Interaction of proteins with spherical polyelectrolyte brushes in solution as studied by small-angle x-ray scattering

We use small-angle x-ray scattering (SAXS) as a tool to study the binding of proteins to spherical polyelectrolyte brushes (SPB) in situ. The SPB consists of a solid core of ∼ 100 nm diam onto which long polyelectrolyte chains [poly(styrene sulfonic acid, PSS) and poly(acrylic acid, PAA)] have been densely grafted. The proteins used in this investigation, Bovine Serum Albumine (BSA) and Bovine Pancreatic Ribonuclease A (RNase A), adsorb strongly to these SPB if the ionic strength is low despite their negative charge. Virtually no adsorption takes place at high ionic strength. SAXS demonstrates that both proteins are distributed within the brush. The findings reported here give further evidence that the strong adsorption of proteins to SPB is due to the “counterions release forces”: The patches of positive charge on the surface of the proteins become multivalent counterions of the polyelectrolyte chains. Thus, a concomitant number of co- and counterions is thereby released and the entropy of the entire system is increased. The repulsive Coulombic interaction as well as the steric repulsion between the proteins and the brush layer are counterbalanced by this effect. The data discussed here demonstrate that the adsorption of proteins in SPB presents a new principle for the immobilization of proteins.

Figure 1

Schematic representation of the spherical polyelectrolyte brushes investigated herein. Two different types of brushes are investigated here: Quenched brushes where poly(styrenesulfonate) chains (PSS) are grafted to the surface (system Q-SPB), and annealed brushes where poly(acrylic acid) chains are affixed to the surface of the poly(styrene) core particles (system A-SPB). The brush thickness $L$ is a function of the ionic strength within the solution [17, 19].

Figure 2

Analysis of the SAXS intensity of free RNase A in aqueous solution. The weight fraction of RNase A is 0.0056. The crosses display the measured scattering intensity whereas the solid line gives the optimal fit by Guinier’s law [Eq. (1)]. The plot shows that Guinier’s law gives a good description of the measured intensities up to $q=1.4{\mathrm{nm}}^{-1}$. The intercept of the solid line yields the molecular weight of the dissolved protein. The value derived from this figure $(13.910\mathrm{g}∕\mathrm{Mol})$ is in excellent agreement with the theoretical value $(13.700\mathrm{g}∕\mathrm{Mol})$.

Figure 3

SAXS intensity of the quenched polyelectrolyte brush Q-SPB (weight fraction: 0.045). The circles give the measured intensity and the solid line shows the optimal fit (see text). The dashed line refers to the contribution to $I\left(q\right)$ deriving from the fluctuations of the brush layer affixed to the surface of the particles (see Fig. 1). The inset presents the excess electron density $\rho \left(r\right)-{\rho }_M$ as the function of the radial distance $r$ which has been obtained from this fit.

Figure 4

Comparison of the SAXS intensities of the spherical polyelectrolyte brush Q-SPB (circles), of the same system loaded with RNase A (quadrangles; 408 mg RNase per g of the carrier particle), and of the free RNase A (crosses; see Fig. 2). Both the scattering curve of the loaded and of the unloaded brush particles has been normalized to the weight fraction. The intensity of the free RNase A has been scaled to the total concentration of the protein adsorbed on the brush particles. The inset shows an enlarged view of the region of smallest angles. Here the shift of the maxima to smaller $q$ values upon absorption of RNase A is clearly seen.

Figure 5

Optimal fit of the SAXS intensity of the quenched polyelectrolyte brush Q-SPB onto which RNase A has been adsorbed. Crosses display the scattering intensity of the unloaded particles whereas circles give the intensity of the loaded particles. The amount of adsorbed RNase A is 408 mg per g of the carrier particle. Both curves have been normalized to the weight fraction of the carrier particles. The inset gives the radial distribution of the excess electron density of the brush particle (thin line) and of the brush particle $+$ RNase (thick line). See text for further explanation.

Figure 6

Locus of adsorption of RNase inside of the brush layer of the quenched spherical polyelectrolyte brush Q-SPB. Circles give the measured scattering intensity of the loaded particles (see Fig. 5). The thin line in the inset gives the radial distribution of the unloaded particle whereas the thick line shows the distribution used for the calculation of the intensity (solid line). In this model, a preferential adsorption near the surface of the core particle was assumed. The obtained fit (solid line) shows poor agreement with the experimental data displayed by circles. Comparing this result to Fig. 5 demonstrates that this model can be ruled out. See text for further explanation.

Figure 7

Adsorption of BSA onto the annealed spherical polyelectrolyte brush A-SPB. The crosses give the scattering intensity of the unloaded particles whereas the circles refer to 296 mg BSA per g of the carrier particles. Both intensities are normalized to the weight fraction of the carrier particles. The inset gives the respective fits of the radial distribution of the excess electron densities. The triangles mark the intensity of an aqueous solution of free BSA scaled up to match the weight concentration of the adsorbed BSA molecules.

Figure 8

Adsorption of BSA onto the annealed spherical polyelectrolyte brush A-SPB. The open squares mark the scattering intensity obtained for the unloaded brush. The crosses refer to 296 mg per g of the carrier particle while the circles refer to 1116 mg per g carrier particle. All intensities have been normalized to the weight fraction of the carrier particles. The inset gives the respective fits of the radial distribution of the excess electron densities.

Figure 9

Schematic representation of the spherical polyelectrolyte brushes loaded with protein. See text for further explanation.

Figure 10

Scheme of the model used for the calculation of the scattering function of the composite system consisting of the SPB and adsorbed protein. See text for further explanation.

Figure 11

Calculation of the scattering cross section of the composite system consisting of the SPB and adsorbed protein. The circles mark the exact $I\left(q\right)$ obtained by the simulation. The triangles mark $I\left(q\right)$ deriving from a ensemble of uncorrelated spheres. The short-dashed line gives the intensity of a core shell system in which the small spheres only constitute a shell of additional electron density around the large sphere. The long-dashed line shows $I\left(q\right)$ of the large sphere in the center. The solid line marks the sum of the core-shell calculation (short-dashed line) and the intensity of the free small spheres (triangles). The inset shows the full $q$ range to demonstrate that the intensity of the small spheres is governing the range of higher scattering angles. See text for further explanation.