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1.
The structure and solubility of helianthinin, the most abundant protein of sunflower seeds, was investigated as a function of pH and temperature. Dissociation of the 11S form (hexamer) into the 7S form (trimer) gradually increased with increasing pH from 5.8 to 9.0. High ionic strength (I = 250 mM) stabilizes the 11S form at pH > 7.0. Heating and low pH resulted in dissociation into the monomeric constituents (2-3S). Next, the 7S and 11S forms of helianthinin were isolated and shown to differ in their secondary and tertiary structure, and to have denaturation temperatures (T(d)) of 65 and 90 degrees C, respectively. Furthermore, the existence of two populations of the monomeric form of helianthinin with denaturation temperatures of 65 and 90 degrees C was described. This leads to the hypothesis that helianthinin can adopt two different conformational states: one with T(d) = 65 degrees C and a second with T(d) = 90 degrees C.  相似文献   

2.
This study describes the relationship between the solubility of glycinin, a major soy protein, and its structural properties at a quaternary, tertiary, and secondary folding level under conditions representative for food products. When the ionic strength is lowered from 0.5 to 0.2 or 0.03, the basic polypeptides shift more to the exterior of the glycinin complex, as determined at pH 7.6 by labeling solvent-exposed lysines, supported by the study of the proteolytic action of clostripain on glycinin. This structural reorganization caused the pH of minimal solubility to shift to higher values. Ultracentrifugational analysis shows that at pH 7.6 and an ionic strength of 0.5 glycinin forms hexameric complexes (11S), whereas at pH 3.8 and at an ionic strength of 0.03 glycinin exists as trimers (7S). Intermediate situations are obtained by modulation of pH and ionic strength. The observed quaternary dissociation correlates with an increased amount of nonstructured protein at a secondary level and with changes in tertiary folding as determined using circular dichroism. Tryptophan fluorescence shows no significant structural changes for different ionic strengths but demonstrates a more tightly packed fluorophore environment when the pH is lowered from 7.6 to 3.8.  相似文献   

3.
Soybean glycinin (11S) and beta-conglycinin (7S) were subjected to select chemical treatments at various concentrations and resulting changes in protein structures were investigated by circular dichroism (CD) and fluorescence spectrometry. Fluorescence quenching results indicated that urea >/=3 M caused significant unfolding of 11S, but not that of 7S. GuHCl was more effective than urea in denaturation of 11S. A two-step transition in 11S structure was observed with a possible existence of a folding intermediate at 2.5 M GuHCl. Sodium dodecyl sulfate (SDS) measurably altered secondary and tertiary structures of 11S and 7S below SDS critical micellar concentration (CMC), possibly due to formation of mixed peptide-SDS micelles. SDS treatment increased alpha-helical and unordered structures of both proteins at the expense of beta-sheet structure. NaCl and CaCl 2 caused a significant decrease in fluorescence intensity without shifting emission lambda max. Exposure of 7S and 11S to NaSCN respectively at >/=0.3 and >/=0.6 M NaSCN caused a significant increase in fluorescence intensity measured at the corresponding lambda max of the protein. beta-Mercaptoethanol (beta-ME), N-ethylmaleimide (NEM), and phytic acid caused variable red shifts, 2.5-4 nm, in the emission lambda max.  相似文献   

4.
Thermal denaturation and hydration of two soybean protein components were studied using differential scanning calorimetry (DSC). Results showed that temperature of denaturation (Td) of both 11S and 7S globulins decreased sharply with an increase in water content. Enthalpy of denaturation (ΔHd) of 11S increased with increasing water content at first, and then leveled off at high water content. However, ΔHd of both 7S and 11S components in 7S samples first increased and then decreased at high water content. The preparation method of samples influenced the ΔHd value significantly but had little effect on the Td. Nonfreezing water was determined from the DSC results. It increased in both 11S and 7S as water content increased but was more abundant in 7S, probably because of different compositions and structures. Threshold value of water content for the appearance of freezing water was 0.30–0.32 h (g of water/g of protein, mass ratio) for 11S. The water absorbed by both 11S and 7S during denaturation increased quickly at low water contents and remained almost constant at high water contents. The results were attributed to different structure and conformation of proteins before and after denaturation.  相似文献   

5.
Glycinin is a hexameric protein composed of five kinds of subunits. The subunits are classified into two groups, group I (A1aB1b, A1bB2, and A2B1a) and group II (A3B4 and A5A4B3). We purified four mutant glycinins composed of only group I subunits (group I-glycinin), only group II subunits (group II-glycinin), only A3B4 (A3B4-glycinin), and only A5A4B3 (A5A4B3-glycinin) from mutant soybean lines. The physicochemical properties of these glycinin samples were compared with those of the normal glycinin (11S) composed of five kinds of subunits. The thermal stabilities (as measured by thermal denaturation midpoint temperatures) of 11S, group I-glycinin, and group II-glycinin were similar to each other, although that of A3B4-glycinin was significantly lower than those of the others. The orders of aromatic and aliphatic surface hydrophobicities were the same: A3B4-glycinin > group II-glycinin > A5A4B3-glycinin > 11S > group I-glycinin. The solubility of 11S as a function of pH at mu = 0.5 was governed by that of group I-glycinin and followed this order at acidic pH: 11S = group I-glycinin > A3B4-glycinin > group II-glycinin = A5A4B3-glycinin. The order of emulsifying abilities was A5A4B3-glycinin > group II-glycinin > A3B4-glycinin > 11S > group I-glycinin. This order was consistent with that of the length of their hypervariable regions. Except for this relationship, there was no significant relationship among the other physicochemical properties of the mutant glycinins.  相似文献   

6.
The influence of pH and ionic strength on gel formation and gel properties of soy protein isolate (SPI) in relation to denaturation and protein aggregation/precipitation was studied. Denaturation proved to be a prerequisite for gel formation under all conditions of pH and ionic strength studied. Gels exhibited a low stiffness at pH >6 and a high stiffness at pH <6. This might be caused by variations in the association/dissociation behavior of the soy proteins on heating as a function of pH, as indicated by the different protein compositions of the dissolved protein after heating. At pH 3-5 all protein seems to participate in the network, whereas at pH >5 less protein and especially fewer acidic polypeptides take part in the network, coinciding with less stiff gels. At pH 7.6, extensive rearrangements in the network structure took place during prolonged heating, whereas at pH 3.8 rearrangements did not occur.  相似文献   

7.
Structural and functional properties of two amaranth protein isolates as a function of pH were studied. Isolates, A9 and A11, were obtained by alkaline extraction at pH 9 and 11, respectively. Gel filtration chromatograms of A9 and A11 showed similar profiles. The A11 isolate contained mainly albumins and globulins, and a small proportion of globulin‐P aggregates, suggesting the presence of species with a higher degree of denaturation compared to A9. Differential scanning calorimetry (DSC) showed that A9 was characterized by two thermal transitions (65.8 and 98°C); A11 exhibited only a small endotherm (66.6°C) and a second, less defined one. DSC analysis of A9 at pH 2–4 did not show endotherms, but at pH 5, some protein structures were observed. A11 showed a greater degree of denaturation. FPLC results showed that the proteins in A9 are more folded and their conformation is closer to the native state than those in A11, which are more unfolded due to pH‐mediated denaturation, mainly in acid media. The surface hydrophobicity of the isolates in acid media was lower than in alkaline media. The fluorescence emission spectra of the isolates showed differences in acidic pH conditions. As expected, the highest solubility was at alkaline pH. The water‐holding capacity was similar for both isolates. The water‐imbibing capacity and speed of foaming was higher for A11 than for A9. In summary, intense pH treatment of amaranth isolates generated partial or total protein denaturation and differences in the functional properties.  相似文献   

8.
Helianthinin, the main storage protein of sunflowers, has low water solubility and does not form a gel when heated; this behavior is different from other 11S globulins and limits its food applications. To understand this particular behavior, changes on helianthinin association-dissociation state induced by modifications in pH and ionic strength were analyzed. The influence of these different medium conditions on its thermal stability and tendency to form aggregates was also studied. Helianthinin behavior at different pH values and ionic strengths is similar to other 11S globulins except that it remains in a trimeric form at pH 11. Helianthinin thermal stability is higher than other 11S globulins but is lower than oat 11S globulin. Alkaline pH produces a 10 degrees C decrease of its denaturation temperature and also of the cooperativity of denaturation process, but it does not affect the denaturation activation energy. The decrease in thermal stability with the pH increase is also manifested by its tendency to form aggregates by SH/SS interchange reactions. When thermal treatments at alkaline pH are performed, all helianthinin subunits form aggregates, characterized by a higher proportion of beta-polypeptides than alpha-polypeptides, which is an indication that aggregation is accompanied by dissociation. Treatments at 80 degrees C are sufficient to induce aggregation but not to produce denaturation, and in these conditions hexameric forms remain after the treatment.  相似文献   

9.
Soybean proteins have shown great potential for applications as renewable and environmentally friendly adhesives. The objective of this work was to study physicochemical and adhesion properties of soy glycinin subunits. Soybean glycinin was extracted from soybean flour and then fractionated into acidic and basic subunits with an estimated purity of 90 and 85%, respectively. Amino acid composition of glycinin subunits was determined. The high hydrophobic amino acid content is a major contributor to the solubility behavior and water resistance of the basic subunits. Acidic subunits and glycinin had similar solubility profiles, showing more than 80% solubility at pH 2.0-4.0 or 6.5-12.0, whereas basic subunits had considerably lower solubility with the minimum at pH 4.5-8.0. Thermal analysis using a differential scanning calorimeter suggested that basic subunits form new oligomeric structures with higher thermal stability than glycinin but no highly ordered structures present in isolated acidic subunits. The wet strength of basic subunits was 160% more than that of acidic subunits prepared at their respective isoelectric points (pI) and cured at 130 degrees C. Both pH and the curing temperature significantly affected adhesive performance. High-adhesion water resistance was usually observed for adhesives from protein prepared at their pI values and cured at elevated temperatures. Basic subunits are responsible for the water resistance of glycinin and are a good starting material for the development of water-resistant adhesives.  相似文献   

10.
In heat denaturation studies conducted in the past the genetic variants of glycinin have been considered as a homogeneous group of proteins. In this work the validity of this assumption was tested. It was found by calorimetric studies that glycinin denatures heterogeneously at pH 7.6. When the temperature of isothermal treatment is increased from 70 to 82 degrees C the proportion of glycinin remaining native progressively decreases from 95% to 5% while the denaturation temperature of the glycinin remaining native increases from 88.5 to 95 degrees C. Similar trends were found for pH 3.8. Fractionation and subsequent analysis (MALDI-TOF and CE) of isothermally treated samples demonstrated that at pH 7.6 the heterogeneous denaturation is caused by differences in thermal stability of the genetic variants of glycinin. The stability increases in the order G2/G3/G1< A(4)< G5 < G4.  相似文献   

11.
Changes induced by high pressure (HP) treatment (200-600 MPa) on soybean protein isolates (SPI) at pH 3 (SPI3) and pH 8 (SPI8) were analyzed. Changes in protein solubility, surface hydrophobicity (Ho), and free sulfhydryl content (SH(F)) were determined. Protein aggregation and denaturation and changes in secondary structure were also studied. An increase in protein Ho and aggregation, a reduction of free SH, and a partial unfolding of 7S and 11S fractions were observed in HP-treated SPI8. Changes in secondary structure were also detected, which led to a more disordered structure. HP-treated SPI3 was partially denatured and presented insoluble aggregates. A major molecular unfolding, a decrease of thermal stability, and an increase of protein solubility and Ho were also detected. At 400 and 600 MPa, a decrease of the SH(F) and a total denaturation were observed.  相似文献   

12.
Two experiments were conducted to study gastric and small intestinal digestion of soybean glycinin and beta-conglycinin in preruminant calves fed milk replacers containing a mixture of skim milk powder and antigenic heated soybean flour. In experiment 1, duodenal passage of immunoreactive beta-conglycinin lasted for a much longer time after the morning meal than that of glycinin. Western blotting revealed the early abomasal outflow of glycinin subunits that associated nearly intact basic polypeptides to partially degraded acidic polypeptides. Intact beta-conglycinin was evidenced at most sampling times. In experiment 2, intact basic glycinin (M(r) = 21000) associated with partially digested acidic glycinin (7000 < M(r) < 25000) was demonstrated in ileal digesta up to 8-10 h after the meal. beta-Conglycinin immunoreactivity could not be evidenced by Western blotting in ileal digesta.  相似文献   

13.
Emulsifying properties of acidic subunits of soy 11S globulin   总被引：3，自引：0，他引：3  
The emulsifying properties of the acidic subunits (AS11S) isolated from soy glycinin (11S) have been studied. The isolated AS11S existed in solution mainly as a dimer species. Circular dichroic analysis indicated only a slight increase in aperiodic structure and no significant difference in beta-sheet structure when compared with those of soy 11S. At similar experimental conditions, the emulsifying properties of AS11S were superior to those of soy 11S and heat-denatured 11S. Emulsions prepared with 1% AS11S remained very stable without any visible oil separation for more than a month under gentle agitating conditions, whereas those prepared with 1% 11S collapsed and separated into phases within 2-3 days. The AS11S-stabilized emulsions were very stable below 0.15 M ionic strength. Studies on the rate of adsorption and surface tension reduction at the air-water interface showed that AS11S was significantly more surface active than soy 11S. It is proposed that, because the mass fraction of acidic subunits in soy 11S is approximately 60% and it is relatively easy to separate the acidic subunits from soy 11S, it may be industrially feasible to develop an economical process to isolate functional acidic subunits for use in emulsion-based food products.  相似文献   

14.
Denaturation of proteins from striated and smooth muscles of scallop (Zygochlamys patagonica) was studied with differential scanning calorimetry (DSC) by monitoring maximum temperatures of transition and denaturation enthalpies. DSC thermograms of both striated and smooth whole muscles showed two transitions: Tmax 55.0, 79.2 degrees C; and Tmax 54.7, 78.7 degrees C, respectively. The DSC thermograms of myofibrils and actomyosin were similar to those corresponding to their respective whole muscles. As pH and ionic strength increased, the thermal stability of whole muscles decreased. The pH increase (5.0-8.0) significantly (p < 0.01) decreased the denaturation enthalpies (deltaH total, deltaH peakI, and deltaH peakII) of whole striated muscles. A significant decrease (p < 0.05) in the deltaH total and the deltaH peakI was also observed in DSC thermograms of smooth muscles at pH 8.0. Denaturation enthalpies (deltaH total and deltaH peakI) significantly decreased (p < 0.01) when the ionic strength increased from 0.05 to 0.5 in both types of muscles. Striated muscles were more affected than smooth muscles by changes in the chemical environment.  相似文献   

15.
Soy protein elastomer (SPE) exhibits elastic, extensible, and sticky properties in its native state and displays great potential as an alternative to wheat gluten. The objective of this study was to better understand the roles of soy protein subunits (polypeptides) contributing to the functional properties of SPE. Six soy protein samples with different subunit compositions were prepared by extracting the proteins at various pH values on the basis of the different solubilities of conglycinin (7S) and glycinin (11S) globulins. Soy protein containing a large amount of high molecular weight aggregates formed from α' and α subunits exhibited stronger viscoelastic solid behavior than other soy protein samples in terms of dynamic elastic and viscous modules. Electrophoresis results revealed that these aggregates are mainly stabilized through disulfide bonds, which also contributed to higher denaturation enthalpy as characterized by DSC and larger size protein aggregates observed by TEM. Besides, the most viscoelastic soy protein sample exhibited flat and smooth surfaces of the protein particles as observed by SEM, whereas other samples had rough and porous particle surfaces. It was proposed that the ability of α' and α to form aggregates and the resultant proper protein-protein interaction in soy proteins are the critical contributions to the continuous network of SPE.  相似文献   

16.
Proteins isolated from blue-green algae Spirulina platensis strain Pacifica were characterized by visible absorption, differential scanning calorimetry (DSC), viscometry, and dynamic oscillatory rheological measurements. Unique thermal unfolding, denaturation, aggregation, and gelation of the algal protein isolate are presented. DSC analysis showed that thermal transitions occur at about 67 and 109 degrees C at neutral pH. Calcium chloride stabilized the quaternary structure against denaturation and shifted the transitions at higher temperatures. Viscometric studies of Spirulina protein isolate as a function of temperature showed that the onset of the viscosity increase is closely related to the dissociation-denaturation process. Lower viscosities were observed for the protein solutions dissolved at pH 9 due to an increased protein solubility. Solutions of Spirulina protein isolate form elastic gels during heating to 90 degrees C. Subsequent cooling at ambient temperatures caused a further pronounced increase in the elastic moduli and network elasticity. Spirulina protein isolate has good gelling properties with fairly low minimum critical gelling concentrations of about 1.5 and 2.5 wt % in 0.1 M Tris buffer, pH 7, and with 0.02 M CaCl(2) in the same buffer, respectively. It is suggested that mainly the interactions of exposed hydrophobic regions generate the molecular association, initial aggregation, and gelation of the protein isolate during the thermal treatment. Hydrogen bonds reinforce the network rigidity of the protein on cooling and further stabilize the structure of Spirulina protein gels but alone are not sufficient to form a network structure. Intermolecular sulfhydryl and disulfide bonds were found to play a minor role for the network strength of Spirulina protein gels but affect the elasticity of the structures formed. Both time and temperature at isothermal heat-induced gelation within 40-80 degrees C affect substantially the network formation and the development of elastic modulus of Spirulina protein gels. This is also attributed to the strong temperature dependence of hydrophobic interactions. The aggregation, denaturation, and gelation properties of Spirulina algal protein isolate are likely to be controlled from protein-protein complexes rather than individual protein molecules.  相似文献   

17.
This study examined the immunogenic response of glycinin under varying conditions of pH and ionic strength using enzyme-linked immunosorbent assay. Differential scanning calorimetric (DSC) analysis and Fourier transform infrared spectroscopy (FTIR) were used to investigate the conformational changes induced as a result of these conditions, and the correlation with the changes observed in glycinin immunoreactivity were determined. A highly purified glycinin obtained by isoelectric precipitation followed by native preparative continuous flow electrophoresis was used for these studies. Purity was confirmed by two-dimensional-polyacrylamide gel electrophoresis and mass spectroscopy. DSC and FTIR results suggest that glycinin immunoreactivity is affected by changes in the tertiary and secondary packing of the protein, when flexibility, stability, and accessibility of certain substructures are modified. Aggregation and/or increased compactness of glycinin subcomponents could have potentially prevented epitopes from reacting with the IgG antibodies.  相似文献   

18.
The soluble potato proteins are mainly composed of patatin and protease inhibitors. Using DSC and both far-UV and near-UV CD spectroscopy, it was shown that potato proteins unfold between 55 and 75 degrees C. Increasing the ionic strength from 15 to 200 mM generally caused an increase in denaturation temperature. It was concluded that either the dimeric protein patatin unfolds in its monomeric state or its monomers are loosely associated and unfold independently. Thermal unfolding of the protease inhibitors was correlated with a decrease in protease inhibitor activities and resulted in an ionic strength dependent loss of protein solubility. Potato proteins were soluble at neutral and strongly acidic pH values. The tertiary structure of patatin was irreversibly altered by precipitation at pH 5. At mildly acidic pH the overall potato protein solubility was dependent on ionic strength and the presence of unfolded patatin.  相似文献   

19.
Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) were used to study changes in the conformation of globulin from common buckwheat (Fagopyrum esculentum Moench) (BWG) under various environmental conditions. The IR spectrum of the native BWG showed several major bands from 1691 to 1636 cm(-1) in the amide I' region, and the secondary structure composition was estimated as 34.5% beta-sheets, 20.0% beta-turns, 16.0% alpha-helices, and 14.4% random coils. Highly acidic and alkaline pH conditions induced decreases in beta-sheet and alpha-helical contents, as well as in denaturation temperature (Td) and enthalpy of denaturation (DeltaH), as shown in the DSC thermograms. Addition of chaotropic salts (1.0 M) caused progressive decreases in ordered structures and thermal stability following the lyotropic series of anions. The presence of several protein structure perturbants also led to changes in IR band intensities and DSC thermal stabilities, suggesting protein unfolding. Intermolecular antiparallel beta-sheet (1620 and 1681 cm(-1)) band intensities started to increase when BWG was heated to 90 degrees C, suggesting the initiation of protein aggregation. Increasing the time of the preheat treatment (at 100 degrees C) caused progressive increases in Td and pronounced decreases in DeltaH, suggesting partial denaturation and reassociation of protein molecules.  相似文献   

20.
Cultivated soybeans (Glycine max) are derived from wild soybeans (Glycine soja) and can be crossed with them to produce fertile offspring. The latter exhibit greater genetic variation than the former, suggesting a possibility that wild soybeans contain storage proteins with properties different from and better than those of cultivated soybeans. To identify a wild soybean suitable for breeding a new soybean cultivar, we analyzed seed proteins from 390 lines of wild soybeans by electrophoresis. We found some lines containing electrophoretic variants of glycinin and beta-conglycinin subunits: one line containing a small alpha' subunit of beta-conglycinin and two and five lines containing small A3 and large A4 polypeptides of glycinin, respectively. Beta-Conglycinin and glycinin containing such variant subunits exhibited solubility and emulsifying ability similar to those of the predominant types of wild and cultivated soybeans. Glycinins containing small A3 and large A4 gave a shoulder derived from the start of denaturation at a temperature 4 degrees C lower than that of glycinin from the predominant types of wild and cultivated soybeans, although their thermal denaturation midpoint temperatures were very similar to each other. Cloning and sequencing of the predominant and variant subunit cDNAs revealed that the small alpha' and the small A3 lacked 24 amino acid residues in the extension region and four amino acid residues in the hypervariable region, respectively, and that the large A4 did not have an insert corresponding to the difference in the electrophoretic mobility but Arg279 and Gln305 were replaced by glutamine and histidine, respectively, in the hypervariable region. These suggest that small differences even in the hypervariable region can affect the thermal stability, as well as the electrophoretic mobilities, of the proteins.  相似文献   

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