Mechanism and kinetics of phase separation in a gelatin/maltodextrin mixture studied by small-angle light scattering

Phase separation mechanisms and kinetics were studied using small-angle light scattering in a gelatin/maltodextrin system where phase separation could be studied in both liquid and gelled states. Nucleation and growth or spinodal decomposition occurred, depending on the quench depth. The transition between the two mechanisms occurred relatively sharply. The different mechanisms were distinguishable by the different behavior of the scattering function even though a peak was observed in both cases. Particular differences were the different evolution of the peak intensity and position, the absence of dynamic scaling of the nucleation and growth scattering function, and the final coarsening exponent of 1/3 that was measured when spinodal decomposition occurred but not for nucleation and growth. Gelation severely reduced the coarsening rate and initially placed the phase compositions far from their equilibrium values. Despite the loss of molecular mobility caused by gelation, the gelled systems did continue to evolve, albeit much more slowly than in the liquid case. Multiple coarsening rates were observed for some of the gelled samples, which were ascribed to the gradual movement of these systems toward the equilibrium compositions.     

Structural investigation of beta-lactoglobulin gelation in ethanol/water solutions.

The aggregation and gelation properties of beta-lactoglobulin (BLG), a globular protein from milk, was studied in hydro-ethanolic solutions (50/50% (v/v)) at room temperature. The phase state diagrams as a function of pH and ethanol concentration showed that a gel structure appeared after a period ranging from 1 min to 1 week depending on the physico-chemical conditions. The aggregation kinetics, studied by infrared spectroscopy and dynamical rheological measurements, highly depended upon the pH; the process being the fastest at pH 7. Alcohol-induced aggregation of BLG was characterized by the formation of intermolecular hydrogen bonded beta-sheet structures. Small angle neutron scattering indicated that the aggregates structures in the final gels were similar at pH 7, 8 and 9. Through the data obtained at the molecular and macroscopic levels, it can be concluded that the kinetics of gelation were pH dependent while the spatial arrangements of the aggregates were similar in the final structures. The heterogeneous structures formed in hydro-ethanolic gels could be analysed in terms of a phase separation, the syneresis being the final visible state.     

Shear-induced mixing and demixing in aqueous methyl hydroxypropyl cellulose solutions

The influence of shear flow on the phase separation of aqueous methyl hydroxypropyl cellulose solutions was investigated by means of rheoturbidity and online rheo small angle light scattering (SALS) experiments. In semidilute solution shear-induced mixing was observed and the cloud curve was shifted to higher temperatures with increasing shear rate. With higher concentrated solutions, however, shear-induced demixing was found. The shear-induced mixing is interpreted as being a disruption of slightly entangled clusters under the influence of the shear energy. The shear demixing appears in line with the observation with other systems. A characteristic butterfly pattern was observed in rheo-SALS.     

"Delayed" phase separation in a gelatin/dextran mixture studied by small-angle light scattering,turbidity, confocal laser scanning microscopy,and polarimetry

Small-angle light scattering, turbidity, and confocal laser scanning microscopy were used to study microstructure formation and evolution in a gelatin/dextran mixture. There was a time-delay of up to tens of minutes between reaching the quench temperature and the onset of phase separation, because demixing only occurred once a certain amount of ordering of the gelatin molecules, measured by polarimetry, was attained. The accompanying phenomenon of gelation retarded the development of the microstructure to different extents, depending on the quench temperature. At low temperatures, the structure was rapidly trapped in a nonequilibrium state with diffuse interfaces, characteristic of the early and intermediate stages of phase separation. At higher temperatures, coarsening continued for a certain amount of time before the structure was trapped. The duration of the coarsening period increased with increasing temperature and the interface between the phases became sharp, characteristic of the late stages of phase separation. Because the ordering process continued after the target quench temperature was reached, the effective quench depth continued to increase after the initial phase separation. At high quench temperatures, the system was able to respond to the thermodynamic requirements of the increasing effective quench depth by undergoing secondary phase separation to form a droplet morphology within the preexisting bicontinuous one.     

Phase separation in a sheared gelatin/maltodextrin mixture studied by small-angle light scattering

The influence of shear on the structure of a gelatin/maltodextrin mixture was investigated using small-angle light scattering both during phase separation and after phase separation was allowed to occur quiescently. In all cases, phase separation occurred via spinodal decomposition to form a droplet morphology, and a characteristic length scale was formed in the structure that was prevalent during shear, as well as in quiescent conditions. Below the critical shear rate for droplet breakup, shear accelerated the coarsening rate of the droplets. A transient regime of rapid hydrodynamic coarsening was present when shear was initiated after phase separation and at late times in all cases once the droplets attained a certain size. At the critical shear rate for droplet breakup (1 s(-1)), the rapid repetition of breakup and coarsening was postulated to occur, which enabled a microstructure consisting of elongated droplets with a narrow size distribution to form. When the shear rate enabled droplets to extend to such an extent that a percolated structure could form (10 s(-1)), then the structure was relatively stable and changed very slowly over time. At very high shear rates (100 s(-1)), droplet breakup was suppressed and a highly fibrillar morphology formed that was stable only while the system was under shear. Cessation of shear at high rates led to fiber breakup and the formation of many small droplets. For a given shear rate, the final microstructure appeared to be independent of the time that shear was started when the structure consisted of discrete droplets or fibers. When a percolated structure could form, however, the shear history appeared to be important.     

Mechanism of formation of amyloid protofibrils of barstar from soluble oligomers: evidence for multiple steps and lateral association coupled to conformational conversion

Understanding the heterogeneity of the soluble oligomers and protofibrillar structures that form initially during the process of amyloid fibril formation is a critical aspect of elucidating the mechanism of amyloid fibril formation by proteins. The small protein barstar offers itself as a good model protein for understanding this aspect of amyloid fibril formation, because it forms a stable soluble oligomer, the A form, at low pH, which can transform into protofibrils. The mechanism of formation of protofibrils from soluble oligomer has been studied by multiple structural probes, including binding to the fluorescent dye thioflavin T, circular dichroism and dynamic light scattering, and at different temperatures and different protein concentrations. The kinetics of the increase in any probe signal are single exponential, and the rate measured depends on the structural probe used to monitor the reaction. Fastest is the rate of increase in the mean hydrodynamic radius, which grows from a value of 6 nm for the A form to 20 nm for the protofibril. Slower is the rate of increase in thioflavin T binding capacity, and slowest is the rate of increase in circular dichroism at 216 nm, which occurs at about the same rate as that of the increase in light scattering intensity. The dynamic light scattering measurements suggest that the A form transforms completely into larger size aggregates at an early stage during the aggregation process. It appears that structural changes within the aggregates occur at the late stages of assembly into protofibrils. For all probes, and at all temperatures, no initial lag phase in protofibril growth is observed for protein concentrations in the range of 1 microM to 50 microM. The absence of a lag phase in the increase of any probe signal suggests that aggregation of the A form to protofibrils is not nucleation dependent. In addition, the absence of a lag phase in the increase of light scattering intensity, which changes the slowest, suggests that protofibril formation occurs through more than one pathway. The rate of aggregation increases with increasing protein concentration, but saturates at high concentrations. An analysis of the dependence of the apparent rates of protofibril formation, determined by the four structural probes, indicates that the slowest step during protofibil formation is lateral association of linear aggregates. Conformational conversion occurs concurrently with lateral association, and does so in two steps leading to the creation of thioflavin T binding sites and then to an increase in beta-sheet structure. Overall, the study indicates that growth during protofibril formation occurs step-wise through progressively larger and larger aggregates, via multiple pathways, and finally through lateral association of critical aggregates.     

The use of small angle light scattering in assessing strain induced collagen degradation in arterial tissue ex vivo

Collagen is the predominant load bearing component in many soft tissues including arterial tissue and is therefore critical in determining the mechanical integrity of such tissues. Degradation of collagen fibres is hypothesized to be a strain dependent process whereby the rate of degradation is affected by the magnitude of strain applied to the collagen fibres. The aim of this study is to investigate the ability of small angle light scattering (SALS) imaging to identify strain dependent degradation of collagen fibres in arterial tissue ex vivo, and determine whether a strain induced protection mechanism exists in arterial tissue as observed in pure collagen and other collagenous tissues. SALS was used in combination with histological and second harmonic generation (SHG) analysis to determine the collagen fibre architecture in arterial tissue subjected to strain directed degradation. SALS alignment analysis identified statistically significant differences in fibre alignment depending on the strain magnitude applied to the tissue. These results were also observed using histology and SHG. Our findings suggest a strain protection mechanism may exist for arterial collagen at intermediate strain magnitudes between 0% and 25%. These findings may have implications for the onset and progression of arterial disease where changes in the mechanical environment of arterial tissue may lead to changes in the collagen degradation rate.     

Insulin particle formation in supersaturated aqueous solutions of poly(ethylene glycol)

Protein microspheres are of particular utility in the field of drug delivery. A novel, completely aqueous, process of microsphere fabrication has been devised based on controlled phase separation of protein from water-soluble polymers such as polyethylene glycols. The fabrication process results in the formation of spherical microparticles with narrow particle size distributions. Cooling of preheated human insulin-poly(ethylene glycol)-water solutions results in the facile formation of insulin particles. To map out the supersaturation conditions conducive to particle nucleation and growth, we determined the temperature- and concentration-dependent boundaries of an equilibrium liquid-solid phase separation. The kinetics of formation of microspheres were followed by dynamic and continuous-angle static light scattering techniques. The presence of PEG at a pH that was close to the protein's isoelectric point resulted in rapid nucleation and growth. The time elapsed from the moment of creation of a supersaturated solution and the detection of a solid phase in the system (the induction period, t(ind)) ranged from tens to several hundreds of seconds. The dependence of t(ind) on supersaturation could be described within the framework of classical nucleation theory, with the time needed for the formation of a critical nucleus (size <10 nm) being much longer than the time of the onset of particle growth. The growth was limited by cluster diffusion kinetics. The interfacial energies of the insulin particles were determined to be 3.2-3.4 and 2.2 mJ/m(2) at equilibrium temperatures of 25 and 37 degrees C, respectively. The insulin particles formed as a result of the process were monodisperse and uniformly spherical, in clear distinction to previously reported processes of microcrystalline insulin particle formation.     

Bovine Serum Albumin protofibril-like aggregates formation: solo but not simple mechanism

We report an experimental study on the model protein Bovine Serum Albumin (BSA), with the aim of elucidating the mechanisms by which a fully folded globular protein undergoes different aggregation pathways leading to the formation of amyloid fibrils or amorphous aggregates. We observe thermally induced formation of fibrillar structures at pH far from the protein isoelectric point. The increase of electrostatic repulsion results in protein destabilization and in modifications of inter and intra-molecular interactions leading to the growth of fibril-like aggregates stabilized by inter-molecular-β sheets. The aggregation kinetics is studied by means of fluorescence techniques, light scattering, Circular Dichroism (CD), infrared spectroscopy (FTIR) and Atomic Force Microscopy (AFM). Changes in protein secondary structures turn out to be the driving mechanism of the observed aggregation and they progress in parallel with the growth of Thioflavin T emission intensity and scattering signal. This concurrent behavior suggests a mutual stabilization of elongated protofibril-like structures and of protein conformational and structural changes, which lead to a more rigid and ordered structures. Our results give new insights on BSA self-assembly process in alkaline conditions clearly providing new pieces of evidences of the interplay of several and interconnected mechanisms occurring on different time and length scales.     

Comparison of two different lysozyme types under native and crystallization conditions using two-dimensional NMR and dynamic light scattering

In order to elucidate differences observed in the aggregation kinetics of hen-egg white lysozyme under crystallization conditions we have undertaken a comparative study of the enzyme marketed by Seikagaku and Sigma companies. When the crystallization of the two lysozyme preparations is followed by time-resolved dynamic light scattering, the structural differences are also observed under native conditions in the early nucleation kinetics. The differences are manifested in the formation rates of macroscopic crystals, but do not influence the morphology of the typical tetragonal lysozyme crystal. Using two-dimensional NMR we have followed the differences in the native-like solution structure of the two preparations, while the primary sequence and molecular mass are identical. According to the published structure of tetragonal lysozyme crystal the largest deviations were found for the residues involved in the intermolecular interactions in crystal structure.     

Formation of stable nanoparticles via electrostatic complexation between sodium caseinate and gum arabic

The formation of electrostatic complexes between sodium caseinate and gum arabic (GA) was studied as a function of pH (2.0-7.0), using slow acidification in situ with glucono-delta-lactone (GDL) or titration with HCl. The colloidal behavior of the complexes under specific conditions was investigated using absorbance measurements (at 515 or 810 nm) and dynamic light scattering (DLS). In contrast to the sudden increase in absorbance and subsequent precipitation of sodium caseinate solutions at pH < 5.4, the absorbance values of mixtures of sodium caseinate and GA increased to a level that was dependent on GA concentration at pH 5.4 (pH(c)). The absorbance values remained constant with further decreases in pH until a sudden increase in absorbance was observed (at pH(phi)). The pH(phi) was also dependent upon the GA concentration. Dynamic light scattering (DLS) data showed that the sizes of the particles formed by the complexation of sodium caseinate and GA between pH(c) and pH(phi) were between 100 and 150 nm and these nanoparticles were visualized using negative staining transmission electron microscopy (TEM). Below pH(phi), the nanoparticles associated to form larger particles, causing phase separation. zeta-Potential measurements of the nanoparticles and chemical analysis after phase separation showed that phase separation was a consequence of charge neutralization. The formation of complexes between sodium caseinate and GA was inhibited at high ionic strength (>50 mM NaCl). It is postulated that the structure of the nanoparticles comprises an aggregated caseinate core, protected from further aggregation by steric repulsion of one, or more, electrostatically attached GA molecules.     

Aggregation kinetics of bovine serum albumin studied by FTIR spectroscopy and light scattering

To investigate which type of structural and conformational changes is involved in the aggregation processes of bovine serum albumin (BSA), we have performed thermal aggregation kinetics in D(2)O solutions of this protein. The tertiary conformational changes are followed by Amide II band, the secondary structural changes and the formation of beta-aggregates by the Amide I' band and, finally, the hydrodynamic radius of aggregates by dynamic light scattering. The results show, as a function of pD, that: tertiary conformational changes are more rapid as pD increases; the aggregation proceeds through formation of ordered aggregates (oligomers) at pD far from the isoelectric point of the protein; disordered structures add as the pD decreases. Moreover, beta-aggregates seem to contribute only to oligomers formation, as showed by the good correlation between kinetics of scattering intensity and IR absorption intensity. These results indicate for BSA a general mechanism of aggregation composed by partial unfolding of the tertiary structure and by the decrease of alpha-helix and random coil contents in favor of beta-sheet aggregates. This mechanism strictly depends on pD and gives rise to almost two distinct types of macromolecular aggregates.     

Magainin 2 and PGLa in Bacterial Membrane Mimics II: Membrane Fusion and Sponge Phase Formation

We studied the synergistic mechanism of equimolar mixtures of magainin 2 (MG2a) and PGLa in phosphatidylethanolamine/phosphatidylglycerol mimics of Gram-negative cytoplasmic membranes. In a preceding article of this series, we reported on the early onset of parallel heterodimer formation of the two antimicrobial peptides already at low concentrations and the resulting defect formation in the membranes. Here, we focus on the structures of the peptide-lipid aggregates occurring in the synergistic regime at elevated peptide concentrations. Using a combination of calorimetric, scattering, electron microscopic, and in silico techniques, we demonstrate that the two peptides, even if applied individually, transform originally large unilamellar vesicles into multilamellar vesicles with a collapsed interbilayer spacing resulting from peptide-induced adhesion. Interestingly, the adhesion does not lead to a peptide-induced lipid separation of charged and charge-neutral species. In addition to this behavior, equimolar mixtures of MG2a and PGLa formed surface-aligned fibril-like structures, which induced adhesion zones between the membranes and the formation of transient fusion stalks in molecular dynamics simulations and a coexisting sponge phase observed by small-angle x-ray scattering. The previously reported increased leakage of lipid vesicles of identical composition in the presence of MG2a/PGLa mixtures is therefore related to a peptide-induced cross-linking of bilayers.     

Cold-Set Whey Protein Gels with Addition of Polysaccharides

Cold-set whey protein (WP) gels with addition of xanthan or guar were evaluated by mechanical properties and scanning electron microscopy. Gels were formed after the addition of different amounts of glucono-δ-lactone to thermally denatured WP solutions, leading to different acidification rates and final pH values. At lower acidification rates and higher final pH, gels showed more discontinuous structure and weaker and less elastic network, which was attributed to a predominance of phase separation during gel formation due to slower gelation kinetics. In contrast, at higher acidification rates and lower final pHs, gelation prevailed over phase separation, favoring the formation of less porous structures, resulting in stronger and more elastic gels. The gels’ fractal dimension (D _f; structure complexity) and lacunarity were also influenced by the simultaneous effects of gelation and phase separation. For systems where phase separation was the prevailing mechanism, greater lacunarity parameters were usually observed, describing the heterogeneity of pore distribution, while the opposite occurred at prevailing gelation conditions. Increase in guar concentration or lower final pH of xanthan gels entailed in D _f reduction, while the increase in xanthan concentration resulted in higher D _f. Such a result suggests that the network contour length was rugged, but this pattern was reduced by the increase of electrostatic interactions among WP and xanthan. Guar addition caused the formation of gel network with smoother surfaces, which could be attributed to the guar–protein excluded volume effects leading to an increase in protein–protein interactions.     

Thermodynamic incompatibility and complex formation in pectin/caseinate mixtures

The instability of polysaccharide/protein mixtures occurs because of either thermodynamic incompatibility or complexation. We studied which instability mechanism dominated given the external conditions. Therefore the effect of temperature, pH, and biopolymer concentration on the phase separation of pectin/caseinate mixtures was investigated. At pH > 6, thermodynamic incompatibility with spinodal decomposition was observed in pectin/caseinate mixtures resulting in the formation of water-in-water emulsions in intermediate stages of the phase separation process. The demixing rate of these emulsions and appearance of two macroscopic phases (lower phase enriched with caseinate and upper phase with pectin) was retarded when the pectin concentration increased or when the storage temperature decreased due to a higher viscosity of the mixtures at those conditions. As the pH of the mixture was lowered below 6, pectin accumulated in the caseinate-rich phase. Complexation of pectin and caseinate led to the formation of microparticles (approximately 3 microm), whose shape depends on the biopolymer concentration ratio and rate of acidification. These pectin/caseinate particles do not coalesce and are insensitive to salt addition.     

Reassembly of brome mosaic virus from dissociated virus

The reconstitution of Brome Mosaic Virus (BMV) has been studied using neutron scattering. Experiments were performed on disassembled virus without subsequent separation of components. Phase diagrams of the disassembly and subsequent reassembly of BMV were established as a function of pH and LiCl molarity by analytical centrifugation and quasi-elastic light scattering. Disassembly occurs at a pH above 6.5 and above 0.8 M LiCl. On reassembly, if the pH is lowered first, capsids are formed without subsequent incorporation of RNA. Neutron scattering was used to investigate the formation of virus particles, when the ionic strength was lowered from 1.4 to 0.1 M LiCl at pH 7.8. The reconstitution was followed continuously. As it was driven by a lowering of the ionic strength the kinetics of the process cannot be studied for short times. However the fact that at any given ionic strength no evolution of the scattering was observed with time implies that the reconstitution is complete within a few minutes. The observations in buffers with various amounts of D₂O lead to the conclusion that the reassembly is achieved by co-condensation of the RNA and of the capsid proteins.     

Growth dynamics of domains in ternary fluid vesicles

We have studied the growth dynamics of domains on ternary fluid vesicles composed of saturated (dipalmitoylphosphatidylcholine), unsaturated (dioleoylphosphatidylcholine) phosphatidylcholine lipids, and cholesterol using a fluorescence microscopy. The domain coarsening processes are classified into two types: normal coarsening and trapped coarsening. For the normal coarsening, the domains having flat circular shape grow in a diffusion-and-coalescence manner and phenomenologically the mean size grows as a power law of approximately t(2/3). The observed growth law is not described by a two-dimensional diffusion-and-coalescence growth mechanism following the Saffman and Delbrück theory, which may originate from the two-body hydrodynamic interactions between domains. For trapped coarsening, on the other hand, the domain coarsening is suppressed at a certain domain size because the repulsive interdomain interactions obstruct the coalescence of domains. The two-color imaging of the trapped domains reveals that the repulsive interactions are induced by the budding of domains. The model free energy consisting of the bending energy of domains, the bending energy of matrix, the line energy of domain boundary, and the translation energy of domains can describe the observed trapped coarsening. The trapping of domains is caused by the coupling between the phase separation and the membrane elasticity under the incompressibility constraint.     

Turbidimetric and morphological studies of type I collagen fibre self assembly in vitro and the influence of fibronectin

Collagen undergoes several stages of self assembly including turbidimetric lag, growth and plateau steps. The later stages of type I collagen self assembly were studied by turbidity—time measurements, low angle laser light scattering and by determination of the birefringence retardation of collagen fibres formed in vitro. These studies were conducted in the presence and absence of fibronectin to evaluate the effect of fibronectin on the kinetics and extent of type I collagen fibrillogenesis. The results of these studies indicate that the collagen fibres observed at the end of the lag phase appear to be identical to fibres seen in the growth phase of turbidity—time curves based on fibre diameter and birefringence retardation measurements. Birefringence retardation measurements suggest that the diffracting unit may be the collagen fibril and that the volume fraction of fibrils in fibres is about 0.95 using a model developed for a series of parallel ellipsoids. Morphological observations suggest that the distribution of fibre diameters formed in vitro during the growth phase is narrow and appears to be independent of time with only the mass of collagen in fibres increasing during the growth phase. During the growth phase, layers of parallel fibres are formed with alternating layers appearing almost orthogonal. In the presence of fibronectin the mechanism of fibre formation appeared unchanged. It was concluded that fibronectin appeared to modify the kinetics of self assembly by preventing collisions between collagen molecules.     

Phase separation and drop size distributions in “homogeneous” Na-alginate/Na-caseinate mixtures

Phase separation and drop size distributions in dilute Na-caseinate/Na-alginate mixtures has been investigated using simultaneously two different measuring techniques: light scattering and image analysis. It has been found that even at very low concentrations of either polymer, where according to literature data the mixture should be homogenous, two phases can be observed. This phase separation was detected by both techniques and in each case, the drop size distributions measured by each of them were in good agreement.     

Kinetics of the cooperative association of T4 tail sheath protein P18 to polysheaths

The polymerization of the monomeric sheath protein P18 to polysheath was followed by light scattering in 1 mM sodium phosphate buffer, pH 7 at a MgCl2 concentration of 5 mM. Sigmoidal kinetics were observed in the case of spontaneous nucleation. These were well fitted by a mechanism involving a slow nucleation step (rate constant kN = 10(-2) M-1 S-1) followed by propagation steps (k = 10(5) M-1 S-1) in which P18 protomers are added to the ends of the polysheath particles. When sonicated polysheaths or contracted sheaths were added as seeds exponential time courses were observed. From the pseudo first order rate constant and the concentration of seeds the above value for the rate constant of propagation was confirmed. The ability of contracted sheaths to nucleate polysheath formation lends support to the conclusion that polysheaths and contracted sheaths have identical structures and differ in their length distributions only. These were measured from electromicrographs and from the distribution of sedimentation coefficients. Poisson type, kinetically controlled size distributions were found after polymerization of polysheath. An extremely slow redistribution towards an exponential distribution was detected. The spontaneous slow formation of polysheaths is much slower than the formation of extended sheath are core baseplates. Extended sheath is a metastable assembly produce of P18 which either dissociates of contracts to form contracted sheath. Polysheaths and contracted sheaths are extremely stable products but their immediate formation is hindered by high nucleation difficulties.