Charge and Interfacial Behavior of Short Side-Chain Heavily Glycosylated Porcine Stomach Mucin Gleb E. Yakubov,* ,† Aristeidis Papagiannopoulos, ‡ Elodie Rat, † and Thomas A. Waigh ‡ Unilever Corporate Research, Colworth Park, Sharnbrook, Beds MK44 1LQ, United Kingdom and Biological Physics, Department of Physics and Astronomy, University of Manchester, Manchester M60 1QD, United Kingdom Received June 28, 2007; Revised Manuscript Received September 20, 2007 The current accepted model for high-molecular-weight gastric mucins of the MUC family is that they adopt a polydisperse coil conformation in bulk solutions. We develop this model using well-characterized highly purified porcine gastric mucin and examine the molecules’ charge and interfacial adsorption. “Orthana” mucin has short side-chains, low levels of sialic acid residues, and includes minute amounts of cystine residues that can be responsible for the self-polymerization of mucin. Atomic force microscopy and transmission electron microscopy are used to examine the interfacial behavior of the mucin and clearly demonstrate the existence of discrete spherical subunits within the mucin molecules, with sizes in agreement with static light scattering, dynamic light scattering, and  potential measurements. Furthermore images indicate the combs are assembled with a beads on a string conformation; the daisy chain model. Zeta potential measurements establish the polyampholyte nature of the mucin molecules, which is used to explain their adsorption behavior on similarly charged surfaces. 1. Introduction Mucins are glycoproteins that are found ubiquitously in animal organisms. 1–3 These mixed protein/carbohydrate mol- ecules have bulk functional properties that give rise to well- optimized viscoelasticities in many biological fluids such as saliva 4,5 or the gelled structure of gastric mucosa linings. 2,3,6–10 Furthermore, at biological interfaces, they greatly facilitate boundary lubrication, reducing damage to soft motile tissues. 11–15 There are a wide variety of examples where mucins demon- strate a dual role functioning in the bulk as well as at the interface. Tooth pellicle is a complex formation comprised of salivary mucins and proline-rich proteins; 13,16–18 oral, GI tract, and respiratory airways have mucosa linings whose behavior is closely related to cilia motility, 3 tear secretions contain ocular mucins, 19,20 and in synovial joints, the active component of the lubricating layer on cartilage surfaces is a mucin-like biopolymer lubricin. 15,21–23 Lubrication is known to be associated with the ability to form a dense hydrated layer at the surfaces of practically any chemistry, 12,16,24–28 including hydroxyapatite, 29 metals, and semiconductors. 17,30 This unique ability to adsorb is associated with the block-copolymer structure of mucins 31,32 that comprise glycosylated hydrated comb-brushes, polyampholyte domains with both positively and negatively charged amino acids, interspersed with domains that are rich in hydrophobic amino acids. 2 When adsorbed, the hydrophilic glycosylated comb sections stretch out into aqueous media due to favorable interactions with water, 6,12,24,33–38 thereby adopting a brush- like architecture even if the whole mucin molecule is adsorbed flat (Figure 1). The interaction between such mucin layers is characterized by a repulsive net force that includes both entropic (depletion) and electrostatic parts. [Comb side-chains of mucins are often negatively charged due to the presence of sialic acid residues (pK a ≈ 2) 8 and/or sulfate groups (pK a ≈ 1) 39 .]; 9,12,15,24,30,34,40–45 both factors are known to be crucial in biolubrication. 14,14,46–49 The actual conformation of the adsorbed layers depends on the radius of gyration (R g ) of the mucin, the type of protein backbone, and the oligosaccharide sequence. Mucins and mucous glycoproteins can adopt either brush-like (h > R g , where h is the layer thickness) 15 or flat architectures (h < R g ). 6,9,24,34,36,40,50–54 The flat layers are reported to correspond either to adsorbed coils, 55,56 extended threads (found prima- rily for ocular mucins 19,20,57–62 ), or complex architectures. 50,53,63,64 In the bulk, hydrophobic domains facilitate mucin self- association, and this aggregation mechanism stands alongside intramolecular disulphide bridging 2,3,5 or calcium-mediated * Author to whom correspondence should be addressed. E-mail: gleb.yakubov@unilever.com. † Unilever Corporate Research. ‡ Biological Physics, Department of Physics and Astronomy, University of Manchester. Figure 1. Conformation of adsorbed layers of mucins: (a) mucin brush; (b) flat conformation of adsorbed mucins with comb side chain forming a secondary brushlike layer. Green is the carbohydrate, red is positively charged protein, and blue is negatively charged protein. Biomacromolecules 2007, 8, 3791–3799 3791 10.1021/bm700721c CCC: $37.00  2007 American Chemical Society Published on Web 11/03/2007