Quantitative and qualitative variability of pea (Pisum sativum L.) protein composition

Smooth and wrinkled pea cultivars were studied to evaluate the protein content of the seeds, the proportion of albumins to globulins and the variability of the vicilin/legumin ratio. Principal components analysis showed a strong negative correlation between albumin and globulin contents. Stepwise discriminant analysis demonstrated that two variables, protein content and vicilin/legumin ratio, divided the samples into smooth and wrinkled cultivars with a percentage of success of 95%. In addition, the vicilin/legumin ratio tended to increase, the lower the protein content is.     

Isolation of faba bean legumin — a comparative study of various methods

Different methods of legumin isolation from faba beans were compared with regard to the removal of vicilin taking into consideration the additional influence of variety. SDS-PAGE, analytical ultracentrifugation and SE-HPLC were used for the analysis of protein composition. SDS-PAGE analysis of the isolated legumin samples revealed, that neither combined salt fraction/isoelectric precipitation nor heating to 73 °C resulted in a complete removal of vicilin. Time-consuming isoelectric zonal precipitation and heating at 83 °C as described by Schlesier et al. (Biochem. Physiol. Pflanzen 180 (1985) 225) provides legumin preparations free of vicilin in low yields. Acceptable yields in shorter time were obtained by combining isoelectric precipitation/salt fractionation with Schlesier's procedure. SDS-PAGE analysis showed a certain dependence on variety of the patterns of legumin α-polypeptide chains.     

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).     

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.     

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.     

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.     

Thermal properties of Phaseolus angularis (red bean) globulin

The thermal properties of Phaseolus angularis (red bean) globulin were studied by differential scanning calorimetry under various medium conditions. Red bean globulin (RGB) was fractionated by ion-exchange chromatography into a major fraction, with SDS–PAGE pattern corresponding to the 7S vicilin, and two minor fractions, probably representing residual vicilin and the 11S legumin, respectively. The thermogram of RBG showed a major endothermic peak at 86.4°C and a minor transition at 92.2°C. Vicilin exhibited two endothermic peaks (87.7 and 94.1°C), while legumin showed one transition at 89.5°C. The progressive increase in denaturation temperature (T_d) with increase in salt concentration, suggests a more compact conformation for RBG with higher thermal stability. Decreases in enthalpy and T_d were observed under the influence of highly acidic and alkaline pHs, chaotropic salts, and protein perturbants such as sodium dodecyl sulfate, urea and ethylene glycol, indicating partial denaturation and decrease in thermal stability. Dithiothreitol and N-ethylmaleimide have little effect on the thermal properties of RBG since the major protein component, vicilin, is devoid of disulfide bonds.     

ISOLATION AND IN VITRO HYDROLYSIS OF GLOBULIN G1 FROM LENTILS (LENS CULINARIS, MEDIK)

The major globulin fraction from lentil seeds was investigated with respect to in vitro hydrolysis by trypsin and chymotrypsin. Globulin was isolated by a NaCl-ascorbate extraction procedure and purified by DEAE-cellulose chromatography and gel filtration chromatography on Sepharose CL-6B. The purity and identification of the protein were performed by PAGE. The native globulin, with a molecular weight of 375 kD, was resolved by SDS-PAGE into twelve polypeptides with molecular weights ranging from 61 to 14.5 kD. Native and heated globulin G1 was hydrolyzed with trypsin and chymotrypsin. SDS-PAGE indicated that native globulin was more resistant to digestion than heated protein. Amino acid analysis of the major globulin revealed that glutamic acid was present in the largest concentration, followed by aspartic acid, arginine and leucine. As is also the case for other legumin-like globulins, lentil G1 was deficient in sulfur-containing amino acids.     

Large-scale purification and characterisation of pea globulins

Pea globulins, vicilin and legumin were isolated by chromatography on DEAE Sepharose and Ultrogel ACA 34. The procedure was carried out on a preparative scale and used to purify about 5 g of each globulin for a separation cycle. The purity of the vicilin and legumin was verified by immunoelectrophoretic and ultracentrifuge analysis.     

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.     

Heat Induced Gelation of Pea (Pisum sativum) Mixed Globulins, Vicilin and Legumin

Mixed globulins (MG) were extracted from ground dry peas (Pisum sativum, B-160) with 0.5M NaCl, 50 mM potassium phosphate, pH 7.2, and isolated by precipitation at pH 4.5. Crude vicilin and legumin were fractionated from the MG by dialysis against 0.2M NaCl, pH 4.8, and centrifugation, then further purified using DEAE-cellulose chromatography. Conditions for maximum gel hardness of heat induced MG gel, as determined with an Instron Universal Testing Machine, were heating for 20 min at pH 7.1 at 87°C. Purified vicilin, but not legumin, formed heat induced gels. The relationship was linear between protein (globulin) concentration and log gel hardness. At all protein concentrations studied, as proportion of legumin decreased, gel hardness increased.     

Gelation Behavior of Protein Isolates Extracted from 5 Cultivars of Pisum sativum L.

ABSTRACT: Protein isolates were extracted from 5 pea ( Pisum ) cultivars and their gelation behaviors were compared at pH 7.6. Gel formation and development was monitored via constant oscillation dynamic measurements. The standard heating and cooling rate was 1.0°/min, but samples were also heated at 0.5°C (and cooled at 1.0°C/min), and others were heated at 1.0°C/min and cooled slowly at 0.2°C/min. When heating more slowly, no changes in gel formation were detected for any of the cultivars. When cooling slowly, the cultivar Solara, with the highest legumin content, formed a stronger gel than all the other cultivars. It did the same when the thiol-blocking agent N -ethylmaleimide (NEM) was added to the system. This indicated that the strengthened gel system formed independently of any disulfide bonds formed by the legumin. The cultivars Supra and Classic formed stronger gels only when cooled slowly in the presence of NEM, and so disulfide bond formation in their gel systems was apparently a factor that prevented gel network strengthening. The cultivars Finale and Espace were unable to form strong and self-supporting gels. This was believed to be because of the repulsive forces on the α-subunits of vicilin, which were at their highest level in the cultivars Finale and Espace. The contribution of legumin to the pea protein isolate gels was shown to be cultivar specific and related to its disulfide bonding ability rather than the absolute amount of legumin protein present.     

Isolation of beer foam polypeptides by hydrophobic interaction chromatography and their partial characterisation

Beer foam polypeptides have been separated into five groups based on their relative hydrophobicity. Foam stability increases with increasing hydrophobicity of the polypeptide groups. The most hydrophobic polypeptide group contains a large proportion of Coomassie blue-binding polypeptides. Analysis by SDS-PAGE reveals that each polypeptide group is composed of several differently-sized polypeptides. Further purification by anion-exchange chromatography results in five fractions, each of which has a different polypeptide profile on SDS-polyacrylamide gels.     

Assessment of protein fractions of three cultivars of <Emphasis Type="Italic">Pisum sativum</Emphasis> L.: effect of germination

Protein composition, nitrogen and amino acid content of Osborne fractions in different cultivars of Pisum sativum L. (cv. ucero, cv. ramrod and cv. agra) seeds were investigated and the effect of germination on these parameters was also analysed. Albumins comprised the main protein fraction in raw seeds, globulins were constituted mainly by vicilin, with a smaller proportion of the glutelin and prolamine fractions. Regarding the amino acid profile of pea protein fractions, although differences among pea cultivars were found, in general albumin, glutelin and prolamine fractions presented Asp, Glu and Gly as the major non-essential amino acids (NEAA) and Lys as the main essential amino acid (EAA). The globulin fraction, however, presented Asp, Glu, Gly and Arg as the major NEAA and Leu, Phe, Lys, and Thr as the main EAA. In general, the albumin fraction accounted for more sulphur amino acids and Lys, followed by the glutelin + prolamine fraction. Germination caused an increase in the total protein content of P. sativum cv. ucero and P. sativum cv. ramrod. In the albumin fraction a wide number of proteins underwent degradation and convicilin disappeared from the globulin fraction of pea sprouts whilst vicilin and legumin decreased slightly. In general, all the Osborne fractions of pea sprouts presented higher EAA contents than raw seeds. The estimated essential amino acid indexes of protein fractions for P. sativum cv. ucero (EAAI_adult and EAAI_egg) improved with the germination process whilst for P. sativum cv. ramrod and P. sativum cv. agra depended on the Osborne fraction.     

Emulsifying Properties of Pea Globulins as Related to Their Adsorption Behaviors

Emulsifying properties of purified pea globulins and of vicilin-legumin mixtures were evaluated through their emulsifying capacity, emulsifying activity index and stability of the resulting emulsions. The results were discussed by reference to the interface adsorption behaviors of these proteins. The influence of the vicilin/legumin ratio on the efficiency of pea isolates as emulsifying agents was also studied. Vi-cilin which has been shown to be more surface active at air/water and dodecane/water interfaces, led also, in both cases, alone or mixed, to better emulsifying properties than legumin. The globulin composition of the isolates did not completely explain their emulsifying behaviors.     

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 include r (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 r or rb. 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, but E. 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 vitro formation 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.     

Characterization of globulin from Phaseolus angularis (red bean)

Phaseolus angularis (red bean) seeds contain about 25% protein (dry basis), almost half of which is globulin. Similar to globulins from other Phaseolus species , 7S vicilin is the major fraction of red bean globulin (RBG), with 11S legumin as a minor component. The amino acid profile of RBG met or exceeded the FAO/WHO standard. Circular dichroism measurements indicate that RBG is a protein rich in α-helical and β-turn structures. RBG exhibited higher protein solubility than Supro 610, a commercial soy protein isolate, especially at acidic pHs, with minimal solubility at around pH 5.0. Compared to Supro 610, RBG had lower water hydration capacity and comparable fat binding capacity, which might be because of its lower surface hydrophobicity. RBG had higher emulsifying activity index and emulsion stability than Supro 610, but with poorer foaming properties.     

CHANGES IN PROTEIN COMPOSITION DURING STRAWBERRY (Fragaria × ananassa Duch.) FRUIT RIPENING1

The changes in strawberry native proteins and polypeptides during ripening were studied. Analysis of protein extracts by nondenaturing electrophoresis showed the presence of a protein species only in the ‘25% Red’ and later ripening stage. This 40 kD polypeptide was hardly detected in ‘Large green’ and ‘White’ fruit extracts. The polypeptide profile, obtained with SDS-PAGE, also changed during ripening. While most polypeptides were found in all ripening stages, the distribution of some of them varied during ripening. Protein synthesis (measured by ³⁵ S-methionine labelling) in strawberry was mainly directed towards the regeneration of previously existing proteins. As ripening proceeded, the synthesis of 67 and 63 kD polypeptides increased, while those of 82, 56 and 25 kD decreased. Three very-abundant polypeptides (36, 24 and 23 kD) were located in the achenes and were not labelled in any ripening stage.     

NMR and FTIR studies of hydrated pea proteins

The effects of increasing D₂O hydration on the plasticization of vicilin, legumin and albumin fractions from peas were investigated using solid state ¹H-NMR transverse relaxation techniques. Measurements showed increases on hydration in the T₂ and intensity of the exponential component of the relaxation decay. However, a Gaussian (more rigid) component remained throughout the sample composition range. This behaviour contrasted with that observed in barley storage proteins and would indicate considerably less plasticization in legume proteins. In ²H-NMR transverse relaxation measurements of a highly D₂O hydrated sample over a large temperature range, vicilin was shown to be hydrophilic in nature. However, the observed absorption of water by vicilin was less than in the HMW subunits of wheat. FTIR spectra show little structural change in vicilin and legumin on hydration, in contrast to changes occurring in the cereal proteins. These differences in behaviour may be ascribed to differences between the globular structure of the legume proteins and the more linear structure of the cereal proteins.