Electrostatic Interactions between a Protein and Oppositely Charged Micelles Patrizia Andreozzi, ²,‡ Adalberto Bonincontro, ‡,§ and Camillo La Mesa* ,²,‡ Dipartimento di Chimica, SOFT-INFM-CNR Research Center, and CNISM-Dipartimento di Fisica, La Sapienza UniVersity, Rome, Italy ReceiVed: June 1, 2007; In Final Form: December 12, 2007 Micellar solutions made of a fully fluorinated surfactant, LiPFN, form water-soluble complexes with lysozyme in a wide concentration range. Such complexes are stabilized by electrostatic and, very presumably, double- layer interactions. The mixtures were investigated by combining electrophoretic mobility, DLS, and dielectric relaxation methods. The former gives information on the surface charge density of protein-micelle complexes and indicates that the resulting adducts retain a negative charge (i.e., charge neutralization is incomplete). The double-layer thickness of proteins, micelles, and protein-micelle complexes is also connected to the dielectric relaxation frequency. Changes in particle size (inferred by DLS), charge density, and double-layer thickness are closely interrelated to each other. A model was developed to quantify such properties. Introduction The interactions between association colloids and polymers are interesting subjects to the investigator, because of their relevance in many fundamental and applied aspects. Electrostatic interactions between the above objects are ubiquitous and control stabilization, ion binding, charge neutralization, coagulation, flocculation, and phase separation. From a biochemical view- point, interest is focused on the stabilization of colloid particles and on their coverage by proteins, 1,2 polysaccharides, 2,3 and polynucleotides. 4 These items are relevant when surface func- tionalization is required, as in the heparinization of surgical implants. 5 Proteins are peculiar block polyelectrolytes. Their properties and biochemical activities are sensitive to modifications in charge distribution, subsequent to adsorption onto surfaces. In some cases proteins lose their native conformations and denature. 6,7 Adsorption is concomitant to changes in surface charge density, σ, and in the electrical double layer thickness, 1/κ, of the particles onto which proteins adsorb. 8,9 Thus, the particle physical state, surface functionality, and conformation of an adsorbed biopolymer are strictly interrelated. From a fundamental viewpoint, micelles and vesicles are much more user-friendly than intrinsic colloids. Studies on such systems offer the opportunity to tune the particle concentration, their size, shape, and charge density. In addition, the soft surfaces peculiar to association colloids are plastic and very similar to biological tissues. That is why micelles and vesicles are used as adsorption sites for biopolymers. 10-16 This possibility opens the way to technological applications, including biomi- metic systems used in controlled release and transfection technologies. The electrochemical behavior of micelles and vesicles is related to the lower or upper limits of the electrical double layer theory, respectively. 17 Micelles and vesicles, in fact, refer to conditions where Hu ¨ckel (for small) or Smoluchowsky ap- proximations (for large particles) hold. Changes in size, or surface charge density, thus may help in clarifying the double- layer theory in a wide range of colloid particle sizes. In previous work we focused on protein binding onto large synthetic vesicles. 14,16 It is interesting, thus, to investigate the binding of small proteins onto micellar aggregates. In such a case, the protein and micelles are in the size range where Hu ¨ckel’s approximation holds. In this contribution the interactions between charged micelles and lysozyme are reported and investigation on particle size, electrophoretic mobility, and dielectric relaxation processes are discussed. The ternary system composed of water, lysozyme (LYSO), and lithium perfluorononanoate (LiPFN) is particularly appealing for the above purposes. 18 LiPFN micelles are char- acterized by small sizes and well-defined surface charge density. 19 The phase diagram of the water-LYSO-LiPFN system has been investigated. 20,21 In such a system molecular solutions, precipitates, protein-surfactant gels, micelle-protein complexes, and micellar solutions are observed, depending on the [protein/surfactant] charge ratio and absolute concentration. Because of their hydrophobic and oleophobic character, 22-25 fluorinated surfactants do not interact with the hydrophobic domains of the protein. Hence electrostatic interactions are dominant in a wide part of the phase diagram. LYSO and oppositely charged micelles exert a mutual electrostatic interac- tion, which is rationalized here. Experimental Section Materials. Chicken egg lysozyme (fraction V, Sigma) was purified as in previous work. 20 Its purity was controlled by the density, ionic conductivity, and viscosity of its aqueous solu- tions. 26 Lysozyme has eight nominal positive charges in the pH conditions used in this work (≈6.5). Unless otherwise indicated, all solutions were prepared in the absence of buffers or without controlling the ionic strength, to avoid drawbacks inherent to modifications in the double-layer thickness of the particles. Water was doubly distilled in an all-glass apparatus. The ionic conductivity of freshly prepared (carbon dioxide free) water, , is 0.7-1.0 µS cm -1 at 25.00 °C. * Corresponding author. Telephone: +39-06-49913707, +39-06-491694. Fax. +39-06-490631. E-mail: camillo.lamesa@uniroma1.it. ² Dipartimento di Chimica. ‡ SOFT-INFM-CNR Research Center. § CNISM-Dipartimento di Fisica. 3339 J. Phys. Chem. B 2008, 112, 3339-3345 10.1021/jp0742618 CCC: $40.75 © 2008 American Chemical Society Published on Web 02/27/2008