Monika Wasilewska

Characterization of globular protein solutions by dynamic light scattering, electrophoretic mobility, and viscosity measurements.

In this work, physicochemical properties of two globular proteinsbovine serum albumin (BSA) having a molecular weight of 67 kDa and human serum albumin (HSA) having a molecular weight of 69 kDawere characterized. The bulk characteristics of these proteins involved the diffusion coefficient (hydrodynamic radius), electrophoretic mobility, and dynamic viscosity as a function of protein solution concentration for various pH values. The hydrodynamic radius data suggested an association of protein molecules, most probably forming compact dimers. Using the hydrodynamic diameter and the electropheretic mobility data allowed the determination of the number of uncompensated (electrokinetic) charges on protein surfaces. The electrophoretic mobility data were converted to zeta potential values, which allowed one to determine the isoelectric point (iep) of these proteins. It was found to be at pH 5.1 for both proteins, in accordance with previous experimental data and theoretical estimations derived from amino acid composition and p K values. To determine further the stability of protein solutions, dynamic viscosity measurements were carried out as a function of their bulk volume concentration for various pH values. The intrinsic viscosity derived from these measurements was interpreted in terms of the Brenner model, which is applicable to hard spheroidal particles. It was found that the experimental values of the intrinsic viscosity of these proteins were in good agreement with this model when assuming protein dimensions of 9.5 x 5 x 5 nm3 (prolate spheroid). The possibility of forming linear aggregates of association degree higher than 2 was excluded by these measurements. It was concluded that the combination of dynamic viscosity and dynamic light scattering can be exploited as a convenient tool for detecting not only the onset of protein aggregation in suspensions but also the form and composition of these aggregates.

Fibrinogen Adsorption on Mica Studied by AFM and in Situ Streaming Potential Measurements

Adsorption of fibrinogen from aqueous solutions on mica was studied using AFM and in situ streaming potential measurements. In the first stage, bulk physicochemical properties of fibrinogen and the mica substrate were characterized for various ionic strength and pH. The zeta potential and number of uncompensated (electrokinetic) charges on the protein surfaces were determined from microelectrophoretic measurements. Analogously, using streaming potential measurements, the electrokinetic charge density of mica was determined for pH range 3-10 and the NaCl background electrolyte concentration of 10(-3) and 10(-2) M. Next, the kinetics of fibrinogen adsorption at pH 3.5 and 7.4 in the diffusion cell was studied using a direct AFM determination of the number of molecules per unit area of the mica substrate. Then, streaming potential measurements were performed to determine the apparent zeta potential of fibrinogen-covered mica for different pH and ionic strength in terms of its surface concentration. A quantitative interpretation of these streaming potential measurements was achieved in terms of the theoretical model postulating a side-on adsorption of fibrinogen molecules as discrete particles. On the basis of these results, the maximum coverage of fibrinogen Θ close to 0.29 was predicted, in accordance with previous theoretical predictions. It was also suggested that anomalous adsorption for pH 7.4, where fibrinogen and the mica substrate were both negatively charged, can be explained in terms of a heterogeneous charge distribution on fibrinogen molecules. It was estimated that the positive charge was 12 e (for NaCl concentration of 10(-2) M and pH 7.4) compared with the net charge of fibrinogen at this pH, equal to -21 e. Results obtained in this work proved that the coverage of fibrinogen can be quantitatively determined using the streaming potential method, especially for Θ < 0.2, where other experimental methods become less accurate.

Structure of Fibrinogen in Electrolyte Solutions Derived from Dynamic Light Scattering (DLS) and Viscosity Measurements

Bulk physicochemical properties of bovine plasma fibrinogen (Fb) in electrolyte solutions were characterized. These comprised determination of the diffusion coefficient (hydrodynamic radius), electrophoretic mobility, and isoelectric point (iep). The hydrodynamic radius of Fb for the ionic strength of 0.15 M was 12.7 nm for pH 7.4 (physiological conditions) and 12 nm for pH 9.5. Using these values, the number of uncompensated (electrokinetic) charges on the protein N(c) was calculated from the electrophoretic mobility data. It was found that for physiological condition (pH 7.4, I = 0.15), N(c) = -7.6. For pH 9.5 and I = 10(-2), N(c) = -26. On the other hand, N(c) became zero independent of the ionic strength at pH 5.8, which was identified as the iep. Consequently, for pH < 5.8, N(c) attained positive values, approaching 26 for lower ionic strength and pH 3.5. It was also found from the hydrodynamic diameter measurements that for a pH range close to the iep, that is, 4-7, the stability of Fb suspension was very low. These physicochemical characteristics were supplemented by dynamic viscosity measurements, carried out as a function of Fb bulk volume concentration, for various pH values. Using these experimental data the contour length of 80 nm was predicted for Fb molecules in electrolyte solutions. On the other hand, the effective length of the molecule was 53-55 nm for physiological conditions, which suggested a collapsed state of the terminal chains. However, for the range of pH outside the iep, its effective length increased to 65-68 nm. This was interpreted in terms of a significant unfolding of the terminal chains of Fb caused by electrostatic repulsion. The effective charge, contour length, and effective length data derived in this work seem to be the first of this type reported in the literature.

Fibrinogen conformations and charge in electrolyte solutions derived from DLS and dynamic viscosity measurements

Hydrodynamic properties of fibrinogen molecules were theoretically calculated. Their shape was approximated by the bead model, considering the presence of flexible side chains of various length and orientation relative to the main body of the molecule. Using the bead model, and the precise many-multipole method of solving the Stokes equations, the mobility coefficients for the fibrinogen molecule were calculated for arbitrary orientations of the arms whose length was varied between 12 and 18 nm. Orientation averaged hydrodynamic radii and intrinsic viscosities were also calculated by considering interactions between the side arms and the core of the fibrinogen molecule. Whereas the hydrodynamic radii changed little with the interaction magnitude, the intrinsic viscosity exhibited considerable variation from 30 to 60 for attractive and repulsive interactions, respectively. These theoretical results were used for the interpretation of experimental data derived from sedimentation and diffusion coefficient measurements as well as dynamic viscosity measurements. Optimum dimensions of the fibrinogen molecule derived in this way were the following: the contour length 84.7 nm, the side arm length 18 nm, and the total volume 470 nm(3), which gives 16% hydration (by volume). Our calculations enabled one to distinguish various conformational states of the fibrinogen molecule, especially the expanded conformation, prevailing for pH<4 and lower ionic strength, characterized by high intrinsic viscosity of 50 and the hydrodynamic radius of 10.6 nm. On the other hand, for the physiological condition, that is, pH=7.4 and the ionic strength of 0.15M NaCl, the semi-collapsed conformation dominates. It is characterized by the average angle equal to =55°, intrinsic viscosity of 35, and the hydrodynamic radius of 10nm. Additionally, the interaction energy between the arms and the body of the molecule was predicted to be -4 kT units, confirming that they are oppositely charged than the central nodule. Results obtained in our work confirm an essential role of the side chains responsible for a highly anisotropic charge distribution in the fibrinogen molecule. These finding can be exploited to explain anomalous adsorption of fibrinogen on various surfaces.

Deposition of colloid particles on protein layers: fibrinogen on mica.

Colloid particle deposition was applied to characterize fibrinogen (Fb) monolayers on mica, which were produced by controlled adsorption under diffusion transport. By adjusting the time of adsorption and the bulk Fb concentration, monolayers of desired surface concentration were obtained. The surface concentration of Fb was determined directly by AFM enumeration of single molecules adsorbed over the substrate surface. It was proven that Fb adsorbed irreversibly on mica both at pH 3.5 and at pH 7.4 with the rate governed by bulk transport. The electrokinetic properties of Fb monolayers produced in this way were studied using the streaming potential method. The dependence of the apparent zeta potential of Fb monolayers was determined as a function of the coverage. It was shown that for pH 3.5 the initial negative zeta potential of the mica substrate was converted to positive for Fb coverage exceeding 0.16. On the other hand, for pH 7.4, the zeta potential of a Fb-covered mica remained negative for the entire coverage range. The charge distribution in Fb monolayers was additionally studied using the colloid deposition method, in which negatively and positively charged polystyrene latex particles (ca. 800 nm in diameter) were used. An anomalous deposition of negative latex particles on substrates exhibiting a negative zeta potential was observed. Results of these experiments were quantitatively interpreted in terms of the fluctuation theory assuming that adsorption sites consisted of two and three Fb molecules, for pH 3.5 and 7.4, respectively. These results suggested that for pH 7.4, the distribution of charge on Fb molecules was heterogeneous, characterized by the presence of positive patches, whereas the average zeta potential was negative, equal to -19 mV. The utility of the colloid deposition method for studying Fb monolayers was further demonstrated in deposition experiments involving positive latex particles. It was shown that for a rather broad range of fibrinogen coverage, both the positive and the negative latex particles can adsorb on surfaces covered by Fb, which behaved, therefore, as superadsorbing surfaces. It was also concluded that the colloid deposition method can be used to determine the Fb bulk concentration for the range inaccessible for other methods.

Mechanisms of fibrinogen adsorption at solid substrates at lower pH.

Adsorption of fibrinogen was theoretically studied using the three-dimensional random sequential adsorption (RSA) model. Fibrinogen molecule shape was approximated by the bead model considering the presence of flexible side arms. Various cases were considered inter alia, the side-on adsorption mechanisms and the simultaneous side-on/end-on adsorption mechanism. The latter mechanisms is pertinent to fibrinogen adsorption at lower pH (below isoelectric point of 5.8) where the entire molecule is positively charged. Extensive calculations enabled one to determine the jamming surface concentration (coverage) of molecules adsorbed under the side-on and end-on orientations as well as the total coverage. For the simultaneous side-on/end-on model the maximum surface concentration was 7.29 × 10(3) μm(-2) corresponding to the protein coverage of 4.12 mg m(-2) (without considering hydration). Additionally, the surface blocking functions for different adsorption regimes were determined and analytically approximated for the entire range of coverage by the interpolating polynomials. Using these blocking functions, fibrinogen adsorption kinetics for diffusion controlled transport conditions was evaluated. Comparison of these theoretical results with experimental data was made. It was demonstrated that the simultaneous side-on/end-on model properly reflects the maximum coverage of fibrinogen adsorbed on latex particles determined via the electrokinetic (electrophoretic mobility) and AFM measurements. Also, streaming potential measurements of fibrinogen adsorption kinetics on mica were successfully interpreted in terms of this model. The theoretical results derived in this work have implications for basic science providing information on mechanisms of anisotropic protein adsorption.

Mechanisms of fibrinogen adsorption at the silica substrate determined by QCM-D measurements.

Adsorption kinetics of fibrinogen at a silica substrate was thoroughly studied in situ using the QCM-D method. Because of low dissipation, the Sauerbreys equation was used for calculating the wet mass per unit area (wet coverage of the protein). Measurements were done for various bulk suspension concentrations, flow rates and pHs. These experimental data were compared with the theoretical dry coverage data derived from the solution of the mass transfer equation. In this way, the hydration functions and water factors of fibrinogen monolayers were quantitatively evaluated for various pHs. In the case of pH 7.4 and ionic strength of 0.15 M, the hydration function changed from 0.75 to 0.6 for the dry coverage Γ(d) equal to 0 and 4 mg m(-2), respectively. Interestingly, for pH 7.4 and 4.5 (ionic strength of 10(-2) M) a minimum of the hydration function appeared at Γ(d) ca. 2 mg m(-2). Analytical polynomial expressions were formulated for the interpolation of the experimental results. By using the hydration functions, the fibrinogen adsorption/desorption runs derived from QCM-D measurements were converted to the Γ(d) vs. the time relationships. This allowed to precisely determine the maximum coverage that varied between 1.2 mg m(-2) at pH 3.5 and 4.2 mg m(-2) at pH 7.4 for ionic strength of 0.15 M. These results agree with theoretical modeling and previous experimental data derived by using ellipsometry, OWLS and TIRF. Various fibrinogen adsorption mechanisms were revealed by exploiting the maximum coverage data whose validity was also confirmed by the dissipation vs. the dry mass relationships. Beside significance to basic science, these results enable to develop a robust technique, based on the QCM-D measurements, suitable for precisely determining the dry mass of protein monolayers adsorbed under various physicochemical conditions.

Mechanisms of fibrinogen adsorption at solid substrates.

The aim of this work was to critically review recent results pertinent to fibrinogen adsorption at solid/electrolyte interfaces with the emphasis focused on a quantitative analysis of these processes in terms of the electrostatic interactions. Accordingly, in the first part, the primary chemical structure of fibrinogen is analyzed. Physicochemical data pertinent to the bulk properties derived from hydrodynamic, dynamic light scattering and micro-electrophoretic measurements aided by theoretical modeling are discussed. Possible conformations and the effective charge distribution over the fibrinogen molecule for various pH an ionic strength are defined, especially the semi-collapsed conformation prevailing at physiological conditions. Adsorption kinetics of fibrinogen at hydrophilic and hydrophobic (polymer modified) substrates determined by various techniques is described. Adsorption at polymeric carrier particles, pertinent to immunological assays, studied in terms of electrokinetic and concentration depletion methods, are also considered. The reversibility of adsorption, fibrinogen molecule orientations and maximum coverages are thoroughly discussed. The stability of fibrinogen monolayers formed at these carrier particles in respect to pH and ionic strength cyclic changes is also discussed. In the final section interactions and deposition of model colloid particles on fibrinogen monolayers are analyzed which allows one to derive valuable information about molecule orientations. Based on the physicochemical data, adsorption kinetics and colloid particle deposition measurements, probable adsorption mechanisms of fibrinogen on solid/electrolyte interfaces are defined.

Fibrinogen monolayer characterization by colloid deposition.

Colloid particle deposition was applied to characterize bovine and human fibrinogen (Fb) monolayers on mica produced by controlled adsorption under diffusion transport at pH 3.5. The surface concentration of Fb was determined by AFM enumeration of single molecules adsorbed over the substrate surface. The electrokinetic properties of Fb monolayers for various ionic strength were studied using the in situ streaming potential measurements. It was shown that Fb adsorbs irreversibly on mica for a broad range of ionic strength of 4 × 10(-4) to 0.15 M, NaCl. The overcharging of initially negative mica surface occurred for fibrinogen surface concentrations higher than 1400 μm(-2). The orientation of fibrinogen molecules in the monolayers was evaluated by the colloid deposition method involving negatively charged polystyrene latex microspheres, 820 nm in diameter. An anomalous deposition of negative latex particles on substrates exhibiting a negative zeta potential was observed, which contradicts the mean-field DLVO predictions. Measurable deposition was observed even at low ionic strength where the minimum approach distance of latex particles to the interface exceeds 70 nm (for 6 × 10(-4) M NaCl). This confirms that, at this pH, fibrinogen molecules adsorb end-on on mica assuming extended conformations with the positive charge located mostly in the end part of the αA chains. This agrees with previous experimental and theoretical results discussed in the literature (Santore, M. M.; Wertz Ch. F. Protein spreading kinetics at liquid-solid interfaces via an adsorption probe method. Langmuir 2005, 21, 10172-10178 (experimental); Adamczyk, Z.; Barbasz, J.; Cieśla, M.; Mechanisms of fibrinogen adsorption at solid substrates. Langmuir, 2011, 25, 6868-6878 (theoretical)). This unusual latex deposition on Fb monolayers was quantitatively interpreted in terms of the model developed in ref 55 (Jin, X.; Wang, N. H. L.; Tarjus, G.; Talbot, J. Irreversible adsorption on nonuniform surfaces: the random site model. J. Phys. Chem. 1993, 97, 4256-4258). It was concluded that the colloid deposition method is an efficient tool for revealing protein adsorption mechanisms at solid/electrolyte interfaces.

Revealing fibrinogen monolayer conformations at different pHs: electrokinetic and colloid deposition studies.

Adsorption mechanism of human fibrinogen on mica at different pHs is studied using the streaming potential and colloid deposition measurements. The fibrinogen monolayers are produced by a controlled adsorption under diffusion transport at pH of 3.5 and 7.4. Initially, the electrokinetic properties of these monolayers and their stability for various ionic strength are determined. It is shown that at pH 3.5 fibrinogen adsorbs irreversibly on mica for ionic strength range of 4×10(-4) to 0.15 M. At pH 7.4, a partial desorption is observed for ionic strength below 10(-2) M. This is attributed to the desorption of the end-on oriented molecules whereas the side-on adsorbed molecules remain irreversibly bound at all ionic strengths. The orientation of molecules and monolayer structure is evaluated by the colloid deposition measurements involving negatively charged polystyrene latex microspheres, 820 nm in diameter. An anomalous deposition of negative latex particles on substrates exhibiting a negative zeta potential is observed. At pH 3.5 measurable deposition of latex is observed even at low ionic strength where the approach distance of latex particles exceeded 70 nm. At pH 7.4 this critical distance is 23 nm. This confirms that fibrinogen monolayers formed at both pHs are characterized by the presence of the side-on and end-on oriented molecules that prevail at higher coverage range. It is also shown that positive charge is located at the end parts of the αA chains of the adsorbed fibrinogen molecules. Therefore, it is concluded that the colloid deposition method is an efficient tool for revealing protein adsorption mechanisms at solid/electrolyte interfaces.