Association between Hydrophobically Modified Polyanions and Negatively Charged Bovine Serum Albumin C. Tribet,* I. Porcar, P. A. Bonnefont, and R. Audebert ² Laboratoire de Physico-chimie Macromole ´ culaire, UniVersite ´ Paris 6, CNRS URA 278, ESPCI, 10 rue Vauquelin, 75231 Paris Cedex 05, France ReceiVed: September 15, 1997; In Final Form: December 4, 1997 Attractive interactions between negatively charged bovine serum albumin (BSA) at pH 7 and 9 and poly- (sodium acrylate) were obtained by substituting a small fraction of acrylic units with alkylacrylamide units. Using light scattering, equilibrium dialysis, and viscometry, we investigated, in dilute solution, the association between BSA and two sets of modified polyacrylates of mean molecular weight 5000 and 150 000, respectively. The formation of complexes was revealed by pronounced increases of the scattering depending on the hydrophobicity of the synthetic polymer. It was not observed with entirely hydrophilic polyacrylates under the same conditions. In the case of long polyacrylates, the apparent hydrodynamic radius of the complexes was slightly larger than that of the free polymer. The polydispersity in size of the complexes seemed low. In the case of short polyacrylates, the complexation can be depicted as the “adsorption” of several polymer chains per protein. In contrast, complexes with long polyacrylates contain a single chain that accommodates several proteins. Introduction Research on the interactions between polymers and proteins has received considerable attention with respect to both practical and fundamental interests of such systems at the boundary between polymer/polymer and polymer/colloid mixtures. 1-16 In the field of biochemistry, these systems can help to model vital phenomena such as protein-mediated polynucleotide behaviors (e.g., DNA/histone collapse). Owing to their interaction with proteins, poly(acrylic acid)s were also found to be antiviral agents. 2 On the other hand, several applications were suggested, such as enzyme stabilization 3 or protein isolation and purification by coprecipitation with synthetic polyelectrolytes or polyam- pholytes. 4,5 Since the pioneering work of Morawetz, 6 a huge majority of these investigations were focused on the formation of soluble or insoluble complexes between a polyelectrolyte and a soluble globular protein. In the latter systems, the Coulombic origin of the associations and the similarities to the usual polyelectrolyte complexes (polyanions/polycations) are now recognized. Only a few papers report the possible influence of driving forces other than Coulombic. At low pH, hydrogen bonds were assumed to explain the observed attraction between pepsin and poly(ethylene glycol). 7 Akiyoshi and colleagues reported the formation of supramolecular assemblies between soluble pro- teins and self-aggregates of pullulan grafted with cholesterol dangling groups. 8,9 They suggested two possible driving forces of the association: a direct participation of the cholesterol groups (hydrophobic association) and hydrogen-bond formation with the backbone of the pullulan. Finally, little attention has been paid to the importance of hydrophobicity even though it was several times assumed to play a major role. 8-11 Among water-soluble proteins studied in the presence of polyelectrolytes, bovine serum albumin (BSA) is one of the more hydrophobic. Owing to the prime importance of BSA in the control of osmotic pressure in vivo and the transport and storage of nutriments or drugs, the structure of the protein and its association with a tremendous number of moleculesssuch as fatty acids, aromatic compoundsswere extensively investi- gated. 17 It can also form complexes with polyanions and polycations, the stability of which is strongly dependent on pH. As shown by several authors, 6,11,12-14 polycations are bound to BSA only above its isoelectric point (pI ≈ 4.3-4.9), when the total charge of the protein is negative. In contrast, complexes with polyanions not only can be formed below the pI but also can be formed a few pH units above it. Under these conditions, a total protein charge up to -25 does not hinder the association with poly(vinyl sulfonate) for instance. 12 On the basis of modification of the intrinsic fluorescence of BSA, Teramoto et al. 11 proposed that at high pH, polyanions interact specifically with binding site II of the protein. Dubin et al.’s measurements by light scattering led to a similar conclusion. The latter group claims the existence of a nonuniform charge distribution and positive charge patches just above the pI, despite a total charge on the order of -10 to -25. This assumption was proposed in the past to explain the adsorption of proteins on ion-exchange resins. 18 It is also supported by electrostatic-potential calcula- tions as a function of pH on RNase, a protein of known structure. 15 Above pH 9, however, all the authors agree that complexes between BSA and polyanions disappear completely. At such a high pH value, the global Coulombic repulsion between the partners must oppose the local attraction with possible residual charge patches or binding sites. In the present paper, we aim at a systematic approach of hydrophobically driven association between a set of modified polyacrylates of adjusted hydrophobicities and bovine serum ² R. Audebert died while the manuscript was being written. He initiated our research on proteins/polyamphiphile interactions. His faith and deter- mination have greatly contributed to the launching and development of the present work. * To whom correspondence should be addressed. Fax: (33) 1 40 79 46 40. E-mail: christophe.tribet@espci.fr. 1327 J. Phys. Chem. B 1998, 102, 1327-1333 S1089-5647(97)03022-8 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/27/1998