Interfacial rheology of surface-active biopolymers: Acacia senegal gum versus hydrophobically modified starch

Acacia gum is a hybrid polyelectrolyte containing both protein and polysaccharide subunits. We study the interfacial rheology of its adsorption layers at the oil/water interface and compare it with adsorbed layers of hydrophobically modified starch, which for economic and political reasons is often used as a substitute for Acacia gum in technological applications. Both the shear and the dilatational rheological responses of the interfaces are considered. In dilatational experiments, the viscoelastic response of the starch derivative is just slightly weaker than that for Acacia gum, whereas we found pronounced differences in shear flow: The interfaces covered with the plant gum flow like a rigid, solidlike material with large storage moduli and a linear viscoelastic regime limited to small shear deformations, above which we observe apparent yielding behavior. In contrast, the films formed by hydrophobically modified starch are predominantly viscous, and the shear moduli are only weakly dependent on the deformation. Concerning their most important technological use as emulsion stabilizers, the dynamic interfacial responses imply not only distinct interfacial dynamics but also different stabilizing mechanisms for these two biopolymers.     

Nanoparticle-induced assembly of hydrophobically modified chitosan

Hydrophobically modified chitosan (HMC) self-assembles in solution to form gels, making it suitable for applications in oil dispersion, hydrogel design and wound dressing. The self-assembly of HMC is driven by the association of hydrophobic moieties that are attached to chitosan monomers along the polymer chain. We present the results of discontinuous molecular dynamics simulations aimed at understanding how the length and density of the hydrophobic modification chains attached to HMC affect self-assembly and the structure of the resulting network. Long modification chains are required to promote the formation of a stable network in solution at a modification density of 5%; the networks form more readily at a modification density of 10%. The pore size distribution of the resulting HMC network is relatively independent of the modification chain length and density. Insertion of different sized hydrophobic nanoparticles into HMC has a significant impact on network formation, with the particles acting as junction points that promote the association of several HMC chains. The networks form faster in the presence of many small nanoparticles than in the presence of few large nanoparticles. We conclude that HMC could be a viable candidate to form hydrogels in solution.     

Layered structures of hydrophobically modified chitosan derivatives

Two series of chitosan derivatives, N-aliphatic acyl chitosans (C_n-CS) and N-aliphatic-O-dicinnamoyl-chitosans (C_n-CinCS) with n=2, 4, 8, 12 and 18, were prepared through regioselective reactions. The solid state structures were studied by FTIR and X-ray diffraction techniques. Two different types of layered structures were found to exist in the powder samples of C_n-CS and C_n-CinCS. The C_n-CS series crystallized into a sheet-type structure, in which all the flexible side chains lied down in the sheet in a direction normal to the backbones and partially interdigitated with each other. For the C_n-CinCS series, the polar backbones were stacked into sheets, and the flexible side chains occupied the space between sheets in a direction inclined to the backbones. The relationship between the structures and the solubilities are discussed.     

Fluorescence characterization of chemical microenvironments in hydrophobically modified chitosan

In this work we use the steady state and time-resolved fluorescence of free and enzyme-bound fluorophores to characterize the binding capacity of both unmodified and hydrophobically modified chitosan polymers. Additionally, fluorescence emission is used to qualitatively characterize the extent to which hydrophobic modification of the chitosan polymer affects the relative polarity of the resultant amphiphillic micelles. In total, these results are used to describe how fluorescence techniques can be used to characterize the chemical microenvironment provided by immobilization polymers such as chitosan. Commentary is also given on how this information can be correlated to enzyme activity and spatial distribution during the immobilization processes.     

Characterisation of a highly hydrophobically modified lactate dehydrogenase

1. Lysine residues of porcine H4 lactate dehydrogenase (L-lactate:NAD+ oxidoreductase EC 1.1.1.27) were modified with methyl-epsilon-(N-2,4-dinitrophenyl)aminocaproimidate - HCl. With increasing incorporation of the reagent a linear decrease of enzymatic activity was noticed. No essential lysyl group with an extraordinary reactivity was modified. 2. The active forms of the modified enzyme with different incorporation values were separated from denatured material by fractional precipitation and gel chromatography. An epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase was obtained with an average incorporation of 38 groups per tetramer and a residual activity of 42%. This material proved to be homogenous in cellulose electrophoresis. 3. The epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase is soluble only in glycine buffer at pH 8 and can be stabilized as ternary complex with NAD+ and sodium sulfite. Gel chromatography and ORD measurements show no strong conformational change. 4. epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase has similar Km values for pyruvate, NADH, lactate and NAD+ as the native enzyme, and shows a lower thermostability due to a diminished stabilization by the hydrate layer on the surface.     

Aqueous solutions of native and hydrophobically modified polysaccharides: temperature effect

Amphiphilic polysaccharides, obtained by the attachment of various hydrocarbon groups onto dextran, are studied in aqueous solutions. The viscosity of their aqueous solutions is examined as a function of concentration and temperature in the range 25-65 degrees C. Varying polymer concentration, viscosity follows a polynomial development of Huggins equation in which the coefficients can be calculated from the Huggins constant determined in the dilute domain (Matsuoka-Cowman equation). For all polymers, the solution viscosity follows an Arrhenius-like variation with temperature. The activation energy of the aqueous solutions is determined as a function of polymer concentration and structural characteristics (nature and amount of grafted hydrocarbon groups). The variation of activation energy with polymer concentration is shown to be consistent with predictions based on the Matsuoka-Cowman equation combined with the equation of Andrade. This conclusion is extended to other polysaccharides using data from the literature.     

Enzymic degradation of starch in cotton leaves

Starch degradation enzymes and their products were determined from chloroplasts and leaves of cotton (Gossypium hirsutum L. cv Coker 100) exposed t     

Autophagic degradation of leaf starch in plants

Autophagy is an evolutionarily conserved process in eukaryotic cells that functions to degrade cytoplasmic components in the vacuole or lysosome. Previous research indicates that the core molecular machinery of autophagosome formation works well in plants, and plant autophagy plays roles in diverse biological processes such as nutrient recycling, development, immunity and responses to a variety of abiotic stresses. Recently, we reported that autophagy contributed to leaf starch degradation, which had been thought to be a process confined to chloroplasts. This finding demonstrated a previously unidentified pathway of leaf starch depletion and a new role of basal autophagy in plants.     

Calcium-induced changes in thyroglobulin conformation

Polyethylene glycol solutions (10% w/v) were used to detect the effect of mono- and divalent cations on some properties of thyroglobulin. It is shown that in presence of 10% w/v polyethylene glycol in 0.01 M Tris-HCl, pH 7.5, calcium (less than 0.05 M) modifies the solubility, the sedimentation rate, and the Stokes' radius of thyroglobulin, while monovalent cations up to 0.6 M do not effect any of these properties. These findings can be explained by an increase in molecular compactness of thyroglobulin. Furthermore, it was shown that a synthetic polymer, polyethylene glycol, could be used to detect conformational changes.     

Effects of degree of deacetylation on enzyme immobilization in hydrophobically modified chitosan

Chitosan is a deacetylated form of the polysaccharide chitin. Over the last decade, researchers have employed reductive amination to hydrophobically modify chitosan to induce a micellar structure. These micellar polymers have been used for a variety of purposes including drug delivery and enzyme immobilization and stabilization. However, commercial sources of chitosan vary in their degree of deacetylation and there remains a paucity of information regarding how this can impact the modified polymer’s functionality for enzyme immobilization. This paper, therefore, evaluates the effect that the degree of deacetylation has on the hydrophobic modification of medium molecular weight chitosan via reductive amination with long chain aldehydes and the resulting changes in enzyme activity after the immobilization of glucose oxidase in the micellar polymeric structure. The chitosan was deacetylated to differing degrees via autoclaving in 40–45% NaOH solutions and characterized using NMR, viscosity measurements, and differential scan calorimetry. Results suggest that a high degree of deacetylation provides optimal enzyme immobilization properties (i.e. high activity), but that the deacetylation method begins to significantly decrease the polymer molecular weight after a 20 min autoclave treatment, which negatively affects immobilized enzyme activity.     

Rheology and nanostructure of hydrophobically modified alginate (HMA) gels and solutions

The effect of hydrophobic modification on the mechanical and structural characteristics of hydrophobically modified alginate (HMA) solutions and hydrogels were evaluated. The HMA systems consisted of alkyl chains, C₈, grafted onto alginate backbones. With an increase in degree of substitution of hydrophobic tails, the association became stronger in solution, but same was not true for gels. The contribution of ionic crosslinking was found to be the dominant factor in determining the mechanical strength of hydrogels. Rheological measurements of 2 wt% HMA gels reveal formation of a strongly crosslinked network with an elastic modulus close to 100 kPa. Small-angle X-ray scattering (SAXS) experiments indicate that HMA assembles into a disordered structure with regions rich in the hydrophobic domain surrounded by a crosslinked hydrophilic network.     

Ultraviolet light-induced changes in ribonuclease conformation

Ultraviolet light-induced inactivation of RNase A is accompanied by a decrease in the circular dichroism (CD) at the extrema observed at 239 nm and 275 nm. As inactivation progresses a new CD band centered near 325 nm also develops. For RNase maintaining up to one half of its original activity an isoelliptic point may be noted near 257 nm. These findings are consistent with initial normalization of a “buried” tyrosyl residue followed by chemical modification of the protein. Further chemical change and extensive conformational reorganization of the protein appear to accompany advanced inactivation.     

pH-induced changes in G-actin conformation and metal affinity

Metal-induced conformational changes in actin at 20 degrees C have been investigated as a function of pH using actin labeled at Cys-374 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. At pH 8, the addition of a high Ca2+ concentration (2 mM) to G-actin gives an instantaneous fluorescence increase while the addition of a high Mg2+ concentration gives both an instantaneous and a slow fluorescence increase. The instantaneous increase is interpreted as divalent cation binding to low-affinity, relatively nonspecific sites, while the slow response is attributed to Mg2+ binding to specific sites of moderate affinity [Zimmerle, C.T., Patane, K., & Frieden, C. (1987) Biochemistry 26, 6545-6552]. The magnitudes of both the instantaneous and slow fluorescence increases associated with Mg2+ addition to G-actin are shown here to decrease as the pH is lowered while the fluorescence of labeled G-actin in the presence of low or moderate Ca2+ concentrations (less than 200 microM) increases. The pH-dependent data suggest that protonation of a single class of residues with an approximate pK of 6.8 alters the immediate environment of the label differently depending upon the cation bound at the moderate-affinity site. The pH-dependent changes in the magnitude of the slow fluorescence response upon Mg2+ addition to Ca2+-actin are not associated with changes in the Mg2+ affinity at the moderate-affinity site but result from protonation altering the fluorescence response to Mg2+ binding. Protonation of this same class of residues is proposed to induce an actin conformation similar to that induced by cation binding at the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Viscometric and spectroscopic studies on the solution behaviour of hydrophobically modified cellulosic polymers

The solution properties of hydroxyethyl cellulose (HEC) and hydrophobically modified hydroxyethyl cellulose (HM-HEC) have been investigated by means of viscometric and spectroscopic techniques involving free radical and fluorescent probes. The greater viscosity of HM-HEC solutions above a critical polymer concentration (C_p) of approximately 0·2% has been interpreted in terms of the formation of a three-dimensional network structure in which the polymer chains are effectively crosslinked by the intermolecular association of neighbouring hydrophobic side chains. C_p is considerably less than the predicted polymer coil overlap concentration (C*) of approximately 1%.

The interaction of the polymers with an anionic surfactant, sodium dodecyl sulphate (SDS) has also been investigated. A mechanism involving the interaction of free surfactant with the regions of intermolecular hydrophobic association is suggested to account for the considerable differences in the rheological behaviour of the polymers in the presence of SDS.     

Estimation of the in situ degradation of the washout fraction of starch by using a modified in situ protocol and in vitro measurements

The in situ degradation of the washout fraction of starch in six feed ingredients (i.e. barley, faba beans, maize, oats, peas and wheat) was studied by using a modified in situ protocol and in vitro measurements. In comparison with the washing machine method, the modified protocol comprises a milder rinsing method to reduce particulate loss during rinsing. The modified method markedly reduced the average washout fraction of starch in these products from 0.333 to 0.042 g/g. Applying the modified rinsing method, the fractional degradation rate (k _d) of starch in barley, oats and wheat decreased from on average 0.327 to 0.144 h⁻¹ whereas for faba beans, peas and maize no differences in k _d were observed compared with the traditional washing machine rinsing. For barley, maize and wheat, the difference in non-fermented starch in the residue between both rinsing methods during the first 4 h of incubation increased, which indicates secondary particle loss. The average effective degradation of starch decreased from 0.761 to 0.572 g/g when using the new rinsing method and to 0.494 g/g when applying a correction for particulate matter loss during incubation. The in vitro k _d of starch in the non-washout fraction did not differ from that in the total product. The calculated ratio between the k _d of starch in the washout and non-washout fraction was on average 1.59 and varied between 0.96 for oats and 2.39 for maize. The fractional rate of gas production was significantly different between the total product and the non-washout fraction. For all products, except oats, this rate of gas production was larger for the total product compared with the non-washout fraction whereas for oats the opposite was observed. The rate of increase in gas production was, especially for grains, strongly correlated with the in vitro k _d of starch. The results of the present study do not support the assumption used in several feed evaluation systems that the degradation of the washout fraction of starch in the rumen is much faster than that of the non-washout fraction.     

Rheological and structural characterization of the interactions between cyclodextrin compounds and hydrophobically modified alginate

Interactions in semidilute solutions of a hydrophobically modified alginate (HM-alginate) in the presence of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) monomer or a beta-cyclodextrin polymer (poly(beta-CD)) have been characterized at different temperatures with the aid of rheology and small-angle neutron scattering (SANS). The viscosity results for the HM-alginate/HP-beta-CD system reveal progressive deactivation of the hydrophobic associations as the concentration of HP-beta-CD increases. For the HM-alginate/poly(beta-CD) system, on the other hand, addition of poly(beta-CD) sets up bridges between adjacent polymer chains and thereby strengthens the associative network. A novel shear-thickening effect is observed at fairly high shear rates for the HM-alginate/poly(beta-CD) system, and this feature is influenced by temperature. Elevated temperature induces higher chain mobility and the formation of weaker network associations. Analyses of the SANS data disclosed that the association strength in HM-alginate/poly(beta-CD) mixtures increases strongly with increasing cosolute concentration, whereas no effect or a moderate weakening of the strength can be traced in HM-alginate/HP-beta-CD solutions upon addition of HP-beta-CD. The value of the correlation length xi is virtually not affected by the addition of cosolute for the HM-alginate/poly(beta-CD) system, whereas the decoupling of hydrophobic moieties of the polymer upon addition of HP-beta-CD gives rise to a smaller value of xi, suggesting that the size of the heterogeneity patches is reduced. The SANS results suggest that compact association structures are formed in the HM-alginate/poly(beta-CD) solutions.     

Sodium-induced conformation changes in membrane transport proteins

In the presence of KCl, tryptic digestion of vesicles derived from pigeon erythrocyte membranes inactivates sodium-dependent uptake of alanine by the vesicles, whereas digestion in the presence of NaCl does not. Extensive degradation of vesicle proteins occurs under both conditions. Similarly, the extent of inhibition by N-ethylmaleimide of the sodium-dependent influxes of both glycine and alanine into human erythrocytes is greater when the cells are exposed to the thiol reagent in the presence of KCl than when NaCl is used. These observations are interpreted as providing evidence for sodium-induced conformation changes in these transport proteins.