1 M salt. Friction between Orthana mucin-coated surfaces showed an especially low friction zone at low Fn, with the zone persisting to greater Fn on HB mica than HP mica. Measurements on HP mica, but not HB mica, showed evidence of hysteresis, whereby Fn was lower and Fs* was higher on decompression than compression. The findings are best considered in terms of the structure of the mucin molecules and their adsorption on the substrate surfaces. As noted, the mucin used in this experiment takes on the shape of a dumbbell,41,61,63 with the large majority of molecules having two ∼20 nm diameter globular regions connected by a 30-50 nm carbohydrate-rich peptide spacer (Figure 1). On both HP and HB mica, adsorbed mucin demonstrated remarkably similar Fn and Fs* behavior in general, notable because across pure water in the absence of mucin, HP and HB mica gave very different force behavior from each other. The merit of investigating mucin on both HP and HB surfaces was that it provided insight into the potential performance of mucin as a lubricant in vivo, where a wide variety of natural and artificial surfaces, both hydrophilic and hydrophobic, may be

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Figure 11. Fs*(vs), HB mica. Several data sets are shown, each at a fixed D and applied Fn. Lines and arrows demonstrate the order in which points in each data set were recorded. “x” marks the first datum in each set. A weak increase in Fs* with vs is visible. Notably, the effect of time in contact is very small compared with observations on HP mica (Figure 10). The nonmonotonic correlation between ÆPæ and Fn is because R and K, the surface radius of curvature and effective bulk modulus, respectively, varied between contact spots, thus affecting the calculated ÆPæ.

encountered. In addition, the amphoteric nature of Orthana mucin enables some basic insight into how such naturally occurring polyampholytes may behave physically on different substrates. The Fn results (Figures 3, 4, and 6) showed that on both types of surface the onset of steric repulsion, in the absence of added salt, was at D ≈ 40 nm (around twice the globule size). It is tempting to attribute this to the mucin molecules adsorbing in a predominantly side-on state with limited end-on adsorption (which would provide an onset of ca. 160 nm). However, it is possible that upon adsorption to the different surfaces the mucin undergoes structural changes that nonetheless lead to similar steric repulsion. The absence of net attractive Fn implies that intersurface osmotic repulsion is the origin of the steric forces, overcoming any effect of attractive intersurface bridging on Fn. The universal reduction in the range of Fn upon the addition of salt is consistent with the shorter screening length and a smaller Rg in the high salt concentration. The shear data on both HP and HB surfaces (Figures 7-9) showed that in all cases a region existed where Fn was appreciable but Fs* was very small. A similar low-friction regime has been observed for model flexible polymers in previous studies;21,47 also lubricin,17 and was attributed to low interpenetration of opposing layers, with energy dissipation arising from the viscous rubbing of segments past each other. The low friction during viscous rubbing, in the case of mucin, is likely due to both limited interpenetration and also “hydration lubrication” arising from bound water surrounding the highly hydrated polar groups in the mucin oligosaccharides; the higher friction at high salt concentration may thus reflect the known dehydration effect of 1047

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Biomacromolecules high salt on the hydration level80 as well as the reduction in electrostatic screening, which may promote more intimate segment-segment contact. The low-friction regime (Figures 8 and 9), where friction coefficients were as low as μ ≈ 0.01 to 0.02, encompasses pressures that are about an order of magnitude lower than those in load-bearing human joints such as hips or knees but comparable to or larger than at other sliding interfaces, such as eyes or oral surfaces, and demonstrates that mucin (and mucin-like molecules) have the potential to be highly effective aqueous boundary lubricants under low to moderate pressures. The lack of linearity in Fs*(Fn) under higher loads (Figures 8 and 9) may arise from molecular rearrangement under strong compression such as bridging effects, which need not be linear with load.21,47,81 The hysteresis in Fn(D)/R on HP mica probably has similar origins to that in Fs*(Fn) and a likely mechanism is bridging: when the surface separation is comparable to or less than the molecular size, molecules from one surface may simultaneously adsorb to the opposing surface as well. Such “bridging” of the gap is a signature of interactions between surfaces bearing adsorbed polymer layers, even under good solvent conditions47,81 and even when the adsorbed chains are charged21 and has been suggested before in normal force studies of mucin.29,35 The effect of bridging on Fn(D)/R is generally to reduce the net osmotic repulsion by providing a compensating attractive component, and so Fn(D)/R on separation is frequently lower than when approaching because molecules in the former case will have had a longer time to form bridges. The effect of bridging on friction is more striking because bridging molecules are dragged along the surfaces during sliding. This effect can strongly influence the friction.21,47 The general effect is probably that the effective viscosity of the confined material and the extent of interlayer interpenetration rises nonlinearly with increasing pressure, for example, because of onset of entangled behavior on HB mica or bridging on HP mica. This is also consistent with the larger low-friction zone on HB mica compared with HP mica. An additional effect that does not involve bridging but may contribute to the hysteresis in Fn(D) is the additional adsorption of each layer onto the substrate induced by compression. Such additional adsorption, seen in other polymeric studies,75,82 may require time to return to equilibrium and so results in more compact layers (and thus lower osmotic repulsion at a given D) on decompression, which manifests as hysteretic behavior. In this present study, initial and subsequent compressions showed the same results, which places an upper limit of ∼1 h (the duration of the most rapid compression/decompression cycles) on the time required for re-equilibration of the surface layer. An unexpected observation, given the similarity of the Fn(D)/ R results for HP and HB mica, was that hysteresis and time effects were almost completely absent from the HB data. Because bridges, which may have given rise to such effects, appeared to form readily over a period of a few minutes on HP mica, this suggests that bridges formed readily on the HP but not the HB mica. This also led to the persistence of the low friction zone to higher loads on HB mica than on HP mica (Figure 9 vs Figure 8) and implies that bridge formation on HB mica is impeded by a kinetic or energetic penalty that does not exist on HP mica. We may attribute this to two possible scenarios: first, although we did not directly measure adsorbance of the mucin onto the mica in this study, previous work34,56 suggests that Orthana mucin adsorbs more to an HB than to an HP surface (∼2.0 mg/m2

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and ∼1.3 mg/m2, respectively). If this also occurred in this present study, the higher adsorbance on HB would make it kinetically more challenging for opposing moieties to form bridges than for the HP case. Second, a greater exposure of highly hydrated molecular moieties to solution on HB mica is possible because more hydrophobic amino acids would be attached by hydrophobic bonding to the substrate, leaving relatively more hydrophilic moieties exposed to the solution. This could account for a greater osmotic penalty for a molecule invading the opposing adsorbed network when HB surfaces were used, leading to lower bridging and consequent lower friction. Indeed, on the basis of these adsorbance values, one may estimate an area per Orthana molecule of ca. 400-600 nm2 . Given the dimensions of the molecules, this implies significant overlap between them on the surface (up to two-fold overlap), and this would readily explain a number of observed features, including the significant compressibility of the adsorbed layers. The different spectrum of solution-exposed moieties may also account for the persistence of lower friction on HB mica in another way: highly hydrated hydrophilic moieties tend to osmotically repel similar moieties74 under the “good solvent” conditions used, whereas attraction between hydrophobic moieties may be expected. On the HB mica, where relatively more solution-exposed hydrophilic moieties are expected than on the HP mica, a greater reduction in layer interpenetration and entanglement would occur owing to this repulsion, resulting in lower friction compared with that of HP mica. It is appropriate to compare some of the results of this investigation with previous studies of mucin, particularly friction studies. Colloidal-probe AFM has been used by Hahn-Berg et al.32 to measure lubrication by bovine submaxillary mucin (BSM) and by Pettersson and Dedinaite53 to measure both BSM lubrication and normal forces. Constant friction coefficients in the region of 0.2 were measured by Hahn-Berg et al. using a 1 mg/mL BSM solution and silica surfaces; lower coefficients of friction ∼0.03 were measured by Pettersson and Dedinaite using a mica substrate and a silica probe up to loads in the region of 40 MPa. This latter study also observed purely repulsive hysteretic normal forces, as observed in this study, although no hysteresis was observed in friction forces. Mucin has been measured using tribometry,16,28,56 and lubrication performance was found to depend on a variety of variables such as pH, salt concentration, and mucin concentration, highlighting the complex nature of mucin interfacial properties. Previous work such as that by Lundin et al.62 and Malmsten et al.29 have shown that the normal force behavior of mucin can be very dependent on the mucin type and the effect of impurities, and so the choice was made in this study to use a “model” mucin that was significantly different from those used in previous (non-Orthana) studies, with relatively short oligosaccharides, a monomeric structure, and a weak negative charge but nonetheless had high purity, a good characterization, and minimal sample variability between experiments. For these reasons, however, we believe that it would be difficult to make a meaningful comparison between our results and previous work.

’ CONCLUSIONS It was found in this work that Orthana mucin adsorbs essentially irreversibly to bare (HP) mica and hydrophobized (HB) mica to form robust layers capable of effective boundary lubrication. The range of steric repulsion reduced in the presence of 0.1 M 1048

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Biomacromolecules salt and suggests a predominantly sideways-on adsorption of the molecule. On HP mica, hysteresis was visible in the normal and shear profiles, but this effect was greatly reduced on HB mica. The hysteresis is attributed to either ready bridging of the gap on HP mica by the mucin molecules or a possible distortion of the adsorbed layers due to compression-induced, slowly relaxing additional adsorption. Both of these effects were absent on HB mica, presumably because of the strong and dense attachment of hydrophobic moieties of the mucin to the HB surface, which suppressed bridging and compression-induced distortion. On initial overlap and compression of the adsorbed layers, low friction barely within the measurable sensitivity of the SFB was measured up to a threshold applied pressure ÆPæ ≈ 0.5 to 1 MPa. At higher pressures, the friction coefficient rose, attributed largely to bridging effects as well as higher viscous dissipation at the interface. Overall lower friction was observed on HB mica compared with HP mica, attributed to the apparent absence of bridging and a relatively larger number of hydrophilic moieties, capable of hydration lubrication, exposed to the solution when mucin adsorbed to the HB mica substrate. This investigation suggests that this mucin has the potential for efficient lubrication up to moderate pressures (and over the range of (low) sliding speeds examined) on both hydrophilic and hydrophobic surfaces.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. )

Present Addresses

School of Chemical Engineering, The University of Queensland, Brisbane, Qld 4072 Australia.

’ ACKNOWLEDGMENT We thank the Biological and Biotechnology Research Council (U.K.), Unilever plc, the Charles W. McCutchen Foundation, the European Research Council, and the Petroleum Research Fund (grant 45694-AC7) for financial support; Dr. Nir Kampf and Mr. Gilad Silbert for STAI samples; and Dr. Wuge Briscoe, Dr. Susan Perkin, Dr. Yael Dror, and the Feel & Function team of Unilever Discovery for helpful discussions and suggestions. ’ REFERENCES (1) Allen, A.; Hutton, D. A.; Pearson, J. P.; Sellers, L. A. Mucus Glycoprotein Structure, Gel Formation and Gastrointestinal Mucus Function. In Mucus and Mucosa; Ciba Foundation Symposium 109; Pitman: London, 1984. (2) Lang, T.; Hansson, G. C.; Samuelsson, T. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16209–16214. (3) Gipson, I. K. Exp. Eye Res. 2004, 78, 379–388. (4) Round, A. N.; Berry, M.; McMaster, T. J.; Stoll, S.; Gowers, D.; Corfield, A. P.; Miles, M. J. Biophys. J. 2002, 83, 1661–1670. (5) Loomis, R. E.; Prakobphol, A.; Levine, M. J.; Reddy, M. S.; Jones, P. C. Arch. Biochem. Biophys. 1987, 258, 452–464. (6) Slomiany, B. L.; Murty, V. L. N.; Piotrowski, J.; Slomiany, A. Gen. Pharmacol. 1996, 27, 761–771. (7) Cao, X.; Bansil, R.; Bhaskar, K. R.; Turner, B. S.; LaMont, J. T.; Niu, N.; Afdhal, N. H. Biophys. J. 1999, 76, 1250–1258. (8) Sheehan, J. K.; Carlstedt, I. Biochem. J. 1984, 221, 499–504. (9) Davies, J. R.; Hovenberg, H. W.; Linden, C.-J.; Howard, R.; Richardson, P. S.; Sheehan, J. K.; Carlstedt, I. Biochem. J. 1996, 313, 413–439.

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