Factors Influencing Conformation Changes in a 7S Protein of Soybean Globulins by Ultracentrifugal Investigations

Effects of pH, ionic strength, kind of salts and disulfide bond cleaving agent (2-mercaptoethanol) on conformation changes revealed on ultracentrifugal patterns of a 7S protein in soybean globulins were investigated. In the solution with lower pH than isoelectric point, this protein dissociated into two components in low ionic strength, but showed a 7S sedimentation pattern in higher ionic strength than 0.1. On the other hand, in the solution with higher pH than isoelectric point, this protoin showed aggregation to a 9S isomer in lower ionic strength than 0.1. Between ionic strength of 0.1 and 0.5, the mixture of 7S and 9S forms existed and in higher ionic strength than 0.5, the protein kept a 7S form stablely. These reactions were reversible and effect of 2-mercaptoethanol was scarcely observed but those of salts were observed.

The molecular weight of the 9S isomer was approximately 370,000 and the s_20,w value was 12.30S. Therefore, the 9S isomer was considered to be a dimer of the 7S protein.     

Gelation of Casein and Soybean Globulins by Transglutaminase

Guinea pig liver transglutaminase is a Ca²⁺ dependent enzyme which catalyzes the formation of inter- and intramolecular ε-(γ-glutamyl)lysyl cross-links between protein molecules. We have found that solutions of several proteins (α_s1-casein, and soybean 11S and 7S globulins) were gelatinized firmly by transglutaminase. The gel formation depended on the protein concentration. In the case of α_s1-casein, a reaction mixture containing below 2% was incapable of gelation. However, above 3%, a firm gel was formed by transglutaminase. As to soybean 11S and 7S globulins, reaction mixtures containing below 5% did not form gels, while, above 8%, firm gels were formed. The protein solutions in the presence of EDTA, an inhibitor of transglutaminase, were not gelatinized on treatment with transglutaminase. Thus, transglutaminase and a higher concentration of a substrate protein are indispensable for firm gel formation. It is supposed that the protein gels are formed through covalent bonds with transglutaminase.     

Relationship between Gelation and Aggregation of Alkali-treated Soybean 7S and 11S Globulins

Gel was obtained when alkaline dope solutions of the 7S and 11S globulins (8% protein concentration) prepared at pH above 11 were dialyzed against phosphate buffer, pH 7.6, µ= 0.3. To make clear the mechanism of gelation, the relationship between changes in viscosity and aggregation phenomena of the neutralized dope solutions was investigated by means of viscosity measurement, disc electrophoresis and gel filtration, comparing the 7S and 11S fractions. In conclusion, it is revealed that the gel is constituted with macromolecule aggregates, and to form the aggregates which are suitable for gelation, all of the following conditions must be satisfied at least : 1); Unfolding and dissociation into subunits once (above pH 11), 2); High ionic strength in the media (µ=0.3), 3); Formation of hydrogen, hydrophobic and disulfide bonds, 4); High protein concentration (above 8%).     

Formation of thermally induced aggregates of the soya globulin beta-conglycinin.

The effect of ionic strength (I) on the formation of thermally induced aggregates by the 7S globular storage protein of soya, beta-conglycinin, has been studied using atomic force microscopy. Aggregates were only apparent when I> or =0.1, and had a fibrous appearance, with a height (diameter) of 8-11 nm. At high ionic strength (I=1.0) the aggregates appeared to associate into clumps. When aggregate formation was studied at I=0.2, it was clear that aggregation only began at temperatures above the main thermal transition for the protein at 75 degrees C, as determined by differential scanning calorimetry. This coincided with a small change in secondary structure, as indicated by circular dichroism spectroscopy, suggesting that a degree of unfolding was necessary for aggregation to proceed. Despite prolonged heating the size of the aggregates did not increase indefinitely, suggesting that certain beta-conglycinin isoforms were able to act as chain terminators. At higher protein concentrations (1% w/v) the linear aggregates appeared to form large macroaggregates, which may be the precursors of protein gel formation. The ability of beta-conglycinin to form such distinctive aggregates is discussed in relation to the presence of acidic inserts in certain of the beta-conglycinin subunits, which may play an important role in limiting aggregate length.     

Simultaneous Determination of Patulin and Penicillic Acid in Grains by Gas Chromatographic Method

Changes in aggregation and properties of alkali-treated soybean 7S and 11S globulins depend upon protein concentration during alkali-treatment. Such variables were investigated by viscosity, electrophoresis, circular dichroism, pulsed NMR, emulsion capacity and CaCl₂ precipitation measurements. In lower protein concentrations, the intrinsic viscosity decreased and the penetrative fractions into electrophoresis gel increased. The reduced contacts of proteins during neutralization resulted in smaller aggregates. Also specific fractions which were more sensitive to protein concentration on aggregation were observed for 11S globulin. The quantity of bound water depended only on pH at 7% concentration treatment. When the gel was formed, the bound water of protein increased, e.g., 0.085 g and 0.135 g/g protein at pH 10.6 and 13.2 treatment, respectively, whereas at 1% treatment, bound water showed almost no pH dependence (about 0.13 g/g protein). Furthermore, proteins prepared at higher protein concentrations were characterized by higher emulsion capacity and CaCI₂ precipitation ability. However, no protein concentration dependence was seen in the secondary structure of the aggregates.     

A Method for Isolation of 2S, 7S and 11S Photeins of Soybean

A metnod is described for the isolation of 2S, 7S and 11S proteins of soybean. The unfractionated proteins are first precipitated with Hg(II) which yields a 11S-rich precipitate and this is further purified by (NH₄)₂SO₄precipitation. From Mg(II)supernatant which is rich in 7S and 2S proteins, they are separated by the use of (NH₄)₂SO₄ and cold ethanol. The 7S ana 11S protein are obtained in a highly purified form as indicated by ultracentrifugation and polyacrylamide gel electrophoresis.     

Isolation and Characterization of A Temperature Sensitive Protein from dog Colostrum And Milk

A temperature sensitive protein (one of several) that was soluble at 2°C but reversibly precipitated upon warming to room temperature was isolated from dog milk by precipitation with 50% saturated ammonium sulfate, ion exchange chromatography, centrifugation at 30°C and preparative isoelectric focusing in polyacrylamide gel. The molecular weight by sedimentation equilibrium was 13, 400; however, by thin layer gel filtration it appeared to be much larger. Ultracentrifu-gation studies at conditions near those at which the protein precipitated revealed no evidence of aggregation. The protein had a fairly high content of nonpolar amino acids with proline being in the highest amount. The effects of pH, ionic strength, heat and ethanol on the solubility of the protein were studied. A circular dichroism spectrum of the temperature sensitive protein indicated that the protein is quite unordered. An antiserum against the temperature sensitive protein reacted with each of the other temperature sensitive proteins from milk but not with dog serum or saliva.     

Conformation of the ribosomal protein S1 of Thermus thermophilus in solution under different ionic conditions

The structure of protein SI of Thermus thermophilus (M = 61 kDa) in solution at low and moderate ionic strengths (0 M and 100 mM NaCl, respectively) has been studied by small-angle X-ray and neutron scattering. It was found that protein S1 has a globular conformation under both ionic conditions. The modelling of different packing of six homologous domains of S1 on the basis of the NMR-resolved structure of one domain showed that the best fit of calculated scattering patterns from such complexes to experimental ones is observed at a compact package of the domains. The calculated value of the radius of gyration of the models is 28-29 angtroms, which is characteristic for globular proteins with a molecular mass of about 60 kDa. It was found that protein S1 has a tendency to form associates, and the type of the associate depends on ionic strength. These associates have, in general, two or three monomers at a moderate ionic strength, while at a low ionic strength the number of monomers exceeds three and they are packed in a compact manner. Strongly elongated associates were observed in neutron experiments at a moderate ionic strength in heavy water. The association of protein molecules was also confirmed by the data of dynamic light scattering. From these data, the translational diffusion coefficient of protein S1 at a moderate ionic strength was calculated to be (D20,w = (2.7 +/- 0.1) x 10(-7)cm2/s). This value is essentially smaller than the expected value (D20,w = (5.8 - 6.0) x 10(-7)cm2/s) for the S1 monomer in the globular conformation, indicating the association of protein molecules under equilibrium conditions.     

Influence of the ionic strength on the heat-induced aggregation of the globular protein beta-lactoglobulin at pH 7

The influence of the ionic strength on the structure of beta-lactoglobulin aggregates formed after heating at pH 7 has been studied using static and dynamic light scattering. The native protein depletion has been monitored using size exclusion chromatography. Above a critical association concentration (CAC) well-defined clusters are formed containing about 100 monomers. The CAC increases with decreasing ionic strength. The so-called primary aggregates associate to form self similar semi-flexible aggregates with a large scale structure that is only weakly dependent on the ionic strength. The local density of the aggregates increases with increasing ionic strength. At a critical gel concentration, Cg, the size of the aggregates diverges. Cg decreases from 100 g/l without added salt to 1 g/l at 0.4M NaCl. For C > Cg the system gels except at high ionic strength close to Cg where the gels collapse under gravity and a precipitate is formed.     

Gas Chromatographic Analysis of Furfural and Hydroxymethyl-furfural in Wine

The thermal denaturation of 7S globulin has been examined as a function of ionic strength from 0 to 4.0 by using immunochemical methods, disc electrophoresis and turbidity. Changes in immunochemical properties were a measure of the extent of denaturation and provided information on the structural changes in 7S globulin at the molecular level. The 7S globulin did not lose its antigenicity even after heating, and both dissociates and aggregates maintained partial cross reactivity against anti-7S. The nature of thermal denaturation was divided into two main types at an ionic strength of approximately 2.0. At an ionic strength near zero, stable dissociation products were actually formed, and at ionic strengths from 0.1 to 2.0, the presence of salts increased in aggregate formation. The stabilizing effect of salt on thermal denaturation appeared at 2.0 ~ 2.2 μ. In the range of 3.5 ~ 4.0 μ, 7S globulin was heated to 100°C for 5min without changes in both antigenic specificity and electrophoresis pattern.     

Interaction of Protein Particles with Lipids in Soybean Milk

The protein in soybean milk exists as 11S and 7S globulins, and the particles formed from them. The lipid content and composition in the protein fractions and effects of defatting on the form of the protein particles were investigated. The size-distribution of protein particles in both raw and heated soybean milk (soymilk) was not influenced by defatting with hexane, but the number of large particles were slightly increased. The protein particles from raw and heated soymilk samples contained 60% and 3% of the total lipid, respectively. Almost all neutral lipid in the particles of raw soymilk was moved to a floating fraction by heating, but a half of the phospholipids was retained in the particles. The protein components from the hexane-defatted meal were similar to those from whole meal, but those from the C-M-de-fatted meal contained remarkably little β-conglycinin. C-M-de-fatting (Removal of polar lipids) caused a reduction in the particulate fraction, and the addition of phospholipids (lecithin) promoted the formation of protein particles.     

Food Processing Characteristics of Soybean Proteins

Relation between sulfhydryl groups in soybean proteins and the physical properties of tofu was studied. Changes in the amount of sulfhydryl groups by heating or treatment with urea were more rapid in 11S protein as compared with 7S protein. Moreover, by changing the amount of sulfhydryl groups in proteins by N-ethylmaleimide, 2-mercapto-ethanol and dithiothreitol, the physical properties of tofu from 11S protein were more significantly effected than that from 7S protein. Namely, tofu-gel from 11S. protein got harder and stronger as the amount of sulfhydryl groups increased.

The results may suggest that tofu prepared from IIS protein has more disulfide bonds in its gel than that from 7S protein.     

Processing and assembly in vitro of engineered soybean beta-conglycinin subunits with the asparagine-glycine proteolytic cleavage site of 11S globulins

A short interdomain sequence between the N- and C-terminal domains of beta-conglycinin, the major 7S seed storage protein of soybean, was selected as a target for insertion of amino acid residues specifically cleaved by an asparaginyl endopeptidase that processes globulins into acidic and basic chains. Modified beta-conglycinin subunits containing the proteolytic cleavage site self-assembled into trimers in vitro at an efficiency similar to that of the unmodified subunit. In contrast to the absence of cleavage of the unmodified subunits, however, the modified beta-conglycinin trimers were processed by purified soybean asparaginyl endopeptidase into two polypeptides, each the size expected for the beta-conglycinin N- and C-terminal domains, respectively. The cleavage did not alter the assembly of mutant beta-conglycinins and the cleaved mutant trimers remained stable to further proteolytic attack. To examine the possibility of coassembly between the cleaved 11S and 7S subunits, in vitro processed mutant beta-conglycinin subunits were mixed with native dissociated 11S globulin preparations. Reassembly at a high ionic condition did not induce the 7S subunits to interact with 11S subunits to form hexameric complexes. Thus, cleavage of 7S globulin subunits into acidic and basic domains may not be sufficient for hexamer assembly to occur. Biotechnological implications of the engineered proteins are discussed.     

Comparative study of the precipitation of low density lipoproteins by aortic proteodermatan sulphate and heparin

The aortic proteoglycans and heparin were shown to form insoluble complexes with human low density lipoproteins (LDL). The effect of temperature, polyethylene glycol and ionic strength on the formation of complexes between porcine aortic proteodermatan sulphate (PDS) and LDL has been studied by laser nephelometry and comparisons made with heparin LDL complexes. Turbidity was a nonlinear function of the quantity of LDL precipitated by PDS. The turbidity of aggregates was constant at temperatures between 2 degrees C and 30 degrees C but increased with temperature above 30 degrees C up to 50 degrees C. The formation of insoluble complexes decreased rapidly with increasing NaCl concentration. Polyethylene glycol enhanced the turbidity at 20 degrees C but not at 37 degrees C. It also increased the resistance of complexes to dissociation by increasing ionic strength. The turbidity of heparin--LDL complexes was linearly correlated with the quantity of precipitated LDL. The heparin-LDL aggregates were less sensitive to modification of temperature and ionic strength than the PDS-LDL aggregates. These results suggest that ionic interactions are weaker in PDS-LDL complexes than in the heparin-LDL complexes. Non-coulombic interactions and/or temperature dependent conformational changes may be involved in the stabilization of supramolecular PDS-LDL aggregates. No such interactions or changes appear to be involved in complex formation between heparin and LDL.     

Isolation and Partial Characterization of Heat-denatured Products of Soybean 11S Globulin and Their Analysis by Electrophoresis

Heat denaturation of soybean 11S globulin was examined at 70° and 100°C in phosphate buffer (pH 7.6), at 0.01 and 0.5 ionic strength. Gel electrophoresis (Davis system) indicated that heat-denatured soybean 11S globulin contained two major components (buffer-soluble form). But they were not identified at 70°C-0.5 ionic strength. Gel filtration followed by SDS-gel electrophoresis showed that the major components were composed of a monomer and at least three of kinds of oligomers containing only an acidic subunit. Gel filtration of the precipitate formed at 100°C at 0.5 ionic strength gave two peaks. SDS-gel electrophoresis indicated that the first peak contained aggregates of highly polymerized subunits, and the second peak contained a monomer of basic subunit and seven kinds of oligomers with various proportions of basic subunits to an acidic subunit.     

Denaturation of Soybean Protein by Freezing

When a solution of soybean acid-precipitated or 11S protein was frozen and stored at ?1 to ?5°C, the protein became partially insoluble after thawing. Ultracentrifugation and disc-electrophoresis of freeze-stored 11S protein solution after removing insoluble components revealed that new components which may be aggregates or associates of the 11S component were formed. When concentrated and stored at 5°C, disc-electrophoresis of 11S component showed that associates were formed. Mercaptoethanol could dissolve the insoluble protein and also convert the associates to the original 11S component. NEM–11S was not insolubilized by frozen storage at ?5°C or storage at 5°C after being concentrated. From these facts it can be concluded that denaturation of soybean protein by freezing may be caused by intermolecular reactions through S-S bonds as a result of concentration by freezing. This may suggest a mechanism of the formation of sponge-like texture in kori-tofu which is made by frozen storage of soybean curd for 15 to 20 days at ?1 to ?3°C.     

Gelation Phenomena Induced by Alkali-alcohol Treatment of 7S and 11S Components in Soybean Globulins

Transparent gels containing about 2% protein were obtained by mixing alkaline dope solution of 7S or 11S soybean proteins with alcohol. The 7S component showed the ability to form a stronger gel than the 11S. This phenomenon depended on pH and alcohol concentration. In 66 % ethanol, the viscosity of the 7S and 11S reached maxima at pH 11.4 and 11.2, respectively. Above these pH levels where further unfolding and dissociation into subunits of the protein molecules occur, the viscosity decreased rather. The effectiveness of alcohol to increase viscosity increased in the order; n-butanol < tert-butanol < n-propanol < iso-propanol < ethanol < methanol. Alcohols having minor hydrophobicity were more effective for increasing viscosity, but ethylene glycol was ineffective. The addition of NaCl or 2-mercaptoethanol to ethanol-mixed alkaline dope solutions resulted in the remarkable increment of the viscosity, especially for the 7S.     

Conformation of a group 2 late embryogenesis abundant protein from soybean. Evidence of poly (L-proline)-type II structure

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic, glycine-rich proteins found in plants, algae, fungi, and bacteria known collectively as hydrophilins that are preferentially expressed in response to dehydration or hyperosmotic stress. Group 2 LEA (dehydrins or responsive to abscisic acid) proteins are postulated to stabilize macromolecules against damage by freezing, dehydration, ionic, or osmotic stress. However, the structural and physicochemical properties of group 2 LEA proteins that account for such functions remain unknown. We have analyzed the structural properties of a recombinant form of a soybean (Glycine max) group 2 LEA (rGmDHN1). Differential scanning calorimetry of purified rGmDHN1 demonstrated that the protein does not display a cooperative unfolding transition upon heating. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein is in a largely hydrated and unstructured conformation in solution. However, ultraviolet absorption and circular dichroism measurements collected at different temperatures showed that the protein exists in equilibrium between two extended conformational states: unordered and left-handed extended helical or poly (L-proline)-type II structures. It is estimated that 27% of the residues of rGmDHN1 adopt or poly (L-proline)-type II-like helical conformation at 12 degrees C. The content of extended helix gradually decreases to 15% as the temperature is increased to 80 degrees C. Studies of the conformation of the protein in solution in the presence of liposomes, trifluoroethanol, and sodium dodecyl sulfate indicated that rGmDHN1 has a very low intrinsic ability to adopt alpha-helical structure and to interact with phospholipid bilayers through amphipathic alpha-helices. The ability of the protein to remain in a highly extended conformation at low temperatures could constitute the basis of the functional role of GmDHN1 in the prevention of freezing, desiccation, ionic, or osmotic stress-related damage to macromolecular structures.     

Gelling properties of soybean beta-conglycinin having different subunit compositions

The effects of protein concentration, and heating temperature and time on the gelling properties of soybean beta-conglycinin (7S globulins) lacking the alpha or alpha' subunit were compared with those of 7S containing all three subunits (alpha, alpha', and beta) to determine whether each subunit contributes equally. In most of the conditions, the relative order of gel hardness was alpha'-lacking > 7S > alpha-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.     

Isolation and Characterization of the Multiple 7S Globulins of Soybean Proteins

Two major proteins (the 7S and 11S globulins) of soybean (Glycine max) were simultaneously isolated by a simple method based on their different solubilities in dilute tris (hydroxymethyl) aminomethane buffers. The purified 7S globulins, which represented essentially the entire 7S soybean protein fraction capable of dimerization at 0.1 ionic strength, were fractionated into five components by diethylaminoethyl Sephadex A-50 column chromatography. The five 7S components were characterized by disc-electrophoresis.