Adsorption of glycinin and β-conglycinin on silica and cellulose: surface interactions as a function of denaturation, pH, and electrolytes

Soybean proteins have found uses in different nonfood applications due to their interesting properties. We report on the kinetics and extent of adsorption on silica and cellulose surfaces of glycinin and β-conglycinin, the main proteins present in soy. Quartz crystal microgravimetry (QCM) experiments indicate that soy protein adsorption is strongly affected by changes in the physicochemical environment. The affinity of glycinin and the mass adsorbed on silica and cellulose increases (by ca. 13 and 89%, respectively) with solution ionic strength (as it increases from 0 to 100 mM NaCl) due to screening of electrostatic interactions. In contrast, β-conglycinin adsorbs on the same substrates to a lower extent and the addition of electrolyte reduces adsorption (by 25 and 57%, respectively). The addition of 10 mM 2-mercaptoethanol, a denaturing agent, reduces the adsorption of both proteins with a significant effect for glycinin. This observation is explained by the cleavage of disulfide bonds which allows unfolding of the molecules and promotes dissociation into subunits that favors more compact adsorbed layer structures. In addition, adsorption of glycinin onto cellulose decreases with lowering the pH from neutral to pH 3 due to dissociation of the macromolecules, resulting in flatter adsorbed layers. The respective adsorption isotherms fit a Langmuir model and QCM shifts in energy dissipation and frequency reveal multiple-step kinetic processes indicative of changes in adlayer structure.     

Association-dissociation of glycinin in urea,guanidine hydrochloride and sodium dodecyl sulphate solutions

The effect of urea, guanidine hydrochloride and sodium dodecyl sulphate on glycinin, the high molecular weight protein fraction from soybean has been investigated by analytical ultracentrifugation. Urea and guanidine hydrochloride dissociate the protein to a ‘2S’ protein through the intermediary 7S and 4S proteins. Howeαer, in sodium dodecyl sulphate the protein directly dissociates to a 2S protein. Analysis of the data by calculation of per cent fraction and S_20,w value indicates that dissociation and denaturation of glycinin occur simultaneously in the presence of the aboαe reagents but to different extents.     

Co-expression of soybean glycinins A1aB1b and A3B4 enhances their accumulation levels in transgenic rice seed

The soybean major storage protein glycinin is encoded by five genes, which are divided into two subfamilies. Expression of A3B4 glycinin in transgenic rice seed reached about 1.5% of total seed protein, even if expressed under the control of strong endosperm-specific promoters. In contrast, expression of A1aB1b glycinin reached about 4% of total seed protein. Co-expression of the two proteins doubled accumulation levels of both A1aB1b and A3B4 glycinins. This increase can be largely accounted for by their aggregation with rice glutelins, self-assembly and inter-glycinin interactions, resulting in the enrichment of globulin and glutelin fractions and a concomitant reduction of the prolamin fraction. Immunoelectron microscopy indicated that the synthesized A1aB1b glycinin was predominantly deposited in protein body-II (PB-II) storage vacuoles, whereas A3B4 glycinin is targeted to both PB-II and endoplasmic reticulum (ER)-derived protein body-I (PB-I) storage structures. Co-expression with A1aB1b facilitated targeting of A3B4 glycinin into PB-II by sequestration with A1aB1b, resulting in an increase in the accumulation of A3B4 glycinin.     

Effects of glycinin on IgE-mediated increase of mast cell numbers and histamine release in the small intestine

Soybean allergy represents a significant health threat to individuals with food allergies. Glycinin, the main storage protein in soybean, has been identified as a major food allergen. The present study was conducted to investigate the mechanism of glycinin-induced hypersensitivity in a swine model. The relationship between glycinin dose and the severity of hypersensitive reactions was also explored. Twenty-four piglets weaned at 18 days were gastric sensitized and subjected to repeated oral challenges with diets containing 0%, 2%, 4% and 8% glycinin. The results showed that dietary supplementation of glycinin reduced piglet performance (P<.01) while increasing occurrence of diarrhea (P<.05) and erythema area (P=.01) in response to an intradermal injection of glycinin. Intestinal mast cell numbers (P<.05) and immunoglobulin E (IgE) levels (P<.05) were increased linearly, whereas the histamine content in intestinal specimens (except in the duodenum) was decreased (P<.01), indicating that more histamine had been released in glycinin-fed piglets than in control. Serum concentrations of total IgE, glycinin-specific IgG1 and interleukin (IL)-4 and IL-10 were also greater (P<.05) in the pigs treated with glycinin. In this study, we found that glycinin-induced hypersensitivity is a predominantly Th2-type immune response, mediated by IgE and associated with increases in intestinal mast cell numbers and histamine release as well as IL-4 and IL-10 concentrations in the serum of sensitized piglets, resulting in diarrhea and reduced performance. The severity of the hypersensitive reactions depends on the dose of glycinin. Higher dose may cause more severe anaphylactic symptoms.     

A Relationship between Papaincatalyzed Aminolysis of Ethyl Hippurate by Amino Acid Esters and Their Incorporation into Ovalbumin Hydrolysate during the Plastein Reaction

The effects of the colloidal properties of emulsion particles and the conformation of tryptic digests of soybean glycinin, the emulsifiers, on emulsion properties were investigated. The digests were separated into some fractions, and the properties of intact glycinin, two kinds of the best were examined. The diameter of the emulsion particles measured by spectroturbidimetry was not very different among the best emulsifiers and intact glycinin in spite of the difference in emulsifying ability; however, a new parameter, flocculation strength, which is the rigidity of the flocculated structure and is defined as the minimum detergent concentration for the dissociation of flocculation, closely and negatively related to the short term emulsion stability. The amount of adsorbed protein on the surface of the emulsion particles was also related to the long term emulsion stability. The two best emulsifiers were analyzed by gel filtration and circular dichroism. The emulsifiers contained large molecular components whose molecular weight and secondary structures were similar to intact glycinin. The conformational stability of the emulsifiers was evaluated by the change in emission maxima of the intrinsic fluorescence of the proteins against changing urea concentration, and the surface hydrophobicity of the proteins was estimated by the binding of l-anilino-8-naphthalene sulfonate (ANS). The emulsion stability increased with decreasing conformational stability and increasing surface hydrophobicity of the emulsifier proteins.     

Foreign gene products can be enhanced by introduction into low storage protein mutants

Transgenic rice expressing soybean glycinin in its endosperm was crossed with two types of low-glutelin mutants to determine how much storage the protein mutants can contribute to increases in glycinin accumulation. The glycinin level (102 microg/100 mg seed) in the parental transgenic line was enhanced to approximately 224-237 microg/100 mg seed within a genetic background deficient in glutelin (i.e. of low glutelins). The enrichment of this foreign gene product was compensated by a decrease in the expression of other endogenous prolamine and globulin storage proteins, resulting in an almost equivalent total amount of seed storage proteins. These results show that low storage protein mutants can provide potentially useful hosts for the expression of foreign genes, allowing a higher-level accumulation, because they can provide wider space for the accumulation of foreign gene products than in the normal host plant.     

Proteomic and genetic analysis of glycinin subunits of sixteen soybean genotypes.

We investigated proteomic and genomic profiles of glycinin, a family of major storage proteins in 16 different soybean genotypes consisting of four groups including wild soybean (Glycine soja), unimproved cultivated soybean landraces from Asia (G. max), ancestors of N. American soybean (G. max), and modern soybean (G. max) genotypes. We observed considerable variation in all five glycinin subunits, G1, G2 G3, G4 and G5 using proteomics and genetic analysis. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) analysis showed that the wild genotypes had a range of 25-29 glycinin protein spots that included both acidic and basic polypeptides followed by the ancestors with 24-28, modern cultivars with 24-25, and landraces with 17-23 protein spots. Overall, the wild genotypes have a higher number of protein spots when compared to the other three genotypes. Major variation was observed in acidic polypeptides of G3, G4 and G5 compared to G1 and G2, and minor variation was observed in basic polypeptides of all subunits. Our data indicated that there are major variations of glycinin subunits between wild and cultivated genotypes rather than within the same groups. Based on Southern blot DNA analysis, we observed genetic polymorphisms in group I genes (G1, G2, and G3) between and within the four genotype groups, but not in group II genes (G4 and G5). This is the first study reporting the comparative analysis of glycinin in a diverse set of soybean genotypes using combined proteomic and genetic analysis.     

Purification and characterization of mRNA from soybean seeds. Identification of glycinin and beta-conglycinin precursors

Poly(A)-rich RNA was isolated from developing soybean seeds (Glycine max (L.) Merr.) and fractionated on linear log sucrose gradients. Two major fractions sedimenting at 18 S and 20 S were separated and then purified by further sucrose gradient fractionation. Both fractions were active as messengers when added to a rabbit reticulocyte lysate protein synthesis system. The 18 S fraction caused proteins migrating primarily to the 60,000-dalton region of a sodium dodecyl sulfate gel to be produced, while translation of the 20 S fraction preferentially directed the synthesis of polypeptides similar in size to the alpha and alpha' subunits of beta-conglycinin. Evidence that many of the 60,000-dalton polypeptides were related to glycinin and the high molecular weight 20 S translation products were related to beta-conglycinin was obtained by immunoprecipitation using monospecific antibodies against glycinin and beta-conglycinin, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated products revealed that the glycinin precursor region contained at least three different size components and that the family of glycinin precursors had larger apparent molecular weight (58,000-63,000) than the disulfide-linked complexes between acidic and basic glycinin subunits (57,000). Unlike the disulfide-linked glycinin complexes which were cleaved by disulfide reduction, glycinin precursors were insensitive to reducing agents. The alpha and alpha' subunits synthesized in vitro also had slightly larger apparent molecular weights than purified alpha and alpha' standards.     

Effects of soybean glycinin on performance and immune function in early weaned pigs

Glycinin, a major storage protein in soybean, has been identified as an important food allergen. The present study was conducted to investigate the effects of soybean glycinin on the local and systemic immune responses using a swine model specific for glycinin allergy. Performance, peripheral blood lymphocyte proliferation and CD4+ and CD8+ subsets in the plasma of naive and sensitised piglets were determined. In addition, the concentrations of IgA, IgG, IgM, interleukin-4 and interleukin-6 in the jejunum mucosa were measured. Our results showed that dietary supplementation of glycinin reduced piglet performance (p < 0.05), while increasing lymphocyte proliferation and CD4+/ CD8+ ratio (p < 0.01). Intestinal mucosal immune responses to glycinin were enhanced with high levels of IgA (p < 0.01) as well as interleukin-4 and interleukin-6 in the jejunum mucosa in glycinin-treated piglets compared with the control (p < 0.01). There were no differences due to treatment in the densities of IgG and IgM. In conclusion, it was found that glycinin stimulated local and systemic immune responses in allergic piglets and had negative effects on piglet performance. The severity of the immune reactions depends on the dose of glycinin with higher doses causing more severe symptoms.     

Structure and flanking regions of soybean seed protein genes

We have characterized the structure and flanking region of genes representing two, coordinately expressed, soybean seed protein gene families. One family directs the synthesis of the major storage protean glycinin; the other encodes a 15.5 kd polypeptide of unknown function. DNA blot hybridization experiments showed approximately three, nonallelic genes in the glycinin family and two in the 15 kd protein family, and showed that these families are not selectively amplified or rearranged during embryogeny. R-loop and S1 nuclease mapping studies demonstrated no detectable introns in the 15 kd protein genes but at least one and possibly two in the glycinin genes. No interfamily clustering of these genes occurs within a 10-15 kb chromosomal domain. Nor are they contiguous to other genes expressed at moderate levels during embryogenesis. Each of them, however, is contiguous to a gene expressed at another developmental period in the leaf. These leaf genes encode rare class messages which constitute only 1 X 10(-5%) of the leaf mRNA, or about one molecule per cell. R-loop analysis of two leaf genes showed that one contains no detectable introns while the other possesses at least three. DNA gel blot studies showed that only one of the seed protein genomic clones contains an interspersed repetitive DNA element. Pairwise cross-hybridization studies did not detect any flanking sequences shared by the 15 kd protein, glycinin and leaf genes.     

Identification of marker proteins for the adulteration of meat products with soybean proteins by multidimensional liquid chromatography-tandem mass spectrometry

Soybean proteins are frequently added to processed meat products for economic reasons and to improve their functional properties. Monitoring of the addition of soybean protein to meat products is of high interest due to the existence of regulations forbidding or limiting the amount of soybean proteins that can be added during the processing of meat products. We have used chromatographic prefractionation on the protein level by perfusion liquid chromatography to isolate peaks of interest from extracts of soybean protein isolate (SPI) and of meat products containing SPI. After enzymatic digestion using trypsin, the collected fractions were analyzed by nanoflow liquid chromatography-tandem mass spectrometry. Several variants and subunits of the major seed proteins, glycinin and beta-conglycinin, were identified in SPI, along with two other proteins. In soybean-protein-containing meat samples, different glycinin A subunits could be identified from the peak discriminating between samples with and without soybean proteins added. Among those, glycinin G4 subunit A4 was consistently found in all samples. Consequently, this protein (subunit) can be used as a target for new analytical techniques in the course of identifying the addition of soybean protein to meat products.     

Accumulation of high levels of free amino acids in soybean seeds through integration of mutations conferring seed protein deficiency

Soybean ( Glycine max [L.] Merr.) seeds are rich in protein, most of which is contributed by the major storage proteins glycinin (11S globulin) and beta-conglycinin (7S globulin). Null mutations for each of the subunits of these storage proteins were integrated by crossbreeding to yield a soybean line that lacks both glycinin and beta-conglycinin components. In spite of the absence of these two major storage proteins, the mutant line grew and reproduced normally, and the nitrogen content of its dry seed was similar to that for wild-type cultivars. However, protein bodies appeared underdeveloped in the cotyledons of the integrated mutant line. Furthermore, whereas free amino acids contribute only 0.3-0.8% of the seed nitrogen content of wild-type varieties, they constituted 4.5-8.2% of the seed nitrogen content in the integrated mutant line, with arginine (Arg) being especially enriched in the mutant seeds. Seeds of the integrated mutant line thus appeared to compensate for the reduced nitrogen content in the form of glycinin and beta-conglycinin by accumulating free amino acids as well as by increasing the expression of certain other seed proteins. These results indicate that soybean seeds are able to store nitrogen mostly in the form of either proteins or free amino acids.     

The amino acid sequence of the A2B1a subunit of glycinin

The amino acid sequences of the acidic and basic components of the A2B1a subunit of glycinin, the major seed reserve protein of the soybean (Glycine max L. Merr.), were determined. They contain 278 and 180 amino acids, respectively, and have molecular weights of 31,600 +/- 100 and 19,900 +/- 100. The molecular weight of the acidic component is considerably less than that estimated by sodium dodecyl sulfate-gel electrophoresis (37,000). Sequence heterogeneity was detected at several positions scattered throughout the primary structures of both components, indicating that the preparation sequenced was composed of several nearly identical polypeptides. These data, in conjunction with a recently determined nucleotide sequence of the 3'-terminal two-thirds of the analogous glycinin subunit gene, illustrate the complexity of the gene family responsible for synthesis of glycinin subunits.     

Genomic organization of glycinin genes in soybean

Glycinin is the predominant seed storage protein in most soybean varieties. Previously, five major genes (designated Gy1 to Gy5) encoding glycinin subunits have been described. In this report two new genes are identified and mapped: a glycinin pseudogene, gy6, and a functional gene, Gy7. Messenger RNA for the gy6 pseudogene is not detected in developing seeds. While Gy7 mRNA was present at the midmaturation stage of seed development in the soybean variety Resnik, the steady state amount of this message was at least an order of magnitude less-prevalent than the mRNA encoding each of the other five glycinin subunits. Even though the amino-acid sequence of the glycinin subunit G7 is related to the other five soybean 11S subunits, it does not fit into either the Group-1 (G1, G2, G3) or the Group-2 (G4, G5) glycinin subunit families. The Gy7 gene is tandemly linked 3' to Gy3 on Linkage Group L (chromosome 19) of the public molecular linkage map. By contrast, the gy6 gene occupies a locus downstream from Gy2 on Linkage Group N (chromosome 3) in a region that is related to the position where Gy7 is located on chromosome 19.     

Expression of soybean glycinin subunit precursor cDNAs in Escherichia coli

As the cDNAs encoding A1aB1b and A2B1a subunit precursors of the glycinin A2 subfamily contain a unique NcoI site sequence, (A)CCATGG, occurring at their translation initiation sites, plasmids were constructed to direct the synthesis of those precursor proteins by inserting NcoI/PstI fragments derived from those cDNA clones into the NcoI/PstI-pKK233-2 expression vector in Escherichia coli MV1190, respectively. The resultant plasmids directed the expression of 57-kDa protein components that have molecular masses in agreement with those of the in vitro translation products directed by glycinin A2 subfamily mRNAs, by the addition of isopropyl beta-D-thiogalactoside. These proteins, which comprised as much as 1% of the total bacterial protein, are immunoprecipitable with rabbit antibodies specific for glycinin subunits. This procedure makes glycinin subunits available as a model for studying structure-function relationships in seed proteins using site-directed mutagenesis. This is the first expression of glycinin-like storage protein in E. coli.     

Studies on the stability of 11S globulin from soybeans by differential scanning microcalorimetry

The scanning microcalorimetry method was applied to the study of thermal denaturation of 11S globulin (glycinin), dodecameric globular protein from soybeans at pH 7.6 and the range of NaCl concentrations from 0 to 0.9 m. The specific enthalpy of denaturation was shown to be the linear function of temperature. The ratio of the calorimetric enthalpy to the effective one (van't Hoff's) per protomer of glycinin was 1.23 ± 0.05. It is concluded that at the first approximation glycinin protomers denaturated independently, in conformity with the two-state model. The plotted temperature-dependent specific free energy of glycinin denaturation at different NaCl concentrations demonstrated that an increase in the salt content brought about the rise of the protein stability. The maximal glycinin stability is reached at about 273 K. The molar free energy of denaturation at 273 K in 1 m NaCl is 1320 kJ/mol.     

Isolation of the acidic and basic subunits of glycinin

The acidic and basic subunits of glycinin—the major storage protein of soybean seeds (Glycine max)—were isolated by a simple ion exchange chromatography method involving a two-step pH change of the elution buffer. Abnormal behaviour of the subunits in dodecyl sulfate polyacrylamide gel electrophoresis is suggested.     

Effects of soybean glycinin on performance and immune function in early weaned pigs

Glycinin, a major storage protein in soybean, has been identified as an important food allergen. The present study was conducted to investigate the effects of soybean glycinin on the local and systemic immune responses using a swine model specific for glycinin allergy. Performance, peripheral blood lymphocyte proliferation and CD4⁺ and CD8⁺  subsets in the plasma of naive and sensitised piglets were determined. In addition, the concentrations of IgA, IgG, IgM, interleukin-4 and interleukin-6 in the jejunum mucosa were measured. Our results showed that dietary supplementation of glycinin reduced piglet performance (p < 0.05), while increasing lymphocyte proliferation and CD4⁺/CD8⁺  ratio (p < 0.01). Intestinal mucosal immune responses to glycinin were enhanced with high levels of IgA (p < 0.01) as well as interleukin-4 and interleukin-6 in the jejunum mucosa in glycinin-treated piglets compared with the control (p < 0.01). There were no differences due to treatment in the densities of IgG and IgM. In conclusion, it was found that glycinin stimulated local and systemic immune responses in allergic piglets and had negative effects on piglet performance. The severity of the immune reactions depends on the dose of glycinin with higher doses causing more severe symptoms.     

Effect of Soy Protein Subunit Composition on the Rheological Properties of Soymilk during Acidification

The effect of soy protein subunit composition on the acid-induced aggregation of soymilk was investigated by preparing soymilk from different soybean lines lacking specific glycinin and β-conglycinin subunits. Acid gelation was induced by glucono-δ-lactone (GDL) and analysis was done using diffusing wave spectroscopy and rheology. Aggregation occurred near pH 5.8 and the increase in radius corresponded to an increase in the elastic modulus measured by small deformation rheology. Diffusing wave spectroscopy was also employed to follow acid gelation, and data indicated that particle interactions start to occur at a higher pH than the pH of onset of gelation (corresponding to the start of the rapid increase in elastic modulus). The protein subunit composition significantly affected the development of structure during acidification. The onset of aggregation occurred at a higher pH for soymilk samples containing group IIb (the acidic subunit A₃) of glycinin, than for samples prepared from Harovinton (a commercial variety containing all subunits) or from genotypes null in glycinin. The gels made from lines containing group I (A₁, A₂) and group IIb (A₃) of glycinin resulted in stiffer acid gels compared to the lines containing only β-conglycinin. These results confirmed that the ratio of glycinin/β-conglycinin has a significant effect on gel structure, with an increase in glycinin causing an increase in gel stiffness. The type of glycinin subunits also affected the aggregation behavior of soymilk.