Barbara Jachimska

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.

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.

Which physical and structural factors of liposome carriers control their drug-loading efficiency?

The correlation between structural and physical properties of lipid membrane and its drug-loading efficiency were studied. The properties of bilayer were altered by incorporation of several lipidic modifiers: cholesterol, oleic acid, methyl oleate, and pegylated lipid. By using the molecular probe technique it was demonstrated that the membrane properties, such as micropolarity, microviscosity and free volume were considerably changed by incorporation of the modifiers. The partitioning of two different porphyrins between the bulk aqueous phase and the modified liposomes was studied using the fluorescence methods, and liposome-binding constants were determined. It was found that cholesterol reduced the partitioning of both porphyrins into liposomal bilayer. On the contrary, the incorporation of methyl oleate and pegylated lipid causes a pronounced increase in the value of the binding constants of both porphyrins. It was concluded that the free volume rather than hydrophobicity of bilayer is a governing factor in the solute partitioning into lipid bilayers.

Influence of adsorption kinetics and bubble motion on stability of the foam films formed at n-octanol, n-hexanol and n-butanol solution surface

Abstract Lifetimes of single bubbles at surface of n-octanol solutions of various concentrations were determined for two different locations of the surface in respect to the capillary orifice at which the bubbles were formed. It was found that when solution surface was located ‘far’ (L=39.5 cm) the average lifetimes of bubbles were shorter than that ones at the solution surface located ‘close’ (L=4 cm) to the point of the bubbles formation. The bubble departing from the capillary orifice has the adsorption coverage determined by the mutual ratio of velocity of its formation and kinetics of surfactant adsorption. The degree of surfactant coverage over the surface of the growing bubble was calculated assuming that the surfactant adsorption at the surface of the departing bubble is governed by convective–diffusion. It was demonstrated that for the time of bubble growth tgrowth=0.16 s encountered in our experiments the departing bubbles had practically equilibrium coverages in n-butanol and n-hexanol solutions, while in n-octanol solutions the adsorption coverage was much smaller than the equilibrium one. Thus, unlike for shorter chain n-alkanols, even for the location ‘close’ the partially non-symmetrical film is formed when in n-octanol solutions the bubble arrives at the free solution surface. For the location ‘far’, the bubble motion through the solution causes further disequilibration of the surfactant coverage over its surface. The bubble reaches the solution surface with its upstream pole practically devoid of surfactant and the fully non-symmetrical film is formed. Such fully non-symmetrical film is less stable than the film formed at the location ‘close’. Our results show that the lifetime of the foam film formed by a single bubble arriving at the solution surface strongly depends on ‘history’ of the bubble formation and motion.

Properties of polyethylene glycol supported tetraarylporphyrin in aqueous solution and its interaction with liposomal membranes.

5,10,15,20-Tetrakis(4-hydroxyphenyl)porphyrin was functionalized by covalent attachment of poly(ethylene glycol) (PEG) chains of various molecular weights, 350, 2000, and 5000 Da. The properties of PEG-functionalized tetraarylporphyrins in aqueous solution and their interactions with liposomes have been studied. Electronic absorption spectroscopy, dynamic light scattering, atomic force microscopy, and fluorescence quenching were used to monitor aggregation of porphyrin chromophores and behavior of the attached PEG chains in the aqueous solution. The tendency for aggregation of porphyrin chromophores in aqueous solution and the efficiency of fluorescence quenching by KI decrease with increasing length of PEG chain linked to the porphyrin ring. The experimental results indicate that polymer clusters are present in aqueous solution of all pegylated porphyrins. The interactions between the pegylated porphyrins and phosphatidylcholine liposomes in the aqueous solution were studied using the fluorescence methods. The apparent binding constants of porphyrin chromophores to liposomes were determined. The degree of binding was found to be dependent on the molecular weight of the attached polymer.

Bovine Serum Albumin Adsorption at a Silica Surface Explored by Simulation and Experiment

Molecular details of BSA adsorption on a silica surface are revealed by fully atomistic molecular dynamics (MD) simulations (with a 0.5 μs trajectory), supported by dynamic light scattering (DLS), zeta potential, multiparametric surface plasmon resonance (MP-SPR), and contact angle experiments. The experimental and theoretical methods complement one another and lead to a wider understanding of the mechanism of BSA adsorption across a range of pH 3-9. The MD results show how the negatively charged BSA at pH7 adsorbs to the negatively charged silica surface, and reveal a unique orientation with preserved secondary and tertiary structure. The experiments then show that the protein forms complete monolayers at ∼ pH6, just above the proteins isoelectric point (pH5.1). The surface contact angle is maximum when it is completely coated with protein, and the hydrophobicity of the surface is understood in terms of the simulated protein conformation. The adsorption behavior at higher pH > 6 is also consistently interpreted using the MD picture; both the contact angle and the adsorbed protein mass density decrease with increasing pH, in line with the increasing magnitude of negative charge on both the protein and the surface. At lower pH < 5 the protein starts to unfold, and the adsorbed mass dramatically decreases. The comprehensive picture that emerges for the formation of oriented protein films with preserved native conformation will help guide efforts to create functional films for new technologies.

'Static' and steady-state foams from ABA triblock copolymers: influence of the type of foam films

Abstract The role of foam films in stability of foams formed from solutions of F108 and P85 (polyethylene oxide–polypropylene oxide–polyethylene oxide (PEO–PPO–PEO)) ABA block copolymers is analyzed. The Π ( h ) isotherms of disjoining pressure are measured for single foam films, under conditions close to those in the foam to clarify the influence of the type of foam films, common thin; black and bilayer sterically stabilized films on foam stability. The experiments with single foam films are performed with the thin liquid film-pressure balance technique (TLF-PBT) developed on the basis of the microinterferometric method of Sheludko–Exerowa. Foam pressure drop technique (FPDT) is applied to determine foam lifetime at constant pressure. This technique involves applying an increased and regulated pressure in the foam liquid phase (plateau borders) which makes it possible to evaluate the effect of foam film type on foam stability under strictly defined capillary pressure values. A combined pneumatic–mechanical method is used to study foam properties under dynamic conditions. Effect of the polymer type and its concentration, velocities of the mixer rotations and gas flow on the foam volume formed and foam stability are studied. The foam lifetime versus applied pressure ( τ p /Δ P ) dependences along with Π ( h ) isotherms indicate that the surface forces (electrostatic, van der Waals and steric), respectively, the type of foam films, play a decisive role in stability of foams from ABA block copolymers. Under identical conditions the copolymer F108 forms more stable foams than those obtained from the P85 solutions. A parallelism between the properties of dynamic foams and single microscopic foam films is observed. When thicker single foam films are formed (from F108 solutions) the steady-state foam column is higher.

Lysozyme adsorption at a silica surface using simulation and experiment: effects of pH on protein layer structure

Hen Egg White Lysozyme (HEWL) is a widely used exemplar to study protein adsorption on surfaces and interfaces. Here we use fully atomistic Molecular Dynamics (MD) simulations, Multi-Parametric Surface Plasmon Resonance (MP-SPR), contact angle and zeta potential measurements to study HEWL adsorption at a silica surface. The simulations provide a detailed description of the adsorption mechanism and indicate that at pH7 the main adsorption driving force is electrostatics, supplemented by weaker hydrophobic forces. Moreover, they reveal the preferred orientation of the adsorbed protein and show that its structure is only slightly altered at the interface with the surface. This provides the basis for interpreting the experimental results, which indicate the surface adsorbs a close-packed monolayer at about pH10 where the surface has a large negative zeta potential and the HEWL is positively charged. At higher pH, the adsorption amount of the protein layer is greatly reduced due to the loss of charge on the protein. At lower pH, the smaller zeta potential of the surface leads to lower HEWL adsorption. These interpretations are complemented by the contact angle measurements that show how the hydrophobicity of the surface is greatest when the surface coverage is highest. The simulations provide details of the hydrophobic residues exposed to solution by the adsorbed HEWL, completing the picture of the protein layer structure.

Human serum albumin encapsulated gold nanoclusters: effects of cluster synthesis on natural protein characteristics

The differences in the physiochemical properties between native Human Serum Albumin (HSA) and HSA encapsulated gold nanoclusters (HSA-AuNCs) are characterised. The light absorbance (UV-Vis), electrophoretic mobility, dynamic viscosity, density, hydrodynamic radius (DLS), absorption (QCM) and chemical bonding (XPS) characteristics of the molecules were studied. The UV-Vis and DLS data show the formation of large aggregates for HSA-AuNCs between pH 4-6 which is not observed for native HSA. This observation was further supported by QCM measurements showing a large increase in mass adsorbed at pH 6 between HSA and HSA-AuNCs. The DLS data also revealed a hydrodynamic diameter of 12 nm for HSA-AuNCs, nearly double that of 7 nm for native HSA at pH higher than 6, suggesting the formation of compact HSA-AuNCs dimers. The electrophoretic mobility data for both HSA-AuNCs and HSA were converted to zeta potentials. The zeta potential of HSA-AuNCs was seen to be more negative between pH 6-12, suggesting that the protein surface interacts with unreacted gold salt anions. Measurements of density and viscosity were also found to be in agreement with previous data suggesting that HSA-AuNCs form aggregates. XPS data also suggest that not all reactants are used up during the HSA-AuNC synthesis and positive side chains play a part in the initial synthesis stages. It was concluded that HSA-AuNCs most likely form dimers at natural and high pH. Between pH 4-6 HSA-AuNCs form very large aggregates limiting their use as fluorescent probes in this pH range. It was also found that the native characteristics of HSA are altered upon HSA-AuNC synthesis which needs to be taken into consideration when applying HSA-AuNCs as fluorescent probes in all fluorescent imaging and sensing.

Effects of motion in n-hexanol solution on the lifetime of bubbles at the solution surface

Lifetimes of single bubbles at the surface of a solution located at two different distances from the capillary orifice, at which the bubbles were formed, were determined for various n-hexanol concentrations. It was found that when the n-hexanol solution surface was located “far” (L = 39.5 cm) the average lifetimes of the bubbles were shorter than for those at the solution surface located “close” (L = 4 cm) to the point of the formation of the bubbles. The bubbles were formed in an identical manner, using the same capillary and the distance between the capillary and solution surface was the only different parameter in the measurements of the lifetimes of the bubbles at these two locations of the interface. This means that the “starting conditions” for the bubbles were identical in both series of experiments. Thus, the fact that the lifetimes of the bubbles at solution surface located far way from the capillary were shorter is an experimental confirmation of the correctness of the theoretical predictions that motion through the solution causes substantial lowering of the adsorption coverage on the upstream part of the floating bubble. When a bubble with lowered adsorption coverage at its upstream pole arrived at the solution surface located “far” then a nonsymmetrical foam film, with a lowered surfactant concentration at one of its interfaces, was formed. When the solution surface was “close”, the distance traveled by the bubble was not long enough for such a nonuniform distribution to develop; hence, a symmetrical foam film, with equilibrium or close-to-equilibrium adsorption coverages on its both interfaces, was formed at the solution surface. Such symmetrical foam films are more stable and, therefore, the lifetimes of the bubbles were longer at the solution surface located close to the capillary orifice. Good agreement was found between the lifetimes of bubbles and the lifetimes of foam films as calculated from a simple theoretical model based on the velocity of thinning of the symmetrical and the fully nonsymmetrical foam film with one surface devoid of n-hexanol molecules.