ISOLATION AND CHARACTERIZATION OF AMARANTIN, THE 11S AMARANTH SEED GLOBULIN

The 11S globulin of seed storage protein of Amaranthus hypochondriacus, termed amarantin, was isolated. This fraction was evaluated for its purity by chromatographic techniques and ultracentrifugation. The 11S globulin was analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) without and with prior reduction of disulfide bonds, and by nondenaturing-PAGE. It exhibited an electrophoretic behavior similar to that of 11S-like proteins in other materials. Its apparent relative molecular weight was estimated to be 389 kDa by gel filtration chromatography at low ionic strength. Ultracentrifugation of the freeze-dried extract gave a sedimentation coefficient of 11S.     

Purification and characterization of a new 11S globulin-like protein from Chinese jujube (<Emphasis Type="Italic">Ziziphus jujube</Emphasis> Mill.) seeds

Around 8 million tons of jujube pomace containing seeds was produced each year in China; so how to utilize these residual matters has been receiving great attentions. The 11S globulins are the major storage proteins, and it can be used as additive agents in many food products with gelling and caking functionality. In the present study, an 11S globulin-like protein (ZSG) was isolated and purified from Chinese jujube (Ziziphus jujube Mill.) seeds by two consecutive anion exchange and size exclusion chromatography. A yield of 8 mg of the ZSG from 1 kg seeds was obtained. The apparent molecular mass of the native ZSG was found to be about 210 kDa. The protein consisted of two subunits with molecular masses of 26.0 kDa and 26.5 kDa, respectively. Amino acid composition analyses indicated that ZSG contained all eight essential amino acids, while lysine was the first limiting amino acids. N-terminal amino acid sequences of 26.0- and 26.5-kDa subunits are GVEETICTLRLLENI and GLEETVCSLRLKENI. The two peptide fragments of 26.0-kDa subunit are ANPDQVLENAFQISR and GVEETICTLR by LC–MS/MS. To the best of our knowledge, this is the first report on the purification of 11S globulin-like protein from Chinese jujube (Ziziphus jujube Mill.) seeds.     

PHYSICAL CHARACTERIZATION OF ALLIINASE, THE FLAVOR GENERATING ENZYME IN ONIONS

The characteristic flavor of onions occurs when the enzyme alliinase (EC 4.4.1.4) hydrolyses the S-alk(en)yl-L-cysteine sulfoxides to form sulfur containing volatiles, pyruvate and ammonia. In this paper we have new evidence on the molecular mass of native onion alliinase, its subunit size and its relationship to the predicted size from translation of the gene. Alliinase from bulbs was purified to homogeneity. Two isoforms of alliinase were detected on ion-exchange chromatography. Both isoforms were analyzed by gel filtration FPLC. Alliinase activity was detected at molecular masses of 59, 166 kDa and 59, 170 and 393 kDa for isoforms 1 and 2, respectively. When analyzed by SDS-PAGE subunit sizes were determined to be 53.3 kDa for isoform 1 and 53.3 and 51.6 kDa for isoform 2. Amino terminal sequencing of both subunits confirmed they were alliinase. Alliinase was deglycosylated and the subunits migrated as a single band indicating a common protein backbone and varying glycosylation. Thus native alliinase is a monomer, trimer and multimer of the subunits. Molecular analysis of alliinase cDNA indicated that two genes and thus two protein subunits were expressed in onion bulb tissue. Translation is likely to have initiated from the third methionine of the leader sequence of the cDNA to give a protein backbone of 53.5 kDa in gradient SDS-PAGE. The functional significance of heterogenous subunits, from different genes, and multimeric native alliinase remains unknown.     

Purification and characterization of a new 11S globulin-like protein from grape (Vitis vinifera L.) seeds

Around 10 million tons of grape pomace containing 38–52% (on a dry matter basis) seeds was produced each year in the world, so how utilize these residual matters has been receiving great attention. In this study, 11S globulin-like protein (GSG) was isolated and purified from grape (Vitis vinifera L.) seeds by two consecutive cation exchange and size exclusion chromatography. A yield of 10 mg the protein from 1 kg seeds was obtained. The apparent molecular mass of the native GSG was found to be about 300 kDa. The protein consisted of two subunits with molecular masses of 25.5 and 40.0 kDa, respectively. MADLI-TOF–MS result showed that they were distinct from each other. N-terminal amino acid sequence of the 40.0 kDa subunit is RQQTSRQQKE. Amino acid composition analyses indicated that GSG contained all eight essential amino acids, while lysine was the first limiting amino acids. To the best of our knowledge, this is the first report on the purification of 11S globulin-like protein from Vitis vinifera L. seeds.     

The relationship between breaking force and hydrophobic interactions or disulfide bonds involved in heat-induced soy protein gels as affected by heating time and temperature

Hydrophobic interactions and disulfide bonds involved in heat-induced soy protein gels were characterised by determining the dissolution kinetics of gels. Reducing SDS-PAGE results revealed that all proteins in gel network could be dissolved simultaneously by 1% (w/v) SDS solution, while a majority of glycinin (11S) A polypeptide and a moderate amount of 11S-B polypeptides, 7S-α′, α, γ, and β subunits were found in 2% (w/v) DTT dissolving samples. Stronger interaction force between proteins in gel network would result in lower dissolution constant rate. The breaking force of soy gels increased from 543 to 2171 g_force with increasing heating temperature from 85 to 100 °C, and denaturation of 11S globulins played an important role in the development of gel network. As increasing heating time from 30 to 120 min, the breaking force of gels increased from 1687 to 2175 g_force, then decreased to 1253 g_force when the time was prolonged to 240 min. Negative correlations were observed between breaking force and dissolution constant rate k_SDS or Δk, which suggested that the strengthening of both hydrophobic interactions and disulfide bonds.     

CHARACTERISTICS OF γ-GLUTAMYL TRANSPEPTIDASE AND ALLIINASE OF ONION AND THEIR EFFECTS ON THE ENHANCEMENT OF PYRUVATE FORMATION IN ONION MACERATES

The activity of alliinase monitored in onions stored for 32 weeks at ambient temperature (mean value of 20C) only slightly increased whereas the activity of γ-glutamyl transpeptidase increased gradually. Purified alliinase and transpeptidase showed single protein bands on SDS-PAGE, with molecular weights between protein markers of 40–69 kDa and 116–205 kDa, respectively. Molecular weights of native transpeptidase and alliinase determined by Sephadex G-200 column chromatography were estimated to be 120 and 200 kDa, respectively. Transpeptidase and alliinase had isoelectric points of 5.2 and 4.8, pH optima of 9.0 and 7.5, temperature optimum of 40C and activation energies (E_a) of 15.8 and 16.6 kJ/mol, respectively. Inhibitor studies suggested that the conditions for optimal alliinase activity did not depress the activity of transpeptidase. Transpeptidase in conjunction with alliinase enhanced pyruvate production in onion macerates over and above that catalyzed by either enzyme alone.     

Characterization,amino acid composition and in vitro digestibility of hemp (Cannabis sativa L.) proteins

The protein constituents and thermal properties of hemp (Cannabis sativa L.) protein isolate (HPI) as well as 11S- and 7S-rich HPIs (HPI-11S and HPI-7S) were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and different scanning calorimetry (DSC), and their amino acid composition and in vitro digestibility were also evaluated, as compared to soy protein isolate (SPI). SDS-PAGE analysis showed that the edestin (consisting of acidic and basic subunits, AS and BS) was the main protein component for HPI and HPI-11S, while HPI-7S was composed of the BS of edestin and a subunit of about 4.8 kDa. DSC analysis characterized thermal transition of the edestin component and the possible present form of different subunits. Except lysine and sulfur-containing amino acids, the essential amino acids of various HPIs met the suggested requirements of FAO/WHO for 2–5 year old infants. The proportion of essential amino acids to the total amino acids (E/T) for HPI (as well as HPI-11S) was significantly higher than that of SPI. In an in vitro digestion model, various protein constituents of various HPIs were much easily digested by pepsin plus trypsin, to release oligo-peptides with molecular weight less than 10.0 kDa (under reduced condition). Only after pepsin digestion, in vitro digestibility of HPIs was comparable to that of SPI, however after pepsin plus trypsin digestion, the digestibility (88–91%) was significantly higher than that (71%) of SPI (P < 0.05). These results suggest that the protein isolates from hempseed are much more nutritional in amino acid nutrition and easily digestible than SPI, and can be utilized as a good source of protein nutrition for human consumption.     

EFFECTS OF HEAT TREATMENTS ON PEANUT ARACHIN AND CONARACHIN

Arachin and conarachin were purified from phosphate buffer (0.20 and 0.05 M, pH 7.9) extracts using ammonium sulfate fractionation at 40 and 60–85% saturation, respectively. Characterization on SDS-PAGE gel revealed that arachin was composed of five major subunits whereas conarachin consisted of one major subunit. Methionine, lysine, and 1/2 cystine content of conarachin was, respectively, about three-, two- and two-fold higher than in arachin. During dry roasting of whole peanut kernels and lyophilization of peanut extracts at 150°C for 60 min, the nitrogen soluble indexes of arachin and conarachin decreased with time. Changes in conarachin were comparatively higher than those in arachin. When buffered extracts of kernels were subjected to boiling water bath cooking for 60 min, the native protein patterns of arachin and conarachin, as shown on gradient PAGE, varied significantly with heating time. Subunits of arachin detected on SDS-PAGE did not qualitatively vary.     

Electrophoretic Characteristics of Gluten Proteins as Influenced by Crop Year and Variety

The glutenin and gliadin fractions in different wheat varieties produced during two different cropping years were determined through SDS-PAGE and Acid-PAGE. It was evident from the electropherograms that greater numbers of polypeptides were present in the region falling under low molecular weight glutenin subunits. The SDS-PAGE patterns of molecular weight of glutenin subunits of different wheat varieties showed the presence of glutenin subunits in the range of 28.23 to 110.89 kDa and 28.29 to 113.51 kDa during the cropping years 2010-11 and 2011-12, respectively. The highest molecular weight glutenin subunit (110.89 kDa) was observed in wheat variety Lassani-08, during the crop year 2010-11, while during the cropping season 2011-12, the highest molecular weight glutenin subunit (113.51 kDa) was found in wheat variety AARI-10. It was also evident from the results that the maximum numbers of gliadin electropherograms were found in wheat variety Lassani-08. The results regarding gliadin bands revealed that total gliadin electropherograms ranged from 31.03 to 89.61 kDa and 32.91 to 92.22 kDa among different wheat varieties, during the crop years 2010-11 and 2011-12, respectively. The polypeptides with molecular weight between 31.03 to 54.50 kDa and 32.91 to 55.21 kDa belongs to α-, β- and γ-subunits of gliadin, during the crop years 2010-11 and 2011-12 respectively. The group of polypeptides with a molecular weight of about 54.50 to 89.61 kDa and 55.21 to 92.22 kDa was composed of subunits of ω-gliadins during the crop years 2010-11 and 2011-12, respectively.     

Heat‐induced denaturation impairs digestibility of legume (Phaseolus vulgaris L and Vicia faba L) 7S and 11S globulins in the small intestine of rat

7S globulin from common bean (Phaseolus vulgaris L) and 11S globulin from faba bean (Vicia faba L) were isolated to over 90% purity and the digestibility of the proteins, either in native or denatured (120 °C, 20 min, 1 atm) state, was tested in the small intestine of growing rats in acute (1 h) experiments. Native globulins were well digested (92 and 95% for 7S and 11S proteins, respectively). However, after thermal denaturation, protein digestibility of 7S globulin was reduced to 88%, while that of 11S globulin to only 79%. SDS‐PAGE revealed that high amounts of the intermediate proteolytic products of phaseolin (MW 22 000–27 000 Da) were present in the small intestine of rats after 1 h digestion of the denatured 7S globulin, while protein material in the high MW range (>55 000 Da) were recovered from the 11S globulin. The overall negative charge of unavailable proteins from the 7S globulin was found by anion exchange–FPLC separation to be higher than that of products from the 11S globulin. MALDI‐MS analysis of proteins in the small intestine confirmed the presence of half‐size phaseolin subunits (MW 23 700 Da) as breakdown products from the denatured 7S globulin, and of highly hydrophobic basic subunits (MW 20 000 Da) from the 11S globulin. Copyright © 2004 Society of Chemical Industry     

Proteomic profiling of the coagulation of soymilk proteins induced by magnesium chloride

The coagulation of soymilk proteins induced by magnesium chloride at 30 °C was investigated using the SDS-PAGE and two-dimensional electrophoresis. Approximately 88.4% of the soymilk proteins were coagulated into the soymilk pellet fraction (SPF) by 5 mM magnesium chloride, and the total protein in the soymilk supernatant fraction (SSF) decreased from 7.80 ± 0.12 mg mL⁻¹ to 0.90 ± 0.18 mg mL⁻¹. SDS-PAGE showed that the total intensities of the protein bands corresponding to the 7S α′ subunit, the 7S α subunit, the 7S β subunit and the 11S A3 subunit were decreased to 10.5 ± 0.3%, 11.8 ± 1.1%, 6.6 ± 0.2% and 15.1 ± 2.2%, respectively. Two-dimensional electrophoresis indicated that most of the soymilk proteins in the SSF, including the 7S subunits, 11S subunits, β-amylase, sucrose binding protein 2, Bd 30K, lectin and trypsin inhibitor A, were coagulated into the SPF by 5 mM magnesium chloride.     

Isolation of conglutin δ, a sulphur-rich protein from the seeds of Lupinus angustifolius L.

Although a 2S globulin class has been observed in salt extracts from seeds of several lupin species, there have been conflicting reports regarding the importance of this class in Lupinus angustifolius. Conglutin δ, a major sulphur-rich protein extracted from mature seeds of L. angustifolius cv. Uniwhite, was separated by gel-filtration into two oligomeric forms. The sedimentation coefficients of conglutin δ₁ (20%) and conglutin δ₂ (80%), were 3.2S and 2.0S respectively. The amino acid compositions of both oligomeric forms of conglutin δ were identical and similar to the compositions published for the 2S globulins in other lupin species. Because of the low level of tyrosine and the absence of tryptophan, conglutins δ₁ and δ₂ showed little absorbance at 280nm (E^1%/1 cm^?23). They were characterised by unusually high levels of glutamic acid and 1/2 cysteine (38.5 and 8.5 residues percent respectively) while methionine was absent. Gradient SDS-PAGE showed that conglutins δ₁ and δ₂ were homogeneous single-subunit species. On reduction, with or without S-carboxymethylation, both the conglutin δ₁ and δ₂ subunits yielded similar pairs of large and small polypeptide chains. Since conglutin δ rarely resolves from conglutin a during electrophoresis on cellulose acetate membranes in phosphate buffer at neutral pH, this method is not as useful for screening lupin cultivars as has been claimed previously.     

Purification and characterisation of basic ascorbate oxidase from satsuma mandarin (Citrus unshiu Marc)

Basic ascorbate oxidase of the multiple enzyme forms existing in young fruit of satsuma mandarin (Citrus unshiu Marc) has been separated and subsequently purified to electrophoretic homogeneity through (NH₄)₂SO₄ fractionation and chromatographies on DEAE-Toyopearl 650M, CM-Sephadex C-50 and Sephadex G-100. The native molecular weight was estimated to be 141 kDa by gel filtration and composed of two non-identical subunits with an apparent mass of 74 kDa and 62 kDa. The optimum pH was found to be 5.5 with reasonable stability between pH 5 and 8. The enzyme had an optimum temperature at 45°C and was stable up to 50°C upon heat treatment for 5 min. The presence of sodium diethyldithiocarbamate, metabisulphite and potassium cyanide completely inhibited the enzyme activity. Fluoride also inhibited the activity substantially at higher concentrations. Other tnonovalent and divalent metal ions did not have inhibitory effects.     

Foaming Characteristics of Commercial Soy Protein Isolate as Influenced by Heat-Induced Aggregation

The foaming properties of commercial soy protein isolate subjected to different temperatures (20–90°C) were assessed. The results revealed that the solubility and surface hydrophobicity of a 5% (w/v) commercial soy protein isolate suspension increased with increasing temperature, which increased foaming capacity and reduced foaming stability. Commercial soy protein isolate supernatant (i.e., soluble fraction) had higher foaming capacity at low temperatures (20–50°C). A high content of commercial soy protein isolate soluble fraction increased foaming capacity but decreased foaming stability. The SDS-PAGE patterns and molecular weight distribution of commercial soy protein isolate revealed that there were soluble, large molecular weight aggregates (>400 kDa) formed mainly from A and B-11S polypeptides of commercial soy protein isolate via disulfide bonds. Additionally, some aggregates also dissociated into small polypeptides and subunits after heat treatment. Commercial soy protein isolate precipitate (i.e., insoluble fraction) had a high content of proline and cysteine, which probably contributed to the foaming stability of commercial soy protein isolate.     

Primary structure of 2S albumin from seeds of Lupinus cosentinii

 The 2S albumin from seeds of Lupinus cosentinii Guss. was purified, and the complete amino acid sequences of the dominating small and large subunit were determined by automated Edman degradation of the reduced and S-pyridylethylated polypeptides and of their enzymatic fragments. The small subunit of the 2S albumin consists of 35 amino acid residues resulting in a molecular mass (M _r) of 4233. The large subunit contains 73 amino acid residues (M _r = 8627). The two polypeptide chains are linked by two interchain disulphide bonds. In addition, the large polypeptide contains two intrachain disulphide bridges and one free sulphydryl group. A high degree of homology (88–89%) exists between the primary structure of the 2S albumin from L. cosentinii and those from other Lupinus species. The positions of the cysteines and of some other amino acids are conserved not only in most of the Dicotyledoneae 2S albumins sequenced so far but also in other storage proteins. Received: 26 May 1997     

Primary structure of 2S albumin from seeds of Lupinus cosentinii

 The 2S albumin from seeds of Lupinus cosentinii Guss. was purified, and the complete amino acid sequences of the dominating small and large subunit were determined by automated Edman degradation of the reduced and S-pyridylethylated polypeptides and of their enzymatic fragments. The small subunit of the 2S albumin consists of 35 amino acid residues resulting in a molecular mass (M _r) of 4233. The large subunit contains 73 amino acid residues (M _r = 8627). The two polypeptide chains are linked by two interchain disulphide bonds. In addition, the large polypeptide contains two intrachain disulphide bridges and one free sulphydryl group. A high degree of homology (88–89%) exists between the primary structure of the 2S albumin from L. cosentinii and those from other Lupinus species. The positions of the cysteines and of some other amino acids are conserved not only in most of the Dicotyledoneae 2S albumins sequenced so far but also in other storage proteins. Received: 26 May 1997     

Relationships between protein and other chemical composition and texture of tofu made from soybeans grown in different locations

Understanding quantitative relationships between protein and other chemical components in diverse soybean genotypes (lines) grown in different locations and the firmness of tofu can provide scientific insight for selecting soybean suitable for tofu making. Locations showed significant effects on seed components, including total protein, major storage proteins, subunits and polypeptides of the major storage proteins, and calcium, but not magnesium or phytic acid. Results showed that 11S content, but not 11S/7S ratio, was only correlated with filled tofu firmness when analyzed over all locations. A strong and positive correlation between firmness and A₃ polypeptide of the 11S protein content was found for both pressed tofu (r = 0.80, p < 0.001) and filled tofu (r = 0.76, p < 0.001) over three locations (overall pooled data) and within most individual locations. The correlation of filled tofu firmness and A₃ polypeptide was significant for each of the three individual locations. However, the correlation of pressed tofu firmness and A₃ polypeptide content was significant at two of three locations. Mean calcium content was positively correlated with mean pressed and filled tofu firmness over all locations, but calcium was not correlated with pressed tofu firmness at any individual location, and only one location showed a significant correlation of calcium and filled tofu firmness. In addition, pressed tofu firmness was found to be negatively correlated with tofu yield. The findings that A₃ polypeptide's strong relationship with tofu firmness within certain locations may be used by the food industry to select proper soybean for manufacturing tofu and to facilitate tofu soybean breeding for tofu making.     

Proteolytic processing of the peanut allergen Ara h 3

The allergen Ara h 3 has been purified recently from peanuts. In contrast to recombinant Ara h 3, a 60 kDa single-chain polypeptide, the allergen isolated from its native source is extensively proteolytically processed. The characteristic proteolytic processing for 11S plant storage proteins of the glycinin family is observed for Ara h 3 yielding an acidic and a basic subunit, bound by a disulfide bridge. In addition to this, proteolytic truncation is observed for the acidic subunit but not for the basic subunit of Ara h 3. A series of Ara h 3 polypeptides ranging from 13-45 kDa was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and each band was digested by trypsin. Peptides related to the bands were identified and a scheme positioning the different polypeptides in the Ara h 3 sequence has been constructed. Peptide analysis showed sequence heterogeneity at two positions indicating the presence of multiple genes encoding variant, but highly homologous Ara h 3 proteins. The pool of Ara h 3 polypeptides from its native source illustrated that allergen from the peanut is much more complex than the recombinant protein used for epitope mapping experiments. From several Ara h 3 truncation products one or more immunoglobulin E (IgE) binding sites had been removed. Characterization of the allergenicity of Ara h 3 should therefore also include IgE-binding studies with peanut-derived Ara h 3, providing the high degree of variation in the Ara h 3 protein structure, as this is what peanut-allergic individuals are confronted with.     

Proteomic characterisation of subclover seed storage proteins during germination

BACKGROUND: Early seedling development is a critical step in the establishment of subclover (Trifolium subterraneum), an economically important and widespread pasture legume. In this study the seed storage proteome of this non‐model species was characterised in mature dry seeds and during imbibition by using two‐dimensional electrophoresis coupled with tandem mass spectrometry. RESULTS: The phenol‐extracted proteome of subclover dry seeds consisted of 97 polypeptide spots displayed within a window of pI 3–10 and molecular mass 10–150 kDa. De novo sequencing coupled with MS BLAST search enabled the confident identification of 61 proteins, which corresponded to 59 7S vicilin‐ and two 11S legumin‐type globulins. The experimental mobility of vicilin isoforms along with peptide mapping indicated that low‐molecular‐mass polypeptides might account for the post‐translational proteolysis of small vicilin subunits according to the model described for those of pea. Analysis of quantitative changes in the seed storage proteome upon imbibition showed that vicilin catabolism according to a site‐specific process was favoured during early seedling growth in T. subterraneum. CONCLUSION: The establishment of a seed proteome map for T. subterraneum pointed to vicilins as dominant proteins in mature seeds whose catabolism features during early seedling growth may be of relevance under environmental conditions. Copyright © 2009 Society of Chemical Industry     

The structure of the 12 S globulin from rapeseed (Brassica napus L.)

The 12 S globulin of rapeseed represents an oligomeric protein with a molecular weight of 300,000. It is composed of 6 subunits, which are arranged in a trigonal antiprism with the point group symmetry 32 (D₃). Each subunit contains smaller units (polypeptide chains) with molecular weights in the range of 18,500 to 31,000. The protein contains the following 4 polypeptide chains differing by their molecular weights in the SDS-electrophoresis; 18,500 ± 800, 21,100 ± 500, 26,800 ± 900, 31,200 ± 1,600. According to its quaternary structure the protein dissociates under milieu conditions. The secondary structure of the protein is characterized by a low (11 %) content of α-helix and a relatively high (31%) content of β-conformation. Owing to its structure and physico-chemical properties the rapeseed protein is closely related to other 11/12 S globulins in the seeds of different plant species and botanical families.