V. Ya. Grinberg

Application of phase-volume-ratio method for determining the phase diagram of water-casein-soybean globulins system

SummaryThe possibility of using the phase-volume method for determining phase diagrams of polymer mixtures in a common solvent has been ascertained. The above method was used to determine a phase diagram of water-casein-soybean globulins system.

Phase equilibria in water-protein-polysaccharide systems: II. Water-casein-neutral polysaccharide systems

SummaryPhase separation and thermodynamic stability of water (W) — casein (C) — neutral polysaccharide (NPS) systems have been studied. Dextrans of different molecular weights D40, D150, D500 and D2000 (10−3Mw=40, 150, 500, 2000), ficoll (10−3Mw=400) and amylopectin (10-−6Mw= 38) have been used as neutral polysaccharides. Phase diagrams of W-C-NPS systems, as well as their effect on the thermodynamic stability of the molecular weight and structural features of polysaccharides, low-molecular salts, pH and temperature have been considered. There has been shown the similarity of conditions under which the stability of W-C-NPS systems is disturbed, resulting in their separation, as well as conditions favorable to self-association of casein. A decrease in the pH value, an increase in the ionic strength and a rise in temperature are favorable both to self-association of casein and separation of W-C-NPS systems. Proceeding from this fact and from the results obtained earlier for W-C-acidic polysaccharide and W-albumin-D-glucan systems, a conclusion is drawn as to the common nature of the relationship between self-association of polymers and their compatibility. At constant temperature and pH, the stability limit of W-C-NPS systems is determined by the concentrations of polymers (w2 andw3) and salt (C4). The totality of cloud points C4* (pH) at givenw2 andw3 being considered as the stability limit. It is also shown that the relationship C4* (pH) is an increasing function within the range of pH values from 6.5 to 11.5 and in all cases where lim C4* (pH)=0. The sequence of values 947-1PH→PH IEP C4* is determined by the nature of the low-molecular salt (Na2SO4 ≊ NaCl ≪ KSCN) and the specific nature of the polysaccharides (D2000≊ amylopectin < D 150 < ficoll < D 40).W-C-NPS-NaCl have been found to possess a lower critical point in a system of coordinates: temperature-composition. W-C-NPS-urea (6M) systems feature, in a system of coordinates: pH-polymer concentration, two areas of separation, overlapping at sufficiently high total concentrations. This fact is indicative of the specific nature of interaction of casein macromolecules capable of association near the isoelectric point due to interaction of charge fluctuations and, in the acid region, due to interaction of non-ionized carboxyl groups. The obtained results are discussed with the stability limit being expressed in terms of second virial coefficients.

Phase equilibria in water-protein-polysaccharide systems

SummaryPhase equilibria in the ternary water-soy bean globulins-polysaccharides systems, containing various acidic and neutral polysaccharides (pectin, sodium alginate, carboxymethylcellulose, arabic gum, dextransulphate, and dextran) were studied. Phase diagrams of the systems were obtained. Effects of pH, concentration of a neutral salt and urea on the compatibility of soy bean globulins with polysaccharides in water media were studied. It has been shown that soy bean globulins could be incompatible with acidic carboxyl-containing polysaccharides in the whole studied range of pH (2–12) and NaCl concentrations (0–1 mol·l−1). Incompatibility of soy bean globulins with sulphated polysaccharides takes place only under condition of high ionic strength independently of pH of the system. Incompatibility of soy bean globulins with neutral polysaccharides at pH ≠ pI occured only at high ionic strength or in the presence of 6 M urea. The change pattern in separation bodies of the water-soy bean globulins-polysaccharide systems at pH < pI differed from that at pH > pI and varied when passing from carboxyl-containing polysaccharides to sulphated or neutral. Under equal conditions compatibility of soy bean globulins with polysaccharides increased in the series: pectin < carboxymethylcellulose < sodium alginate < arabic gum < dextran < dextransulphate.ZusammenfassungEs wurden Phasengleichgewichtsprozesse von Dreikomponentensystemen Wasser-Sojabohnenglobuline-Polysaccharid untersucht, die verschiedene saure und neutrale Polysaccharide enthalten: Pektin, Natriumalginat, Karboxymethylzellulose, arabisches Gummi, Dextransulfat und Dextran. Es wurden isotherme Phasendiagramme der aufgezeigten Systeme erhalten. Man untersuchte den Einfluß des pH-Wertes, der Konzentration des neutralen Salzes und des Harnstoffes auf die Kompatibilität (Verträglichkeit) der Sojabohnenglobuline mit Polysacchariden in wäßrigen Medien. Es wurde gezeigt, daß die Sojabohnenglobuline mit sauren karboxylhaltigen Polysacchariden im gesamten untersuchten Bereich der pH-Werte (2...12) und NaCl-Konzentrationen (0...1 Mol/l) inkompatibel sein können. Die Inkompatibilität der Sojabohnenglobuline mit sulfatierten Polysacchariden liegt nur bei einer großen Ionenkraft oder unabhängig vom pH-Wert des Systems vor. Die Inkompatibilität der Sojabohnenglobuline mit neutralen Polysacchariden bei einem pH-Wert ≠ dem isoelektrischen Punkt des Eiweißes liegt nur bei einer großen Ionenkraft oder in Gegenwart von Harnstoff (6 Mol/l) vor. Die Tendenzen einer Veränderung des Aufspaltungskörpers der Systeme Wasser-Sojabohnenglobuline-Polysaccharid Σ =F (W3,W3,C4,C5,C5,t) = 0 sind unterschiedlich bei einer Veränderung der VariablenW2,W3,C4 unter den Bedingungen, daß der pH-Wert höher als der isoelektrische Punkt und niedriger als der isoelektrische Punkt des Eiweißes liegt sowie beim Übergang von karboxylhaltigen sauren Polysacchariden zu sulfatierten sauren oder neutralen Polysacchariden. Bei gleichen übrigen Bedingungen erhöht sich die Kompatibilität der Sojabohnenglobuline mit Polysacchariden in folgender Reihenfolge: Pektin < Karboxymethylzellulose < Natriumalginat < arabisches Gummi < Dextran < Dextransulfat.

On the Concentration Dependence of the Elasticity Modulus of Soybean Globulin Gels

SummaryThe concentration dependence of the elasticity modulus (G) of gels produced by heating soybean globulins (SBG) has been investigated in the concentration range C=7.5–58.4%. The tests included constant rate loading and stress relaxation. The experimental results are presented in the form of a modulus reduction parameter b(C)=G(C) / G(C∘), where C∘=15% corresponds to the standard reference state. Whatever the testing conditions, the results are described by a single smooth curve similar, to a constant factor, to the concentration dependence of the equilibrium elasticity modulus of SBG gels. The experimental relationship b (C) agrees with the theoretical concentration dependence of the equilibrium polymer gel modulus according to Hermanns if it is assumed that the gel-point of SBG, Co=6.6 %. Within an order of magnitude this value of Co is consistent with the experimental data. We have to thank the European Branch Director of Central Soya Intern., Inc., Mr.J.F.Casey for kindly providing us defatted soya flour.

A study on gelation of soybean globulin solutions

SummaryA study has been undertaken on the linear viscoelastic properties (stress relaxation) of thermotropic gels of soybean globulins (SBG) over a wide range of concentration and temperature. Experimental data on isothermal stress relaxation for SBG gels of various concentrations were generalized in the form of a concentration-invariant relaxation curve using the reduction by modulus, but without the reduction by time. The concentration dependence of the modulus reduction parameter was shown to be similar to that of the equilibrium gel modulus with an accuracy of up to a constant factor. The temperature-invariant relaxation curve of 17.5% gel was obtained in the traditional way in the range of reduced time equal to about seven decimal orders. Its form is characteristic of ordinary cross-linked high elastic polymers at the end of the transition zone and the beginning of plateau zone. The activation energy for relaxation of SBG gels amounts to 136 kJ/mol irrespective of their concentration. Based on assaying of sol-fraction content and its composition, it has been found that 7 and 11 S SBG fractions play a predominant part in gelation. SBG gel are soluble in 8 M urea, but insoluble in 0.01 M mercaptoethanol. Under the action of urea the gels and native SBG are decomposed into practically identical molecular fragments (subunits). These facts attest that the hydrophobic interaction of subunits play a leading role in SBG gelation. Gelation is not accompanied by changes in the integral intensity of a comparatively well resolved leucine band in PMR (proton magnetic resonance) spectrum of SBG at 100 MHz. This fact serves as a basis for assumption that the packing densities of polypeptide chains in the structural elements of gels and native molecules of SBG are comparable. The relaxation properties of gels at the end of the transition zone and the beginning of the plateau zone are determined predominantly by the “internal viscosity” of their elastic structural elements. It should be noted in this connection that the role of the “local viscosity” as an effective criterium for hydrodynamic interaction of these elements is apparently small due to their limited flexibility. The elastic elements of gels are long-term fluctuations of protein concentration. In the event of gel with 17.5% concentration one elastic element consists of approximately 20 subunits. In their thermo-rheological behaviour SBG gels can be classed with the systems having entropic elasticity.

Limited Thermodynamic Compatibility of Proteins in Aqueous Solutions

SummaryConditions are established for limited thermodynamic compatibility of proteins of different classes, as distinguished by Osborne, in aqueous solutions. The result obtained are in good agreement with the concept known as Δx —effect.

Studies on gelation of soybean globulin solutions

Thermal denaturation of soybean globulin fraction (SBGF) in diluted solution (protein concentration 0.15–0.63%) has been studied by the method of differential adiabatic scanning calorimetry. SBGF thermograms have two maxima. The low temperature maximum is consistent with denaturation of 7S component, while the high temperature maximum with denaturation of 11S components of this fraction. In the investigated range of protein concentrations the thermodynamic parameters (temperature and enthalpy) of denaturation of SBGF and its main components are constant. This fact suggests that differential adiabatic scanning calorimetry gives information purporting a change in the protein state at molecular level. The temperatures and enthalpies of denaturation of the main SBGF components linearly rise with increase of NaCl concentration. The slope of dependences of denaturation temperature on salt concentration,Ks, is extremely large (nearly 20 K · l/mole). The elementary thermodynamic theory of lyotropic effects in thermal denaturation of proteins has been developed based on the two-state model and linear approximation of protein-salt interactions by means of the corresponding second virial coefficient. It shows that the dependences of thermodynamic parameters of thermal denaturation on salt concentration should be linear in the initial section. This conclusion is consistent with the experiment. The differences of enthalpies and entropies of transferring denatured and native forms of the main SBGF components from water into NaCl solution have been determined. They are positive and their quantity increases linearly with salt concentration. This fact is consistent with the concept to the effect that the main factor of salt influence on thermal denaturation of SBGF is confined to a decrease of protein hydration. The effect of protein nature on the quantity of lyotropic effect in thermal denaturation has been considered. Using simple considerations as a basis, the dependence of the ratio betweenKs and the denaturation temperature in water has been obtained, which characterizes the lyotropic effect, on the molar fraction of hydrophobic residues in the protein molecule. This dependence is linear and the lyotropic effect rises with increase in the content of hydrophobic residues. It is satisfactorily consistent with the experimental data on NaCl effect on thermal denaturation temperature for ichthyocol gelatin, ribonuclease, lysozyme, 7S and 11S SBGF components. An extraordinary strong influence of NaCl on thermal denaturation temperatures for the main SBGF components can be accounted for by a relatively high content of hydrophobic residues.

Thermotropic gelation of ovalbumin 1. Viscoelastic properties of gels as a function of heating conditions and protein concentration at various pH values

A study has been undertaken of stress relaxation in ovalbumin thermotropic gels with a concentration of 8–20%, depending on time and temperature of heating (respectively, 20–60 min, 70°–110°C), at pH 2.5–10.0. In all instances, the dependence of the initial gel elasticity modulus on heating has a single maximum. Gelation conditions corresponding to this maximum are considered optimal. Optimal gelation time is 30 min, regardless of pH. On the other hand, the optimal heating temperature depends on pH. To the right and left of the isoelectric point of protein (2.5≤pH<4.0 and 5.5<pH≤10.0) the optimal temperature is 80°. However, in the vicinity of the isoelectric point (4.0≤pH ≤5.5) the optimal temperature rises considerably. Gels produced to the right and left of the isoelectric point belong to the homologous group A, while gels produced in the point vicinity belong to another homologous group B. Light scattering data proves group A gels to be more homogeneous optically. These gels may be supposed a single-phase system, while group B gels are two-phase-systems. For each group, the dependence of the relaxation modulus (G) of gels on heating conditions, pH and protein concentration (X1,X2,X3,X4), as well as on time of relaxation (t) may be generally described asG(X1,X1,X1,X1,t)=Ge(X1,X2,X3,X4)f(t), whereGe is the equilibrium value of the elasticity modulus, and f(t) the relaxation function. Thus, a change in the parameters only affects the value of the equilibrium elasticity modulus, and exerts no effect on the relaxation time spectrum. For this reason, all the relaxation curves obtained may be transformed into two normalized relaxation functions:∼f(t)=f(t)/f(1)=G(X1,X2,X3,X4,t)/G(X1,X2,X3,X4, 1)Each of these normalized functions corresponds to one of the homologous groups. Rheological similarity of gels in each homologous group evidently points to their structural similarity. Invariance of the gel relaxationproperties with regard to protein concentration, leads to a concentration dependence of the equilibrium modulus at various pH values. These dependences are curvilinear on a double logarithmic scale. The slope of the curve exceeds 2 in the entire concentration interval studied. In other words, the dependences obtained cannot be described by the usual “law of squares”. On the other hand, they adequately match Hermans theoretical relation for a network formed by random association of identical polyfunctional particles without cyclization. This simple model evidently gives a true picture of the major regularities of thermotropic gelation for ovalbumin. An agreement between this theory and experiment was achieved for a protein concentration ofC*=6.0±1.0% at the gel point regardless of pH. Invariance of gelpoint position with regards to pH demands further confirmation.

On the possibility of estimating weak interactions of macromolecules in solutions from the experimental viscous flow activation energies data

SummaryThe initial viscosity and activation energy in viscous flow of the systems: water(W)-casein(C)-polysaccharide(PS) (gum arabic(G),de-xtran(D), extran sulfat (DS)) have been determined for various ionic strengths corresponding to total or limited thermodynamic compatibility of macrocomponents. Excess activation energy ΔHηE due to the protein-polysaccharide interactions has been calculated. It is positive for systems with total compatibility and negative for systems with limited compatibility. Moreover, it yields information on type of the protein-polysaccharide interactions. Negative ΔHηE means that repulsive forces are dominant, while positive ΔHηE means that attractive forces are dominant. Since the properties of the systems W-C-D and W-C-DS are similar, it is believed that C-D complexes can possibly be formed with an energy ∼ 2 kT(10 mJ/g).