Amaranth (Amaranthus hypochondriacus) vicilin subunit structure

The 7S-globulin fraction is a minor component of the amaranth storage proteins. The present work provides new information about this protein. The amaranth 7S-globulin or vicilin presented a sedimentation coefficient of 8.6 ± 0.6 S and was composed of main subunits of 66, 52, 38, and 16 kDa. On the basis of mass spectrometry (MS) analysis of tryptic fragments, the 52, 38, and 16 kDa subunits presented sequence homology with sesame vicilin, whereas the 66 kDa subunit showed sequence similarity with a putative vicilin. Several characteristics of the 66 kDa subunit were similar to members of the convicilin family. Results support the hypothesis that the 7S-globulin molecules are composed of subunits coming from at least two gene families with primary products of 66 and 52 kDa, respectively. According to the present information, amaranth vicilin may be classified into the vicilin group that includes pea, broad bean, and sesame vicilins, among others.     

Characterization of Protein Fractions of Rice Bran to Devise Effective Methods of Protein Solubilization

Proteins from the defatted brans of representative rice cultivars were fractionated into albumins, globulins, prolamins, and acid-soluble glutelins, accounting for 34, 15, 6, and 11% of the total bran proteins, respectively. The remaining insoluble residue protein, after treatment with 0.1M sodium hydroxide, resulted in the solubilization of 95% of the residue protein, representing 32% of the total bran protein. The relative molecular mass (M_r) values determined by size-exclusion HPLC were 10–100 kDa, 10–150 kDa, 33–150 kDa, and 25–100 kDa for the fully dissociated polypeptides of albumins, globulins, prolamins, and acid-soluble glutelins, respectively. Despite a breakdown of disulfide bonds of the residue protein during sodium hydroxide solubilization, the M_r of the majority of the fully dissociated polypeptides of this fraction ranged from 45 to 150 kDa. Insolubility of residue protein was due mainly to its strong aggregation and extensive disulfide bond cross-linking. Efficient methods may be developed for solubilizing up to 98% of rice bran protein by the use of dissociating and disulfide breaking agents currently in use in the food industry.     

Amaranth globulin structure modifications induced by enzymatic proteolysis

Globulin-P was partially hydrolyzed with papain under specific conditions to study the resulting structural modifications. Under mild hydrolytic conditions, globulin-P polymers were cleaved to render their unitary constituents (280 kDa molecules). Under stronger hydrolytic conditions these unitary molecules were 13% smaller than those from nonhydrolyzed globulin. Moreover, these molecules remained assembled even though they contained degraded polypeptides. The monomeric (M) subunit and the A chains were preferentially cleaved under mild and intermediate hydrolytic conditions, whereas B chains remained with the same size. These results suggest that the M and A polypeptides might be located at an exposed site of the molecules resembling the structure of the legumins. The M subunit may be participating in the stabilization of globulin-P polymers, on the basis that these two species disappeared under the same hydrolytic conditions. Similar events such as those described in this paper might be taking place on globulin-P during germination of amaranth grain.     

Characterization of yam bean (Pachyrhizus erosus) proteins

Seed proteins from Mexican yam bean seeds (Pachyrhizus erosus L.) were sequentially extracted according to the Osborne classification. Albumins were the major fraction (52.1-31.0%), followed by globulins (30.7-27.5%). The minor protein fraction was prolamins (0.8%). Defatting with chloroform/methanol remarkably affected the distribution of protein solubility classes; albumins were the most affected fraction (4.3-17.5%). Electrophoretic patterns of albumins showed bands at 55, 40, 35, and 31 kDa. After reduction of the globulin fraction exhibited two triplets, one from 35 to 31 kDa and the second from 19 to 21 kDa, these could be compared to the acid and basic polypeptides of 11S-like proteins. Prolamins showed one band at 31 kDa, and glutelins after reduction showed three main bands at 52, 27, and 14 kDa. Trypsin inhibitors were assayed in saline extracts; the values found (1232-2608 IU/g of meal) were lower than those of other legumes. In general, yam bean seed proteins showed an excellent balance of all essential amino acids; albumins contain the highest amount of essential amino acids.     

Storage proteins from Lathyrus sativus seeds

The proteins from Lathyrus sativus Linn. (chickling vetch or grass pea) seeds were investigated. Protein constitutes approximately 20% of the seed dry weight, >60% of which is composed by globulins and 30% by albumins. A single, 24 kDa polypeptide comprises more than half of the protein present in the albumin fraction. The globulins may be fractionated into three main components, which were named alpha-lathyrin (the major globulin), beta-lathyrin, and gamma-lathyrin. alpha-Lathyrin, with a sedimentation coefficient of approximately 18S, is composed of three main types of unglycosylated subunits (50-66 kDa), each of which produce, upon reduction, a heavy and a light polypeptide chain, by analogy with 11S. beta-Lathyrin, with a sedimentation coefficient of 13S, is composed by a relatively large number of subunits (8-66 kDa). Two major polypeptides are glycosylated and exhibit structural similarity with beta-conglutin from Lupinus albus. One of these possesses an internal disulfide bond. gamma-Lathyrin, with a sedimentation coefficient of approximately 5S, contains two interacting, unglycosylated polypeptides, with no disulfide bonds: the major 24 kDa albumin and the heavier (20 kDa) polypeptide chain of La. sativus lectin.     

Jojoba seed meal proteins associated with proteolytic and protease inhibitory activities

The jojoba, Simmondsia chinensis, is a characteristic desert plant native to the Sonoran desert. The jojoba meal after oil extraction is rich in protein. The major jojoba proteins were albumins (79%) and globulins (21%), which have similar amino acid compositions and also showed a labile thrombin-inhibitory activity. SDS-PAGE showed two major proteins at 50 kDa and 25 kDa both in the albumins and in the globulins. The 25 kDa protein has trypsin- and chymotrypsin-inhibitory activities. In vitro digestibility of the globulins and albumins resembled that of casein and soybean protein concentrates and was increased after heat treatment. The increased digestibility achieved by boiling may be attributed to inactivation of the protease inhibitors and denaturation of proteins.     

Influence of the extracting solvent upon the structural properties of Amaranth (Amaranthus hypochondriacus) glutelin

Two amaranth glutelin preparations, Gt-bo extracted with borate buffer at pH 10 and Gt-na extracted with 0.1 N NaOH, were characterized and compared with the amaranth polymerized 11S globulin (Gp, globulin-P). Gt-bo and Gt-na presented very similar polypeptidic composition and a similar reactivity against an anti-Gp polyclonal antibody, although lower than that of Gp. It is demonstrated that Gt-na is composed of denatured and dissociated molecules, whereas Gt-bo consists of folded molecules. The size, polypeptidic composition, thermal stability, and denaturation enthalpy of Gt-bo molecules were similar to those of Gp subjected to a borate treatment at pH 10. The Gp immunoreactivity decreased to the level of Gt reactivity when subjected to alkaline treatment; this could be due to conformational changes. Results suggest that, like Gp, amaranth Gt molecules may be hexameric oligomers of approximately 300 kDa. They would be partially unfolded during the alkaline extraction.     

Biochemical characterization of soluble proteins in pecan [Carya illinoinensis (Wangenh.) K. Koch

Pecans (cv. Desirable) contained approximately 10% protein on a dry weight basis. The minimum nitrogen solubility (5.9-7.5%) at 0.25-0.75 M trichloroacetic acid represented the nonprotein nitrogen. Among the solvents assessed for protein solubilization, 0.1 M NaOH was the most effective, while borate saline buffer (pH 8.45) was judged to be optimal for protein solubilization. The protein solubility was minimal in the pH range of 3-7 and significantly increased on either side of this pH range. Increasing the NaCl concentration from 0 to 4 M significantly improved ( approximately 8-fold increase) protein solubilization. Following Osborne protein fractionation, the alkali-soluble glutelin fraction (60.1%) accounted for a major portion of pecan proteins followed by globulin (31.5%), prolamin (3.4%), and albumin (1.5%), respectively. The majority of pecan polypeptides were in the molecular mass range of 12-66 kDa and in the pI range of 4.0-8.3. The pecan globulin fraction was characterized by the presence of several glycoprotein polypeptides. Lysine was the first limiting essential amino acid in the defatted flour, globulin, prolamin, and alkaline glutelin fractions. Leucine and tryptophan were the first limiting essential amino acids in albumin and acid glutelin fractions, respectively. Rabbit polyclonal antibodies detected a range of pecan polypeptides in the 12-60 kDa range, of which the globulin fraction contained the most reactive polypeptides.     

Effect of Transglutaminase on Protein Electrophoretic Pattern of Rice,Soybean, and Rice‐Soybean Blends

The interactions taking place in composite dough containing rice flour and soybean proteins (5% w/w) in the presence of transglutaminase, an enzyme with cross‐linking activity, were studied using different electrophoretic analyses. The interaction between rice proteins and soybean proteins was intensified by the formation of new intermolecular covalent bonds catalyzed by transglutaminase and the indirect formation of disulfide bonds among proteins. The main protein fractions involved in those interactions were both β‐conglycinin and glycinin of soybean and the glutelins of the rice flour, although albumins and globulins were also cross‐linked. The addition of soybean proteins to rice flour improves the amino acid balance and they also might play an important role on the rice dough properties because soybean proteins interact with rice proteins, yielding protein aggregates of high molecular weight.     

Characterization of globulins from common vetch (Vicia sativa L.)

The proteins from Vicia sativa L. (common vetch) seeds were investigated. Protein comprises approximately 11.4% of the seed fresh weight, >50.8% of which is composed by globulins and 43.6% by albumins. The globulins may be fractionated into two main components, which were named alpha-vicinin (comprising 73% of the total globulin fraction, and hence >37% of the total seed protein) and beta-vicinin. Two minor globulin components are also present, gamma-vicinin and delta-vicinin. alpha-Vicinin, the legumin-like globulin, with a sedimentation coefficient of 10.6 S, is a nonglycosylated, disulfide-bond-containing globulin, composed of a group of subunits with molecular masses ranging from 50 to 78 kDa. Upon reduction, each of these subunits releases a heavy polypeptide chain (34-66 kDa) and a light polypeptide chain (21-23 kDa). beta-Vicinin, the vicilin-like globulin, with a sedimentation coefficient of 7.7 S, is a nonglycosylated globulin that contains no disulfide bonds and consists of two major polypeptides with molecular masses of 58 and 66 kDa. gamma-Vicinin is a minor, glycosylated, disulfide-bond-containing globulin. In the reduced form, it comprises six polypeptide chains with molecular masses of 12, 19, 21, 22, 23, and 31 kDa. Finally, delta-vicinin is a minor, highly glycosylated globulin that exhibits hemagglutinating activity. It is composed of a major 47 kDa polypeptide and two minor (33 and 38 kDa) polypeptides. N-terminal sequencing of the delta-vicinin 47 kDa polypeptide revealed no homology to any other known storage protein.     

Characterization of the proteins from Vigna unguiculata seeds

The proteins from Vigna unguiculata (L.) Walp. (cowpea) seeds were investigated. Globulins constitute over 51% of the total seed protein, with albumins composing approximately 45%. The globulins may be fractionated by native electrophoresis or anion exchange chromatography into three main components, which were termed (in decreasing order of anodic mobility) alpha-vignin, beta-vignin, and gamma-vignin. alpha-Vignin, with a sedimentation coefficient of 16.5S, is a major, nonglycosylated globulin, composed of a major 80 kDa subunit, which upon reduction, produces two polypeptides (20 and 60 kDa). beta-Vignin, with a sedimentation coefficient of 13S, is a major, glycosylated globulin, composed of two main polypeptides (55 and 60 kDa) with no disulfide bonds. Finally, gamma-vignin, a minor globulin, is composed by one main type of subunit (22 kDa), which upon reduction, is converted into a single, apparently heavier polypeptide chain (30 kDa) due to the presence of an internal disulfide bond. Immunological analyses revealed structural homology between beta-vignin and beta-conglutin (the vicilin from Lupinus seeds) but not between alpha- or gamma-vignins and their Lupinus counterparts. Haemagglutination activity toward trypsinized rabbit erythrocytes was found exclusively in the albumin fraction and was strongly inhibited by N-acetylglucosamine or chitin.     

2S albumin from buckwheat (Fagopyrum esculentum moench) seeds

Sucrose density gradient centrifugation showed that approximately 30% of total buckwheat proteins migrated with a 2S sedimentation coefficient. The main part of that fraction, polypeptides in the range of molecular mass from 8 to 16 kDa, were water soluble and represented albumins. SDS-PAGE analysis in nonreducing and reducing conditions showed that these polypeptides were not linked by disulfide bonds. The albumins make 25% of total salt soluble proteins, but that content is dramatically reduced under S-deficiency conditions. Determination of amino acid composition showed high methionine (9.2%) and lysine (5.6%) contents. That characteristic offers the possibility of transfer of the genes for individual albumin polypeptides to legumes and cereals limited in those essential amino acids to improve their nutritional quality.     

Influence of pH on Structure and Function of Amaranth (Amaranthus hypochondriacus) Protein Isolates

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.     

油菜花粉谷蛋白酶解物的制备

以陈放1年的油菜蜂花粉为原料,对破壁脱脂后的花粉中各组分蛋白含量进行了研究,其中谷蛋白的含量约占总蛋白含量的55.7%,清蛋白为39.O%,球蛋白和醇蛋白只有3.2%和2.1%.以谷蛋白为原料,在单因素试验的基础上,利用响应面分析法对油菜花粉谷蛋白最佳酶解条件进行了研究.结果表明,Alcalase碱性蛋白酶水解油菜花粉谷蛋白的最佳水解条件为底物浓度6%,pH值9.0,水解温度60℃,酶底比1460U/g,水解时间2h,谷蛋白酶法改性后抗羟基自由基的活性能达到70.0%.     

Bioactive peptides in amaranth (Amaranthus hypochondriacus) seed

Amaranth seeds are rich in protein with a high nutritional value, but little is known about their bioactive compounds that could benefit health. The objectives of this research were to investigate the presence, characterization, and the anticarcinogenic properties of the peptide lunasin in amaranth seeds. Furthermore, to predict and identify other peptides in amaranth seed with potential biological activities. ELISA showed an average concentration of 11.1 microg lunasin equivalent/g total extracted protein in four genotypes of mature amaranth seeds. Glutelin fraction had the highest lunasin concentration (3.0 microg/g). Lunasin was also identified in albumin, prolamin and globulin amaranth protein fractions and even in popped amaranth seeds. Western blot analysis revealed a band at 18.5 kDa, and MALDI-TOF analysis showed that this peptide matched more than 60% of the soybean lunasin peptide sequence. Glutelin extracts digested with trypsin, showed the induction of apoptosis against HeLa cells. Prediction of other bioactive peptides in amaranth globulins and glutelins were mainly antihypertensive. This is the first study that reports the presence of a lunasin-like peptide and other potentially bioactive peptides in amaranth protein fractions.     

Identification and variation of glutelin alpha polypeptides in the genus Oryza assessed by two-dimensional electrophoresis and step-by-step immunodetection

To obtain fundamental information for nutritional improvement of rice (Oryza sativa) seed proteins, the alpha polypeptides of the major storage protein glutelin varied over the genus Oryza were qualitatively and quantitatively characterized with unique methods. The polypeptides were maximally separated by two-dimensional electrophoresis (2D-PAGE) composed of nonequilibrium pH gradient gel electrophoresis (NEPHGE) and higher temperature SDS-PAGE. Then the subunit for each polypeptide spot was identified with the sequential immunodetection called a step-by-step detection method, making use of highly subunit-specific antibodies. The comparative analysis showed considerable variation in the accumulation level of A-type and B-type glutelin subunits and found unknown glutelin subunits that were unable to be identified with the antibodies used. Wild species accumulating a high amount of lysine-rich B-type glutelin subunits and unknown unique subunits were identified as they might play a crucial role in nutritional quality improvement of the cultivated rice.     

Nutritional responses of rats to diets based on chickpea ( Cicer arietinum L.) seed meal or its protein fractions

The aim of this study was to isolate the protein fractions from chickpea, var. IAC-Marrocos, as well as to evaluate its in vivo nutritional protein quality. Among the proteins, albumins showed better nutritional value in the in vivo assays and amino acid contents, despite their higher trypsin inhibitor contents. Trypsin inhibitors were found to be heat labile in all samples, but the digestibility results for unheated and heated flour and albumins suggest that their contents are not very decisive. The PER values for casein (not supplemented) were very similar to those of heated flour and unheated or heated albumin and total globulins. The albumin and glutelin fractions showed the best results for PDCAAS, however, lower than those of casein. Despite the high digestibility of the globulin the very low essential amino acid content lowered its PDCAAS, and it had the lowest values.     

Physicochemical properties of soy protein: effects of subunit composition

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.     

Proteolysis in model sourdough fermentations

Model wheat doughs started with six different lactic acid bacteria (LAB), with or without a commercial baker's yeast culture, were used to study proteolysis in sourdough fermentations. Cell counts, pH, and free amino acid concentration were measured. Sequential extraction of dough samples was performed to separate wheat proteins. The salt-soluble protein fraction (albumins and globulins) was analyzed by RP-HPLC and SDS-PAGE, whereas propanol-soluble (gliadins) and insoluble (glutenins) protein fractions were analyzed by SDS-PAGE only. Multivariate statistical methods were used for the analysis of results. The presence of yeasts and LAB affected RP-HPLC and SDS-PAGE patterns of the salt-soluble fraction in a complex way. The only changes in the gluten proteins that could be related to the presence of LAB were the appearance of new protein fragments (20 and 27 kDa) from gliadins and the degradation of high molecular weight glutenin subunits.     

Proteome profiling of seed storage proteins reveals the nutritional potential of Salicornia brachiata Roxb., an extreme halophyte

Salicornia brachiata is an extreme halophyte that grows in salty marshes and is considered to be a potential alternative crop for seawater agriculture. Salicornia seeds are rich in protein, and its tender shoots are eaten as salad greens. Seed storage proteins were fractionated by sequential extraction using different solvents, including distilled water for albumins, NaCl (1.0 M) for globulins, NaOH (0.1 N) for glutelins, and ethanol (70% v/v) for prolamins. Globulins accounted for 54.75% of the total seed storage proteins followed by albumins (34.30%) and glutelins (8.70%). The fractionated proteins were characterized using 2D-diagonal SDS-PAGE and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. The globulin fraction, composed of seven intermolecular disulfide-linked polypeptide pairs of molecular mass 63.5, 62.5, 54.7, 53.0, 43.2, 38.5, and 35.1 kDa, encompassed a basic and an acidic subunit. Two-dimensional gels revealed approximately 32 spots, with isoelectric points and molecular masses ranging from 4.93 to 11.6 and from ～5.2 to ～109.4 kDa, respectively. Protein spots were identified by MALDI-TOF MS peptide mass fingerprint analysis and further classified. Homology analysis demonstrated that 19% of the proteins were involved in metabolism, 16% were involved in signaling, and 15% were regulatory proteins. Peptide mass fingerprint analysis confirmed the presence of inter- and intramolecular disulfide linkages in the globulin fraction. Sulfur-rich proteins are of high nutritional value, and disulfides make S. brachiata a potential source of dietary supplementation.