Chemical and gel electrophoretic characterization of acetylated faba bean protein isolates

The extent of modification of amino and hydroxyl groups in acetylated faba bean protein isolates was determined. Gel electrophoretic studies of unmodified and acetylated faba bean legumin and protein isolates were carried out in acidic and alkaline buffer systems as well as in a SDS-containing system. A remarkable increase of O-acetylation was found after the N-acetylation has reached a degree of about 60%. Structure changes of the proteins were indicated from the gel electrophoresis patterns in the same region of modification. The quantity of acetyl residues fixed on hydroxyl groups in exhaustively modified protein isolates corresponds to 40 % of the total amount of acetyl groups introduced into the proteins. The SDS electrophoresis of the acetylated legumin and protein isolates revealed a remarkable increase of the molecular mass of the acidic α-polypeptide chains only, which is an experimental proof of a preferential acetylation of the latter ones. This gives confirmation of the structural model in which the α-chains are proposed to be situated on the surface of the protein.     

Reflections about the functional potential of legume proteins A Review

The term “functional potential” was introduced to better approach to the understanding of the relationships between the structure and the functional properties of food proteins. Although in practice the complex of structural features of a protein contributes to its functionality, it is very useful to regard the functional potential of the protein surface on the one side and to look on special effects of protein conformation (role of compact or unfolded state) on the functionality on the other side. This point of view may help to design the best strategy of modification of the protein structure and functionality. The special situation of the oligomeric legume storage protein, i. e. legumin‐like 11 S globulins and vicilin‐like 7 S globulin, and the structural and functional modification of 11 S globulin by limited tryptic hydrolysis and by acylation are discussed in this paper.     

Expression of legumin and vicilin genes in pea mutants and the production of legumin in transgenic plants

Pea seeds contain two major storage proteins, legumin and vicilin, in proportions that are genetically and environmentally determined. They are synthesized from at least 40 genes and at least 10 different genetic loci. Mutant alleles at loci involved in starch synthesis, which result in perturbations in starch accumulation, also affect the expression of legumin genes, thereby influencing the legumin : vicilin ratio within the total seed protein. Examples of such alleles includer(starch‐branching enzyme) and rb(ADP‐glucose pyrophosphorylase( both of which result in a reduction in legumin synthesis; double mutants (rrb) show a particularly severe reduction in the amount of legumin. The effects of such mutations are specific to legumins. The amounts of vicilin are unaffected by mutations at rorrb. One of the consequences of the production of legumin from many genes is structural heterogeneity that is believed to preclude the purification of homogeneous legumin for crystallization and 3D‐structure determination. Expression of cloned legumin cDNA in E. coli can result in sequence homogeneity, butE. coli is unable to carry out the normal proteolytic processing of legumin precursors and consequently such material is different from that produced in pea seeds. This paper describes the high‐level synthesis, processing and assembly of pea legumin in transgenic wheat seeds, leading to the spontaneous in vitroformation of paracrystalline arrays of legumin, which may be attributed to the fact that the legumin consists of a single type of subunit. Such material might be used as a source of single‐sequence, processed and assembled pea legumin for structural investigation.     

Effect of high moisture extrusion cooking on protein–protein interactions of pea (Pisum sativum L.) protein isolates

This study focuses on protein alterations in fibrous meat substitutes produced by high moisture extrusion cooking of pea protein isolates. Three commercially available pea protein isolates and their respective extrudates were evaluated regarding their amino acid composition, molecular weight distribution and protein–protein interactions. Extrusion had no effect on the degree of hydrolysis and amino acid composition indicating that the thermal and mechanical energy during extrusion did not cause the formation of peptide bonds or the degradation of amino acids due to Maillard reactions. Decrease of protein solubilised from extrudates in a buffered solution containing urea indicated that the structural integrity of extrudates could be attributed mainly to covalent disulphide bonding and, to a smaller extent, to non‐covalent interactions. Additionally, the disappearance of legumin bands in extrudates as determined by electrophoresis could be explained by its participation in a macromolecular network that was aggregated and cross‐linked via disulphide bonds. This study contributes to a better understanding of the way the proteins interact during extrusion of pea protein isolates.     

Restricted proteolysis of legumin of broad beans: Effect on thermodynamic properties of aqueous solutions and interaction with ficoll

Thermodynamic properties of aqueous solutions of both native and modified legumin of broad beans (Vicia faba L.) have been examined. A restricted trypsin-induced proteolysis was used to modify protein structure. Evaluation of protein affinity to ficoll in aqueous solutions showed that modified protein possessed higher hydrophilicity. Thermodynamic properties of diluted solutions were used to predict the phase behaviour in concentrated systems containing protein and ficoll. At specific concentrations of native legumin, the system can separate in two phases, whereas in the case of modified protein the single-phase behaviour of the system was predicted for any concentrations of both components. The experimental data obtained in concentrated systems confirmed predictions of thermodynamic analysis of diluted solutions.     

Isolation and Characterization of Chickpea (Cicer arietinum L.) Seed Protein Fractions

Proteins of ground chickpea seeds were extracted with sodium hydroxide (NaOH) solution and precipitated with addition of acid (isoelectric precipitate (C-IP)) and by cryoprecipitation (cryoprecipitate (C-CP)). The protein isolates were characterized by Native PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high performance liquid chromatography (RP-HPLC) and electrospray-ionization mass spectrometry (ESI/MS). Both the isoelectric precipitate and cryoprecipitate contained the globulin protein 11S legumins and 7S vicilins as the major protein fractions and 2S albumin proteins as a minor protein fraction. The major subunits of RP-HPLC protein fractions from both cryoprecipitate and isoelectric precipitate were found to contain subunits of both legumins and vicilins. SDS-PAGE identified legumin α-subunits with MW 40.6 and 39.5 kDa and legumin β- subunits with MW 23.5 and 22.5 kDa, and vicilin subunits with MW 70.2, 50.7, 35.0, 33.6, 18.9 and 15.5 kDa. ESI-MS molecular weights 35,366, 35,268 and 14,648 Da are likely vicilin subunits while the 24,894 Da is a legumin β-subunit.     

Seed globulins from legumes. III. Seed globulins from broad bean (Vicia faba L.)]

The seed globulins from Vicia faba predominantly consist of two components, vicilin and legumin, which are exclusively deposited within the protein bodies of the storage cotyledons. The globulin biosynthesis commences with high intensity within a distinct phase during seed development as a consequence of reactivated genetic information. Isolation and purification of vicilin and legumin were achieved by a combination of zone precipitation, ion exchange chromatography, and gel filtration. Purity was controlled by disc electrophoresis on polyacrylamide e gels at pH 4.3 and by two dimensional immunoelectrophoresis, respectively. Vicilin prepared by zone precipitation and ion exchange chromatography consists of several serologically different proteins. One of them occupies the legumin position on polyacrylamide gels, although not identic with legumin, as revealed by tandem immunoelectrophoresis. The serological nonrelationship of vicilin and legumin was confirmed. Vicilin is characterized by micro heterogeneity which seems to indicate a molecular polymorphism.     

Differential scanning calorimetric study of meals and constituents of some food grain Legumes

Two thermal transitions were observed in soya bean meal by differential scanning calorimetry (DSC) and on the basis of previous data were ascribed to the denaturation of the 11S globulin, glycinin, and the 7S globulin, β-conglycinin. Three DSC transitions were apparent in Vicia faba meal; one was associated with starch gelatinisation, and was absent from thermograms of soya bean because the latter stores oil rather than starch. The remaining two transitions were identified with the 11S globulin, legumin, and a 7S globulin, vicilin. The denaturation parameters of legumin and glycinin were very similar, in contrast to those of vicilin and β-conglycinin. From this it was concluded that legumin and glycinin probably represent homologous proteins, whereas the two 7S proteins possess intrinsically different structures to one another. Cowpeas (Vigna unguiculata) and dry beans (Phaseolus vulgaris) afforded two major transitions which were assigned to 7S globulins and starch. From its DSC profile, the 7S globulins of cowpea appeared heterogeneous, but nevertheless possessed denaturation characteristics similar to those of vicilin of V. faba. In contrast, the thermal behaviour of the 7S globulin of dry beans (glyco-protein II) was distinct from that of any of the other 7S globulins investigated. Three protein transitions were observed in the three lupin species examined. As in the case of soya bean, no starch transition was present. On the basis of the correspondence in transition temperature, one of the protein transitions was assigned to the 11S globulin component. Overall, this study indicates the potential of DSC as a means for obtaining data on seed protein homology from whole meals rather than the extracted proteins.     

Quantitative studies on the cotyledonary proteins in the genus Pisum

Forty-five lines of peas including primitive or wild forms, field peas, and round and wrinkled garden peas, were grown under uniform conditions and the seeds examined for variation in protein characteristics likely to influence nutritional value. The characters measured were crude protein, extractable protein, globulins and albumins, the percentages of legumin, total sulphur and protein sulphur, carbon: nitrogen and nitrogen: sulphur ratios. The extractable protein was separated quantitatively into an albumin fraction (20–35%) and a globulin fraction (legumin and vicilin). Without exception lines high in albumin content were low in legumin content (correlation coefficient r= ?0.757). As both the albumin fraction and legumin are rich in sulphur amino acids, this negative correlation has important implications for attempts through plant breeding to improve the nutritional quality of legume seed proteins, by increasing the sulphur amino acid content. Total sulphur was not correlated with any other protein character.     

Interfacial behaviour of succinylated faba bean legumin at low ionic strength

Changes in the tensio-active properties of the main storage protein from faba beans (legumin) after succinylation were studied at a low salt concentration. Surface tension, surface dilatational properties of monolayers and emulsifying activity were measured at a ionic strength of I = 0.02. The results were compared with those at a high ionic strength of I = 0.3. Parameters of the Gibbs' adsorption isotherm indicate that the most surface-active derivatives are legumins with a moderate degree of succinylation (34% and 65%). The equilibrium surface pressure, π_e, inreased from 18.47 (native legumin) to 20.72 mN/m (65% succinylation). The critical association concentration, CAC, i. e., the subphase concentration at which the plateau of π_e was reached, decreased from 15.9·10^–6 to 7.12·10^–6 g/ml after 34% succinylation. The film forming properties differed from the adsorption behaviour. Only monolayers of the 65% succinylated legumin exhibited viscoelastic behaviour. By contrast, the emulsifying activity, EAI, reached the highest values for the 65% and 95% succinylated legumins. Low salt concentrations favour the adsorption of the native legumin and reduce the surface activity of succinylated legumin. Monolayer formation and especially the ability to form elastic networks seems to be diminished by the repulsive interaction of like-charged molecules. The emulsifying properties of the higher succinylated legumins are not influenced by the ionic strength whereas those of the native and low succinylated legumin are distinctly lower at I = 0.02. This result points to different adsorption and stabilizing processes during emulsion formation.     

Plant protein interactions with polysaccharides and their influence on legume protein functionality A Review

Associative interactions between proteins and polysaccharides, both coulombic and non‐coulombic, lead to the formation of interpolymer complexes. Complex formation with charged polysaccharides, either anionic or cationic, imparts solubility to seed globulins in the vicinity of their isoelectric points. This has been shown for the complexes “sunflower 11 S globulin (helianthinin) – sodium alginate” and “faba bean legumin (or the product of its limited proteolysis with trypsin – legumin‐T) – chitosan”. Hysteresis effects allow to control the solubility of seed globulins in weakly acid or weakly basic media. Formation of soluble complexes of faba bean legumin or legumin‐T with chitosan substantially increases the emulsion stability of the both proteins.     

Structural and functional changes of faba bean legumin during super-limited tryptic hydrolysis

The influence of a super-limited tryptic hydrolysis on physicochemical and surface functional properties of faba bean legumin has been studied using size-exclusion HPLC, SDS-PAGE, UV and fluorescence spectroscopy, fluorescence probe techniques, surface tension measurements as well as determination of emulsifying activity index (EAI) and emulsion droplets diameter (D). The extent of legumin hydrolysis comprised the range between about 14 and 60 split peptide bonds per molecule resulting in a stepwise decrease of legumin molecular weight to 240 kDa (legumin-T) via discrete intermediates with characteristic subunit patterns. These changes are accompanied by an increase in the surface hydrophobicity and the exposure of aromatic chromophores. No differences were found in the surface tension between the variously hydrolyzed legumin samples. Best emulsifying properties (highest EAI and lowest D values) were attained after a rather low tryptic hydrolysis (about 30 split peptide bonds per mol). Further hydrolysis impaired the emulsifying parameter which were, however, higher (EAI) or lower (D) than those for native legumin.     

Amino acid composition of storage proteins of a promising chickpea (Cicer arietinum L) cultivar

The amino acid compositions of flours made from the cotyledons and from the whole seeds of a disease-resistant, stable and high-yielding cultivar of chickpea (Cicer arietinum L) cv H75–35, known locally as Gaurav, have been analysed. These, together with similar analyses of the albumin, globulin, legumin and vicilin protein fractions, have been compared with those of other legumes. Seed protein content was 19·8 % with globulin constituting 62·4 % of the total seed protein. The ratios of albumin to globulin and legumin to vicilin were 1:4 and 6:1, respectively. The proportion of basic amino acids in the albumin was low whereas the reverse was true in the globulin. The legumin fraction seems to be superior in terms of total essential amino acids to those from other sources. Sulphur amino acids were the most limiting, followed by tryptophan or threonine depending on the fraction. However, the ratio of methionine to cystine was high (2·76:1). The extent of the sulphur amino acid deficiency was assessed, and possible approaches for its improvement are outlined.     

Preliminary study of the influence of dextran on the precipitation of legumin from aqueous salt solutions

Ultrasound has been used to study the behaviour of dextran, legumin (11s globulin of the broad bean Vicia faba ) and their mixtures in aqueous salt solutions under different physico-chemical and thermodynamic conditions. The attenuation coefficient of the solutions was measured as a function of pH (3–10) and ionic strength (0, 0.025, 0.05 M NaCl). The attenuation in the dextran solutions was independent of pH and ionic strength, whereas that in the solutions containing legumin had a maximum value near to the isoelectric point of the legumin (∼φ 5). The magnitude of this maximum depended on the ionic strength of the solution. The cause of the observed attenuation was scattering of ultrasound by precipitated protein. The addition of dextran to solutions where the biopolymers are thermodynamically compatible decreases the attenuation, whereas its addition to solutions where the biopolymers are incompatible increases it. This is because protein precipitation is greater in incompatible systems.     

Composition of marama bean protein

The protein composition of marama beans, an indigenous African oilseed legume, was determined in comparison with soya beans. Marama bean protein contained a substantial amount of tyrosine compared with soya bean protein. It was slightly richer in proline than was soya. By SDS–PAGE, marama protein contained fewer protein bands than did soya. The patterns of these bands in marama under non-reducing and reducing conditions were similar, suggesting an absence of disulphide bonds. The vicilin (7S) and acidic 11S subunits seemed to be absent in marama. This is most unusual in legume proteins. Only a major basic legumin (11S) (20 kDa), medium (63 kDa) and high (148 kDa) molecular weight protein bands were separated for marama. Most polypeptides in the marama proteome map are basic compared with soya. Only one polypeptide match, comparable to soya, was tentatively identified. Marama protein composition is very different from that of soya.     

Foaming and emulsifying properties of pea albumin fractions and partial characterisation of surface‐active components

Whole albumin of pea seed (Palb) was extracted from pea flour (var Frilene) by solubilisation at pH 4.9 and dialysis against water. Palb was fractionated by differential solubilisation in 60% methanol, yielding a soluble (S60) and an insoluble (IS60) fraction. The composition of the fractions was determined by SDS‐PAGE and RP‐HPLC. Albumins exhibited a wide variability of surface hydrophobicity. Albumin PA2 was present as a major component in Palb and IS60. Low‐MW hydrophobic albumins were concentrated in S60. Foaming and emulsifying properties of the three extracts were determined in model conditions. Functionality of albumins was highest at acid pH, but the fractions differed in their properties. The presence of PA2 albumin resulted in the best foaming and emulsifying properties. This protein was shown to adsorb preferentially at air/water and oil/water interfaces, whereas low‐MW hydrophilic albumins did not. Low‐MW hydrophobic albumins were also adsorbed at interfaces, as shown by the behaviour of the S60 fraction, but they formed more fragile films than did PA2. This was related to the structure of the polypeptide chains. © 2000 Society of Chemical Industry     

Thermal aggregation of globulin from an indigenous Chinese legume,Phaseolus angularis (red bean)

The thermal aggregation behavior of red bean (Phaseolus angularis) globulin (RBG) was studied at ≈1% (w/v) protein concentration in 0.01 M phosphate buffer, pH 7.4. The percentage of protein precipitated was affected by heating temperature, heating time and salt concentration. The influences of several salts of the chaotropic series and protein structure-modifying agents on thermal coagulation of RBG were also investigated. The effects of chaotropic salts did not follow the lyotropic series of anions. Sodium dodecyl sulfate caused a more pronounced reduction in heat-induced aggregation of RBG than did dithiothreitol, while N-ethylmaleimide did not affect aggregation until after a long heating period. Differential scanning calorimetric (DSC) data showed that heat aggregation of RBG was preceded by thermal denaturation. SDS-PAGE showed that heating led to the disappearance of some protein bands, and the basic polypeptide of 11S globulin (legumin) was not found in the buffer-soluble aggregates. Heating caused increases of surface hydrophobicity, again suggesting protein unfolding prior to aggregate formation. The buffer-insoluble aggregates did not show any DSC response, indicating extensive denaturation, and had a lower surface hydrophobicity and higher disulfide content than the buffer-soluble aggregates. The data suggest that electrostatic and hydrophobic interactions may play an important role in thermal aggregation of RBG, with disulfide bonds playing a limited role.     

Partial characterization of proteins from baru (Dipteryx alata Vog) seeds

BACKGROUND: Baru (Dipteryx alata Vog.) is a fruit distributed throughout the Brazilian savanna and contains a seed with a high protein content, whose properties have been rarely explored. The purpose of this study was to characterize this protein, especially by isolation and quantifying its fractions and measuring some of its molecular properties. RESULTS: Baru seeds contain 244 g kg^?1 protein on a dry weight basis. Solubility profiles showed a preponderance of globulins. This fraction dominated the seed composition, with 61.7 wt% of the total soluble proteins. Albumins and glutelins accounted for 14 and 3.3 wt%, respectively. SDS‐PAGE resolution of albumin and globulin showed main bands with molecular weights of 84 kDa and 64, 66 and 73 kDa, respectively. The total protein of the flour and the globulin showed values of in vitro digestibility of 85.59% and 90.54%, relative to casein. Total globulin produced only one chromatographic peak, both on Sepharose CL‐6B gel filtration and on DEAE‐cellulose ion‐exchange columns, eluted at a concentration of 0.12 mol L^?1 NaCl. CONCLUSION: The baru seed had high protein content with large quantities of storage proteins. The chromatographic and solubility profiles indicate the predominance of a fraction with characteristics of a legumin‐type protein. Copyright © 2011 Society of Chemical Industry     

Characterization of protein fractions from chickpea (Cicer arietinum L.) and oat (Avena sativa L.) seeds using proteomic techniques

Albumin, globulin and glutelin fractions were prepared from chickpea and oat seeds using sequential extractions. Molecular characteristics of individual protein fractions were investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in combination with proteomic techniques. SDS-PAGE results revealed that chickpea albumin and globulin fractions (C-Ab and C-Gb) showed protein bands with molecular weights (MWs) related to subunits of legumin (11S globulin) and pea vicilin (7S globulin); oat protein fractions (O-Ab, O-Gb and O-Gt) showed most protein bands with MWs related to subunits of oat 12S globulin (avenalin). With proteomic analysis, eighteen tryptic peptides from chickpea globulin fraction showed sequence homology that corresponded to chickpea legumin α- and β-subunit (NCBI accession number: gi|6273402; theoretical mass 56,216 Da) while sixteen tryptic peptides from chickpea albumin fraction (C-Ab) were identified as chickpea provicilin precursor (NCBI accession number: gi|82173888; theoretical mass 51,390 Da); fifteen tryptic peptides from oat protein fractions were identified with origin from oat 12S seed storage globulin 1 (NCBI accession number: gi|134918; theoretical mass 58,508 Da). The identified tryptic peptide, ALIVPQNFAIAAK, was commonly found in chickpea glutelin fraction (C-Gt), rice glutelin fraction (R-Gt), and oat albumin, globulin and glutelin fractions (O-Ab, O-Gb and O-Gt).     

Role of Noncovalent Forces in Micellization Using Legumin from Vicia faba as a Study System

The seed storage protein, legumin, from Vicia faba interacted to form micelles and elaborate protein networks under varying conditions. Several molecular parameters correlated with observed micelle interaction parameters; these included thermal properties as indicators of protein stability and surface hydrophobicity as an assessment for potential hydrophobic interactions. The optimal pH for micelle formation ranged from 5.5 to 6.5, values at which electrostatic repulsions were minimal and surface hydrophobicites were adequate to allow hydrophobic interactions. The micelle response was affected by anions both in terms of concentration and type of anion. Finally, gradual denaturation of legumin with increasing urea concentrations had a negative impact on the micelle response.