Thermodynamics of conformational ordering of iota-carrageenan in KCl solutions using high-sensitivity differential scanning calorimetry.

Thermodynamic properties of aqueous solutions of iota-carrageenan as affected by KCl (0.15-1.2 M) or iota-carrageenan (0.5-6 mg/mL) content were studied by high-sensitivity differential scanning calorimetry. The polysaccharide was found to undergo two consecutive cooperative conformational transitions, which can be represented by the scheme: [H2]2<-->2H2<-->4C where C is the random coil, H2 is the double helix, and [H2]2 is the double helix dimer. The first transition follows by the "all or none" mechanism. The profile of the second transition resembles that of a second-order phase transition. The parameter sigma (of order 1), estimated for this latter transition, suggests that the stacking effect in helices of iota-carrageenan is rather small. The cooperativity of the transition is mainly defined by the loop factor. Free energies of both transitions at 273 K were calculated as a function of salt concentration. These experimental data were found to agree with Manning's theory.

Ethanol-dependent heat-induced dissociation of casein micelles.

The dissociation of casein micelles when heated to approximately 65 degrees C in the presence of ethanol [1:1 mixture (v/v) of milk and 65% (w/w) aqueous ethanol] was investigated using L* values and transmission measurements. Mixtures of milk and ethanol became transparent on heating, which suggests dissociation of casein micelles. Results of experiments using confocal laser scanning microscopy, light scattering (static and dynamic), and dialysis to examine the changes of milk during heating in the presence of ethanol supported the assertion that such treatments result in dissociation of casein micelles, as did studies of model beta-casein micellar systems.

Mechanism for the ethanol-dependent heat-induced dissociation of casein micelles.

An explanation as to how casein micelles dissociate when heated in the presence of ethanol is presented. Dissociation of casein micelles in milk-ethanol mixtures was studied using (1)H NMR, and the effects of addition of CaCl(2), NaCl, or EDTA or alteration of milk pH on this dissociation were studied. It is proposed that at low temperatures, ethanol reduces the solvent quality of milk serum, but above a critical temperature (approximately 30 degrees C in a 35% ethanol solution), ethanol enhances solvent quality and dissociates the casein micelles. Ethanol reduced protein hydrophobicity and increased the pK(a) value of phosphoserine, effects that are likely to be significant in the dissociating effect of ethanol at elevated temperatures.

Acid-induced gelation of heat-treated milk studied by diffusing wave spectroscopy.

Fresh skim milk is a stable colloidal system containing casein micelles and whey proteins. By decreasing the pH, the casein micelles become unstable and a gel is formed. During heat treatment at temperatures higher than 70 degrees C, the major whey proteins, e.g. alpha-lactalbumin and beta-lactoglobulin denature and start to interact with each other and with casein micelles. This changes the colloidal properties of the casein micelles. In this article, the pH-induced gel formation of heat-treated milk and the role of whey proteins was studied. Heat treatment in the range 70-90 degrees C induced a shift in gelation pH of skim milk to more alkaline pH values. This shift was directly related to whey protein denaturation. By using WPF milk it was shown that beta-lactoglobulin is principally responsible for the shift in gelation pH. alpha-lactalbumin caused neither alone nor in combination with beta-lg, an effect on the gelation pH. Heat treatment of milk for 10 min at 90 degrees C resulted in complete denaturation of the beta-lg present in skim milk but it is estimated that the casein micelles are coated only up to 40% by whey proteins when compared with pure whey protein aggregates.

Thermal aggregation of patatin studied in situ.

In this work dynamic light scattering was used to study the thermal aggregation of patatin in situ, to elucidate the physical aggregation mechanism of the protein and to be able to relate the aggregation behavior to its structural properties. The dependence of the aggregation rates on the temperature and the ionic strength suggested a mechanism of slow coagulation, being both diffusion and chemically limited. The aggregation rate dependence on the protein concentration was in accordance with the mechanism proposed. The aggregation rates as obtained at temperatures ranging from 40 to 65 degrees C correlated well with unfolding of the protein at a secondary level. Small-angle neutron scattering and dynamic light scattering results were in good accordance; they revealed that native patatin has a cylindrical shape with a diameter and length of 5 and 9.8 nm, respectively.

Equilibrium unfolding CD studies of bovine beta-lactoglobulin and its 14-52 fragment at acidic pH.

Bovine beta-lactoglobulin represents an interesting example of context-dependent secondary structure induction. In fact, secondary structure predictions indicated that this beta-barrel protein has a surprisingly high alpha-helical preference, which was retained for short fragments. Cooperative transitions from the native beta-sheet to alpha-helical structures were additionally induced by organic solvents, in particular trifluoroethanol. As a result of this high alpha-helical preference, it has been proposed that non-native alpha-helical intermediates could be formed in the unfolding pathway of this protein. In order to provide a better understanding of the processes that underlie conformational plasticity in this protein, CD measurements in the presence of increasing amounts of urea and in the presence of organic solvents were performed. Urea unfolding studies, performed at pH 2.1 and 37 degrees C, revealed an apparent two-state transition, and afforded no evidence of non native alpha-helical intermediates. The protein treated with up to 6M urea, refolded to the native structure, while treatment with higher molar concentration urea, lead to partial misfolding. A 29-mer peptide covering the region of strands a and b of the intact protein, characterized by the presence of 4/3 heptad repeats, was synthesized and studied by CD in the presence of different solvents. On the basis of the obtained results, a mechanism was proposed to explain the structural transition from the beta to alpha structure, provoked by organic solvents in the intact protein.

Association behavior of native beta-lactoglobulin.

The association behavior of beta-lactoglobulin has been studied by small-angle neutron scattering as a function of protein concentration, temperature, pH, and NaCl concentration of the solution. By indirect Fourier transformation of the spectra, pair-distance distribution functions for the various samples were obtained. These functions provided information on the maximum size, the weight-averaged molecular mass, and the z-averaged radius of gyration of the beta-lactoglobulin particles. At room temperature and pH values below 4 and above 5.2 the protein consists predominantly of monomers and dimers, consistent with literature. In these pH regimes the formation of dimers is favored upon increasing ionic strength and decreasing protein charge (pH values closer to the isoelectric point of the protein). Around pH 4.7, larger oligomeric structures are formed, enhanced by a decrease in temperature and a decrease in ionic strength. beta-Lactoglobulin A associates more strongly than beta-lactoglobulin B. Surprisingly, at pH 6.9 larger structures than dimers seem to be formed at high protein concentrations (> 30 mg mL-1).

Monomeric bovine beta-lactoglobulin adopts a beta-barrel fold at pH 2.

We have determined a crude structure of the apo form of bovine beta-lactoglobulin, a protein of 162 amino acid residues with a molecular mass of 18 kDa, at a low pH on the basis of data collected using only homonuclear 1H NMR spectroscopy. An ensemble of protein conformations was calculated with the distance-geometry algorithm for NMR applications (DYANA). The monomeric protein at low pH adopts a beta-barrel fold, well-superimposable on the structure determined by X-ray crystallography for the dimer at physiological pH. NMR evidence suggests the presence of disordered loop regions and terminal segments. Structural differences between the monomer at pH 2 and the dimer at pH 7, obtained by X-ray crystallography, are discussed, paying particular attention to surface electrostatic properties, in view of the high charge state of the protein at low pH.

Aggregation of beta-lactoglobulin and influence of D2O.

The conformational stability of beta-lactoglobulin increases in D2O over that in H2O. This is concluded from an increase in peak temperature by about 3 degrees C of differential scanning calorimetry (DSC) thermograms and from a decrease in overall aggregation rate. However, effects of pH and salt concentration on the heat-induced aggregation (reaction kinetics, DSC thermograms and aggregate growth) are similar in H2O and D2O. This indicates that the mechanism of heat-induced aggregation of beta-lactoglobulin is not significantly affected by replacement of H2O with D2O.

Gelation of proteins from milk.

We give a quantitative treatment of the destabilization of three types of milk protein dispersions. For this we consider the proteins as adhesive-hard-sphere bio-colloids. If the attractive interactions become strong enough the system passes the percolation threshold and gels. Macroscopic properties of these gels are studied by measuring viscoelasticity and permeability coefficients. These coefficients are related to structural (fractal) properties of the gels which were measured using scattering and confocal scanning laser microscopy (CLSM) techniques. The behaviour of the protein gels can be understood on a qualitative level.

A model for the denaturation and aggregation of beta-lactoglobulin.

A quantitatively correct kinetic model for the temperature-induced denaturation and aggregation of beta-lactoglobulin is presented. The model recognizes an initiation, a propagation and a termination step by analogy with polymer radical chemistry. The decrease in native beta-lactoglobulin is predicted to follow order 3/2, in agreement with experimental results. The size of the protein polymer particles is predicted to be proportional to the square root of the initial beta-lactoglobulin concentration. The scattered light intensity is proportional to the product of concentration and size of the protein polymer particles. The initial increase in scattering intensity of the particles therefore scales with the initial squared beta-lactoglobulin concentration. The influence of other reaction conditions, e.g. ionic strength and pH, can be incorporated via the reaction constants of the reaction kinetic pathway.