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J. Phys. Chem. B 1998, 102, 7906-7909
Formation of Complexes between Protein Particles and Long Amphiphilic Polymers: Binding Isotherms versus Size and Surface of the Particles Iolanda Porcar,*,† Pierre Gareil,‡ and Christophe Tribet† Laboratoire de Physico-Chimie des Polyme` res UMR 7615, ESPCI, CNRS, UniVersity Paris 6, 10 Rue Vauquelin, 75231 Paris Cedex 05, France, and Laboratoire d’Electrochimie et de Chimie Analytique, CNRS UMR 7575, ENSCP, 11 Rue Pierre et Marie Curie, 75231 Paris Cedex 05, France ReceiVed: June 17, 1998
Typical isotherms of protein bindingshere three serum albuminssto hydrophobically modified polyacrylates (HMPA) were obtained by separation of free proteins from bound ones using capillary electrophoresis and the frontal analysis method. The main qualitative features of this association were anticooperativity, high sensitivity to the hydrophobicity of HMPAs, no sensitivity to the size of the proteins, and subtle differences related to small variations in primary structure. Keeping the structure of the protein surface unmodified, monomers and covalent dimers of the protein were compared. In terms of molar ratio, isotherms appeared to be not markedly dependent on the protein size. On the contrary, despite their similarity in terms of molecular weight, tertiary structure, surface, and binding to fatty acids, human and bovine serum albumins show marked differences in the composition of complexes. Short-range interactions, at the surface of the particles, dominate the association with long HMPA.
Introduction In many industrial fields, the addition of macromolecules enables controlling of rheology and stabilizing various colloidal dispersions,1 preferably in water as a “green” solvent. The interactions between synthetic macromolecules and particles, such as pigments, micelles,2,3 vesicles,4 or proteins,5 have been shown to modify the macroscopic behaviors profoundly, which results in demixing, gelation, or stabilization of dispersions. For instance, a protective polymer layer surrounding an enzyme can slow down aging processes and enhance thermal stability.6 Protein/polyelectrolyte complexes have also been extensively studied with the aim of protein purification by selective precipitation7 mainly focused on Coulombic interactions. Hydrophobicity can, however, play a major role in these waterbased systems.8 To this point of view, the use of proteins as model amphiphilic particles offers interesting opportunities for studying the physical chemistry of the association. The advantages of proteins include their monodispersity in size, their well-defined structure down to the scale of angstrom, and the access to a variety of very similar particles through the use of proteins from different species. Serum albumin, a multifunctional transport protein, is especially the principal carrier of fatty acids and other hydrophobic molecules in circulating plasma.9 Its structure has been recently resolved at the angstrom level.10 The studies of the interactions between small molecules and this protein have provided prolific binding data11 as well as useful information about locations and characteristics of different binding sites. The interactions exerted between this protein and macromolecules have also attracted much attention with regard to the understanding of many biological systems as well as the immobilization of proteins in biotechnology.12 However, most of the studies so * Corresponding author. Fax: (33) 1 40 79 46 40. E-mail: iolanda.
[email protected]. † CNRS UMR 7615. ‡ CNRS UMR 7575.
far have dealt with the formation of complexes with polyelectrolytes, stressing the main role of the Coulombic forces in the association. Only a small number of studies involving polymers with hydrophobic groups have addressed the importance of hydrophobic interactions.13 To our knowledge, the influence of the protein state (monomer, homodimer, oligomers) in the interaction with polyelectrolytes has not yet been regarded, although it is likely to reveal the sensitivity of the binding to size effects. In this study we report on the interaction between hydrophobically modified poly(sodium acrylate) (HMPA) and serum albumins (monomer or dimer bovine (BSA), human (HSA)). Binding isotherms were obtained using frontal analysis continuous capillary electrophoresis (FACCE), a method recently applied by Dubin et al. to protein/polyelectrolyte complexes.14 The comparison between BSA monomer and dimer enables us to discuss size effects and the number of binding sites per particle. On the other hand, the comparison between HSA and BSA emphasizes the sensitivity to minor structural differences located at the surface of closely related particles. Two polymers differing in their molar percentage of dangling groups were implemented in order to ensure that the results being obtained do not correspond to a specific behavior of one HMPA. Material and Methods Materials. Bovine serum albumin, monomer and dimer, at respectively 98 and 90% purity, and human albumin at 99% purity, were purchased from Sigma (St. Louis, MO). Poly(acrylic acid), of claimed molecular weight (Mw) 150 000, was supplied by Polysciences (Warrington, PA). Gel permeation chromatography measurements performed in NaNO3 solution gave a number average Mw of 130 000 and a polydispersity index of 4 for the sodium polyacrylate. The reaction for the synthesis of the hydrophobically modified poly(acrylic acids) has been described previously.15 They were obtained in the sodium salt form, as
S1089-5647(98)02662-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/12/1998
Complexes of Protein Particles and Long Polymers
where x is the molar modification ratio and R is an alkyl chain with y carbon atoms (y ) 12 or 18). In the following, a polymer sample containing x mol % of CyH2y+1 dangling groups, randomly anchored to the precursor, is denoted 150-xCy. Aqueous stock solutions of protein were daily prepared to prevent bacterial growth and dialyzed against water to remove the possible presence of small oligopeptides or additional salts. Polymer stock solutions were prepared at least 18 h before use. Sample mixtures were made up from the stock solutions and kept at room temperature 1 h at least before study. Capillary Electrophoresis. Experiments were carried out with an Applied Biosystems (ABI) Model 270A instrument equipped with a UV detector (Santa Clara, CA). Bare silica capillaries of 50 µm × 45 cm (effective length, 25 cm) from Supelco (Bellefonte, PA) were used. Data acquisition was performed using a Spectra-Physics Model SP4400 Integrator (San Jose, CA). The experiments were conducted at a constant voltage of 10 kV and at 25 °C. The detection was performed at 279 nm, where only the proteins absorb. The running electrolyte was 30 mM sodium borate buffer, pH 9.2. It was filtered prior to use through a 0.45 µm membrane (Millipore). Mesityl oxide (0.1% (v/v) in borate buffer) was used as a neutral marker. The capillary was flushed daily with 0.1 M NaOH for 10 min, followed by a water rinse for 5 min, and finally was allowed to equilibrate with the run buffer for 5 min. Frontal analysis experiments were initiated by immersing the inlet of the capillary in the sample vial. A positive voltage was then applied at the inlet of the capillary to achieve continuous sample introduction and a simultaneous separation process. According to their mobilities, the sample species were detected as two discrete and adjacent plateaus (see Figure 1). The first one corresponded to the free protein, whereas the second one was representative of the mixture continuously introduced. The concentration of the free protein was determined from the height of the first plateau through a plateau height calibration that was acquired by injecting known concentrations of neat protein before the sample runs. The bound BSA concentrations were derived for each sample by subtracting the free from the total protein concentration. Note that since the method is based on the continuous sampling, and the introduction and separation of the sample are integrated into the same process, the binding equilibrium is not perturbed, and there is no dilution of the proteins as they migrate through the columns. Then, the first plateau gives the “true” free protein concentration. Moreover, the height of the second plateau, being proportional to the total protein concentration, allowed us to check the absence of adsorption or dilution of BSA during the run. After each electrophoretic run, the capillary was rinsed with the run buffer for 4 min to restore the surface state of the capillary that might have been altered by protein adsorption. Then, a short plug of the mesityl oxide solution was hydrodynamically injected into the capillary (negative pressure of 16.7 kPa for 1 s) to check the electroosmotic flow (meo ) 6.7 × 10-4 cm2 V-1 s-1 under these conditions), thus ensuring that the surface conditions stayed unchanged at the start of each run. Polyacrylamide Gel Electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments were performed using a single sided vertical gel electrophoresis system (Owl Scientific, Woburn, MA). Poly-
J. Phys. Chem. B, Vol. 102, No. 40, 1998 7907
Figure 1. Schematics of the FACCE method and the electropherogram obtained for 1% BSA + 1% 150-3C18 in 30 mM borate buffer, pH 9.2.
acrylamide stacking (5% T) and resolving (8% T) gels were prepared according to the Laemmli formulation described elsewhere.16 The gels were stained with Coomassie brilliant blue. β-Mercaptoethanol was not added to the denaturing buffer in order to avoid the disruption of disulfide bridges holding the dimer structure. The same amount of protein was loaded in each lane. For the mixtures containing HMPA, the polymer content was high enough to achieve a majority of bound protein. After 1 h of incubation, samples were diluted and SDS added before being loaded into the gel. The molecular weight corresponding to the different bands was ascertained by comparing their location with those obtained with standard molecular weight markers previously run. Results and Discussion Binding Isotherms. Upon addition of BSA into dilute solutions of a long HMPA (
