Salt Concentration and pH-Dependent Adsorption of Two Polypeptides on Planar and Divided Alumina Surfaces. In Situ IR Investigations C. M. Pradier,* V. Humblot, L. Stievano, C. Me ´thivier, and J. F. Lambert Laboratoire de Re ´ actiVite ´ de Surface, CNRS UMR 7609, UniVersite ´ Pierre et Marie Curie, 4 place Jussieu, Case 178, 75252 Paris Cedex 05, France ReceiVed July 27, 2006. In Final Form: December 12, 2006 The adsorption of proteins is the first process to take place when a solid is immersed in a biological fluid; though not yet thoroughly understood at a molecular level, this process is also known to be strongly influenced by the presence of salt in solution or by pH changes. In the present work, poly-L-glutamic acid (PG) and poly-L-lysine (PL) were selected to mimic the behavior of some protein fragments. Their adsorption was investigated by infrared spectroscopy in various modes, both on planar and on divided (powder) surfaces of aluminum oxide. These two peptides were shown to have different behaviors when adsorbed from solutions with or without CaCl 2 and at various pH values. Polarization modulation-reflection absorption infrared spectroscopy, applied in a special cell designed to characterize the solid surface in contact with the liquid, enabled the observation of the influence of pH and salts upon polypeptide adsorption. At pH values higher than 5 and in the presence of CaCl 2 in solution, a net increase of the PG adsorbed amount is observed, whereas no such effect could be detected for PL. Specific interactions between the COO - groups on the side chains and the surface, or between those of two different molecules, was inferred. Interestingly, similar conclusions could be drawn for the surface of alumina powders contacted with solutions of PG and PL and characterized by attenuated total reflectance IR. This work demonstrates the potential for IR investigations of solid oxide-liquid interfaces combining the study of planar and finely divided surfaces. 1. Introduction The mechanism of protein aggregation in a biological medium has been widely studied for its importance in various domains like separation, blood coagulation, biofilm growth, food, or in vivo applications. In particular, poly-L-Lysine (PLL or PL) and poly-L-glutamic acid (PLGA or PG) have often been used as protein models because their molecular weights are close to that of several common proteins. They have contrasting chemical structures: PL has basic amine groups on its side chain, whereas PG has carboxylic acid groups (see Scheme 1). They have been previously used to illustrate and study attractive or repulsive long-range interactions in colloidal solutions as a function of pH, 1 but they have been used also for their prospective applications as the layer-by-layer deposition of PL and PG is a well-explored route to the obtention of nanostructured films. 2-7 Studies of the topic, however, usually focus on the global properties of multilayer films, while the initial step of polyelectrolyte adsorption on the substrate is less well understood. It has been known for a long time that divalent cations, and in particular calcium ions, favor protein aggregation in solution 8 but the reason for that remains somewhat controversial with the direct bridging between negatively charged sites in protein molecules and Ca 2+ cations being often invoked 9 but never demonstrated at a molecular level. Caseinate solutions were studied in the presence of monovalent or divalent cations; in addition to an effect on the solution viscosity, divalent ions rapidly induced protein aggregation. 10 Several studies reported compara- tive investigations of the respective roles of monovalent and divalent cations. Although monovalent ions, such as Na + or K + , do increase the viscosity of a protein solution, the influence on aggregation is always significantly stronger with divalent ions. The ion strength is highly important in calcium binding to proteins; there is a competition between Ca 2+ and Mg 2+ , both having similar affinity for the albumin binding sites regardless of their different sizes. 11 On the other hand, the turbidity of a soy protein isolate was higher with Ca 2+ than with Mg 2+ at similar concentrations indicating Ca 2+ binding selectivity. 12 The interaction between proteins in solution and solid surfaces shows phenomena that are similar to those observed in protein aggregation. For instance, the adsorption of proteins on hydro- phobic media has been investigated in the presence of salts at various concentrations to understand the operation of gradient elution chromatography. 13 In NaCl solutions, the amount of adsorbed BSA on a polymeric membrane decreased when the salt concentration increased (50-150 mM), which was attributed to shielded electrostatic interactions with the solid phase. 14 A model of this elution behavior of proteins on a hydrophobic medium, based on a two-state protein (hydrated or not), was recently proposed by Chen and Sun, leading to protein adsorption * Corresponding author. E-mail: pradier@ccr.jussieu.fr. (1) Watson, G. S.; Blach, J. A.; Cahill, C.; Nicolau, D. V.; Pham, D. K.; Wright, J.; Myhra, S. Biosens. Bioelectron. 2004, 19, 1355. (2) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3422. (3) Elbert, D. L.; Herbert, C. B.; Hubbell, J. A. Langmuir 1999, 15, 5355. (4) (4) Zhi, Z. L.; Haynie, D. T. Macromolecules 2004, 37, 8668. (5) Bouldemais, F.; Frisch, B.; Etienne, O.; Lavalle, P.; Picart, C.; Ogier, J.; Voegel, J. C.; Schaaf, P.; Egles, C. Biomaterials 2004, 25, 2003. (6) Haynie, D. T.; Zhang, L.; Rudra, J. S.; Zhao, W. H.; Zhong, Y.; Palath, N. Biomacromolecules 2005, 6, 2895. (7) Haynie, D. T. J. Biomed. Mater. Res. B 2006, 78, 243. (8) Barbut, S.; Foegeding, E. A. J. Food Sci. 1993, 5, 867. (9) Roefs, S. P. F. M.; Peppelman, H. A. Food Colloids: Fundamentals of Formulation; Royal Society of Chemistry: Cambridge, UK, 2001; Chapter 12. (10) Carr, A. J.; Munro, P. A.; Campanella, O. H. Int. Dairy J. 2002, 12, 487. (11) Pedersen, K. O. Scan. J. Clin. Lab. InVest. 1972, 29, 427. (12) Molina, M. I.; Wagner, J. R. Food Res. Int. 1999, 32, 135. (13) Oscarsson, S.; Karsnas, P. J. Chromatogr., A 1998, 803, 83. (14) Hunter, A. K.; Carta, G. J. Chromatogr., A 2001, 930, 79. Scheme 1. (a) Schematic Representation of PG; (b) Schematic Representation of PL 2463 Langmuir 2007, 23, 2463-2471 10.1021/la062208p CCC: $37.00 © 2007 American Chemical Society Published on Web 02/03/2007