Analysis of Light Scattering Data on the Calcium Ion Sensitivity of Caseinate Solution Thermodynamics: Relationship to Emulsion Flocculation

We describe the quantitative interrelation between the thermodynamic parameters of caseinate submicelles in the presence of calcium ions (0-14 mM) in aqueous medium and the capacity of the protein to induce depletion flocculation in oil-in-water emulsions at pH 7.0 and ionic strength 0.05 mol dm(-3). Measurements have been made by static and dynamic multiangle laser light scattering of the weight-average molecular weight, the radius of gyration, the hydrodynamic radius, and the second virial coefficient of caseinate submicelles in aqueous solution. Successive thermodynamic approximations with and without consideration of correlations between caseinate submicelles have been used to calculate the osmotic pressure in caseinate aqueous solutions and the free energy of the depletion interaction between droplets in oil-in-water emulsions stabilized by caseinate. Numerical results from both thermodynamic approximations are in reasonably good agreement with experiment, predicting a pronounced decrease in the strength of the depletion attraction at concentrations of Ca(2+) in the range 4-8 mM (with a minimum value at 8 mM). This correlates well with the great enhancement of stability of these emulsions with respect to flocculation in comparison with systems having no added ionic calcium and emulsions with lower (2 mM) or higher (10 mM) Ca(2+) contents. Nevertheless, the allowance for interactive correlations between caseinate submicelles seems to lead to a better prediction of emulsion flocculation on a qualitative level over the whole range of Ca(2+) concentrations studied (2-14 mM). The calculated pronounced decrease in depletion interaction strength is attributable to marked changes in weight-average molecular weight and mean size of aggregates, and to more positive values of the second virial coefficient of caseinate submicelles with increasing Ca(2+) content. Finally, we discuss the part played by the electrical charge on the protein in determining the overall strength of the flocculation-inducing attractive interactions between droplets. Copyright 2001 Academic Press.     

A laser light scattering study of the interaction between human serum albumin and ampicillin sodium

1 Introduction Studies on the interaction of the complexes formed between proteins and amphiphilic molecules in aque- ous solutions have become a new focus and great pro- gress has been made in recent years[1―5]. An under- standing of these systems is of great importance in many biological processes and clinical use of drugs. The globular anionic protein human serum albumin (HSA) is widely used as a protein model in many studies[1―4,6]. Its principal function is to transport fatty acids an…     

聚羧酸系高性能减水剂分子在稀溶液中尺寸及其在体积排除色谱表征中表观分子量的模型研究

针对被称为"第一代聚羧酸高性能减水剂"(以下简称为MPEG-type PCE)的甲基丙烯酸(MAA)/烯酸甲酯(MAA-MPEG)梳状共聚物分子,从高分子物理基础理论出发,构建等效自由连接链模型,结合前人的理论结果和实验数据,得到了MPEG-type PCE分子的回转半径、流体力学半径及其相应的支化参数的数学表达式.在此基础上,报道了以下三方面的工作:首先,将计算结果与文献中的实验结果进行比较,检验模型的合理性;其次,利用所建立的数学模型考察主链分子量、侧链分子量和侧链接枝密度对PCE分子的回转半径和流体力学半径的影响;最后,结合近年来发展的体积排除色谱分离理论,对PCE分子的真实分子量与其常规体积排除色谱"表观分子量"(又被称为GPC分子量)两者之间的差异进行了分析.本文所提出的计算模型和数学表达式没有不确定的指前因子,可用来估算MPEG-type PCE分子在稀水溶液中的尺寸以及根据其GPC分子量估算真实分子量.     

Preparative isoelectric focusing of reduced wheat gluten proteins

Proteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2-mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3-10 gradient containing 50% v/v 1-propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1-propanol was comparable to that obtained on two-dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.     

Light Scattering Study of the Antibody–Poly(methacrylic acid) and Antibody–Poly(acrylic acid) Conjugates in Aqueous Solutions

The effect of the conformational state of the polymer coil on the properties of protein–polymer conjugates has been studied for the conjugates of antibody (monoclonal antibody from 6C5 clone against inactivated rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase; Ab) with poly(methacrylic acid) (PMAA) or poly‐(acrylic acid) (PAA). The pH‐dependencies of molecular properties and structural parameters of aqueous solutions (radius of gyration, intensity of scattered light, hydrodynamic diameter, and polydispersity index) of Ab, PMAA, and PAA and their conjugates, i. e., Ab‐PMAA and Ab‐PAA, have been studied using static and dynamic light scattering techniques. While free Ab aggregates in solution and precipitates at its isoelectric point, the covalent attachment of a charged polymer to Ab prevents its association and shifts the precipitation point towards more acidic values (from pH 5.95 for Ab to pH ˜ 4.8 for Ab‐PMAA). The predominant role of the conformational status of the polymer in the process of conjugate precipitation has been considered. Contrary to the precipitation of Ab‐PMAA, the formation of stable colloidal particles was suggested for Ab‐PAA at pH < 4.8. In the conjugates, polymer chains surround the protein globule in an extremely compact manner while Ab significantly affects the polymer conformation. The essentially larger hydrodynamic radii of conjugates, when compared with their radii of gyration, confirm the strong interaction of conjugates with solvent molecules.     

Structure of gum arabic and its configuration in solution

Gum arabic was found to have an osmotic molecular weight of 250,000, in agreement with earlier determinations. A molecular weight of 365,000 was found by light scattering, somewhat higher than obtained earlier by sedimentation equilibrium analysis but lower than light-scattering values reported by other investigators. The M?_w/M _n ratio, 1.46, is quite low in gum arabic. The angular dependence of light scattering exhibited the upward curvature to be expected of a spherical molecule and a radius of gyration of about 100 A. or less, as estimated from a Zimm plot. Fractionation of the original gum arabic was done by precipitation of a 0.5% solution in aqueous 0.5% NaCl with acetone. Comparison of the curves of viscosity versus molecular weight and the estimated radius of gyration shows that the hydrodynamic volume is less than that of branched dextran of similar molecular weight. The electroviscous effects for gum arabic in aqueous solution were shown by reduced viscosity curves at various acidities and in salt. The degree of dissociation was calculated for each pH level. The minimum intrinsic viscosity was found in 0.04N HCl where the degree of dissociation at pH 1.5 was found to be 0.049. When the acidity was increased, further reduction in viscosity was found to be negligible. Routine determination of the viscosity and molecular weight of the fractions was done in 0.35M NaCl at pH 10 to which 0.25% of the sodium salt of ethylenediaminetetraacetic acid was added as a sequestrant. The intrinsic viscosity in this solvent was nearly as low as in 0.04N HCl. Light-scattering dissymmetries in water and in 0.35M NaCl plus EDTA at pH 10 were similar, 1.13 and 1.09, respectively, which showed that actual expansion of the macroion is not the cause of the large increase in viscosity of gum arabic when the ionic strength of the solvent is reduced. Periodate oxidation of the polymer confirmed the existence of a 1–3-linked backbone of galactose. Subsequent treatment of the oxidized polymer with alkali reduced the osmotic molecular weight to 45,000 but failed to remove oxidized side branches. The oxidized polymer was fractionated by gel permeation chromatography and the intrinsie viscosity–molecular weight relation compared with relations for fractions of the unoxidized polymer and for other branched and crosslinked polymers.     

Formation of intra- and interparticle polyelectrolyte complexes between cationic nanogel and strong polyanion

Polyelectrolyte complex formation of a strong polyanion, potassium poly(vinyl alcohol) sulfate (KPVS), with positively charged nanogels was studied at 25 degrees C in aqueous solutions with different KCl concentrations (C(s)) as a function of the polyion-nanogel mixing ratio based on moles of anions versus cations. Used as the gel sample was a polyampholytic nanogel consisting of lightly cross-linked terpolymer chains of N-isopropylacrylamide, acrylic acid, and 1-vinylimidazole; thus, the complexation was performed at pH 3 at which the imidazole groups are fully protonated to generate positive charges. Turbidimetric titration was employed to vary the mixing ratio. Also employed for studies of the resulting complexes at different stages of the titration were dynamic light scattering (DLS) and static light scattering (SLS) techniques. It was found from the titration as well as DLS and SLS that there is a critical mixing ratio (cmr) at which both the size and molar mass of the complexed gel particles abruptly increase. The value of the cmr at C(s) = 0 or 0.01 M (mol/L) was observed at approximately 1:1 mixing ratio of anions versus cations but at lower mixing ratios than the 1:1 ratio under conditions of C(s) = 0.05 and 0.1 M. At the mixing ratios less than the cmr, the molar mass of the complex agrees with that of one gel particle with the calculated amount of the bound KPVS ions, indicating the formation of an "intraparticle" KPVS-nanogel complex, by the aggregation of which an "interparticle" complex is formed at the cmr. During the process of the intraparticle complex formation, both the hydrodynamic radius by DLS and the radius gyration by SLS decreased with increasing mixing ratio, demonstrating the gel collapse due to the complexation. At C(s) = 0 or 0.01 M and under conditions where the amount of KPVS bindings was less than half of the nanogel cations, however, the decrease of the hydrodynamic radius was very small, while the radius gyration fell monotonically. These results were discussed in connection with a collapse of dangling chains attached to the nanogel surface by the binding of KPVS.     

Microstructure of acid-induced caseinate gels containing sucrose: quantification from confocal microscopy and image analysis

We investigate the effect of sucrose on the microstructure of sodium caseinate gels induced by acidification. The average pore size and the fractal properties of two-dimensional slices of the gels are studied using confocal scanning laser microscopy and image analysis. The addition of sucrose promotes stronger and more fine-stranded gels while re-arrangements of the network tend to be prevented. Whereas the fractal dimension itself is not sensitive to changes in the gel microstructure upon addition of sucrose, the maximum cut-off distance, up to which fractal scaling behaviour applies, is substantially reduced, as is the average pore size. The overall microstructural changes seem to be consistent with previous rheological and light scattering studies of the same system.     

Effects of spray drying on physicochemical properties of milk protein-stabilised emulsions

The effect of spray drying and reconstitution has been studied for oil-in-water emulsions (20.6% maltodextrin, 20% soybean oil, 2.4% protein, 0.13 M NaCl, pH 6.7) with varying ratios of sodium caseinate and whey protein, but with equal size distribution (d₃₂=0.77 μm). When the concentration of sodium caseinate in the emulsion was high enough to entirely cover the oil–water interface, the particle size distribution was hardly affected by spray drying and reconstitution. However, for emulsions of which the total protein consisted of more than 70% whey protein, spray drying resulted in a strong increase of the droplet size distribution. The adsorbed amount of protein ranged from 3 mg m⁻² for casein-stabilised emulsions to 4 mg m⁻² for whey protein-stabilised emulsions with a maximum of 4.2 mg m⁻² for emulsions containing 80% whey protein on total protein, which means that for all these emulsions about one quarter of the available protein was adsorbed at the oil–water interface. The adsorbed amount of protein was hardly affected by spray drying. After emulsion preparation casein proteins adsorbed preferentially at the oil–water interface. As a result of spray drying, the relative amount of β-lactoglobulin in the adsorbed layer increased strongly at the expense of _s1-casein and β-casein. Percentages of _s2-casein and κ-casein in the adsorbed layer remained largely unchanged. The changes in the protein composition of the adsorbed layer as a result of spray drying and reconstitution were the largest when beforehand hardly any whey protein was present in the adsorbed layer and hardly any sodium caseinate in the aqueous phase. Apparently, during spray drying conditions have been such that β-lactoglobulin could unfold, aggregate, and react with other cystein-containing proteins changing the particle size distribution of the emulsions and the composition of the adsorbed layer. It seemed, however, that non-adsorbed sodium caseinate in some way was able to protect the adsorbed casein proteins from being displaced by aggregating whey protein.     

Retention behaviour of amylopectins in asymmetrical flow field-flow fractionation studied by multi-angle light scattering detection

Asymmetrical flow field-flow fractionation (FFF) with multi-angle light scattering (MALS) detection was applied for the fractionation of amylopectins from four different sources. Samples originated from genetically modified potatoes and waxy maize. Amylopectins were dissolved in a 1 mol l(-1) sodium hydroxide solution or water. With an injected mass of 0.2 microg, well below overloading conditions, a decrease of the apparent hydrodynamic radius with increasing inlet flow-rate was observed. Moreover, a decrease of the radius of gyration with increasing elution volume was recorded by the MALS detector. Steric/hyperlayer effects are a feasible explanation for this behaviour. The observed radius of gyration at the steric inversion point was in the order of 0.3 microm, which is smaller than the theoretically calculated inversion point. Apparently, the amylopectin behave as macromolecules with a larger hydrodynamic radius than expected on basis of their radius of gyration and are subjected to significant lift forces. The results were confirmed by four fractionations with varying flow-rates but constant ratio of cross to outlet-flow. In contrast to the normal mode operation, the retention of the amylopectins depended strongly on the applied flow-rates and was close to that of a much smaller 10 kDa dextran. Apparent molar masses in the order of between 10(7) and 10(9) g mol(-1) were obtained. The results are contrasted with enzymatically degraded and oxidised starch samples that were fractionated in the normal mode.     

Hydrodynamic Radius Characterization of a Vinyl-Type Polynorbornene by Size-Exclusion Chromatography with a Static and Dynamic Laser Light Scattering Detector

Both absolute molecular weight and molecular sizes (radius of gyration and hydrodynamic radius) of a vinyl-type polynorbornene eluting from size-exclusion chromatography columns were determined by combined with a static and dynamic laser light scattering detector. The hydrodynamic radius of polymer fraction eluting from size-exclusion chromatography columns was obtained from dynamic laser light scattering measurements at only a single angle of 90° by introducing a correction factor. According to the scaling relationship between molecular sizes and molecular weight and the ratio between radius of gyration and hydrodynamic radius, the vinyl-type polynorbornene took a random coil conformation in 1,2,4-trichlorobenzene at 150 °C.     

Single step hybrid coating process to enhance the electrosteric stabilization of inorganic particles

We report on a single-step coating process and the resulting colloidal stability of silica-coated spindle-type hematite nanoparticles (NPs) decorated with a layer of poly(acrylic acid) (PAA) polyelectrolyte chains that are partially incorporated into the silica shell. The stability of PAA coated NPs as a function of pH and salt concentration in water was compared to bare hematite particles and simple silica-coated hematite NPs, studying their electrophoretic mobility and the hydrodynamic radius by dynamic light scattering. Particles coated with this method were found to be more stable upon the addition of salt at pH 7, and their aggregation at the pH of the isoelectric point is reversible. The hybrid coating appears to increase the colloidal stability in aqueous media due to the combination of the decrease of the isoelectric point and the electrosteric stabilization. This coating method is not limited to hematite particles but can easily be adapted to any silica-coatable particle.     

Electrical Conductivity and Complexation of Sodium Borate in Trehalose and Sucrose Aqueous Solutions

The formation equilibrium constants for the esterification reaction between sucrose or trehalose and borate ion have been determined by measuring the pH changes produced when the corresponding sugar is added to sodium borate solutions. The speciation of the ternary sugar–borate–water system was calculated from this equilibrium information and used to separate the contribution of each ionic species to the electrical conductivity of the system. From the analysis of the results it is concluded that, in the viscosity region studied, the complex borate–trehalose and borate–sucrose ions obey Walden's rule, while the behavior of the smaller borate ion is not clear. The dramatic difference of the infinite dilution molar conductivity, and consequently of the hydrodynamic radius, of the trehalose– and sucrose–borate ester ions is ascribed to the formation of an intramolecular diester in aqueous trehalose.     

On relationships between molecular structure, interaction and surface behavior in mixture: small-molecule surfactant+protein

We report on the effect of distinct in nature small-molecule surfactants (model, a sodium salt of capric acid, Na-caprate; and commercially important, a citric acid ester of monoglyceride, CITREM; a sodium salt of stearol-lactoyl lactic acid, SSL (Na(+)); polyglycerol ester, PGE (080)) on molecular properties in a bulk and at the air-water interface of globular legumin and random-coiled micellar sodium caseinate. The role of the structure of both proteins and small-molecule surfactants in the effect studied has been elucidated by measurements in a bulk aqueous medium of the enthalpy of their interaction from mixing calorimetry, the change in value of weight average molecular weight of the proteins and the thermodynamics of the pair protein-protein interactions from laser static light scattering as well as, in addition, by measurements of the change in hydrodynamic radius for micellar sodium caseinate from laser dynamic light scattering. The effect of the small-molecule surfactants on the thermodynamics of the protein heat denaturation and thereby on the protein conformational stability has been studied by differential scanning calorimetry in the case of globular legumin. The interrelation between the effects of the small-molecule surfactants on the properties of the proteins in a bulk and at the planar air-water interface has been elucidated by tensiometry. The combined data of mixing calorimetry, differential scanning calorimetry and laser light scattering suggest some complex formation between the small-molecule surfactants and the proteins in a bulk aqueous medium. Predominantly hydrophobic interaction along with electrostatic and hydrogen bonding form the basis of the complex formation. The found effect of the small-molecule surfactants on the surface activity of their mixtures with proteins is governed primarily by both the extent of the protein association, resulting in specific hydrophobicity/hydrophilicity of the surface of the protein associates, and the specific protein conformational stability, for the globular protein, produced by the interaction between the proteins and the small-molecule surfactants.     

Ostwald ripening of protein-stabilized emulsions: effect of transglutaminase crosslinking

Ostwald ripening in n-alkane oil-in-water emulsions stabilized by sodium caseinate at neutral pH has been studied by monitoring time-dependent changes in the number-average droplet diameter and the droplet-size distribution. In qualitative agreement with theory, the destabilization rate has been shown to increase with reduction of the n-alkane chain length and on addition of ethanol to the aqueous phase. Replacement of caseinate by β-lactoglobulin also leads to improved stability, but addition of calcium ions does not. The potential use of transglutaminase-induced crosslinking of adsorbed protein as a way of inhibiting the Ostwald ripening of caseinate-stabilized emulsions has been examined. It is shown that enzymic crosslinking before emulsification can lead to a modest reduction in the coarsening rate at long storage times. Crosslinking of caseinate after emulsification produces enhanced stability at short times, but there is a catastrophic loss of stability at long times due to droplet coalescence.     

Hydrodynamic radius of polyethylene glycol in solution obtained by dynamic light scattering

In order to compare the size characterizations in poly(ethylene glycol) (PEG) obtained by dynamic light scattering (DLS) and small angle neutron scattering (SANS), DLS experiments were performed in various PEG solutions to ascertain the hydrodynamic radius. Data from the experiments were analyzed by using a method to eliminate effects of PEG aggregation on dynamic correlation functions. The results of the analysis were then compared to the radii of gyration reported from SANS experiments. The relation between the hydrodynamic radius, obtained by DLS, and the radius of gyration, obtained by SANS, in PEG in solution was found to be in agreement with a previously obtained relation for PEG, where the radius of gyration was found by static light scattering.     

Dynamic light scattering from non-entangled wormlike micellar solutions

The fuzzy cylinder theory, originally proposed for conventional polymer solutions, was applied to wormlike micellar solutions to take into account effects of the intermicellar collision and hydrodynamic interaction on the self-diffusion of wormlike micelles in solution at finite concentrations. Previously reported apparent hydrodynamic radius data obtained by dynamic light scattering for non-entangled wormlike micelles formed in aqueous solution by non-ionic surfactants, polyoxyethylene monoalkyl ethers C(i)E(j), were analyzed by this theory to estimate the persistence length q of the wormlike micelles. The results of q estimated were consistent with those obtained from radius of gyration data obtained by static light scattering.     

Trypsin activity assay in substrate-specific one- and two-dimensional gels: a powerful method to separate and characterize novel proteases in active form in biological samples

To separate and identify the proteases, a substrate-specific, sensitive assay in sodium dodecyl sulfate (SDS)-polyacrylamide gels after two-dimensional (2-D) electrophoresis has been developed. This method allows simultaneous determination of protease cleavage specificity, molecular weight, isoelectric point, and if necessary, amino acid sequencing. After isoelectric focusing in immobilized pH gradient (IPG) strips (pH 6-11) (first dimension), trypsin was electrophoresed in 12% SDS polyacrylamide gels (second dimension) copolymerized with Boc-Gln-Ala-Arg-MCA (4-methyl-coumaryl-7-amide). The gels were washed in cold 2.5% Triton X-100 and water, and incubated in assay buffer (6.3 mM Bicine, 100 mM NaCl). Trypsin cleavage of the peptide-MCA generated fluorescent 7-amino-4-methyl-coumarin. In 1-D gels, as low as 500 pg trypsin could be detected and trypsin band volumes correlated linearly with the amounts of trypsin (R(2) = 0.999). In 2-D gels, the lowest amount of trypsin detected was 1 ng. The linear regression of spot volume and loading amount was still good (R(2) = 0.974). To optimize renaturation conditions, 5x5 min washes with 2.5% Triton X-100 and water, respectively, gave the strongest band volume. For fluorescence development, an assay buffer at pH 9 was the best; incubation at 37 degrees C for 30 min was sufficient. The method has application for identifying novel proteases as it does not rely on antibodies.     

Preparation and characterization of nanoparticles formed by chitosan-caseinate interactions

Intermacromolecular complexation between chitosan and sodium caseinate in aqueous solutions was studied as a function of pH (3–6.5), using absorbance measurements (at 600 nm), dynamic light scattering (DLS), and transmission electron microscopy (TEM). The chitosan–caseinate complexes formed were stable and soluble in the pH range 4.8–6.0. In this pH range, the biopolymers had opposite charges. At higher concentrations of chitosan (0.15 wt%), the soluble complexes associated to form larger particles. DLS data showed that, between pH 4.8 and 6.0, the particles formed by the complexation of chitosan and caseinate had sizes between 250 and 350 nm and these nanoparticles were visualized using negative staining TEM. Above pH 6.0, the nanoparticles associated to form larger particles, causing phase separation. Addition of NaCl increased the particle size. The pH dependence of the zeta potential of the mixture solutions was appreciably different from that of the pure protein and pure chitosan solutions.     

Mean square radius of gyration and hydrodynamic radius of jointed star (dumbbell) and H-comb polymers

Equations for the mean square radius of gyration and the hydrodynamic radius for jointed stars (dumbbells) and H-combs are derived, based on random flight statistics for each subchain. Comparision with literature data on computer simulations and experimental data for H-combs show good agreement for the g-value of the mean square radius of gyration even in good solvents. This suggests that for the mean square radius of gyration the relative dimension of a H-comb relative to the linear molecule of the same degree of polymerization is not altered significantly by long range interactions, as in the case of star polymers. For the hydrodynamic radius the situation is different. Fair agreement is found when comparing our results with viscosity measurements under θ conditions, while in good solvents the viscosity of the branched molecule is more reduced than predicted by our results.