Quasihomogeneous nucleation of amyloid beta yields numerical bounds for the critical radius, the surface tension, and the free energy barrier for nucleus formation

Amyloid aggregates are believed to grow through a nucleation mediated pathway, but important aggregation parameters, such as the nucleation radius, the surface tension of the aggregate, and the free energy barrier toward aggregation, have remained difficult to measure. Homogeneous nucleation theory, if applicable, can directly relate these parameters to measurable quantities. We employ fluorescence correlation spectroscopy to measure the particle size distribution in an aggregating solution of Alzheimer's amyloid beta molecule (Abeta(1-40)) and analyze the data from a homogeneous nucleation theory perspective. We observe a reproducible saturation concentration and a critical dependence of various aspects of the aggregation process on this saturation concentration, which supports the applicability of the nucleation theory to Abeta aggregation. The measured size distributions show a valley between two peaks ranging from 5 to 50 nm, which defines a boundary for the value of the nucleation radius. By carefully controlling the conditions to inhibit heterogeneous nucleation, we can hold off nucleation in a 25 times supersaturated solution for at least up to 3 h at room temperature. This quasi-homogeneous kinetics implies that at room temperature, the surface energy of the Abeta/water interface is > or =4.8 mJ/m(2), the free energy barrier to nucleation (at 25 times supersaturation) is > or =1.93x10(-19) J, and the number of monomers in the nucleus is > or =29.     

Influence of the Secondary Interaction Energy Minimum on the Early Stages of Colloidal Aggregation

The pair interaction energy of charged colloidal particles in electrolyte solutions can exhibit a large barrier as well as a pronounced secondary minimum. We discuss the effect of a secondary energy minimum on aggregation kinetics by modeling irreversible dimer formation as a two-step process in which charged colloidal particles in electrolyte solutions first aggregate reversibly into the secondary minimum before they can cross the energy barrier. In the classical regime of slow aggregation, the secondary minimum is seen to have a pronounced effect if either the ionic strength of the solution is high (e.g., 0.1 M for particles of 150-nm radius) or particles are large (>/=350-nm radius for an ionic strength of 0.01 M). Under these conditions, our calculations predict a transient period of fast aggregation into the secondary minimum followed by slow primary aggregation. The aggregation in this second regime is found to take place at a lower rate than what would be expected in the absence of the secondary minimum or from an earlier linearized model for secondary aggregation. The crossover time between the two regimes strongly depends on the particle size but not on the particle concentration, which however determines the degree of aggregation reached within the fast regime. We also conclude that a previously observed severe discrepancy between measured and predicted aggregation rate constants for submicron particles is not due to the neglect of secondary aggregation in the theoretical treatment. Copyright 2000 Academic Press.     

Comparative DSC study of human and bovine serum albumin

The thermal denaturation process of bovine and human both fatty acid containing and fatty acid free albumins in aqueous solution was studied by use of differential scanning calorimetry. Human serum albumins were found to be more stable than their bovine counterparts. Fatty acid free albumins were characterized as generally less stable, more susceptible to aggregation, their unfolding endothermic transition was less cooperative and with the smaller degree of reversibility. Deconvolution analysis with using a non-two-state model with two component transitions showed essential differences in the thermodynamic parameters between all studied albumins, particularly regarding the high-temperature component transition.     

Aggregation Behavior of Poly(Acrylic acid-co-Octadecyl Methacrylate) and Bovine Serum Albumin in Aqueous Solutions

Polymer-protein complexing systems have been extensively studied because of their wide application in biomedicine and industry. Here, we studied the aggregation behavior of the hydrophobically associating water-soluble polymer poly(acrylic acid-co-octadecyl methacrylate) [P(AA-co-OMA)] prepared with nonionic surfactant as an emulsifier and bovine serum albumin (BSA) in aqueous solution. We identified the optimal composite conditions of P(AA-co-OMA) and BSA aqueous solution. We measured the zeta potential, dynamic light-scattering particle size, and surface tension of P(AA-co-OMA) and BSA mixed aqueous solution. The results showed that the aggregation behavior between the polymer and BSA relied mainly on the hydrophobic interactions between the molecules. In addition, the best compounding condition was 8 wt.% of P(AA-co-OMA) content. The structure of hydrophobically associating polymer P(AA-co-OMA) and its aggregation with BSA were characterized by Fourier-transform infrared spectroscopy. The infrared spectroscopy results identified the hydrogen bonding behavior of the amino and carboxyl groups between the polymer and BSA. This behavior was also confirmed using thermogravimetric analysis and differential scanning calorimetry. The thermal decomposition temperature and melting temperature of BSA changed before and after it was combined with the polymer. We measured the morphology of the polymer BSA aggregate with 8 % polymer content by transmission electron microscopy. The binding mechanism was investigated, as well.     

Light scattering study of complex formation between protein and polyelectrolyte at various ionic strengths

Formation of protein-polyelectrolyte complexes (PPCs) between bovine serum albumin (BSA) and potassium poly (vinyl alcohol) sulfate (KPVS) was studied at pH 3 as a function of ionic strength. Turbidimetric titration was employed by a combination of dynamic light scattering (DLS) and electrophoretic light scattering (ELS). The formal charge (Z(PPC)) of the resulting PPCs at different ionic strengths were estimated from ELS data by assuming the free draining and the non-free draining model. The radius of a BSA molecule in the complex was used in the former model for calculation of Z(PPC) with the Henry's equation, while in the latter case the hydrodynamic radius of a PPC particle determined from DLS was employed. The results obtained were compared with the Z(PPC) values calculated using a relation of Z(PPC)=n(b)Z(BSA)+alphaZ(KPVS), where Z(BSA) (> or =0) and Z(KPVS) (< or =0) denote the formal charge of BSA and KPVS, respectively. Moreover, n(b) is the number of bound proteins per complex composed of alpha polymer chains. It was suggested that the PPC between BSA and KPVS behaves as a free draining molecule during the electrophoresis, at least at a high ionic strength. Also suggested is that the PPC formation at low ionic strength follows a 1:1 stoichiometry in the charge neutralization.     

Synthesis and characterization of carbon nanotubes covalently functionalized with amphiphilic polymer coated superparamagnetic nanocrystals

The kinetic investigations of oxidation of tris(1,10-phenanthroline)iron(II) by oxone have been studied spectrophotometrically in phosphate buffer medium of pH 6.8, temperature 308 K, and ionic strength 0.25 mol L(-1). The reactions were also carried out in presence of globular transport protein, bovine serum albumin (BSA) having isoelectric point 4.9, anionic surfactant sodium dodecyl sulfate (SDS), and their mixtures. The critical aggregation concentration (CAC) and critical micelle concentration (CMC) of SDS in presence of BSA have been determined using conductivity and kinetic measurement techniques. The secondary structure of BSA was examined by Circular Dichroism (CD) measurement at 308 K. The helix nature of BSA decreases with increase of SDS concentration. The effect of pH on rate in presence of BSA is opposite to its absence, and the effect of urea on rate in presence of BSA indicates the denaturation of BSA. The results depict that amphiphile SDS interacts with BSA and different molecular events, for example, specific binding, cooperative binding, protein unfolding, and micelle formation act. Activation parameters of the reaction in different environments have been determined.     

Protein type effects on steady-state crossflow membrane ultrafiltration fluxes and protein transmission

The permeate fluxes and percent protein transmission were evaluated for steady-state crossflow ultrafiltration of two proteins of different composition: bovine serum albumin (BSA), containing fatty acid, and “fatty-acid-poor” BSA, from which most of the fatty acids had been removed (BSA/FAP). The influences of protein concentration up to 6.5 percent w/v, transmembrane pressure, ionic environment and membrane type (i.e. nominal molecular weight cut-off) were investigated. For both BSA and BSA/FAP, the fluxes and the protein transmission were dependent on the amount of salt present. The higher fatty acid content in the BSA apparently enhanced protein-protein interaction, resulting in a more cohesive and resistant fouling layer; permeate fluxes were lower with BSA/FAP than with BSA at otherwise corresponding operating conditions. A hysteresis behaviour of the flux (J)-transmembrane pressure (TMP) relationship was observed whenever the ultrafiltration unit was operated at a TMP less than some higher value to which the membrane previously had been exposed.     

Stability of bovine serum albumin at different pH

Summary The effect of pH on the thermal denaturation of BSA containing fatty acids was studied by use of differential scanning calorimetry (DSC). Thermal scanning of BSA aqueous solutions gave various types of DSC curves depending on the protein concentration and on the pH. The broad bimodal endothermic transition was suggested to be connected with loose protein structure in contradistinction to single peak for compact molecule structure. The propensity toward precipitation at pH conditions ranging from 3.8 to 5 was observed. A scan-rate independent and partly reversible behavior of the thermal heating of BSA was found. Deconvolution of DSC traces in non-two-state model with assumption of two- or three-component transition allowed to study the effect of pH on different parts of BSA molecule.     

A comparative study on specific and nonspecific interactions in bovine serum albumin: thermal and volume effects of halothane and palmitic acid

Interaction modes of halothane and palmitic acid with bovine serum albumin (BSA) were studied from the thermal and volumetric viewpoints. The thermal stability of BSA was increased by increasing both ligand concentrations. However, the stronger effect of palmitic acid than halothane on BSA was observed at lower concentrations irrespective of the pH-dependent BSA structure. On the other hand, the volume of BSA in the solution shrunk by adding halothane independent of its structure while it expanded by adding palmitic acid. The molar ratio of halothane to BSA at the effective concentration was not consistent with the binding numbers on human serum albumin determined from the X-ray analysis, whereas that of palmitic acid was in good agreement with the numbers. We judged from these facts that halothane is a nonspecific binder to BSA; by contrast, palmitic acid is a specific binder. The stabilization mechanisms of the BSA structure were also revealed.     

Thermal aggregation of bovine serum albumin studied by asymmetrical flow field-flow fractionation

The use of asymmetrical flow field-flow fractionation (AsFlFFF) in the study of heat-induced aggregation of proteins is demonstrated with bovine serum albumin (BSA) as a model analyte. The hydrodynamic diameter (d_h), the molar mass of heat-induced aggregates, and the radius of gyration (R_g) were calculated in order to get more detailed understanding of the conformational changes of BSA upon heating. The hydrodynamic diameter of native BSA at ambient temperature was ∼7 nm. The particle size was relatively stable up to 60 °C; above 63 °C, however, BSA underwent aggregation (growth of hydrodynamic diameter). The hydrodynamic diameters of the aggregated particles, heated to 80 °C, ranged from 15 to 149 nm depending on the BSA concentration, duration of incubation, and the ionic strength of the solvent. Heating of BSA in the presence of sodium dodecyl sulfate (1.7 or 17 mM) did not lead to aggregation. The heat-induced aggregates were characterized in terms of their molar mass and particle size together with their respective distributions with a hyphenated technique consisting of an asymmetrical field-flow fractionation device and a multi-angle light scattering detector and a UV-detector. The carrier solution comprised 8.5 mM phosphate and 150 mM sodium chloride at pH 7.4. The weight-average molar mass (M_w) of native BSA at ambient temperature is 6.6 × 10⁴ g mol⁻¹. Incubation of solutions with BSA concentrations of 1.0 and 2.5 mg mL⁻¹ at 80 °C for 1 h resulted in aggregates with M_w 1.2 × 10⁶ and 1.9 × 10⁶ g mol⁻¹, respectively. The average radius of gyration and the average hydrodynamic radius of the heat-induced aggregate samples were calculated and compared to the values obtained from the size distributions measured by AsFlFFF. For comparison static light scattering measurements were carried out and the corresponding average molar mass distributions of solutions with BSA concentrations of 1.0 and 2.5 mg mL⁻¹ at 80 °C for 1 h gave aggregates with M_w 1.7 × 10⁶ and 3.5 × 10⁶ g mol⁻¹, respectively.     

Supercritical fluid extraction of grape seed oil and subsequent separation of free fatty acids by high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of grape seed oil was performed to study the effect of various parameters such as pressure, temperature and the particle size of the sample on the yield and composition of oil using an analytical-scale SFE system. Then the extraction was scaled up by 125 times using a preparative SFE system under the optimized conditions of high pressure (30-40 MPa) and low temperature (35-40 degrees C) with medium particle size (20-40 mesh). The maximum yield of the oil can reach 6.2% with pure supercritical CO2 and 4.0% more oil can be obtained by adding 10% of ethanol as modifier. The unsaturated fatty acids (UFSs) make up about 70% in the oil on the basis of free fatty acids. The grape seed oil was then subjected to separation and purification for free fatty acids after saponification by high-speed counter-current chromatography coupled with evaporative light scattering detection (ELSD). The separation of 1.0 g of oil can yield about 430 mg pure linoleic acid at 99% purity. The fatty acids were analyzed by HPLC-ELSD.     

Improved gas chromatographic method for the determination of the individual free fatty acids in cheese using a capillary column and a PTV injector

Summary A gas chromatographic method with a capillary column and a programmed temperature vaporizer injector has been used to analyze the individual free fatty acids in cheese. The lipids were extracted from an acidified cheese slurry with diethyl ether and treated with tetramethylamonium hydroxide (TMAH) to convert the free fatty acids to tetramethylammonium soaps (TMA-soaps), which were subsequently pyrolyzed to methyl esters in the injector. Carrying out injection at the initial column temperature resulted in lower dispersion of the results, but the solvent front prevented quantitative determination of butyric and caproic acids, and an injector temperature of 300°C was therefore employed. Under the conditions tested, trimethylamine (tma) flash-off did not affect the determinations. The accuracy of the method improved at higher free fatty acid contents (coefficient of variation of 0.53% for a total free fatty acid content of 9000 mg/kg as opposed to 7.0% for a total free fatty acid content of 1400 mg/kg). The recovery rate for individual free fatty acids ranged between 91 and 103%.     

Precipitation of Zinc Sulfide Particles from Homogeneous Solutions

Zinc sulfide particles were homogeneously precipitated by thermal decomposition of thioacetamide in acidic aqueous solutions in a one-step process. The influence of the operating conditions (initial concentration of zinc ion and TAA) on the nucleation time and number concentration of the generated particles was investigated. The experimental results show that the model of homogeneous nucleation previously developed and successfully tested for silver particle generation by a chemical reduction method can also be applied to the formation of zinc sulfide particles by homogeneous precipitation. Furthermore, in the particle formation method in which the nucleation time t* can be measured, the particle number concentration n* can be predicted by the simple relation n*=1/(4pir*Dt*) (r* is the critical nucleus radius, and D the monomer diffusion coefficient). Thus the particle number concentration can be easily predicted even if the rate expression and the critical supersaturation concentration are unknown. Copyright 2000 Academic Press.     

Release characteristics of a free polyunsaturated fatty acid from an oily lymphographic agent

The release profiles of a free polyunsaturated fatty acid, alpha-linolenic acid, from solutions in an oily lymphographic agent Lipiodol-Ultra-Fluid (Lipiodol), to rabbit and human plasma, phosphate buffer solution (PBS), and PBS containing bovine serum albumin (BSA) were examined in vitro. The times required for 50% release of alpha-linolenic acid from Lipiodol were about 1 and 1.5 h in the rabbit and human plasma, respectively. Although only a slight amount of alpha-linolenic acid was released from Lipiodol to PBS after 24 h incubation at 37 degrees C, release was markedly enhanced by the addition of BSA to PBS. The amount of alpha-linolenic acid released from Lipiodol into PBS containing 5% BSA increased as the alpha-linolenic acid content in Lipiodol was increased. In all experiments, the release had stopped before all alpha-linolenic acid had been released. The prolongation of alpha-linolenic acid release from Lipiodol is considered a requisite for a selective anticancer effect of Lipiodol containing a free fatty acid on liver cancer.     

Impact of High Temperature on Ethanol Fermentation by Kluyveromyces marxianus Immobilized on Banana Leaf Sheath Pieces

Ethanol fermentation was carried out with Kluyveromyces marxianus cells at various temperatures (30, 35, 40, and 45 °C). Fermentation performance of the immobilized yeast on banana leaf sheath pieces and the free yeast were evaluated and compared. Generally, ethanol production of the immobilized and free yeast was stable in a temperature range of 30–40 °C. Temperature of 45 °C restricted yeast growth and lengthened the fermentation. The immobilized yeast demonstrated faster sugar assimilation and higher ethanol level in the fermentation broth in comparison with the free yeast at all fermentation temperatures. Change in fatty acid level in cellular membrane was determined to clarify the response of the free and immobilized yeast to thermal stress. The free cells of K. marxianus responded to temperature increase by increasing saturated fatty acid (C16:0 and C18:0) level and by decreasing unsaturated fatty acid (C18:1 and C18:2) level in cellular membrane. For fermentation at 40 °C with immobilized cells of K. marxianus, however, the changes were not observed in both saturated fatty acid (C16:0) and unsaturated fatty acid (C18:1 and C18:2) level.     

Configuration of bovine serum albumin adsorbed on polymer particles with grafted dextran corona

The configuration of BSA macromolecules adsorbed on the surfaces of poly(alkylcyanoacrylate) nanoparticles has been determined using small angle neutron scattering (SANS). The nanoparticles were made by anionic emulsion polymerization (AEP) and self-assembly of dextran–poly(isobutylcyanoacrylate) (PICBA) copolymers. They have a hydrophobic PICBA core and a hydrophilic dextran corona. In vivo, they are recognized by the macrophages of the mononuclear phagocyte system. The amount of BSA bound to the particles, at adsorption equilibrium, has been determined through immunodiffusion, immunoelectrophoresis, and SANS. For particles with a radius of 25.3 nm, the adsorption was found to saturate at 64 adsorbed BSA molecules per particle. The configuration of the adsorbed BSA molecules was determined from the SANS scattering curves, first at full contrast, and then at contrast match. Both experiments indicate that the BSA molecules are adsorbed on the PICBA core, in a flat configuration. This result may be important for understanding the in vivo opsonization mechanisms of nanoparticles and their resulting biodistribution.     

Effect of surfactant agents and lipids on optical resolution of amino acid by ultrafiltration membranes containing bovine serum albumin

Ultrafiltration experiments for the optical resolution of racemic phenylalanine were performed in a solution system containing bovine serum albumin (BSA) and surfactant agents (Triton X-100, Tween 20, sodium dodecyl sulfate), lipid (phosphaticylcholine) and fatty acid (palmitic acid sodium salt). It was found that -phenylalanine preferentially existed in the permeate at pH 7.0 due to the binding of BSA to -phenylalanine in the feed and that the separation factors (=concentration ratio of -isomer to -isomer in the permeate) increased with a decrease in the BSA solution containing no additives and in the BSA solution containing Triton X-100 or Tween 20. The unusual tendency that the separation factors were less than unity was observed and the separation factors decreased with a decrease in the feed concentration of phenylalanine during the ultrafiltration containing the palmitic acid sodium salt or the phosphatidylcholine. This is caused by the fact that the binding constants of -phenylalanine to BSA are higher than those of -phenylalanine in the BSA solution containing the palmitic acid sodium salt or phosphatidylcholine. Since there were found conformational changes of BSA in the presence of palmitic acid sodium salt based on circular dichroism measurements of BSA solution, the conformational changes of BSA were attributed to the higher affinity of -phenylalanine to BSA than that of -phenylalanine in the BSA solution containing the palmitic acid sodium salt or phosphatidylcholine.     

Lag phase alteration in the modified bovine serum albumin under the inducing and inhibitory effect of vitamin C

Lag phase as the initial stage of protein aggregation has an important role in fibril formation and contributes to pathogenesis of many human diseases. Harsh environments provided by some additives can change the rate and the amounts of protein abnormalities. Here we studied the lag phase as the first stage of fibrillation process of bovine serum albumin (BSA) and determined the effects of potassium sorbate (PS) a widespread industrial preservative and vitamin C as an important antioxidant on these changes. Thioflavin T binding assay, circular dichroism, intrinsic and extrinsic fluorescence spectroscopy, and atomic force microscopy techniques were used for these assessments. Our results indicated that the lag phase of BSA fibrillation was affected under different conditions. The length of lag phase in BSA protein aggregation process was short due to the generation of the advanced glycation end products (AGEs) by PS. In contrast, vitamin C by its antioxidant properties and chaperone ability dramatically prolonged BSA lag phase, preserved BSA structural changes that lead to aggregation, and dramatically decreased formation of AGEs and the amount of fibrillation.     

Small-angle X-ray scattering and electron paramagnetic resonance study of the interaction of bovine serum albumin with ionic surfactants

Small-angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR) techniques have been used to monitor the interaction of bovine serum albumin (BSA) with ionic surfactants such as anionic sodium dodecyl sulfate (SDS), zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propane sulfonate (HPS), and cationic cethyltrimethylammonium chloride (CTAC) at pH 7.0. The SAXS results have shown that in the presence of 5 mM SDS and HPS the radius of gyration (Rg) almost does not change as compared to the BSA free-surfactant solution; its value is ca. 30 Angstroms. In the presence of 5 mM CTAC the SAXS data indicate the presence of a particle with a Rg of at least 63 Angstroms, suggesting that in this case, a kind of protein aggregation takes place. In the presence of SDS and HPS surfactants at concentrations above 10 mM, a characteristic broad peak in the region of 0.12-0.18 Angstroms(-1) indicates the presence of micelle-like aggregates in solution. The SAXS curves are consistent with the "pearl necklace" model, where micelle-like aggregates are randomly distributed around the polypeptide chain. EPR results using 5-DSA and 16-DSA spin labels show that in the presence of BSA the EPR spectra are composed of two label populations, one contacting the protein and a second one due to label localization in the micelles. Evidence is also obtained for a competition of the surfactants with the spin labels for the high-affinity binding sites of the stearic acid spin labels as monitored by changes in the fractions of the two label populations as the surfactant concentration is increased. The effect of SDS seems to be stronger in the sense that increased SDS concentration leads to a complete transfer of spin labels from close protein contact sites to micelles, while for HPS, a significant immobilization of probe apparently remains even at higher surfactant concentrations. These two techniques are quite useful since SAXS monitors the overall properties of the scattering particle, while EPR gives information on the dynamics inside this particle and associated with label localization and motion.     

Polyacrylamide gel method: synthesis and property of BeO nanopowders

Effects of monomer (AM) concentration, monomer/crosslinker (AM/MBAM) ratio and salt concentration on the thermal behavior of precursor gel and the properties of BeO nanopowder synthesized by polyacrylamide gel method were investigated. The decomposition process of precursor gel was also studied. The decomposition process of precursor gel is that, first, the extraction of free and crystallized water, and then the thermal degradation of polymeric network under temperature higher than 600 °C, final, the decomposition of nanoscale beryllium sulfate to BeO nanopowder. As the monomer concentration increases, the calcination temperature of precursor gel decreases due to more compact network structure of gel and thus smaller size of salt in nanocaves in gel. The average particle size of nanopowder reduces correspondingly. The AM/MBAM ratio also has significant effect on the thermal behavior of precursor gel and the average particle size of product. When the ratio of AM to MBAM is 6, the calcination temperature of precursor gel is the lowest, the average particle size of powders is the smallest, because the network structures of gel is the tightest and thus the sizes of salts in precursor gels are the smallest. As the AM/MBAM ratio deviates from this value, the network structures of gel becomes looser and thus the size of salt in precursor gel becomes larger, so the calcination temperature increases and the average particle size of powders becomes larger certainly. For the same reason, both the calcination temperature and the average particle size of powders increases with increasing the salt concentration. The synthesis conditions have no effect on the particle size distribution of the final product due to the natural random distribution of porosity in gel.