Colloids and Surfaces A: Physicochem. Eng. Aspects 343 (2009) 118–126 Contents lists available at ScienceDirect Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa Calcium uptake by casein embedded in polyelectrolyte multilayer Lilianna Szyk-Warszy ´ nska a,∗ , Csilla Gergely b , Ewelina Jarek a , Frédéric Cuisinier c , Robert P. Socha a , Piotr Warszy ´ nski a a Institute of Catalysis and Surface Chemistry PAS, ut. Niezapomianjek 8, 30-239 Krakow, Poland b Groupe d’étude des semi-conducteurs Université Montpellier II, UMR 5650 CNRS, Place Eugéne Bataillon, 34095 Montpellier Cedex 05, France c UFR Odontologie, Université Montpellier I, 545 Avenue prof. Viala, 34193 Montpellier Cedex, France article info Article history: Received 30 September 2008 Received in revised form 5 January 2009 Accepted 30 January 2009 Available online 5 April 2009 Keywords: Casein Layer-by-layer Polyelectrolyte films Calcium binding Multilayers abstract The aim of our work was to investigate formation of polyelectrolyte multilayer films containing - and -casein. Since in neutral pH casein is negatively charged (as verified by electrophoretic mobility measure- ments) it has been used as a polyanionic layer for the film build-up. Casein containing films were formed on Si/SiO 2 wafers. The formation of the film was investigated by liquid cell ellipsometry. After the multi- layer films were formed they were contacted with solution containing calcium ions and changes in the film thickness were monitored. Additionally the surfaces of casein containing multilayers were analyzed with AFM for the structural changes within the films occurring after binding of calcium ions. Presence of calcium ions bound in the film was also monitored by XPS. We concluded that casein embedded in the polyelectrolyte multilayers preserves its ability to bind calcium ions. © 2009 Elsevier B.V. All rights reserved. 1. Introduction In recent years there has been an increased interest in intrinsi- cally unstructured proteins (IUP), i.e., proteins that in their natural state do not adopt stable, folded structures [1–4]. Proteins of this type, abundant in nature, play an important role in living organisms. However, despite their function is well understood, our knowledge concerning adsorption and self-organisation is rather limited. A characteristic feature of IUP’s is an open structure, which becomes preserved even after ligand binding. Casein is one of the most com- mon IUP’s. It is a phosphoprotein present in mammalian milk and its products, where it occurs in a micellar form made of four major types [5,6]:  s1 -,  s2 -, -, -casein.  s1 - and  s2 -caseins comprise 40% and 10%, respectively, of the whole casein content of milk and are usually called  s -caseins [7]. -casein comprise 38% of total casein contents [8,9]. The function of caseins is to store and transport bio-available metal ions (especially, Ca(II) and Mg(II)) by sequestering and transporting them from mother to the neonates [2,7,10,11]. In aqueous solution casein is surface active [11–14] and forms micellar aggregates. Single protein behaves as flexible, disordered, polyelectrolyte-like molecule [15], therefore, it should be easily integrated into polyelectrolyte films. Casein has ability to bind calcium ions and therefore, it can be used in biotechnology and ∗ Corresponding author. Fax: +48 12 4251923. E-mail address: ncszyk@cyf-kr.edu.pl (L. Szyk-Warszy ´ nska). in biomedical applications. Films containing caseins are used as paints, which may be diluted with water or adhesives for labeling of glass containers [16]. Materials covered with casein containing films can be also applied in diary industry for the prevention of calcium deposit formation. Molecular structures of two types of caseins used in our investi- gations are illustrated in Fig. 1. (s1)-casein has molecular weight (MW) 23,000. The chain is build from 199 amino acids with 17 proline residues. It has two hydrophobic regions, containing all the proline residues, separated by a polar region, which con- tains all but one of eight phosphate groups and is highly charged. Molecular diameter of (s1)-casein is 9 nm. (s2)-casein: MW 24,000; 207 residues, 10 prolines. Negative charges are concen- trated near N-terminus and positive charges near C-terminus. Both caseins can be precipitated at very low levels of calcium. Molec- ular weight of -casein is 24,000. It has 209 residues and 35 prolines. N-terminal region is highly charged, hydrophilic and C- terminal region is a hydrophobic. This amphiphilic protein acts like a detergent molecule. It is less sensitive to calcium precipi- tation. Its molecular diameter is 7.5nm. MW of -casein is 19,000, 169 residues with 20 prolines. This casein is very resistant to cal- cium precipitation and it stabilizes other proteins and their micellar aggregates [20–22]. Proline acts as a structural disruptor in the middle of regular sec- ondary structure elements such as alpha helices and beta sheets. However, proline is also commonly found as a first residues of an alpha helix and also in the edge strands of beta sheets. As proline lacks a hydrogen on the amino group, it cannot act as a hydrogen 0927-7757/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2009.01.038