DOI: 10.1021/la902267c 4901 Langmuir 2010, 26(7), 4901–4908 Published on Web 02/25/2010 pubs.acs.org/Langmuir © 2010 American Chemical Society Sequential Adsorption of Bovine Mucin and Lactoperoxidase to Various Substrates Studied with Quartz Crystal Microbalance with Dissipation Tobias J. Halthur,* ,†,§ Thomas Arnebrant, † Lubica Macakova, ‡ and Adam Feiler ‡ † Biomedical Laboratory Science and Technology, Faculty of Health and Society, Malm€ o University, SE-205 06 Malm€ o, Sweden, and ‡ YKI, Institute for Surface Chemistry, Box 5607, SE-114 86 Stockholm, Sweden. § Present address: Colloidal Resource AB, Box 124, SE-221 00 Lund, Sweden. Received June 24, 2009. Revised Manuscript Received February 12, 2010 Mucin and lactoperoxidase are both natively present in the human saliva. Mucin provides lubricating and antiadhesive function, while lactoperoxidase has antimicrobial activity. We propose that combined films of the two proteins can be used as a strategy for surface modification in biomedical applications such as implants or biosensors. In order to design and ultilize mixed protein films, it is necessary to understand the variation in adsorption behavior of the proteins onto different surfaces and how it affects their interaction. The quartz crystal microbalance with dissipation (QCM-D) technique has been used to extract information of the adsorption properties of bovine mucin (BSM) and lactoperoxidase (LPO) to gold, silica, and hydrophobized silica surfaces. The information has further been used to retrieve information of the viscoelastic properties of the adsorbed film. The adsorption and compaction of BSM were found to vary depending on the nature of the underlying bare surface, adsorbing as a thick highly hydrated film with loops and tails extending out in the bulk on gold and as a thinner film with much lower adsorbed amount on silica; and on hydrophobic surfaces, BSM adsorbs as a flat and much more compact layer. On gold and silica, the highly hydrated BSM film is cross-linked and compacted by the addition of LPO, whereas the compaction is not as pronounced on the already more compact film formed on hydrophobic surfaces. The adsorption of LPO to bare surfaces also varied depending on the type of surface. The adsorption profile of BSM onto LPO-coated surfaces mimicked the adsorption to the underlying surface, implying little interaction between the LPO and BSM. The interaction between the protein layers was interpreted as a combination of electrostatic and hydrophobic interactions, which was in turn influenced by the interaction of the proteins with the different substrates. Introduction It is well-known that the mucosal membranes present in the respiratory tract and the gastrointestinal tract act as a protective barrier (being antiadhesive and yet acting as a selective mediator) between the external environment and the body. The outermost layer of these membranes is the highly hydrated (approximately 90% water content) 1,2 viscous mucus in which the mucin proteins act as a scaffold. Although mucin by itself forms a viscous hydrogel, it is believed that the interaction of mucin with smaller proteins and salts affects the structure of mucin and gives mucus its exceptionally high viscosity, 3,4 and also affects its lubricating effect in saliva. 5,6 Mucin is a diverse goup of proteins that vary in size and composition depending on the source (human or animal) and type (membrane or secreted). 2 Mucin consists of large macromolecule monomers with a polypeptide backbone which contains one or more heavily glycosylated domains, rich in serine and threonine residues which serve as anchoring points for the oligosaccha- ride side chains. These glycosylated domains are separated by short “naked” nonglycosylated patches. The carbohydrate weight fraction is substantial, and values between 68 and 81% have been reported. 7,8 Because of the high concentration of oligosaccha- rides, the glycosylated domains are hydrophilic; they are also negatively charged due to the presence of sialic acid residues and sometimes also due to the presence of sulfated sugars. The “naked” patches and the end terminals (which are also nongly- cosylated regions) on the other hand contain a normal distri- bution of amino acid residues and are mostly hydrophobic. Furthermore, cystein residues are located in the end terminals, which provides for intra- and intermolecular disulfide bonds. According to a model for human cervical mucin first proposed by Carlstedt and Sheehan, several mucin monomers are linked together by disulfide bonds in a linear chain. This particular mucin carried four monomers on average, and each monomer contained four to five glycosylated domains. 9 Lactoperoxidase is a cationic enzyme, with an isoelectric point of 8.3 and a net charge of þ4 eq/molecule at pH 7.0, that catalyzes the oxidation of halides and pseudohalides by the aid of hydrogen peroxide, and generates highly reactive products with a wide antimicrobial activity. 10 The polypeptide backbone consists of a single polypeptide chain of 612 amino acids with a molecular mass of 78.5 kDa and a carbohydrate content of about 10%. It also contains 14 cystein residues, out of which 12 are involved in internal disulfide bridges and two are free sulfhydryl groups. 11 Lactoperoxidase has been found to be very stable and keeps its *To whom correspondence should be addressed. E-mail: tobias@ halthur.com. (1) Matthes, I.; Nimmerfall, F.; Sucker, H. Pharmazie 1992, 47, 609–613. (2) Strous, G. J.; Dekker, J. Crit. Rev. Biochem. Mol. Biol. 1992, 27, 57–92. 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