Yasuki Matsumura

Proteins at liquid interfaces: Role of the molten globule state

Abstract Recent advances in understanding the structure and dynamics of proteins at liquid interfaces are reviewed with particular reference to adsorbed layers at the oil—water interface in protein-stabilized emulsions. We discuss the importance of molecular flexibility in determining the properties of adsorbed layers and the ease of exchange of protein molecules between bulk and surface in mixed systems. While a statistical model of a nearly random copolymer can be used to describe the adsorption of a disordered protein such as β-casein, such a representation is unrealistic for an adsorbed globular protein, which is typically a compact deformable macromolecular particle having a structure lying somewhere between the native state and the completely unfolded form. It is proposed that such a state of an adsorbed globular protein at a liquid interface is close to what is now called the “molten globule” state. This is the partially denatured state of a globular protein which retains the ordered secondary structure but not the tertiary structure of the native protein. We describe the various ways of producing the molten globule state, and we review the experimental evidence for the molten globule state of α-lactalbumin in some detail. In the final part of the paper, we discuss some new results on the surface activity of α-lactalbumin and the competitive adsorption of α-lactalbumin and β-lactoglobulin in emulsions at acidic pH or in the presence of ethylenediaminetetraacetic acid. This discussion shows how the concept of the molten globule state provides new insight into the relationship between protein structure and the properties of adsorbed layers at liquid interfaces.

Enhanced susceptibility to transglutaminase reaction of α-lactalbumin in the molten globule state

The susceptibility of alpha-lactalbumin to transglutaminase reactions was studied using an enzyme from Streptoverticillium which can catalyze the reactions irrespective of the presence or absence of Ca2+. Transglutaminase-catalyzed polymerization of alpha-lactalbumin in the native state occurred to a very limited extent. Transformation from the native state to the molten globule state brought about by Ca(2+)-removal from holo-alpha-lactalbumin enhanced the polymerization of the protein catalyzed by transglutaminase. The incorporation of Carbobenzoxy-Gln-Gly into alpha-lactalbumin through the enzyme reaction was investigated to determine the amounts of lysine residues which are present at molecular surface and available to the enzyme. There was no significant difference in the amount of available lysine residues between the native and the molten globule molecule. However, the amount of surface glutamine residues incorporated with monodansylcadaverine by transglutaminase was remarkably higher in the molten globule state than that in the native state. The monodansylcadaverine-incorporated site of alpha-lactalbumin in the molten globule state was identified as Gln-54 by amino-acid sequence analysis of fluorescence-labeled peptides separated from chymotryptic digests of the protein. Possible reason for selective labeling of Gln-54 in molten globule alpha-lactalbumin was proposed.

Characterization of texture and mechanical properties of heat-induced soy protein gels

Heat-set gels were prepared from acid-precipitated soybean proteins at various heating temperatures (80–100°C), protein concentrations (18–20%), and proportions of glycinin. The gels were evaluated for mechanical parameters by means of a compression-decompression test. Gels formed at higher heating temperature and protein concentration were firm, tough and unfracturable. The elasticities of the gels were similar at all protein concentrations and were lower when heated at higher temperature. Heating above 93°C was necessary for formation of rigid gels. The glycinin/β-conglycinin ratio affected the texture of the gels. Three-dimensional representation of the gels through factor analysis of instrumental data and calculation of factor scores was useful to evaluate the texture of the gels.

Inhibitory effects of peptide-bound polysaccharides on lipid oxidation in emulsions

Abstract The inhibitory effects of polysaccharides on lipid oxidation in emulsions were investigated. Methyl linoleate was emulsified by β-casein or surfactants to prepare emulsions, and the oxidation was induced by an azo-compound or FeSO4 addition. The initial oxidation was followed by measuring the oxygen consumption in the emulsions for 30 min, and the extent of the lipid oxidation over a long time (∼48 h) was evaluated by the determination of the unoxidized lipid by gas chromatography and the thiobarbituric acid test. Peptide-bound polysaccharides, such as gum arabic and soluble soybean polysaccharides (SSPS), showed an ability to inhibit lipid oxidation, whereas maltodextrin and pullulan exhibited no inhibitory effects. Especially, SSPS almost perfectly suppressed the lipid oxidation from the initial to the late stages, irrespective of the emulsifiers and oxidation-inducing reagents. The mechanism whereby the peptide-bound polysaccharides inhibited the lipid oxidation in the emulsions is considered in terms of the chemical composition and other properties of the polysaccharides.

Filler effects of oil droplets on the viscoelastic properties of emulsion gels

Abstract Filler effects of oil droplets on the viscoelastic properties of emulsion gels were investigated by small deformation mechanical measurements. The emulsions were made with soya oil and soy 11S globulin (15 wt% oil, 1.5–7% protein concentrations). The emulsion and soy 11S globulin solutions were gelled using Ca ++ -independent transglutaminase from a microorganism. The shear storage modulus (G′) and loss modulus (G″) of the proteinous gels and emulsion gels depended on the protein concentration. The concentration dependence of the modulus was approximated by an exponential function of the form: G ∝ C n , n = 4 and n = 2 being found in the cases of the proteinous gels and the emulsion gels, respectively. Shear moduli of the emulsion gels were much higher than those of the proteinous gels. Gels made from a fine emulsion (containing smaller oil droplets) exhibited higher G′ and G″ values than corresponding gels from a coarse emulsion. Addition of Tween 20 was found to reduce G′ and G″ of emulsion gels even when the protein matrix was not significantly displaced from the oil—water interface by the surfactant.

Disaggregation and Reaggregation of Gluten Proteins by Sodium Chloride

This study showed that gluten proteins were extracted with distilled water from dough prepared in the presence of NaCl. To elucidate the interrelationship of NaCl and gluten proteins in dough, the extracted proteins were characterized. These proteins were primarily found to be soluble gliadin monomers by N-terminal amino acid sequencing and analytical ultracentrifugation. Extracted proteins were aggregated by the addition of NaCl at concentrations of >10 mM. A decrease in beta-turn structures, which expose tryptophan residues to an aqueous environment in the presence of NaCl, was revealed by Fourier transform infrared analysis and scanning of fluorescence spectra. In addition, cross-linking experiments with disuccinimidyl tartrate showed that a large amount of protein was cross-linked in the dough only in the presence of NaCl. These results suggest that both interactions and distances between proteins were altered by the addition of NaCl.

Adsorption and Structural Change of β-Lactoglobulin at the Diacylglycerol−Water Interface

Diacylglycerol (DAG)/water and triacylglycerol (TAG)/water emulsions were prepared using beta-lactoglobulin (beta-LG) as an emulsifier. The oil phase (20% in emulsion) was mixed with beta-LG solution (1% beta-LG in water, pH 7) to prepare the emulsions. A fine oil-in-water emulsion was produced from both DAG and TAG oils. The interfacial protein concentration of the TAG emulsion was higher than that of the DAG emulsion. The zeta potential of the DAG oil droplet was higher than that of the TAG oil droplet. The front-surface fluorescence spectroscopy results revealed that tryptophan residues in beta-LG moved to the more hydrophobic environment during the adsorption of protein on the oil droplet surfaces. Changes in secondary structure of beta-LG during the adsorption were determined by FT-IR spectroscopy. Decreases in the beta-sheet content concomitant with increases in the alpha-helix content were observed during the adsorption to the oil droplets, and the degree of structural change was greater for beta-LG in the TAG emulsion than in the DAG emulsion, indicating the increased unfolding of adsorbed beta-LG on the TAG oil droplet surface. Results of interfacial tension measurement supported this speculation, that is, the increased unfolding of the protein at the TAG-water interface. Trypsin- and proteinase K-catalyzed proteolysis was used to probe the topography of the adsorbed beta-LG on the oil droplet surface. SDS-PAGE analyses of liberated peptides after the proteolysis indicated the higher susceptibility of beta-LG adsorbed on the DAG oil droplet surface than on the TAG oil droplet surface. On the basis of all the results, we discussed the conformation of the adsorbed beta-LG on the two oil droplet surfaces.

Interaction of Gum Arabic, Maltodextrin and Pullulan with Lipids in Emulsions

The interaction of gum arabic, maltodextrin and pullulan with lipids in emulsion systems was investigated. Interfacial tension and interfacial viscosity measurements revealed that only gum arabic could adsorb and form a viscoelastic film at the oil-water interface. Good emulsifying activity was demonstrated for gum arabic, whereas fine emulsions could not be produced from the other polysaccharide solutions and oil. Frequency-dependent increases in the storage and loss moduli were observed for all the polysaccharide solutions. Such rheological behavior did not substantially change when maltodextrin and pullulan were mixed with oil to form emulsions. However, the frequency-dependence of the dynamic moduli disappeared in the gum arabic-stabilized emulsion, suggesting the formation of a network structure in which oil droplets could form junctions with gum arabic chains. The results on the inhibition of lipid oxidation by polysaccharides suggest that gum arabic protected lipids from the attack of lipoxygenase and free radicals by adsorbing at the oil droplet surface.

Competitive adsorption of α-lactalbumin in the molten globule state

The molten globule state of α-lactalbumin is a partially denatured form with native-like secondary structure and disordered tertiary structure. Using circular dichroism measurements, it was demonstrated that the molten globule state was produced by decreasing the pH to 2.0 at 25°C or by removing bound Ca2+ by treatment with ethylenediamine—tetraacetic acid (EDTA) at pH 7.5 and 40°C. Tension measurements showed that α-lactalbumin in the molten globule state is more easily unfolded at liquid interfaces than is the native protein. Results of competitive adsorption experiments involving α-lactalbumin and β-lactoglobulin at the oil droplet surface in emulsions are consistent with preferential adsorption of α-lactalbumin during emulsification when it is in the molten globule state. In contrast to the difficulty of exchange between α-lactalbumin and β-lactoglobulin at the oil-water interface in emulsions at 25°C, it has been found that the two whey proteins are able partially to displace one another from the oil—water interface at 40°C. While native α-lactalbumin was found to be readily displaced from the oil—water interface by β-lactoglobulin at 40°C, it was found that α-lactalbumin in the molten globule state in the presence of EDTA at 40°C had itself the capacity for displacing β-lactoglobulin from the interface.

Gelling Properties of Soybean β-Conglycinin Having Different Subunit Compositions

The effects of protein concentration, and heating temperature and time on the gelling properties of soybean β-conglycinin (7S globulins) lacking the α or α′ subunit were compared with those of 7S containing all three subunits (α, α′, and β) to determine whether each subunit contributes equally. In most of the conditions, the relative order of gel hardness was α′-lacking > 7S > α-lacking. From Fourier transform infrared studies, the secondary structure change after heating was very similar among the three samples; thus, the secondary structural change is not the reason for the differences in gel hardness. By using scanning electron microscopy, we observed differences in strand thickness and the density of the gel network among the three samples. These differences correlated well with the differences in gel hardness.