Structural studies on native and chemically modified storage proteins from rapeseed (Brassica napus L.) and related plant proteins

Recent data on the structure and chemical modification of the two main storage proteins of rapeseed, the high-molecular mass 12 S globulin and the low-molecular mass 2 S protein (napin) are summarized and compared with those of related seed proteins. The 12 S globulin is built up of six subunits forming a quaternary structure which can be approximated by the model of a trigonal antiprism. The subunits, composed of a larger and a smaller polypeptide chain each, have a two-domain structure which is typical for all related plant proteins. These are characterized by a sedimentation coefficient of 11-13 S, a molecular mass of 300,000-360,000 g/mol and a high percentage of beta-sheet conformation. Increasing succinylation results in a step-by-step dissociation up to the subunits and to an unfolding of the latter at a critical level of modification amounting to 60-70%. These structural changes affect the functional properties remarkably. The napin fraction comprises a group of closely related and highly basic proteins with molecular masses of 12,000-14,000 g/mol, a high content of sulphur-containing amino acids and rich in helical conformation. They are built up of a larger and a smaller disulphide bridged polypeptide chain. Acylation does not abolish the secondary or tertiary structure which are stabilized by inter- and intrachain disulphide bonds. Acylation results, however, in a stabilization of the protein against heat-induced aggregation.     

Purification of trypsin/chymotrypsin inhibitor from jack fruit seeds

A trypsin/chymotrypsin inhibitor (JSTI) was isolated from jack fruit seeds (Artocarpus integrifolia Hook f) by ammonium sulphate fractionation and chromatography on DEAE–cellulose and Sephadex G-100. During all stages of purification, the ratio of trypsin and chymotrypsin inhibitory activities remained constant. The purified preparation was found to be homogeneous by gel filtration, polyacrylamide gel electrophoresis (PAGE) and ultra-centrifugation. From the sedimentation coefficient, S _20w value of 3·5 ± 0·15 S. the molecular weight of JSTI was calculated to be 30·00 ± 2·50 kamu. The inhibitor showed a molecular weight of 24·55 kamu on a Sephadex G-75 column when eluted with 6 M guanidine hydrochloride, Under non-denaturing conditions, JSTI exhibited anomalous behaviour on a Sephadex G-200 column. On SDS–PAGE, the inhibitor showed two major bands with molecular weights of 26·30 and 15·00 kamu and two minor bands with molecular weights of 19·50 and 12·00 kamu. The carboxyamidomethylated JSTI showed three trypsin inhibitory activity bands on PAGE, suggesting the presence of isoinhibitors.     

Molecular Weight of αs3- and αs4-Caseins by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

Molecular weights of both bovine α_S3- and α_s4-casein were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Analysis by least squares of natural logarithm of molecular weight versus distance of migration gave a molecular weight of 27,800 ± 900 daltons for each protein. The value agrees closely with the molecular weight (26,000 daltons) for bovine α_S3-casein reported by Richardson and Creamer and is less than 31,800 daltons for reduced α_S5-casein reported by Toma and Nakai.     

Primary structure of 2S albumin from seeds of Lupinus albus

 The 2S albumin from the seeds of white lupin (Lupinus albus L.) was purified by ammonium sulfate precipitation and ultrafiltration followed by anion-exchange and reversed-phase HPLC. The complete amino acid sequences of the large and the dominating small 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 (a) consists of 37 amino acid residues resulting in a molecular mass M _r of 4407. The large subunit (b) contains 75 amino acid residues (M _r=8827). The two polypeptide chains are linked by two interchain disulfide bonds (Cys^a8-Cys^b24 and Cys^a20-Cys^b13 or, more probably, Cys^a20-Cys^b12). In addition, the large polypeptide contains two intrachain disulfide bridges (Cys^b26-Cys^b68 and Cys^b12-Cys^b60 or, more probably, Cys^b13-Cys^b60) and one free sulfhydryl group (Cys^b40). A high degree of homology (88%) exists between the primary structure of the 2S albumin from L. albus and that of a comparable 2S protein from L. angustifolius, designated conglutin δ. Received: 4 April 1996     

Primary structure of 2S albumin from seeds of Lupinus albus

 The 2S albumin from the seeds of white lupin (Lupinus albus L.) was purified by ammonium sulfate precipitation and ultrafiltration followed by anion-exchange and reversed-phase HPLC. The complete amino acid sequences of the large and the dominating small 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 (a) consists of 37 amino acid residues resulting in a molecular mass M _r of 4407. The large subunit (b) contains 75 amino acid residues (M _r=8827). The two polypeptide chains are linked by two interchain disulfide bonds (Cys^a8-Cys^b24 and Cys^a20-Cys^b13 or, more probably, Cys^a20-Cys^b12). In addition, the large polypeptide contains two intrachain disulfide bridges (Cys^b26-Cys^b68 and Cys^b12-Cys^b60 or, more probably, Cys^b13-Cys^b60) and one free sulfhydryl group (Cys^b40). A high degree of homology (88%) exists between the primary structure of the 2S albumin from L. albus and that of a comparable 2S protein from L. angustifolius, designated conglutin δ. Received: 4 April 1996     

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     

沙蒿籽胶多糖的分级纯化和结构分析

采用0.1mol/L的氢氧化钾对沙蒿籽胶多糖进行组分分级得到水溶多糖(AG1)和水不溶多糖(AG2)。利用离子交换柱层析从AG1中分离得到酸性多糖(AG11)和中性多糖(AG12)。经凝胶过滤色谱检测,AG11和AG12为均一组分。化学组成分析表明,AG1为葡萄糖糖基组成的葡聚糖,并含有糖醛酸;AG11和AG12分别为阿拉伯糖、鼠李糖、木糖、甘露糖、葡萄糖和半乳糖等糖基组成的杂多糖,并含有糖醛酸,相对分子质量分别为8.63×105Da和3.46×105Da。红外光谱测定表明,AG11、AG12和AG2具有多糖的特征吸收,并且其糖环均为吡喃环。     

Study on Antioxidant Activity and Amino Acid Analysis of Rapeseed Protein Hydrolysates

Alkaline extraction followed by acid precipitation were employed to extract rapeseed protein and, Alcalase 2.4 L was used to obtain rapeseed protein hydrolysates. Three groups of rapeseed protein hydrolysates were obtained by purifying with membrane ultrafiltration and a Sephacryl S-100HR gel column. The antioxidant activities were then determined. Group 3 had the best antioxidant activities according to the oxygen radical absorbance capacity, peroxyl radical-scavenging capacity, and cellular antioxidant activity assays, with the following antioxidant values: an oxygen radical absorbance capacity value of 1610 ± 113 µmol TE/(g sample), a peroxyl radical-scavenging capacity value of 622 ± 30 mg VC/(100 g sample), and a cellular antioxidant activity value of 25 ± 2 µmol QE/(g sample) and corresponding EC₅₀ value of 58 ± 3 µg/mL. Six peaks of group 3 were collected and well separated by reversed phase–high-performance liquid chromatography. Peak 5 were identified to exhibit a higher antioxidant activity, the amino acid sequence of which was found to be Trp-Ile (Leu)-Tyr, as determined by liquid chromatography–mass spectrometry.     

Immunocytochemical and biochemical studies of the mobilisation of storage oil-bodies and proteins in germinating cotyledons of oilseed rape,Brassica napus

Seeds of Brassica napus L cv Mikado contain about 25% w/w protein in addition to 40–50% w/w storage oil. About 50% of the seed protein is the legumin-like neutral protein, cruciferin, and a further 20% is the small basic protein, napin. The only other major seed protein (20% of total) is a polypeptide of apparent molecular mass (M_r) 19 000±200, which is associated with the membranes of the storage oil-bodies. The purification of this protein and preparation of monospecific antibodies have recently been reported. The kinetics of protein and oil mobilisation and the subcellular distribution of the M_r 19 000 oil-body protein have been studied by techniques including sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), gas-liquid chromatography (GLC), sucrose density gradient fractionation, and electron microscop-immunocytochemistry. The results show that the mobilisation of the storage products of rapeseed occurs in at least three distinct phases: (1) a lag phase of 10–15 h, (2) breakdown of cruciferin and napin from 12 h until day 3, (3) breakdown of storage oil and oil-body membranes from day 2 until day 7. The M_r 19000 protein was localised on oil-body membranes in early stages of germination but was later associated with a light membrane fraction, which probably contained oil-body ghosts. Relatively little difference in the kinetics of the mobilisation of storage oils and proteins was found whether seedlings were grown in the light or in the dark. The implications of these results for the mechanism of storage oil mobilisation in oilseeds are discussed.     

Further studies on SS bonds in cereal glutelins

Flours of wheat, rye, barley, oats, maize and sorghum have been extracted to remove albumins, globulins and prolamins. The SS bonds of the starch-glutelin residues (4 to 8% protein) have been reduced with sulphite and titrated with phenyl mercury acetate (PMA) in the presence of 3 M -guanidine hydrochloride at 37 °C and, in its absence, at 2 and 37 °C. Attention has been paid to several possible sources of error including oxidation and the rate of diffusion of PMA into protein particles. Approximate values for the diffusion coefficients of PMA through cellulose and protein films were obtained. Titrations at 37 °C in the absence of guanidine hydrochloride are unsatisfactory due to reaction of intrachain SS links. Evidence in the literature suggests that the SS bonds titratable at 2 °C are inter-chain, possibly includingsomestrained intra-chain bonds. The results imply that most of the major polypeptide chains in the cereal glutelins examined, apart from barley and sorghum, contain two such bonds. In the case of barley glutelin probably less than half the chains have two labile SS bonds. Most of the chains in sorghum glutelin appear to have a single labile bond and the polymers may contain only a few chains. The molecular weights (in thousands) of the principal polypeptide chains of the glutelins, deduced from gel electrophoresis in sodium dodecyl sulphate (SDS), are: Cappelle-Desprez wheat 44, 41; Manitoba wheat, rye and barley, 44; oats, 33, 23; maize, 23, 19; sorghum, 22, 18.     

Physicochemical and Functional Properties of Tea Protein

Tea protein has received much attention due to its potential health functions. This article studied the molecular weight, foaming capacity, foam stability, emulsifying capacity, emulsion stability, and oil absorption of tea protein prepared from tea residue by alkali extraction. The results showed that tea protein contained two components with different molecular weights (1.26 × 10⁶ ± 1.3 × 10⁴ and 2.4 × 10⁴ ± 1.1 × 10³). The content of the lower molecular component in tea protein was higher than the higher molecular one. When the concentration of tea protein was 4 mg/mL, its sparkling ability was satisfied, while the foaming capacity and foam stability was the best, respectively, at the conditions of pH 9 and 7. The emulsifying capacity was 62 ± 2% as well as 99 ± 1% of emulsion stability, and oil absorption was 170 ± 7%.     

The subunit structure and stability of conglutin δ, a sulphurrich protein from the seeds of Lupinus angustifolius L.

The oligomeric structure and stability of conglutin δ, the 2S sulphur-rich lupin seed protein, has been studied. Molecular weights were determined by sedimentation equilibrium methods in the analytical ultracentrifuge. Conglutin δ₂ (M_r 14 000, 2S), the most abundant form, was composed of one large (?9600) and one small polypeptide chain linked by disulphide bonds. The minor oligomeric form, conglutin δ₁ (M_r 28 000, 2.8S), was a disulphide-linked dimer of conglutin δ₂. Each form was capable of reversible association and at low ionic strength (neutral pH), the conglutin δ₁ momomer associated to a homogeneous dimer (M_r 56 000, 4.1S). Calculated frictional ratios (f/f₀ ? 1.28–1.49) suggested that the three different forms were asymmetric. Spectral studies (ORD/CD) showed that conglutins δ₁ and δ₂ were rich in alpha-helix (?38%) unlike the major 7S and 11S legume seed storage proteins. The helical structure was unusually stable both to heat and chemical denaturants; below 60°C (at neutral pH) the helix remained unaffected and only partial denaturation occurred at higher temperatures. Nevertheless, complete denaturation was achieved (at 20°C) in high concentrations of guanidine hydrochloride (greater than 6.5 M). The stability was due to the presence of disulphide cross-links; with the addition of reducing agent, as little as 1 M guanidine hydrochloride (GuHCl) was sufficient for denaturation of the helical structure. Titration experiments showed a single buried tyrosine (pK 11.4) which could be exposed (pK 10.2) in 6 M GuHCl.     

On seed proteins Part 11. Purification,Chemical Composition,and Some Physico-chemical Properties of the 11 S Globulin (Helianthinin) in Sunflower Seed

An improved procedure for the isolation and purification of the 11 S globulin from sunflower seeds (helianthinin) is described, including a combined purification by gel chromatography and ionexchange chromatography. The protein has a sedimentation constant of s_{20, w} = 12.8 x 10^?13 s, a Stokes radius of 57 Å and a diffusion constant of 3.76 x 10^?7 cm² s^?1 (the last two derived from gel chromatographic analysis). Hence it follows a molecular weight of M_{s, D} = 305000. The isoelectric point determined by isoelectric focusing lies at pH 4.7. High contents of glutamic (26%) and aspartic (14%) acid and arginine (9.7%) as well as a low content of sulphur containing amino acids are characteristic for the amino acid composition. 59% of the acidic amino acids are present in an amidated form. The globulin contains 12 disulphide bridges per molecule.     

Physicochemical, functional and angiotensin converting enzyme inhibitory properties of amaranth (Amaranthus hypochondriacus) 7S globulin

BACKGROUND: Amaranth 7S globulin is a minor globulin component and its impact on the properties of an amaranth protein ingredient depends on its proportion in the variety of amaranth being considered. Some physicochemical, functional and angiotesin I‐converting enzyme (ACE) inhibitory properties of amaranth vicilin were studied in this work and compared with the 11S globulin. RESULTS: Fluorescence spectroscopy results indicated that 7S globulin tryptophans were more exposed to the solvent and, by calorimetry, the 7S globulin denaturation temperature (T_d) was found lower than the 11S globulin T_d, suggesting a more flexible structure. The 7S globulin surface hydrophobicity was higher than that of the 11S globulin, which is in agreement with the better emulsifying properties of the 7S globulin. The solubility in neutral buffer of the 7S globulin (851 ± 25 g kg^?1) was also higher than that of the 11S globulin (195 ± 6 g kg^?1). Bioinformatic analyses showed the presence of ACE inhibitory peptides encrypted in 7S tryptic sequences and peptides released after in vitro gastrointestinal digestion showed a high ACE‐inhibitory capacity (IC₅₀ = 0.17 g L^?1), similar to that of 11S globulin peptides. CONCLUSION: Compared with the 11S globulin, the 7S globulin presents similar ACE inhibitory activity and some functional advantages, better solubility and emulsifying activity, which suits some food requirements. The functional behavior has been related with the structural properties. Copyright © 2011 Society of Chemical Industry     

Molecular structure and rheological character of high-amylose water caltrop (Trapa bispinosa Roxb.) starch

The molecular structure and rheological properties of high-amylose water caltrop (Trapa bispinosa Roxb.) starch cultivated in Vietnam were investigated. The water caltrop starch had 47.1% amylose and its molecular weight (Mw) was (4.77±0.27)×10⁶ g/mol, whereas the Mw was (2.07±0.10)×10⁷ g/mol for amylopectin. The average chain length of amylopectin was DP_n=19.0 and the proportions of A, B₁, B₂, and B₃ chains were 28.2, 50.3, 13.1, and 8.5%, respectively. The DSC thermogram of the water caltrop starch was broadly endothermic, with 2 distinct endothermic peaks at 73.6 and 80.7°C. Gel formation of water caltrop starch occurred rapidly, with an extremely high storage modulus up to approximately 1,200 Pa. High-amylose water caltrop starch paste had an extremely high final viscosity compared to that of other cereal starches. These rheological behaviors may have been due to the extremely high amylose content.     

Structural and Functional Analysis of Various Globulin Proteins from Soy Seed

Storage proteins of soybean mostly consist of globulins, which are classified according to their sedimentation coefficient. Among 4 major types: 2S, 7S, 11S, and 15S of globulins, 7S and 11S constitute major fraction. The 11S fraction consists only of glycinin and 7S fraction majorly consists of β-conglycinin, small amounts of γ-conglycinin and basic 7S globulin (Bg7S). Glycinin exist as a hexamer while β-conglycinin as a trimer and Bg7S as a tetramer. Glycinin subunits are coded by 5 genes of a family, whereas about 15 genes are present for β-conglycinin subunits. Bg7S gene is present in four copies in soybean genome. Synthesis of all proteins takes place as a single polypeptide chain, which is cleaved after folding to yield different chains or subunits. Glycinin and β-Conglycinin are made for storage purpose. However, Bg7S has potential xylanase inhibition activity and protein kinase activity. Primary structure of Bg7S reveals 12 conserved cysteine residues involved in forming 6 disulfide bonds, which provides appreciable stability to protein. Secondary structure is predominately rich in β-sheets with few alpha helices. Bg7S shares structural similarity with various aspartic-proteases. In this review, our aim is to discuss sequence, structure, and function of various globulins present in Glycine max.     

Composition of Saccharomyces fragilis biomass grown on lactose permeate

Saccharomyces fragilis (NRRL y 1109) was grown on a medium composed of Cheddar cheese whey ultrafiltration permeate, supplemented with inorganic nitrogen. The biomass had a crude protein content of 49.5% and an ash content of 7.6%. The “true protein” content, based on a N fractionation procedure was 37.2% and the total nucleic acid content was 5.7%. Qualitatively, the amino acid composition of S. fragilis was similar to that reported for other whey-yeasts. The quantity of individual essential amino acids was greater in the permeate grown S. fragilis. The sulphur containing amino acids, methionine and cyst(e)ine were present at a combined level of 3.2 g/16g N, compared with 2.2g/16 g N in a commercial whey-yeast product. The chemical score (CS) and essential amino acid index (EAAI) were 58.2 and 76.0 respectively. Rats fed on a diet containing S. fragilis biomass as the sole source of dietary protein showed a net weight increase. NPU_carcass for S. fragilis protein was 67.0±3.9 compared with a value of 75.3±3.0 for a casein control. Supplementation of S. fragilis biomass with DL-methionine(2 g/16 g N) raised the NPU_carcass value to 84.4 ± 2.8.     

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.     

The Effect of High-Pressure Carbon Dioxide on the Skeletal Muscle Myoglobin

High-pressure carbon dioxide (HPCD) is widely used in the food industry, but some scientific issue is still unclear, especially the effect on the proteins. In this work, the effect of HPCD on the color, polypeptide chains, α-helix contents, and spectroscopy of amino acid residues of myoglobin (Mb) was investigated by a comparison that the samples were treated with different pressures of 7, 21, and 35 MPa at 35 °C for 30 min. The results showed that during the HPCD treating, there is relative α-helix content increase sharply from 17.02 to 21.81 % for all the treated samples with increasing CO₂ pressures, while the fluorescence and synchronous fluorescence spectroscopy analysis showed that the intrinsic relative fluorescence intensity of amino acid residues increases. Interestingly, it is found that there is no direct effect of HPCD on polypeptide chains and interior structure of Mb.