Structural and Mechanical Properties of Thin Films of Bovine Submaxillary Mucin versus Porcine Gastric Mucin on a Hydrophobic Surface in Aqueous Solutions.

The structural and mechanical properties of thin films generated from two types of mucins, namely, bovine submaxillary mucin (BSM) and porcine gastric mucin (PGM) in aqueous environment were investigated with several bulk and surface analytical techniques. Both mucins generated hydrated films on hydrophobic polydimethylsiloxane (PDMS) surfaces from spontaneous adsorption arising from their amphiphilic characteristic. However, BSM formed more elastic films than PGM at neutral pH condition. This structural difference was manifested from the initial film formation processes to the responses to shear stresses applied to the films. Acidification of environmental pH led to strengthening the elastic character of BSM films with increased adsorbed mass, whereas an opposite trend was observed for PGM films. We propose that this contrast originates from that negatively charged motifs are present for both the central and terminal regions of BSM molecule, whereas a similar magnitude of negative charges is localized at the termini of PGM molecule. Given that hydrophobic motifs acting as an anchor are also localized in the terminal region, electrostatic repulsion between anchoring units of PGM molecules on a nonpolar PDMS surface leads to weakening of the mechanical integrity of the films.

Proteolytic Degradation of Bovine Submaxillary Mucin (BSM) and Its Impact on Adsorption and Lubrication at a Hydrophobic Surface.

The effects of proteolytic digestion on bovine submaxillary mucin (BSM) were investigated in terms of changes in size, secondary structure, surface adsorption, and lubricating properties. Two proteases with distinctly different cleavage specificities, namely trypsin and pepsin, were employed. SDS-PAGE analysis with staining for proteins and carbohydrate moieties showed that only the unglycosylated terminal regions of BSM were degraded by the proteases. Size exclusion chromatography (SEC) and dynamic light scattering (DLS) analyses indicated that tryptic digestion mainly led to the reduction in size, whereas pepsin digestion rather caused an increase in the size of BSM. Less complete cleavage in terminal peptide regions by pepsin and subsequent aggregation were possibly responsible for the increased size. Far-UV circular dichroism (CD) spectra of the protease-treated BSM showed a slight change in the secondary structure owing to the removal of terminal domains, but the overall random coil conformation adopted by the central glycosylated domain remained dominant and essentially unchanged. Surface adsorption properties as characterized by optical waveguide lightmode spectroscopy (OWLS) showed that tryptic digestion of BSM resulted in a decrease in the adsorbed mass, but pepsin digestion led to a slight increase in the adsorbed mass onto a hydrophobic surface compared to intact BSM. This is in agreement with the partial preservation of peptide segments in the terminal regions after pepsin digestion as confirmed by SEC and DLS studies. Despite a contrast in the adsorbed amount of the protease-treated BSMs onto the surface, both proteases substantially deteriorated the lubricating capabilities of BSM at a hydrophobic interface. The present study supports the notion that the terminal domains of BSM are critical to the adsorption and lubricating properties of BSM at hydrophobic interfaces.

A simplified chromatographic approach to purify commercially available bovine submaxillary mucins (BSM).

In this study, a simple purification protocol is developed to reduce the bovine serum albumin (BSA) content in commercially available bovine submaxillary mucin (BSM). This involved purification of the BSM by one-column anion-exchange chromatography protocol resulting in BSM with greatly reduced BSA content and homogeneously distributed size, and in a high yield of ∼43% from BSM as received from the manufacturer. The purity and composition of commercially acquired BSM were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry, which verified that BSA is the most abundant nonmucinous protein component. The purification effect was evident from a significantly altered circular dichroism (CD) spectrum of BSM after anion-exchange chromatography.

Influence of impurities and contact scale on the lubricating properties of bovine submaxillary mucin (BSM) films on a hydrophobic surface.

Lubricating properties of bovine submaxillary mucin (BSM) on a compliant, hydrophobic surface were studied as influenced by impurities, in particular bovine serum albumin (BSA), at macro and nanoscale contacts by means of pin-on-disk tribometry and friction force microscopy (FFM), respectively. At both contact scales, the purity of BSM and the presence of BSA were quantitatively discriminated. The presence of BSA was responsible for higher frictional forces observed from BSM samples containing relatively larger amount of BSA. But, the mechanisms contributing to higher friction forces by BSA were different at different contact scales. At the macroscale contact, higher friction forces were caused by faster and dominant adsorption of BSA into the contacting area under a continuous cycle of desorption and re-adsorption of the macromolecules from tribostress. Nevertheless, all BSMs lowered the interfacial friction forces due to large contact area and a large number of BSM molecules in the contact area. At the nanoscale contact, however, no significant desorption of the macromolecules is expected in tribological contacts because of too small contact area and extremely small number of BSM molecules involved in the contact area. Instead, increasingly higher friction forces with increasing amount of BSA in BSM layer are attributed to higher viscosity caused by BSA in the layer. Comparable size of AFM probes with BSM molecules allowed them to penetrate through the BSM layers and to scratch on the underlying substrates, and thus induced higher friction forces compared to the sliding contact on bare substrates.

Adsorption and nanowear properties of bovine submaxillary mucin films on solid surfaces: influence of solution pH and substrate hydrophobicity.

The adsorption and mechanical stability of bovine submaxillary mucins (BSM) films at solid-liquid interfaces were studied with respect to both substrate hydrophobicity and solution pH. Dynamic light scattering revealed a single peak distribution in neutral aqueous solution (pH 7.4) and a small fraction with enhanced aggregation was observed in acidic solution (pH 3.8). Both substrate hydrophobicity and solution pH were found to affect the spontaneous adsorption of BSM onto solid surfaces; BSM adsorbed more onto hydrophobic surfaces than hydrophilic ones, and adsorbed more at pH 3.8 than at pH 7.4. Thus, the highest "dry" adsorbed mass was observed for hydrophobic surfaces in pH 3.8 solution. However, a highest "wet" adsorbed mass, i.e. which includes the solvent coupled to the film, was observed for hydrophobic surfaces at pH 7.4. The mechanical stability of the films was studied at the nanoscale with an atomic force microscope operated in the friction force spectroscopy mode. Results revealed that BSM films formed on hydrophobic substrates were stronger than those formed on hydrophilic ones. Moreover, the film stability also depended on the ambient pH and stronger films were formed at acidic conditions, i.e. close to the BSM isoelectric point.

Thermostability of bovine submaxillary mucin (BSM) in bulk solution and at a sliding interface.

Thermostability of bovine submaxillary mucin (BSM) was studied in terms of its structure, hydrodynamic size, surface adsorption, and lubricating properties in the temperature range of 5-85°C. The overall random coil structure of BSM showed a gradual loosening with increasing temperature as characterized by circular dichroism (CD) spectroscopy, but this change was fully reversible upon lowering temperature. Extended heating up to 120 min at 80°C did not make any appreciable changes in the structure of BSM when it was cooled to room temperature. The hydrodynamic size of BSM, as studied by dynamic light scattering (DLS), showed a slight increase after heating at high temperature (80°C). Optical waveguide lightmode spectroscopy (OWLS) studies showed facile adsorption of BSM onto poly(dimethylsiloxane) (PDMS) surface (>180 ng/cm(2)) at room temperature due to its amphiphilic characteristics. Adsorbed mass of BSM was noticeably reduced after heating at 80°C, possibly resulting from its aggregation. BSM showed excellent lubricity at self-mated sliding contacts between PDMS at room temperature or lower (friction coefficient≈0.02), even when BSM solution was pre-heated up to 120 min at 80°C. Gradual degradation of lubricity of BSM was observed with increasing temperature, but it was also reversibly recovered with decreasing temperature. Structural and functional stability of BSM against heating is proposed to originate from heavy glycosylation and lack of higher degree of protein structure in BSM.