Protein/polyelectrolyte coacervation: Investigating its occurrence in the lysozyme- carboxymethylcellulose system.

The coacervation of carboxymethylcellulose (CMC) and hen egg white lysozyme (HEWL) was investigated. The work focused on the effects of pH, ionic strength, I, temperature, T, and mass fraction of the macromolecular components on the coacervation process by spectrophotometry, and on characteristics of the resulting coacervate phase by rheology in the linear and non-linear regimes. Coacervation was found to be highest at HEWL mass fraction ≈0.25 with very slight dependence on pH in the range from 5 to 9. The process was favored at I < 0.075 mol·L-1 for NaCl, KCl and NaBr. For BaCl2, the dependence was markedly distinct, reflecting differences brought about by a bivalent cation on the Debye length of the electric double layer. The coacervation process has been characterized as endothermic and entropically driven. Rheological analysis of the coacervates indicated a rather low limit of the linear viscoelastic region (0.1% strain) and the prevalence of the elastic over the viscous contribution (G´â€¯> G´´). Despite configuring rather fragile structures, the presence of a network of strong interactions has been inferred from the observed pattern of variation of the moduli with frequency. In the non-linear regime, pseudoplasticity was observed, pointing out to the capacity of these structures in complying with the flow lines of the circumventing environment. Both apparently opposed characteristics suggest a possible evolutionary advantage of such structures in the prebiotic era.

Non-ideal behavior of binary aqueous mixtures of some urea derivatives and their capacity to induce lysozyme gelation.

The urea derivatives, namely, ethylurea (EU), 1,3 dimethylurea (1,3-DMU) and 1,1 diethylurea (1,1-DEU), in the limiting regions of their solubilities in water, and tetramethylurea (TMU) at w≥0.65 were investigated in relation to their capacity of inducing hen egg white lysozyme (HEWL) physical (non-covalent) gelation. Protein transparent gels were generated out of TMU/H2O and 1,1-DEU/H2O, respectively, whereas an intensively turbid gel resulted from sol-gel transition taking place in EU/H2O. Oscillatory rheology revealed distinctions in the gels' structural and dynamic characteristics. Hydration patterns of the derivatives in solution, sizes of their non-polar domains and supramolecular symmetry features played a central role in their capacity of gel formation and in the gels' rheological behavior and morphology. Effects on gel characteristics of distinctively positioned ions in the Hofmeister series showed that SCN- disrupted water H-bonding interconnectivity in TMU lysozyme gel, strengthening gel structure, yet maintaining gel transparency. Citrate enhanced system elasticity albeit causing intense turbidity and leading to phase separation. Larger values of the storage modulus, G', were verified for gels generated from binary mixtures containing urea derivatives with higher dipole moments.

Chemistry and applications of polysaccharide solutions in strong electrolytes/dipolar aprotic solvents: an overview.

Biopolymers and their derivatives are being actively investigated as substitutes for petroleum-based polymers. This has generated an intense interest in investigating new solvents, in particular for cellulose, chitin/chitosan, and starch. This overview focuses on recent advances in the dissolution and derivatization of these polysaccharides in solutions of strong electrolytes in dipolar aprotic solvents. A brief description of the molecular structures of these biopolymers is given, with emphases on the properties that are relevant to derivatization, namely crystallinity and accessibility. The mechanism of cellulose dissolution is then discussed, followed by a description of the strategies employed for the synthesis of cellulose derivatives (carboxylic acid esters, and ethers) under homogeneous reaction conditions. The same sequence of presentation has been followed for chitin/chitosan and starch. Future perspectives for this subject are summarized, in particular with regard to compliance with the principles of green chemistry.

DMSO-induced denaturation of hen egg white lysozyme.

We report on the size, shape, structure, and interactions of lysozyme in the ternary system lysozyme/DMSO/water at low protein concentrations. Three structural regimes have been identified, which we term the "folded" (0 < φ(DMSO) < 0.7), "unfolded" (0.7 ≤ φ(DMSO) < 0.9), and "partially collapsed" (0.9 ≤ φ(DMSO) < 1.0) regime. Lysozyme resides in a folded conformation with an average radius of gyration of 1.3 ± 0.1 nm for φ(DMSO) < 0.7 and unfolds (average R(g) of 2.4 ± 0.1 nm) above φ(DMSO) > 0.7. This drastic change in the protein's size coincides with a loss of the characteristic tertiary structure. It is preceded by a compaction of the local environment of the tryptophan residues and accompanied by a large increase in the protein's overall flexibility. In terms of secondary structure, there is a gradual loss of α-helix and concomitant increase of ß-sheet structural elements toward φ(DMSO) = 0.7, while an increase in φ(DMSO) at even higher DMSO volume fractions reduces the presence of both α-helix and ß-sheet secondary structural elements. Protein-protein interactions remain overall repulsive for all values of φ(DMSO). An attempt is made to relate these structural changes to the three most important physical mechanisms that underlie them: the DMSO/water microstructure is strongly dependent on the DMSO volume fraction, DMSO acts as a strong H-bond acceptor, and DMSO is a bad solvent for the protein backbone and a number of relatively polar side groups, but a good solvent for relatively apolar side groups, such as tryptophan.

Solvent-induced lysozyme gels: effects of system composition and temperature on structural and dynamic characteristics.

The gelation process of lysozyme in water/tetramethylurea in the presence of salt was investigated as a function of temperature and system composition by rheology, infrared spectroscopy, and microcalorimetry. Times and temperatures of gelation were determined from the variation of the storage (G') and loss (G'') moduli. It was found that gelation times follow exponential decays with both protein and tetramethylurea (TMU) concentrations and with temperature. The activation energy for the overall process shows a linear dependence on TMU mass fraction. A strongly increased beta-sheet content and reduced alpha-helix occur with the increase of TMU concentration in the binary solvent. Also, a linear decrease of lysozyme denaturation temperature and enthalpy on TMU concentration is found for the TMU mass fraction up to 0.5, above which no denaturation signal can be detected.

Low-resolution 1H spin-spin relaxation of n-decane/water emulsions stabilized by beta-casein.

A low-resolution 1H NMR relaxometry study on the dynamics of an n-decane/water emulsion stabilized by beta-casein is presented. Spin-spin (transverse) relaxation time constants (T2) were used to assess relative mobilities of emulsion components, by a selective deuteration procedure. Data analysis allowed the emulsion investigated to be described by a heterogeneous collection of dynamically distinct populations. A major population of n-decane molecules presented an average mobility that very nearly approached that of pure solvent, which is compatible with its occurrence in the emulsion continuous microphase. beta-Casein molecules displayed a prevalent population with significantly decreased mobility as compared to the free protein in solution, which is in accordance with the protein location at the oil/water interface. Also, a major H2O population with significantly lower average T2 as compared to the pure liquid was detected and has been assigned to interfacial water.

Solvent-induced lysozyme gels: rheology, fractal analysis, and sol-gel kinetics.

In this work, the gelation kinetics and fractal character of lysozyme gel matrices developed in tetramethylurea (TMU)-water media were investigated. Gelation times were determined from the temporal crossover point between the storage, G', and loss, G'', moduli, as a function of the binary solvent composition and of protein concentration. The inverse dependence of the upper limit of the linear viscoelastic region (gamma0) on protein concentration indicate that the lysozyme gels belong to the "strong link" kind, a gel category where interparticle links are stronger than intraparticle ones. Lysozyme gel fractal dimensions (Df) were determined from the analysis of rheological data according to a scaling theory by Shih et al. [Phys. Rev. A 42 (1990) 4772-4779] and were found to be compatible with a diffusion-limited cluster-aggregation kinetics (DLCA) for lysozyme gels formed at the TMU mass fraction in the binary organic-aqueous solvent, wTMU=0.9, and with a reaction-limited cluster aggregation kinetics (RLCA) for wTMU in the 0.6< or =wTMU< or =0.8 range.

Gliadin effect on fluctuation properties of phospholipid giant vesicles.

Gliadin is a fraction of wheat gluten, a protein supramolecular complex known for its remarkable and biotechnologically relevant viscoelastic properties. Very high molecular mass characterise these systems, thus hindering high-resolution structural investigations. It is known, however, that these proteins comprise rather extended, extensively interassociated structures, which respond for their peculiar mechanical behaviour. Besides these properties, some of gluten's fractions, such as gliadin, are also known to be involved in a nutritionally relevant pathology of auto-immune character, the celiac disease, supposedly related to some unusual structural features of the protein. Despite its medical relevance, however, the role played by gliadin in the etiology of the celiac disease is not sufficiently understood to date. In this work, we investigated the role of gliadin on mechanical properties of a membrane model of dioleoylphosphatidylcholine (DOPC) giant unilamellar vesicles. The technique of micropipette aspiration, coupled to videomicroscopy, was applied. The microvesicles, produced by electric field pulsing over metal-covered plates, were suctioned into the micropipettes under varying applied pressures. A significant increase in the values of the bilayer curvature constant, k(c), was observed, with a saturation effect being verified at around 0.02-0.03 gliadin/DOPC mass ratio, indicating that the membrane becomes less elastic in the presence of the protein. Possible correlations between the observed membrane fluctuation properties and the celiac disease etiology are suggested and discussed.

Rheological study on lysozyme/tetramethylurea viscoelastic matrices.

Rheological properties of lysozyme viscoelastic matrices resulting from a sol-gel transition taking place in organic/aqueous media at room temperature were investigated. Gel-like structures, of transparent appearance, developed out of lysozyme (5.0 mmol/dm(3)) dispersed in tetramethylurea (TMU)/water binary mixtures, at TMU mass fraction (w) ranging from w(TMU) 0.6 to 0.9. The wide linear viscoelastic region (LVR) observed, up to strains of 10%, was invariant throughout the TMU concentration range investigated, indicating that the 3D structures of protein matrices, although fragile, are quite flexible and able to withstand great deformation before rupture. Storage (G') and loss (G") moduli continuously increased with increasing TMU concentration, the former at a greater rate, consequently leading systems to a decrease in the loss angle, tandelta. For gels developed out of binary systems at w(TMU)=0.9, creep curves revealed behaviour that very nearly approaches that of a perfect elastic solid. Although gelification under the experimental conditions employed is macroscopically accomplished in a time interval that does not exceed 24 h (for the gel developed out of the solvent mixture of lowest TMU concentration, w(TMU)=0.6), a slight decrease in loss angle can still be detected after that period. Such changes, however, have no effect on the LVR. Relaxation tests indicate that systems comprise at least two dynamically distinct contributions.

Lysozyme viscoelastic matrices in tetramethylurea/water media: a small angle X-ray scattering study.

Semi-solid viscoelastic matrices produced out of lysozyme in organic/aqueous media [tetramethylurea (TMU)/water] were characterized by small angle X-ray scattering (SAXS). The scattering curves were modeled in their form and interference factors. Radii of gyration of scattering particles were found to undergo a dramatic increase from 14 A in water to approximately 44 A in the matrices. Average correlation distances d=155 A were consistently verified for the scattering particles in the matrices, irrespective of solvent composition (in the 0.6