Effects of protein-polyelectrolyte affinity and polyelectrolyte molecular weight on dynamic properties of bovine serum albumin-poly(diallyldimethylammonium chloride) coacervates

Bovine serum albumin (BSA) and poly(diallyldimethylammonium chloride) (PDADMAC) spontaneously form, over a range of ionic strength I and pH, dense fluids rich in both macroions. To study their nanostructure, these coacervates were prepared at low I and high pH (strong interaction) or at high I and lower pH (weaker interaction), with polymer MWs ranging from 90K to 700K, and then examined by dynamic light scattering (DLS) and rheology. DLS shows a dominant and surprisingly fast protein diffusional mode independent of polymer MW; accompanied by robust slow modes, slower by 1-2 orders of magnitude, which are also insensitive to MW and are present regardless of I, pH, and sample aging. High MW sensitivity was observed by rheology for the terminal time (order of milliseconds), which increased as well with the strength of polyelectrolyte-protein interaction. Viscoelastic behavior also indicated a tenuous network, solidlike at low strain but re-forming after breakage by shear. Two models, both of which have strengths and defects, are put forward: (I) macroion-rich domains dispersed in a continuum of macroion-poor domains near the percolation limit and (II) a semidilute solution of PDADMAC chains with interchain friction modulated by transient BSA-PDADMAC association.     

Self-healing fish gelatin/sodium montmorillonite biohybrid coacervates: structural and rheological characterization

Complex coacervation driven by associative electrostatic interactions was studied in mixtures of exfoliated sodium-montmorillonite (Na(+)-MMT) nanoplatelets and fish gelatin, at a specific mixing ratio and room temperature. Structural and viscoelastic properties of the coacervate phase were investigated as a function of pH by means of different complementary techniques. Independent of the technique used, the results consistently showed that there is an optimum pH value at which the coacervate phase shows the tightest structure with highest elasticity. The solid-like coacervates showed an obvious shear-thinning behavior and network fracture but immediately recovered back into their original elastic character upon removal of the shear strain. The nonlinear mechanical response characterized by single step stress relaxation experiments revealed the same trend for the yield stress and isochronal shear modulus of the coacervates as a function of pH with a maximum at pH 3.0 and lower values at 2.5 and 3.5 pHs, followed by a very sharp drop at pH 4.0. Finally, small-angle X-ray scattering (SAXS) data confirmed that at pHs lower than 4.0 the coacervate phases were dense and structured with a characteristic length scale (ξ(SAXS)) of ~7-9 nm. Comparing the ξ(SAXS) with rheological characteristic length (ξ(rheol)) estimated from low-frequency linear viscoelastic data and network theory, it was concluded that both the strength of the electrostatic interactions and the conformation of the gelatin chains before and during of the coacervation process are responsible for the structure and rigidity of the coacervates.     

Composition and structure of whey protein/gum arabic coacervates

Complex coacervation in whey protein/gum arabic (WP/GA) mixtures was studied as a function of three main key parameters: pH, initial protein to polysaccharide mixing ratio (Pr:Ps)(ini), and ionic strength. Previous studies had already revealed under which conditions a coacervate phase was obtained. This study is aimed at understanding how these parameters influence the phase separation kinetics, the coacervate composition, and the internal coacervate structure. At a defined (Pr:Ps)(ini), an optimum pH of complex coacervation was found (pH(opt)), at which the strength of electrostatic interaction was maximum. For (Pr:Ps)(ini) = 2:1, the phase separation occurred the fastest and the final coacervate volume was the largest at pH(opt) = 4.0. The composition of the coacervate phase was determined after 48 h of phase separation and revealed that, at pH(opt), the coacervate phase was the most concentrated. Varying the (Pr:Ps)(ini) shifted the pH(opt) to higher values when (Pr:Ps)(ini) was increased and to lower values when (Pr:Ps)(ini) was decreased. This phenomenon was due to the level of charge compensation of the WP/GA complexes. Finally, the structure of the coacervate phase was studied with small-angle X-ray scattering (SAXS). SAXS data confirmed that at pH(opt) the coacervate phase was dense and structured. Model calculations revealed that the structure factor of WP induced a peak at Q = 0.7 nm(-1), illustrating that the coacervate phase was more structured, inducing the stronger correlation length of WP molecules. When the pH was changed to more acidic values, the correlation peak faded away, due to a more open structure of the coacervate. A shoulder in the scattering pattern of the coacervates was visible at small Q. This peak was attributed to the presence of residual charges on the GA. The peak intensity was reduced when the strength of interaction was increased, highlighting a greater charge compensation of the polyelectrolyte. Finally, increasing the ionic strength led to a less concentrated, a more heterogeneous, and a less structured coacervate phase, induced by the screening of the electrostatic interactions.     

Complexation of trypsin and alcohol dehydrogenase with poly(diallyldimethylammonium chloride)

Complexation of alcohol dehydrogenase (ADH) and trypsin with poly(diallyldimethyl-ammonium chloride) (PDADMAC) in dilute electrolyte solution was studied by turbidimetric titration, quasi-elastic light scattering (QELS), and electrophoretic light scattering (ELS). Both QELS and turbidimetric titration show that PDADMAC forms complexes with ADH and trypsin in 0.01M NaCl solution at pH ≥ 6.8 and pH ≥ 9.2, respectively. These complexes take the form of stable coacervates in 0.01M, pH 11.0, phosphate buffer solution. QELS shows sizes of 400 and 315 nm for the coacervates of ADH-PDMDAAC and trypsin-PDMDAAC, respectively, while ELS reveals that these coacervates carry a net positive charge. Activity measurements show that both ADH and trypsin are enzymatically active in their coacervated states. Complexation of trypsin and PDADMAC was also studied by fluorescence in 0.01M, pH 11.0, phosphate buffer, and the protein emission was found to be quenched by complexation. The fluorescence quenching data show that trypsin retains its three-dimensional structure in the complex. These and other results are consistent with the quenching of the two tryptophans on the protein surface, but not the interior ones.© 1997 John Wiley & Sons, Inc.     

Rheological interfacial properties of plant protein-arabic gum coacervates at the oil-water interface

This study concerns the interfacial properties of the plant proteins-arabic gum coacervates, which are involved in encapsulation processes based on complex coacervation. The results make it possible to deduce the prerequisite characteristics of the protein, which are involved in the coacervate interfacial properties. The influence of pH and concentration on protein interfacial properties was also studied so as to enable us to predict the best conditions to achieve encapsulation. It has been established that, to obtain a good encapsulation yield, the coacervate must show high surface-active properties and its adsorption on the oil droplets must be favored compared to the free protein adsorption. On the other hand, mechanical properties of the interfacial film made of the coacervate, appear to be a key parameter, as reflected by the dilational viscoelasticity measurements. When compared to the properties of the proteins films, an increase of the rigidity of the interfacial film was shown with the coacervates. It was also observed that viscoelastic properties of the coacervate film were strongly reduced, as well as the associated relaxation times. In acidic conditions, the coacervates containing alpha-gliadin are characterized by an interfacial viscoelastic behavior. This behavior reflects the softness of the interfacial film. This viscoelasticity allows also the formation of a continuous layer around the oil droplets to be encapsulated. Drop tensiometry is shown to be a method that could allow the most adapted protein to be selected and the conditions of the coacervation process to be optimized with regard to concentration and pH.     

Composition and rheological properties of beta-Lactoglobulin/pectin coacervates: effects of salt concentration and initial protein/polysaccharide ratio

The composition and rheological properties of beta-lactoglobulin/pectin coacervates have shown significant correlations with sodium chloride concentration (C(NaCl)) and initial protein/polysaccharide ratio (r). An increase of C(NaCl) from 0.01 to 0.21 M at r = 5:1 leads to the increase in both beta-lactoglobulin and pectin contents in the coacervates, which can be explained in terms of salt-enhanced effect at lower salt concentrations. Further increase of C(NaCl) from 0.21 to 0.41 M decreases the proportions of these two biopolymers in the coacervates, exhibiting salt-reduced effect at higher salt concentrations. Moreover, the stronger self-aggregation of beta-lactoglobulin with increasing salt concentration gives rise to a decreasing actual protein/polysaccharide ratio in the coacervates at 0.01-0.21 M C(NaCl) and r = 5:1. An increase of r from 5:1 to 40:1 often increases the actual amount of pectin chains in beta-lactoglobulin/pectin coacervates, but it exhibits a maximum in beta-lactoglobulin content at r = 20:1. A much higher storage modulus (G') than loss modulus (G' ') for all beta-lactoglobulin/pectin coacervates suggests the formation of highly interconnected gel-like structure. The values of G' increase as C(NaCl) increases from 0.01 to 0.21 M, whereas a further increase of C(NaCl) from 0.21 to 0.41 M causes G' values to decrease to much lower values. These results further disclose the salt-enhanced effect and the salt-reduced effect at low and high salt concentrations, respectively. On the other hand, increasing r from 5:1 to 40:1 favors the formation of stronger gel-like beta-lactoglobulin/pectin coacervates, which mainly originates from the higher actual amount of pectin chains in beta-lactoglobulin/pectin coacervates at higher r values.     

Functional properties of hemoglobin immobilized in coacervates prepared from gelatin A and polyanionic carbohydrates

Complex coacervation is a phenomenon of phase separation that may occur in a solution of positively and negatively charged polyions. The resulting two phases are distinguished by the total concentration of both polyions, with the concentrated phase often containing vesicular structures composed of the two polyelectrolytes. We have used this phenomenon in an attempt to-prepare a hemoglobin-based red blood cell analog. Hemoglobin-containing coacervate vesicles have been prepared from gelatin A and the polyanionic carbohydrates acacia, pectin, or dextranstilfate. Hemoglobin seems to be anchored into the vesicle walls through interaction of its polyanion binding site with the negatively charged residues on the carbohydrates. Oxygen binding by the immobilized HbA is reversible and cooperative, with p50 values at 20 degrees C of 2.8, 6, and 24 mm Hg for the acacia- (pH 7.5), pectin- (pH 6.6), and dextransulfate-(pH 6.6) derived coacervates. Kinetic studies on CO binding show that the rate of CO uptake by the coacervates (t((1/2)) = 13-27 ms at 0.5 mM CO) is similar to that of human erythrocytes.The HbA-containing coacervates slowly dissolve in isotonic salt solutions (145 mM NaCl, pH 7.4), but they can be stabilized by treatment with glutaraldehyde. Oxygen binding by HbA incorporated into the stabilized coacervates derived from dextran sulfate is very similar to oxy gen binding by human red blood cells: p50 = 26 mm Hg and n = 1.89 at 37 degrees C in isotonic salt. These results show how a novel approach, based on an old concept, has led to the preparation of immobilized HbA, with functional properties similar to those of intraerythrocytic HbA.     

SANS/SAXS study of the BSA solvation properties in aqueous urea solutions via a global fit approach

We report on the solvation properties and intermolecular interactions of a model protein (bovine serum albumine, BSA) in urea aqueous solutions, as obtained by combining small-angle neutron and X-ray scattering experiments. According to a global fit strategy, all the whole set of scattering curves are analysed by considering a unique model which includes the BSA structure, the protein-protein interactions and the thermodynamic exchange process of water/urea molecules at the protein solvent interface. As a main result, the equilibrium constant that accounts for the difference in composition between the bulk solvent and the protein solvation layer is derived. Results confirm that urea preferentially sticks to the protein surface, inducing a noticeable change in both the repulsive and the attractive interaction potentials.     

Structural studies of agar-gelatin complex coacervates by small angle neutron scattering, rheology and differential scanning calorimetry

Agar-gelatin complex coacervates are studied by small angle neutron scattering (SANS), rheology (in both flow and temperature scan modes) and differential scanning calorimetry (DSC) in order to probe the microscopic structure of this dense protein-polysaccharide-rich phase. DSC and isochronal temperature sweep (rheology) experiments yielded a characteristic temperature at approximately 35+/-2 degrees C. Rheology data revealed a second characteristic temperature at approximately 75+/-5 degrees C which was absent in DSC thermograms. In the flow mode, shear viscosity (eta) was found to scale with (Carreau model) applied shear rate (gamma ) as: eta(gamma ) approximately (gamma )(-k) with k=1.2+/-0.2 indicating non-Newtonian and shear-thinning features independent of ionic strength. The static structure factor S(q) deduced from SANS data in the low wave vector (0.018 A(-1)相似文献   

Effects of Lipid Interactions on Model Vesicle Engulfment by Alveolar Macrophages

The engulfment function of macrophages relies on complex molecular interactions involving both lipids and proteins. In particular, the clearance of apoptotic bodies (efferocytosis) is enabled by externalization on the cell target of phosphatidylserine lipids, which activate receptors on macrophages, suggesting that (local) specific lipid-protein interactions are required at least for the initiation of efferocytosis. However, in addition to apoptotic cells, macrophages can engulf foreign bodies that vary substantially in size from a few nanometers to microns, suggesting that nonspecific interactions over a wide range of length scales could be relevant. Here, we use model lipid membranes (made of phosphatidylcholine, phosphatidylserine, and ceramide) and rat alveolar macrophages to show how lipid bilayer properties probed by small-angle x-ray scattering and solid-state ²H NMR correlate with engulfment rates measured by flow cytometry. We find that engulfment of protein-free model lipid vesicles is promoted by the presence of phosphatidylserine lipids but inhibited by ceramide, in accord with a previous study of apoptotic cells. We conclude that the roles of phosphatidylserine and ceramide in phagocytosis is based, at least in part, on lipid-mediated modification of membrane physical properties, including interactions at large length scales as well as local lipid ordering and possible domain formation.     

Effects of Lipid Interactions on Model Vesicle Engulfment by Alveolar Macrophages

The engulfment function of macrophages relies on complex molecular interactions involving both lipids and proteins. In particular, the clearance of apoptotic bodies (efferocytosis) is enabled by externalization on the cell target of phosphatidylserine lipids, which activate receptors on macrophages, suggesting that (local) specific lipid-protein interactions are required at least for the initiation of efferocytosis. However, in addition to apoptotic cells, macrophages can engulf foreign bodies that vary substantially in size from a few nanometers to microns, suggesting that nonspecific interactions over a wide range of length scales could be relevant. Here, we use model lipid membranes (made of phosphatidylcholine, phosphatidylserine, and ceramide) and rat alveolar macrophages to show how lipid bilayer properties probed by small-angle x-ray scattering and solid-state ²H NMR correlate with engulfment rates measured by flow cytometry. We find that engulfment of protein-free model lipid vesicles is promoted by the presence of phosphatidylserine lipids but inhibited by ceramide, in accord with a previous study of apoptotic cells. We conclude that the roles of phosphatidylserine and ceramide in phagocytosis is based, at least in part, on lipid-mediated modification of membrane physical properties, including interactions at large length scales as well as local lipid ordering and possible domain formation.     

Influence of processing conditions on the physical properties,retention rate,and antimicrobial activity of cinnamaldehyde loaded in gelatin/pectin complex coacervates

Food Biophysics - To enhance the release characteristics and antimicrobial properties of cinnamaldehyde microcapsules based on gelatin and pectin coacervates, microcapsules were prepared using...     

Swelling and mechanical properties of biopolymer hydrogels containing chitosan and bovine serum albumin

Experimental and theoretical investigations of the swelling and mechanical properties of hydrogels formed from chitosan, bovine serum albumin (BSA), and chitosan/BSA mixtures cross-linked with genipin were performed. The properties of cross-linked chitosan hydrogels were explained in terms of its polyelectrolyte behavior, which led to a gradual increase in swelling ratio below the pK value, but whereby its swelling ability was eliminated by the presence of salt that screened the charges. Comparison of theoretical and experimental calculations of the swelling ratio, however, indicated that complications arising from wastage of cross-links, and formation of polymerized genipin cross-links must be considered before quantitative prediction can be achieved. Cross-linked BSA hydrogels swelled even in the presence of salt, and a marked increase in swelling was observed below pH = 3 that was explained as the result of an acid induced denaturation of the protein that led to unfolding of the molecule. Swollen BSA hydrogels were mechanically weak, however. Composite gels made from a cross-linked mixture of chitosan and BSA exhibited the swelling behavior of BSA combined with the mechanical properties of chitosan and were therefore considered most suitable for use in a gastric environment.     

Effect of chitosan degradation on its interaction with β-lactoglobulin

Complexes between chitosan and β-lactoglobulin (β-Lg) were investigated, and their formation was found to depend on pH and ionic strength. The electrostatic attraction between the cationic polysaccharide and the negatively charged protein above its isoelectric point has been identified as the main driving force in the molecular recognition process. At low protein concentration, soluble complexes were shown to be formed, and their structural features were characterized by circular dichroism (CD) and steady-state fluorescence. Both the overall secondary structure of the protein and the local environment probed by its tryptophan residues are not affected by the presence of chitosan in the complex. Furthermore, the formation of the complex does not lead to a net stabilization of the native state of the protein over its denatured state due to formation of a similarly stable complex between the polyelectrolyte and the denatured state of the protein. The formation of coacervates between β-Lg and chitosan was also characterized as a function of average molecular weight of chitosan (subjected to ultrasonication for different periods of time: 0, 5, 15, and 30 min) by means of both turbidimetric and calorimetric techniques. The combination of turbidimetric as well as isothermal calorimetric titrations have allowed the deconvolution of two processes usually coupled in the formation of protein-polyelectrolyte coacervates: the formation of complex coacervates as the protein sites become saturated by polyelectrolyte molecules and the redissolution of the coacervates as the polyelectrolyte-to-protein ratio increases.     

Large size fibrillar bundles of the Alzheimer amyloid β-protein

Self-assembly of amyloid β-protein (Aβ) and its deposition into senile plaques are distinctive features of Alzheimer’s disease. Aβ forms typical linear aggregates known as amyloid fibrils, with a diameter of a few tens of nanometers and a length spanning from hundreds of nanometers to micrometers. Fibrils eventually assemble into large size clusters and precipitate in vivo in the brain deposits. Here, we study the late stage of aggregation of Aβ(1–40) in vitro at pH 3.1. We characterize the structure of fibrillar aggregates by a combined use of different experimental techniques. Small angle light scattering, heterodyne near field scattering, large angle light scattering, ultra small angle X-ray scattering and small angle X-ray scattering measurements have been performed to highlight the structural features of amyloid bundles over several lengthscales, from nanometers to tens of micrometers. Phase contrast optical microscopy has been used to complement scattering measurements and directly visualize some morphological details. We show that elongated fibrils of Aβ with a diameter of a few nanometers are packed into large size compact bundles having a typical size of tens of micrometers. The linear morphology of fibrils is reflected in the elongated shape of bundles. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Polyelectrolyte nanoparticles based on water-soluble chitosan-poly(l-aspartic acid)-polyethylene glycol for controlled protein release

Water-soluble chitosan (WSC)-poly(l-aspartic acid) (PASP)-polyethylene glycol (PEG) nanoparticles (CPP nanoparticles) were prepared spontaneously under quite mild conditions by polyelectrolyte complexation. These nanoparticles were well dispersed and stable in aqueous solution, and their physicochemical properties were characterized by turbidity, FTIR spectroscopy, dynamic light scattering (DLS), transmission electron microscope (TEM), and zeta potential. PEG was chosen to modify WSC-PASP nanoparticles to make a protein-protective agent. Investigation on the encapsulation efficiency and loading capacity of the bovine serum albumin (BSA)-loaded CPP nanoparticles was also conducted. Encapsulation efficiency was obviously decreased with the increase of initial BSA concentration. Furthermore, its in vitro release characteristics were evaluated at pH 1.2, 2.5, and 7.4. In vitro release showed that these nanoparticles provided an initial burst release, followed by a slowly sustained release for more than 24 h. The BSA released from CPP nanoparticles showed no significant conformational change compared with native BSA, which is superior to the BSA released from nanoparticles without PEG. A cell viability study suggested that the nanoparticles had good biocompatibility. This nanoparticle system was considered promising as an advanced drug delivery system for the peptide and protein drug delivery.     

STUDIES ON THE INTRACELLULAR DIGESTIVE PROCESS IN MAMMALIAN TISSUE CULTURE CELLS

DNA-protein coacervates containing colloidal gold particles were readily phagocytized by strain L fibroblasts. During the subsequent digestion process, the gold particles served as markers which permitted the demonstration of the evolution of digestive vacuoles to multivesicular bodies and finally to dense bodies. Acid phosphatase and esterolytic activity was present in these structures. The hydrolytic enzymes were apparently brought to the phagocytotic vacuoles in small vesicles originating in the Golgi region. These vesicles entered the vacuoles prior to the digestion of the coacervates and the appearance of positive cytochemical reactions. The cytoplasmic dense bodies frequently merged with the phagocytotic vacuoles. This was demonstrated by prelabeling the dense bodies with colloidal iron prior to phagocytosis of the coacervates. In addition, evidence is presented for the interrelationship of the phagocytotic and autophagic pathways.     

Electrospun Chitosan Microspheres for Complete Encapsulation of Anionic Proteins: Controlling Particle Size and Encapsulation Efficiency

Electrospinning was employed to fabricate chitosan microspheres by a single-step encapsulation of proteins without organic solvents. Chitosan in acetic acid was electrospun toward a grounded sodium carbonate solution at various electric potential and feeding rates. Electrospun microspheres became insoluble and solidified in the sodium carbonate solution by neutralization of chitosan acetate. When the freeze-dried microspheres were examined by scanning electron microscopy, the small particle size was obtained at higher voltages. This is explained by the chitosan droplet size at the electrospinning needle was clearly controllable by the electric potential. The recovery yield of chitosan microspheres was dependent on the concentration of chitosan solution due to the viscosity is the major factor affecting formation of chitosan droplet during curling of the electrospinning jets. For protein encapsulation, fluorescently labeled bovine serum albumin (BSA) was codissolved with chitosan in the solution and electrospun. At higher concentration of sodium carbonate solution and longer solidification time in the solution, the encapsulation efficiency of the protein was confirmed to be significantly high. The high encapsulation efficiency was achievable by instant solidification of microspheres and electrostatic interactions between chitosan and BSA. Release profiles of BSA from the microspheres showed that the protein release was faster in acidic solution due to dissolution of chitosan. Reversed-phase chromatography of the released fractions confirmed that exposure of BSA to acidic solution during the electrospinning did not result in structural changes of the encapsulated protein.     

Partitioning and Enhanced Self-Assembly of Actin in Polypeptide Coacervates

Biomolecules exist and function in cellular microenvironments that control their spatial organization, local concentration, and biochemical reactivity. Due to the complexity of native cytoplasm, the development of artificial bioreactors and cellular mimics to compartmentalize, concentrate, and control the local physico-chemical properties is of great interest. Here, we employ self-assembling polypeptide coacervates to explore the partitioning of the ubiquitous cytoskeletal protein actin into liquid polymer-rich droplets. We find that actin spontaneously partitions into coacervate droplets and is enriched by up to ～30-fold. Actin polymerizes into micrometer-long filaments and, in contrast to the globular protein BSA, these filaments localize predominately to the droplet periphery. We observe up to a 50-fold enhancement in the actin filament assembly rate inside coacervate droplets, consistent with the enrichment of actin within the coacervate phase. Together these results suggest that coacervates can serve as a versatile platform in which to localize and enrich biomolecules to study their reactivity in physiological environments.     

Influence of the NaCl or CaCl2 concentration on the structure of heat-set bovine serum albumin gels at pH 7

The structure of heat-set systems of the globular protein bovine serum albumin (BSA) was investigated at pH 7 in different salt conditions (NaCl or CaCl(2)) using light scattering. Cross-correlation dynamic light scattering was used to correct for multiple scattering from turbid samples. After heat treatment, aggregates are formed whose size increases as the protein concentration increases. Beyond a critical concentration that decreases with increasing salt concentration, gels are formed. The heterogeneity and the reduced turbidity of the gels were found to increase with increasing salt concentration and to decrease with increasing protein concentration. The structure of the gels is determined by the strength of the repulsive electrostatic interactions between the aggregated proteins. The results obtained in NaCl are similar to those reported in previous studies for other globular proteins. CaCl(2) was found to be much more efficient in reducing electrostatic interactions than NaCl at the same ionic strength.