Vicilin Subunits Research Articles

Unpicking the changes in content, structural properties and in vitro digestibility of pigeon pea seed proteins as influenced by imbibition and heating in sodium bicarbonate

BackgroundPigeon pea proteins are underutilised mainly because of the long cooking time of the legume seeds. Traditionally, this is mitigated by cooking pigeon pea seeds in sodium bicarbonate (NaHCO3). This study investigated whether, and to what degree, directly cooking pigeon pea seeds in water (CS0W) or NaHCO3 (CS0N), soaking in water or NaHCO3 for 4 h (CS4W and CS4N, respectively) or for 24 h (CS24W and CS24N, respectively) before cooking in water or NaHCO3 influenced the protein content, structural property and in vitro digestibility. ResultsIn vitro protein digestibility of pigeon pea seed flour was lowest in the raw seeds and increased with CS0W<CS4W<S24CW and CS0N<CS4N<S24CN. The α-helix content of the samples also decreased in the same order. SDS-PAGE showed ∼90% decrease in the 7S vicilin subunits (65 and 47 kDa) in CS24N and a corresponding 4-fold increase in protein bands at 10–15 kDa compared to raw seeds. NaHCO3 treatments decreased the protein isolate yields due to protein losses (up to 7.5-folds) in the cooking fluids. ConclusionThe findings showed that NaHCO3 improved protein digestibility by degrading the higher molecular weight proteins, but also increased protein diffusion into the cooking fluid. Therefore, the traditional method of cooking pigeon pea seeds needs optimization to maximize the protein accessibility.

Structure – Function relationship of Australian Acacia seed protein concentrates: Amino acid composition, in vitro protein digestibility and molecular properties

The nutritional, functional, in vitro protein digestibility (PD), structural, and molecular properties of Acacia seed protein concentrates (ASPC) from three Acacia species (Acacia victoriae, A. coriacea and A. cowleana) were investigated and compared with a commercial soy protein isolate (SPI). The essential amino acid profile in ASPC, except for methionine, exceeded the minimum daily requirements for adults recommended by the FAO/WHO. The ASPC PD, particularly from A. victoriae and A. coriacea, was considerably high (64.2–79.3%), but lower than SPI (89.5%). ASPC proteins had higher molecular weights ranging from 50 to 65 kDa (associated with 7S vicilin subunit), better protein solubility and oil absorption capacity than SPI. Emulsifying properties were comparable between ASPC and SPI. Among the Acacia species, the proteins from A. victoriae and A. cowleana exhibited higher protein quality, foaming capacity and solubility compared to A. coriacea. Overall, ASPC from A. victoriae and A. cowleana showed potential as ingredients in food processing.

Mechanism of high-moisture extruded protein fibrous structure formation based on the interactions among pea protein, amylopectin, and stearic acid

During high-moisture extrusion (HME) processing for plant protein texturization, the interactions among proteins, starch and lipids should determine the formation of fibrous structures, which has not been confirmed systematically. In this study, the mechanism of protein fibrous structure formation during HME processing (58% moisture) was investigated based on the intermolecular interactions among the pea protein, amylopectin and stearic acid. Results suggested that the amylopectin and stearic acid synergistically contributed to improve the fibrous structures such as the hardness and fibrous degree in pea protein extrudate. In the extruder barrel, the complexation among the protein with amylopectin and stearic acid gave rise to the formation of aggregates with higher thermal stability and delayed the formation of protein gel network. In the die, the amylopectin and stearic acid weakened the hydrogen bonds between proteins and hindered the aggregation of legumin and vicilin subunits. In the cooling zone and extrudate, the amylopectin and stearic acid promoted the formation of fibrous structures through an “anchor orientation and flexible cross-linking” mechanism. Namely, the stearic acid as anchors hindered the refolding of protein molecular chains, while the amylopectin promoted the rearrangement, cross-linking and aggregation of protein molecules. The amylopectin and stearic acid synergistically weakened the interaction forces between proteins and led to the formation of more flexible structures in aggregates, which was favorable for the orientation and formation of anisotropic fibrous structures in extrudates. This study provided a theoretical basis for the development and improvement of the plant-based meat substitutes.

Influence of Chitosan and Glucono-δ-Lactone on the Gel Properties, Microstructural and Textural Modification of Pea-Based Tofu-Type Product

This study investigated the effects of the addition of chitosan (0–1.0%) or glucono-δ-lactone (GDL) (0–60 mM) on the gel properties, microstructure, and texture of pea-based tofu-type product. Following the addition of 0.5% chitosan or 20 mM GDL, we observed a significant decrease in the hardness and cohesiveness of the tofu, resulting in a slightly discontinuous network structure with pores smaller than those in samples without chitosan or GDL. SDS-PAGE analysis revealed the induced aggregation of pea legumin (11S) and vicilin (7S) subunits (30, 34, and 50 kDa), legumin α subunit (40 kDa), and legumin β subunit (20 kDa) by chitosan or GDL. It appears that chitosan and GDL could potentially be used as food additives for the development of texture-modified pea-based tofu-type products.

Effect of fatty acid saturation degree on the rheological properties of pea protein and its high-moisture extruded product quality

To provide a deep insight into the application of lipid in plant protein-based meat substitutes through high-moisture extrusion (HME) process, the effect of fatty acid saturation degree (stearic acid, oleic acid and linoleic acid) on the rheological and structural properties of pea protein isolate (PPI), and its relationship with the extrusion system parameters and extrudate qualities were investigated. The oleic acid and linoleic acid significantly decreased the apparent viscosity of PPI dispersion and thus reduced the die pressure and torque during HME processing. Electrophoresis and Fourier transform infrared spectroscopy results revealed that fatty acids inhibited the aggregation of legumin and vicilin subunits at 50 and 20 kDa, and promoted the conversion of α-helix and β-sheet to β-turn and random coil. The macro- and micro-structure observation and texture analysis suggested that the fatty acids with higher unsaturation degree were not advantageous for the pea protein fibrous structure improvement during HME process.

Comparative Study of Structural and Physicochemical Properties of Pigeon Pea (Cajanus cajan L.) Protein Isolates and its Major Protein Fractions.

Pigeon pea protein isolates (PPI) are an option to obtain a high yield of good quality proteins and represent a great potential for the food industry. In this work, physicochemical and structural properties of albumin (ALB), globulin (GLB), and PPI obtained at different pHs (8, 9, 10, and 11) were studied to deepen the knowledge of these proteins for future application. GLB presented protein aggregates and polypeptides characteristics of 7S vicilin subunits while ALB presented polypeptides with low molecular masses. GLB showed a more compact and less flexible structure than ALB fraction due to the distinct conformational characteristics found in DSC, fluorescence spectroscopy, Ho. These structural characteristics conferred GLB greater conformational stability (∆GH2O) than ALB fraction. The latter presented a higher proportion of β-strand in aggregated structures. PPI11 showed the highest protein recovery, but the least So with more presence of protein aggregates with the least proportion of β-strands in aggregated structures. A higher percentage of protein unfolding and exposure of hydrophobic residues to solvent was observed as the extraction pH of the isolates increased. Enthalpy change of transition decreased, and the maximum emission wavelength shifted to red in fluorescence spectroscopy. However, PPI11 showed only a slight increase in Ho (10%) with respect to PPI8. The variation in pH for protein extraction constitutes a simple, rapid, and low-cost method to obtain PPI with physicochemical and structural properties that will determine its functional properties and their use as food ingredients.

Germination alters the microstructure, in vitro protein digestibility, α‐glucosidase and dipeptidyl peptidase‐IV inhibitory activities of bioaccessible fraction of pigeon pea (Cajanus cajan) seeds

AbstractGermination was investigated as a bioprocess for enhancing the digestibility and bioactivity of pigeon pea (Cajanus cajan). Pigeon pea seeds were germinated for 0, 4, 24 and 48 h. Electrophoresis revealed that the 7S vicilin subunits were the most abundant proteins in control and germinated seeds. FTIR showed a loss of β sheet and a gain of α‐helix contents, and microscopy showed cell wall degradation in germinated seeds. Germination decreased the seed protein yield due to partial hydrolysis of proteins. After in vitro digestion, 48‐h germination increased the protein digestibility‐corrected amino acid score of the pigeon pea flour and isolated protein. Germination also enhanced the inhibitory activity of the seed digesta against α‐glucosidase but not dipeptidyl peptidase IV. Taken together, germination could be used to enhance the nutritional quality and bioactivity of pigeon pea towards improving its future utilization as novel healthy food for mitigating food insecurity.

Microwave treatment increased protein digestibility of pigeon pea (Cajanus cajan) flour: Elucidation of underlying mechanisms.

Pigeon pea is rich in proteins but has low protein digestibility like other legumes. This work investigated the effects of processing, including soaking, grinding, ultrasound and microwave, on the protein digestibility of pigeon pea flour. Only microwave treatment significantly increased in vitro protein digestibility from 54.4±2.5% to 71.6±4.2%. SDS-PAGE showed that the most abundant proteins in all samples were the 7S vicilin subunits. After microwave treatment, the starch granular structures of pigeon pea flour changed to clusters, and protein secondary structures lost 5% β-sheet and gained 5% random coil, which contributed to the increased protein digestibility. Microwave decreased protein water solubility from 94.4±0.8% to 48.1±6.5% and increased the disulfide bond content by 42%. The increased protein digestibility is attributable to the relatively reduced particle size (166.6±38.6nm) and increased zeta potential (-35.2±2.6mV) of the microwave-treated sample. Therefore, microwave is a promising approach for increasing pigeon pea flour protein quality and utilisation.

Structure, composition and functional properties of storage proteins extracted from bambara groundnut (Vigna subterranea) landraces

AbstractBambara groundnut is a protein‐rich traditional legume. In this study, storage proteins were isolated from three bambara landraces. Bambara protein revealed four major protein bands: one broad band at 55 kDa, two medium bands at 62 kDa and 80 kDa and a high molecular weight (HMW) protein at 141 kDa. The vicilin (7S) subunits with molecular weight of 55 kDa and 62 kDa were major fractions in bambara storage proteins. Bambara proteins showed two endothermic peaks ranging from 64 to 69 °C and 76 to 90 °C, respectively. Bambara protein isolates had well‐defined tertiary and secondary structures, respectively, at pH 3.0, and this well‐defined structure decreased slightly at higher pH values. The isolates revealed a strong secondary structure dominated by α‐helical conformation. Foaming capacities of bambara proteins were dependent on pH with maximum percentage FC observed at pH 3.0, while the emulsion activity increased with increasing pH for all the isolates. Vicilin (7S) fraction seems to be the major storage protein fraction of bambara. Bambara proteins could serve as excellent ingredients for the formulation of food foams and emulsions.

Biochemical fate of vicilin storage protein during fermentation and drying of cocoa beans

Key cocoa-specific aroma precursors are generated during the fermentation of cocoa beans via the proteolysis of the vicilin-like globulin. Previous studies had shown that degradation of this particular 566 amino acid-long storage protein leads to three distinct subunits with different molecular masses. Although oligopeptides generated from the proteolysis of vicilin-like globulin have been studied previously, changes occurring to vicilin at different stages of fermentation have not yet been explored in detail. The aim of this study was to investigate the fate of vicilin protein from the non-fermented stage up to the dried cocoa beans. Our results showed a remarkable shift in the electrophoretic mobility of vicilin towards higher pI during the onset of fermentation. The pI-shifted subunit was found susceptible to further degradation into a lower-molecular-weight vicilin subunit. The observed pI shift correlated with, but did not depend on protein phosphorylation. Glycosylation of some but not all vicilin subunits occurred at different stages of the fermentation process. Peptides generated from vicilin throughout fermentation were analyzed by UHPLC-ESI-MS/MS revealing an initial increase and subsequent decrease in the diversity of peptides with an increasing degree of fermentation. We furthermore describe the rate of degradation of different vicilin subunits. The detected diversity and dynamics of vicilin peptides will help to define biochemical markers of distinct steps of the fermentation process.

Mobilization of Reserve Proteins and Activities of Cysteine Peptidases During Germinative and Post-Germinative Events of Cowpea Seeds

Cysteine peptidases are the best characterized peptidases among those involved with storage protein mobilization during seed germination. In the present work we show two major groups of cysteine peptidase activities, one of higher (55 to 97 kDa) and other with lower (15 to 20 kDa) molecular masses which are temporally activated after 24 and 48 HAI, respectively, in germinating cowpeas. The former group is found both in protein bodies and in cytoplasmic fraction, while the latter is mostly present outside protein bodies. A third cysteine peptidase activity of ~37 kDa was specifically active at quiescent cotyledons and at 12 and 60 hours after imbibition (HAI). Main peptidase activities of albumin fractions were synchronizedly detected with radicle emergence at 36 HAI. Major vicilin mobilization was more pronounced from 60 HAI onwards and steadily increased until 144 HAI, when low levels of the smallest vicilin subunit were present. Cysteine peptidases were susceptible to iodoacetamide, E-64, iodoacetic acid, pCMB and β-mercaptoethanol, except for the ~37 kDa peptidase, which was not affected by any of the inhibitors. By a two-dimensional native/SDS-PAGE combination it was observed an apparent linear arrangement of protein breakdown products as well as of peptidase activity spots. The finding may indicate a complex set of sequential proteolytic events where peptidases induce or activate new peptidases, which may act upon different aggregates or zymogens, and these hydrolysis products appear in a line of constant decreasing Rf x Mr ratio.

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).

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.

Proteomic characterisation of subclover seed storage proteins during germination

AbstractBACKGROUND: 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

Subunit structure of the vicilin-like globular storage protein of cocoa seeds and the origin of cocoa- and chocolate-specific aroma precursors

Essential cocoa-specific aroma precursors are generated during fermentation of cocoa seeds by proteolysis of the vicilin-like globulin. This particular storage protein consists of three subunits with apparent molecular masses of 47 kDa, 31 kDa, and 15 kDa, respectively, which are derived from a common 66-kDa precursor. Since all these subunits resist N-terminal sequencing by Edman degradation, we have analysed the localisation of these vicilin subunits on their common precursor sequence by MALDI-TOF-MS analyses of their tryptic fragments. The results were corroborated by epitope mapping of polyclonal antibodies using 185 overlapping pentadeca-peptides covering the whole sequence of the precursor. The results show that the cocoa vicilin is encoded by a single gene and that heterogeneity of the vicilin subunits must be attributed to statistical post-translational modifications. Localisation of the precursor regions, from which the various subunits are derived, will be helpful for the future identification of aroma-relevant peptides generated during the fermentation process.

Vicilin and convicilin are potential major allergens from pea.

Allergic reactions to pea (Pisum sativum) ingestion are frequently associated with lentil allergy in the Spanish population. Vicilin have been described as a major lentil allergen. To identify the main IgE binding components from pea seeds and to study their potential cross-reactivity with lentil vicilin. A serum pool or individual sera from 18 patients with pea allergy were used to detect IgE binding proteins from pea seeds by immunodetection and immunoblot inhibition assays. Protein preparations enriched in pea vicilin were obtained by gel filtration chromatography followed by reverse-phase high-performance liquid chromatography (HPLC). IgE binding components were identified by means of N-terminal amino acid sequencing. Complete cDNAs encoding pea vicilin were isolated by PCR, using primers based on the amino acid sequence of the reactive proteins. IgE immunodetection of crude pea extracts revealed that convicilin (63 kDa), as well as vicilin (44 kDa) and one of its proteolytic fragments (32 kDa), reacted with more than 50% of the individual sera tested. Additional proteolytic subunits of vicilin (36, 16 and 13 kDa) bound IgE from approximately 20% of the sera. The lentil vicilin allergen Len c 1 strongly inhibited the IgE binding to all components mentioned above. The characterization of cDNA clones encoding pea vicilin has allowed the deduction of its complete amino acid sequence (90% of sequence identity to Len c 1), as well as those of its reactive proteolytic processed subunits. Vicilin and convicilin are potential major allergens from pea seeds. Furthermore, proteolytic fragments from vicilin are also relevant IgE binding pea components. All these proteins cross-react with the major lentil allergen Len c 1.

Effect of sugars on the association between cowpea vicilin (7S storage proteins) and fungal cells

Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. In the present work vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia. Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the vicilins to the surface of cells of S. cerevisiae and F. oxysporum treated with this protein. After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of vicilins associated to chitinous structures present in yeast cells. Our results showed that vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine.

Selective Neoglycosylation Increases the Structural Stability of Vicilin, the 7S Storage Globulin from Pea Seeds

The effects of glycosylation on the stability and subunit interactions of vicilin, the major storage protein in pea seeds, were investigated. Glycosylated vicilin derivatives were prepared by alkylation of lysine ϵ-amino groups with various carbohydrates. Average modification levels of 13.4 ± 3.0, 11.1 ± 3.6, 7.5 ± 4.2, and 4.7 ± 0.3 moles of carbohydrate/mol of vicilin were obtained with glucose, galactose, galacturonic acid, and lactose, respectively. Nondenaturing polyacrylamide gel electrophoresis and size-exclusion chromatography indicated that the quaternary structure and hydrodynamic radius of vicilin were not affected by glycosylation at the levels used. We have previously shown that application of hydrostatic pressure causes dissociation of vicilin subunits [C. Pedrosa and S. T. Ferreira (1994) Biochemistry 33, 4046–4055]. Analysis of pressure dissociation data allowed determination of the Gibbs free energy change (ΔGdiss) and molar volume change (ΔVdiss) of dissociation of vicilin subunits. For unmodified vicilin, ΔGdiss = 18.2 kcal/mol and ΔVdiss = −102 ml/mol. Glycosylated vicilin derivatives were significantly stabilized against subunit dissociation, with ΔGdiss of 19.4, 19.2, 20.6, and 22.1 kcal/mol for glucose, galactose, lactose, and galacturonic acid derivatives, respectively. No changes in ΔVdiss were found for the glucose and galactose derivatives, whereas ΔVdiss of −128 and −135 ml/mol, respectively, were found for the lactose and galacturonic acid derivatives. The glycosylated derivatives also appeared more resistant to unfolding by guanidine hydrochloride than unmodified vicilin. Intrinsic fluorescence lifetime measurements showed that glycosylation caused a significant increase in heterogeneity of the fluorescence decay, possibly reflecting increased conformational heterogeneity of glycosylated derivatives relative to unmodified vicilin. These results indicate that the stability and subunit interactions of vicilin may be modulated by mild, selective glycosylation at low modification levels, an effect that may be of interest in the study of other oligomeric proteins.

Deterministic pressure-induced dissociation of vicilin, the 7S storage globulin from pea seeds: effects of pH and cosolvents on oligomer stability.

A thermodynamic characterization of subunit association of vicilin, a storage protein from pea seeds, was performed using a combination of hydrostatic pressure and fluorescence spectroscopy. Application of pressure up to 2.4 kbar caused dissociation of vicilin subunits, as revealed by (1) size-exclusion PPLC of pressurized samples, (2) fluorescence anisotropy measurements of a dansyl-vicilin conjugate under pressure, and (3) quenching of the intrinsic fluorescence of vicilin. Pressure dissociation data were well described by a model for dissociation of a trimer. This enabled calculation of the standard molar volume change of association (delta Vo) and the equilibrium dissociation constant at atmospheric pressure (Ko); at pH 10 these were found to be delta Vo = 146 mL/mol and Ko = 2.2 x 10(-15) M2, respectively, corresponding to C1/2 (the concentration of protein at 50% dissociation at atmospheric pressure) = 18 nM and a stabilization free energy of -19.6 kcal/mol for the oligomer. Vicilin exhibited an anomalously low dependence on protein concentration for pressure dissociation. This appeared related to conformational changes in the dissociated subunits, which caused a loss of ca. 5 kcal/mol in the free energy of association and to structural/energetic heterogeneity in the population of oligomers. Pressure dissociation was markedly pH-dependent, with a stabilization free energy loss of 3.4 kcal/mol upon raising pH from 9 to 10. Circular dichroism and intrinsic fluorescence lifetime measurements at atmospheric pressure showed that the structure of vicilin was largely unaffected by pH in the range investigated. These results suggest that the effect of pH may involve deprotonation of lysine residues participating in salt bridges between vicilin subunits. Pressure dissociation of vicilin was significantly inhibited by addition of salts (NaCl, KCl, LiCl) or glycerol. Dissociation curves obtained in the presence of salts enabled calculation of the free energies of stabilization (ranging from ca. -1.2 to -2.4 kcal/mol) of the vicilin oligomers by these cosolvents. The similar effects of salts or glycerol suggest a common mechanism of stabilization of the oligomer involving exclusion of the cosolvents from the protein interface and preferential hydration of the protein.

Molecular heterogeneity and genetics of Vicia faba seed storage proteins

Legumin and vicilin were purified from seeds of Vicia faba L. var. Scuro, characterized in different electrophoretic systems, and used to produce polyclonal antibodies in rabbits. Two-dimensional electrophoretic studies showed a wide range of heterogeneity in the subunits of both legumin and vicilin. Legumin was found to be composed of 29 disulphide-linked subunit pairs with different molecular weight and/or isoelectric point. Western blot analysis of legumin of several mutants revealed molecular polymorphism based on a corresponding gene family. Three different α-major legumin patterns were found, and inheritance studies showed that the 34.3-kD legumin polypeptide is the product of one locus, Lg-1α, which is the first legumin genetic locus described in Vicia faba. Vicilin was found to be composed of as many as 59 subunits distributed in a molecular weight range of 65.7 to 42.8 kD (major polypeptides) and 37.2 to 15.2 kD (minor polypeptides), with different isoelectric points. A model is proposed that explains the possible formation of the minor subunits and the major subunits of 48.2 and 46 kD molecular weight (MW) from proteolytic cleavages and/or glycosilation of precursor polypeptides. Ten different vicilin electrophoretic patterns were observed among the analyzed accessions, which showed large molecular polymorphism that proved to be under genetic control.