Purification and properties of three albumins from Triticum aestivum seeds

Three albumins were isolated from bread wheat seeds by gel filtration on Sephadex G-100 and differential preparative disc-electrophoresis on polyacrylamide gel. The proteins were obtained in sufficient yield and purity to carry out characterisation studies. The electrophoretic mobilities of the three albumins in polyacrylamide discs in glycine—Tris medium, pH 9·5, were 0·28, 0·34 and 0·39 (referred to that of bromophenol blue taken as 1). The isoelectric points were 6·40, 6·40 and 5·35 and the molecular weights 17, 700, 18, 200 and 18, 900, respectively. The amino acid compositions of the purified albumins were very similar. The correlation of the purified albumins with those isolated from bread wheats by other authors is discussed.     

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.     

Relationship between proportion and composition of albumins,and in vitro protein digestibility of raw and cooked pea seeds (Pisum sativum L.)

BACKGROUND: Peas provide an excellent plant protein resource for human diets, but their proteins are less readily digestible than animal proteins. To identify the relationship between composition and in vitro digestibility of pea protein, eight pea varieties with a wide range of protein content (157.3–272.7 g kg^?1) were determined for the proportion of albumins and globulins, their compositions using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and in vitro protein digestibility (IVPD) before and after heat treatment using a multi‐enzyme (trypsin, chymotrypsin and peptidase) method. RESULTS: The proportion of albumins based on total seed protein content decreased from 229 to 147 g kg^?1 as seed protein content increased from 157.3 to 272.7 g kg^?1, while the proportion of globulins increased from 483 to 590 g kg^?1. The IVPDs of eight raw pea seeds were 79.9–83.5%, with significant varietal variations, and those were improved to 85.9–86.8% by cooking. Albumins, including (pea albumins 2) PA2, trypsin inhibitor, lectin and lipoxygenase, were identified as proteolytic resistant proteins. Globulins were mostly digested by protease treatment after heating. CONCLUSION: The quantitative ratio of albumins and globulins, and the quantitative variations of albumin protein components, including lipoxygenase, PA2, lectins and trypsin inhibitors, appear to influence the protein digestibility of both raw and cooked pea seeds. Copyright © 2010 Society of Chemical Industry     

Isoelectric fractionation and some properties of a protease from soyabean seeds

A protease, capable of hydrolysing benzoyl DL -arginine p-nitroanilide(BAPA), and L-amino acid β-naphthylamide derivatives, was purified, by isoelectric focusing in the region pH 3–6, from dormant and 6-day germinated soyabean seeds. The enzyme was focused at pH 4·80. The K_m value using BAPA as substrate was found to be 5·03 × 10⁻⁴M . Maximum activity of the enzyme towards BAPA was obtained in the pH 8·2–8–5 region. Slight activation was observed in the presence of 0·05 M concentration of Ca²⁺ and Mg²⁺ ions. The protease lacked caseinolytic activity, and was not inhibited by Kunitz soyabean trypsin inhibitor.     

Characterization and insecticidal properties of globulins and albumins from Luetzelburgia auriculata (Allemao) Ducke seeds towards Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)

We evaluated the development of Callosobruchus maculatus growing in artificial seeds composed of Vigna unguiculata (cowpea) seed flour mixed with exogenous proteins from Luetzelburgia auriculata. Albumin and globulin fractions from Luetzelburgia auriculata were characterized in terms of protein content, amino acid composition and antimetabolic proteins (trypsin/chymotrypsin inhibitory, porcine pancreatic alpha-amylase inhibitory, lectin activity and presence of chitin-binding proteins). Both fractions were distinct in terms of protein content and diversity as determined by electrophoresis. Lectin activity was present only in the globulins. Neither fraction exhibited inhibitory activity towards porcine pancreatic alpha-amylase but trypsin inhibition was observed. Interestingly, chitin-binding proteins were detected in both protein classes. Albumins had a severe effect upon larval development and were detrimental to insect emergence (LD₅₀=0.4%) while globulins displayed slight toxicity upon larval development and no effect towards insect emergence. The presence of serine proteinase inhibitory activity and chitin binding proteins could explain, at least in part, the harmful effects on C. maculatus development while lectin activity and amino acid availability seem not to correlate with any deleterious effects. Luetzelburgia auriculata would be an interesting source of seed proteins to study behavior of C. maculatus upon infestation and genes coding for insecticidal proteins could become candidates for molecular biology programs devoted to producing transgenic seeds expressing resistance towards the beetle.     

Effect of non-enzymatic glycosylation of pea albumins on their immunoreactive properties

The aim of this study was to determine the effect of non-enzymatic glycosylation of pea proteins on their immunoreactive properties. Extracted total pea albumins were glycated. No changes were found in molecular weight distribution of total pea albumins before and after glycation using size exclusion chromatography and SDS–PAGE methods. SDS–PAGE GLYCO test stained the glycated proteins and OPA method showed 15% progress in glycation. Glycated and unglycated pea albumins were orally and intraperitoneally administered to Balb/C mice. Serum specific IgG and IgA and sIgA were determined. No difference in serum specific IgG level was found after oral mice immunization with TA and GTA. In the presence of antigen SPL lymphocytes culture showed higher proliferation activity as compared to the culture without the antigen addition. The glycation does not change the immunoreactivity of proteins significantly. During the presented route of immunization with TA and GTA specific tolerance mechanism could be induced.     

Carbohydrates in raw and germinated seeds from mung bean and chick pea

The contents of hydrophilic extractives, lipids, protein, starch, non-starch polysaccharides, Klason lignin and ash were determined in raw and germinated seeds of mung bean (Phaseolus aureus) and chick pea (Cicer arietinum). Several low molecular weight carbohydrates were found in the hydrophilic extractives. During germination, due mainly to their use as an easily available source of germinating energy, there was a rapid decrease of the raffinose family oligosaccharides in mung bean and a somewhat slower decrease in chick pea. Changes in the content and composition of dietary fibres during germination was small.     

A study of lipoxidase in pea seeds and seedlings

Lipoxidase enzyme was isolated and partly purified from pea seeds by ultracentrifugation, ammonium sulphate precipitation, Sephadex and DEAE-Cellulose column chromatography. The pH optimum of the enzyme was 7.2 and the Michaelis–Menton constant 2.3 × 10^?3 M . Disc gel electrophoresis revealed the presence of 3 to 4 isoenzymes, while the molecular weight determination in the presence of sodium dodecyl sulphate gave a value of 7.4 × 10⁴. The presence of lipoxidase in pea mitochondria and in the peroxisome-like bodies was demonstrated. A low enzyme activity was found with the purified chloroplasts but a much higher activity was found in the plastids and in the cytoplasm of the etiolated tissue. The enzyme was found not to be compartmentalised in any particulate fraction of the pea seedlings investigated.     

Primary structure and polymorphism of 2S albumins from seeds of Andean lupin (Lupinus mutabilis Sweet)

 2S albumins were isolated from seeds of Andean lupin (Lupinus mutabilis Sweet) by buffer extraction, ammonium sulphate precipitation and ultrafiltration followed ion-exchange and reversed-phase HPLC. The 2S albumin preparation (LM2S) contained eight albumins. The complete amino acid sequences of small and large subunits of three major albumins (LM2S-4, -5 and -6) were determined by automated Edman degradation of S-pyridylethylated polypeptides and peptides obtained from them by enzymatic digestions. The small subunit of the dominant 2S albumin (LM2S-4) contains 39 amino acid residues and has a molecular mass of 4731 Da. The large subunit of LM2S-4 contains 74 amino acid residues (molecular mass=8708 Da). Two 2S albumin isoforms (LM2S-4 and -6) are due to the expression of two distinct genes; LM2S-6 isoform has eight amino acid replacements when its sequence is compared with the sequence of LM2S-4. The LM2S-5 isoform contains an identical small subunit to LM2S-4, and has in comparison with LM2S-4 two additional amino acid residues at the N-terminus of the large subunit. The amino acid sequences of 2S isoforms from L. mutabilis showed high homology (78–83% identity) with 2S albumins from different Old World Lupinus species. Received: 15 December 1998 / Revised version: 9 February 1999     

Subtilisin inhibitors from legume seeds: A purification procedure

Subtilisin inhibitors were prepared from ethanol-acetone precipitated seed extracts of chick peas, jack beans and broad beans, heated to destroy endogenous proteases. Trypsin inhibitors were complexed with trypsin and separated from subtilisin inhibitors by molecular sieve chromatography. The eluates were again heated to avoid tryptic degradation. The procedure was easy to handle and gave satisfactory yields. In a final step two subtilisin isoinhibitors from chick peas were purified to electrophoretic homogeneity. They constitute 0.01% of the defatted dry weight.     

Effects of water blanching on pea seeds.

Laboratory scale experiments were conducted to elucidate the main mechanisms responsible for changes in the proportions of ascorbic acid (AA) and dehydroascorbic acid(DHA) in peas during water blanching. Studies utilized hand-harvested peas from specially grown Dark Skinned Perfection (DSP), Swan and Swift cultivars. The influence of pea size/maturity, blanch time and damage to the pea were studied over the temperature range 35–97°C.
With increasing DSP pea size/maturity, the proportion of AA oxidized increased, and the proportion of AA leached into the water decreased when blanching between 45 and 65°C. Maximum AA oxidation occurred at 60°C and leaching became the prime mode of loss above 70°C. Leaching of AA from DSP peas increased almost linearly from 40 to 97°C.
Damaging peas by bruising and slitting the testa, induced enhanced AA oxidation below 60°C and allowed immediate leaching of vitamin C largely as DHA even at the lowest blanch temperatures. Results suggested that the oxygen content of the tissues was a factor limiting the amount of AA oxidation. Cultivars Swift and Swan contained higher proportions of DHA particularly in the testa tissues, and calculations indicated that greater proportions of vitamin C were leached as DHA. A negligible proportion of AA was oxidized and some 28% of the initial AA was leached into the water when undamaged DSP peas were blanched at 97°C for 1 min. Bruised plus slit peas lost significantly more AA than undamaged peas when blanched at 97°C. Further evidence indicated that the micropyle serves as a major pathway for leaching losses.     

A study of stability of O/W emulsions stabilized by soybean and pea globulins

Flotation stability of O/W emulsions, stabilized by total globulins of soybean and peas increases with an increase in protein concentration and diminishes with NaCI concentration. Stability increases significantly as a result of long emulsion storage (24 h and 72 h) at room temperature, that is most likely caused by the flocculation process. Heating of emulsions, stabilized by soybean globulins at 100 °C and 120 °C results in an increase in their flotation stability. Emulsion heating during the first 10 min is associated with diminishing in stability to coalescence, that becomes more pronounced as protein concentration decreases. At further heating the resistance to coalescence remains unchanged apparently because of interface adsorption layers gelation.     

Heterogeneity of subunit composition in lupin globulins. Comparison of proteins from a single seed and from a number of seeds

The subunit compositions of a legumin-like (globulin 8) and a vicilin-like protein (globulin 4) extracted from a plurality of seeds and from a single seed both in commercial and in a selected cultivar of Lupinus albus were studied. In the case of globulin 4, a very similar number of bands were observed in SDS-PAGE, in the protein extracted from a batch of seed and in the same globulin isolated from a single seed. For globulin 8 the SDS-PAGE pattern showed fewer subunits in the protein from the batch of seeds than in that from a single seed. This is more pronounced in the single seed and in the plurality of seeds of a selected cultivar. Reasons for this behaviour are discussed.     

Functional properties of protein hydrolysates from pea (Pisum sativum,L) seeds

The aim of this study was to investigate the effects of partial enzymatic hydrolysis on functional properties of two different pea protein isolates obtained from two pea genotypes, Maja and L1. Papain and commercial protease (Streptomyces griseus protease) were used for protein modification. Solubility, emulsifying and foaming properties were estimated at four different pH values (3.0, 5.0, 7.0 and 8.0). Papain increased solubility of L1 pea protein isolate at pH 3.0, 5.0 and 8.0, emulsifying properties and foaming capacity at all pH values. Otherwise, papain increased solubility of Maja pea protein isolate only at pH 8.0. This pea protein isolate modified with both enzymes formed emulsions with improved stability at lower pH (3.0, 5.0). The commercial protease‐prepared pea protein isolates showed generally low solubility and different emulsifying and foaming properties. Proper selection of enzyme, conditions of hydrolysis and genotypes could result in production of pea protein isolates with desirable functional properties.