Insecticidal components from field pea extracts: isolation and separation of peptide mixtures related to pea albumin 1b

Chromatographic fractionation of crude extracts (C8 extracts) from the protein-enriched flour of commercial field peas (Pisum sativum L.) has been shown here to yield peptide mixtures related to the pea albumin 1b (PA1b) family of cysteine-rich plant peptides. The mixtures were obtained initially by flash chromatography with silica gel. Following elution of soyasaponins and lysolecithins, the end fractions obtained with the use of two flash chromatographic solvent systems displayed activity in a flour disk antifeedant bioassay with the rice weevil [Sitophilus oryzae (L.)]. Chemical properties of these mixtures were compared by thin-layer chromatography, high-performance liquid chromatography (HPLC), IR, MS, and amino acid analyses. The major peptides of C8 extracts, with average masses of 3752, 3757, and 3805 Da, were isolated by anion exchange chromatography. Samples enriched in the peptide of mass 3752 were isolated by cation exchange chromatography. Reduction plus alkylation experiments in combination with electrospray ionization mass spectrometry showed that C8 extracts contained about 10 peptides and, like PA1b, each peptide possessed six cysteine residues (three disulfide bonds). Disulfide bond reduction with 2-mercaptoethanol destroyed the antifeedant activity. The native peptides of C8 extracts were found to be resolved into nine peaks with XTerra HPLC columns operating at alkaline pH. These columns were employed to assess the distribution of pea peptides in the isolated fractions, with photodiode array and electrospray detection.     

High molecular weight glutenin subunits in some durum wheat cultivars investigated by means of mass spectrometric techniques

The primary structures of high molecular weight glutenin subunits (HMW-GS) of 5 Triticum durum Desf. cultivars (Simeto, Svevo, Duilio, Bronte, and Sant'Agata), largely cultivated in the south of Italy, and of 13 populations of the old spring Sicilian durum wheat landrace Timilia (Triticum durum Desf.) (accession nos. 1, 2, 3, 4, 7, 8, 9, 13, 14, 15, SG1, SG2, and SG3) were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high performance liquid chromatography/nanoelectrospray ionization mass spectrometry (RP-HPLC/nESI-MS/MS). M(r) of the intact proteins determined by MALDI mass spectrometry showed that all the 13 populations of Timilia contained the same two HMW-GS with 75.2 kDa and 86.4 kDa, whereas the other durum wheat cultivars showed the presence of the expected HMW-GS 1By8 and 1Bx7 at 75.1 kDa and 83.1 kDa, respectively. By MALDI mass spectrometry of the tryptic digestion peptides of the isolated HMW-GS of Timilia, the 1Bx and 1By subunits were identified as the NCBInr Acc. No AAQ93629, and AAQ93633, respectively. Sequence verification for HMW-GS 1Bx and 1By both in Simeto and Timilia was obtained by MALDI mass mapping and HPLC/nESI-MSMS of the tryptic peptides. The Bx subunit of Timila presents a sequence similarity of 96% with respect to Simeto, with differences in the insertion of 3 peptides of 5, 9, and 15 amino acids, for a total insertion of 29 amino acids and 25 amino acid substitutions. These differences in the amino acidic sequence account for the determined Δm of 3294 Da between the M(r) of the 1Bx subunits in Timilia and Simeto. Sequence alignment between the two By subunits shows 10 amino acid substitutions and is consistent with the Δm of 148 Da found in the MALDI mass spectra of the intact subunits.     

Fractionation of lentil seeds (Lens culinaris Medik.) for insecticidal and flavonol tetraglycoside components

Crude methanol extracts from four cultivated varieties of mature lentil seeds (Lens culinaris Medik.) were found to possess antifeedant and insecticidal properties in laboratory tests with the rice weevil (Sitophilus oryzae L.), an insect pest of stored products. Flash chromatography with silica gel on active Diaion HP-20 methanol extracts gave flavonol, lysolecithin, soyasaponin, and peptide fractions, as determined by HPLC and electrospray ionization LC/MS. The flavonol fraction was shown by high-resolution NMR experiments to contain a mixture of kaempferol 3-O-beta-glucopyranosyl(1-->2)-O-[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside-7-O-alpha-rhamnopyranoside and, tentatively, kaempferol 3-O-beta-glucopyranosyl(1-->2)-O-[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside-7-O-alpha-rhamnopyranoside. These inactive tetraglycosides, although inseparable under the reported HPLC conditions, were detected by NMR spectroscopy in nearly equal proportions. Three lysolecithins were identical to those previously identified in pea extracts. Soyasaponin I (soyasaponin Bb) and soyasaponin VI (soyasaponin betag) were found in Diaion HP-20 methanol extracts. An insecticidal lentil peptide with a mass of 3881 Da, isolated from an Eston variety in small quantities by anion exchange chromatography, was related to the cysteine-rich pea albumin 1b class of botanical insecticides. Binary mixtures of the insecticidal lentil peptide and soybean soyasaponin I were synergistic in tests with S. oryzae.     

Insecticidal components from field pea extracts: soyasaponins and lysolecithins

Extracts from field peas (Pisum sativum L.) have previously been shown to have a utility to control insect pests. To identify potentially new bioinsecticides in field crops, we describe the fractionation of impure extracts (C8 extracts) derived from protein-rich fractions of commercial pea flour. The activity of separated fractions was determined by a flour disk antifeedant bioassay with the rice weevil [Sitophilus oryzae (L.)], an insect pest of stored products. Bioassay-guided fractionation showed that the triterpenoid saponin fractions were partly responsible for the antifeedant activity of C8 extracts. Soyasaponin I (soyasaponin Bb), isolated from peas and soybeans, and mixtures of soyasaponins, comprised of soyasaponins I-III and isolated from soybeans, were inactive antifeedants, but dehydrosoyasaponin I (the C-22 ketone derivative of soyasaponin I), a minor component found in C8 extracts, was shown to be an active component. Dehydrosoyasaponin I (soyasaponin Be) and soyasaponin VI (soyasaponin betag) coeluted under conditions of silica gel thin-layer chromatography and C18 high-performance liquid chromatography. However, dehydrosoyasaponin I could be isolated from saponin-enriched fractions with a reversed phase column of styrene/divinylbenzene operated at alkaline pH. Phospholipids of the lysolecithin type were also identified in saponin fractions of C8 extracts from peas. Three of the lysolecithins were inactive alone against rice weevils, but mixtures of these phospholipids enhanced the insecticidal activity of dehydrosoyasaponin I.     

Isolation of oligopeptides from the water-soluble extract of goat cheese and their identification by mass spectrometry

A procedure for the separation and identification of small peptides from the water-soluble fraction of a goat cheese was developed. The water-soluble extract was ultrafiltered (1000 Da membrane cutoff), and peptides were isolated by sequential chromatography: size exclusion chromatography (HPLC-grade water), anion exchange chromatography (phosphate buffer gradient), and semipreparative reverse-phase high-performance liquid chromatography (water/acetonitrile gradient). The fractions obtained were analyzed by combined mass spectrometry methods including electrospray ionization, liquid secondary ionization, and tandem mass spectrometry to identify and to confirm the sequences of 28 tri- to octapeptides naturally appearing in goat cheese during ripening. Among these peptides, 26 are produced by degradation of caseins but do not correspond to the known specific cleavages due to chymosin. Only low correlation was found between hydrophobicity of peptides and HPLC elution time with acetonitrile gradient.     

Enzyme-induced gelation of extensively hydrolyzed whey proteins by Alcalase: peptide identification and determination of enzyme specificity

Extensive hydrolysis of whey protein isolate by Alcalase was shown to induce gelation mainly via hydrophobic interactions. The aim of this work was to characterize the peptides released in order to better understand this phenomenon. The apparent molecular mass distribution indicated that aggregates were formed by small molecular mass peptides (<2000 Da). One hundred and thirty peptides with various lengths were identified by reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Alcalase was observed to have a high specificity for aromatic (Phe, Trp, and Tyr), acidic (Glu), sulfur-containing (Met), aliphatic (Leu and Ala), hydroxyl (Ser), and basic (Lys) residues. Most peptides had an average hydrophobicity of 1-1.5 kcal/residue and a net charge of 0 at the pH at which gelation occurred (6.0). Therefore, an intermolecular attractive force such as hydrophobic interaction suggests the formation of aggregates that further leads to the formation of a gel.     

Relationship between MALDI-TOF analysis of beta-CN f193-209 concentration and sensory evaluation of bitterness intensity of aged cheddar cheese

An internal standard method was previously developed to measure the concentration of a synthetic bitter peptide, beta-CN f193-209, by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The objective of this study was to evaluate the relationship between beta-CN f193-209 concentration in an aqueous extract of aged Cheddar cheese and bitterness intensity of the cheese. Concentrations of beta-CN f193-209 in cheese extracts were determined by MALDI-TOF at 0, 120, 180, and 270 days. Trained panels evaluated the bitterness intensity of the cheeses at 180 and 270 days. Correlation coefficients between MALDI and sensory data at 180 and 270 days were 0.803 and 0.554, respectively. The decreased correlation may be due to the presence of other bitter peptides more responsible for bitterness at longer aging or the production of compounds that mask bitterness intensity.     

Identification of a new form of lipid transfer protein (LTP1) in wheat seeds

Recently, this laboratory has isolated from barley and beer extract an isoform of lipid transfer protein (LTP1), which was not fully sequenced (Jégou, S.; Douliez, J. P.; Mollé, D.; Boivin, P.; Marion, D. J. Agric. Food Chem. 2000, 48, 5023--5029). It was named LTP1b and exhibited a molecular weight 294 Da higher than that of the known LTP1. This paper reports the finding of an LTP1 isoform in wheat that also exhibits an excess of 294 Da compared to the native protein. Amino acid sequencing, reduction and alkylation, and mass spectrometry showed that this new LTP1b possesses the same N-terminal sequence as the native LTP1, suggesting that the difference resides in the binding of an adduct which has a molecular weight of 294 Da. The aim of the present paper is to highlight various biophysical techniques that afford the identification of such an isoform-like LTP1 and to correlate this finding with other isoforms of LTP1 that were isolated from other plants but not fully sequenced.     

Determination of exposed sulfhydryl groups in heated beta-lactoglobulin A using IAEDANS and mass spectrometry

This paper takes a new approach to determining which sulfhydryl groups are exposed during the heat denaturation of bovine beta-lactoglobulin A. The sulfhydryl groups exposed after heating were blocked with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS). The results show that IAEDANS is a suitable blocking agent, and its absorbance at 336 nm enabled the quantification of exposed sulfhydryl groups in a mixture of protein species by gel permeation chromatography. Combined with the specific fragmentation of bound IAEDANS by matrix-assisted laser desorption ionization (MALDI) MS/MS in negative ionization mode, this facilitated the identification of peptides that contained blocked cysteines after enzymatic digestion of the protein. During MALDI MS/MS of the peptides, in positive ionization mode, the IAEDANS molecule remained bound to the cysteines, making it possible to identify exactly which cysteine had been exposed after heating. In beta-lactoglobulin A it was found that cysteine 66 and cysteine 160 were predominantly exposed regardless of the length of exposure to heat.     

Ethanol Fermentation of Starch from Field Peas

Field peas (Pisum sativum) were evaluated as a potential feedstock for ethanol production. Ground peas were dry‐milled and separated into starch, protein, and fibrous fractions by air classification. Starch‐enriched fractions prepared from whole peas and dehulled peas contained 73.7% wt and 77.8% wt starch, respectively, a nearly two‐fold enrichment compared with whole peas. The fractions were liquefied and saccharified using industrial α‐amylase and glucoamylase at recommended enzyme loadings. A final ethanol concentration of 11.0% (w/v) was obtained in 48–52 hr, with yields of 0.43–0.48 g of ethanol/g of glucose. Starch present in whole ground peas was also saccharified and fermented, with 97% of the starch fermented when an autoclaving step was included in the liquefaction stage.     

Sulphur amino acid content and nutritive value of pea and cauliflower crude protein as influenced by Sulphur deficiency

Cystine concentrations (g/16g N) in plant tissue declined more under S deficiency conditions than methionine. In N-balance trials with rats the negative effect of S deficiency on S amino acid composition of peas and cauliflower resulted in a corresponding decrease of the biological value of the protein.     

Hydrophobicity of bitter peptides from soy protein hydrolysates

Soy peptides were characterized for flavor, chemical properties, and hydrophobicity to investigate their relationships with bitterness. Five peptide fractions ranging in average molecular mass from 580 to 11300 Da were fractionated by ultrafiltration from two commercial soy protein hydrolysates. The bitterness of fractionated peptides was related to molecular mass, with maximum bitterness observed at approximately 4000 Da for one hydrolysate and 2000 Da for the other. The bitterness increased as the peptide M(w) decreased to 3000 Da for the first hydrolysate and to 2000 Da for the second one and then decreased as the peptide M(w) decreased below 1000 Da. The peptide fraction with molecular mass of <1000 Da showed the lowest bitterness for both. The hydrophobicity data based on Q values do not support Ney's Q rule as a predictor of bitterness for soy peptides.     

Purification and characterization of novel proteinase inhibitors from dried figs

Two types of the natural organic matter P and B were isolated from dried figs by gel permeation and high-performance liquid chromatography. The characterizations of their molecular structures were also performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and infrared absorption spectrometry. As a result, these samples were revealed to inhibit the serine and cysteine proteinases chymotrypsin and papain (K(i) = 10(-)(6)-10(-)(4) M). The optimal inhibitory pH values of the P and B samples were observed to be approximately 5.5 and 5.0, respectively. The analyses of their UV-vis absorption spectra and infrared absorption spectra indicated that they would be a kind of humic substance. The mass spectrometry analyses showed that they possessed relatively small heterogeneous molecules with molecular masses around 692, 845, and 1389 Da for the P sample and around 551, 704, and 909 Da for the B sample.     

Isolation and characterization of 2S cocoa seed albumin storage polypeptide and the corresponding cDNA

The amine pool of cocoa is known to be an essential component for the development of the typical cocoa flavor. To better understand and to produce an intense in vitro cocoa flavor, identification of the polypeptides that are the source of the amine flavor precursor pool is essential. Chromatographic analysis of the polypeptide profile of unfermented cocoa resulted in identification of a novel storage polypeptide of M(r) 8515. The N-terminal sequence of the first 34 residues of the purified polypeptide shows similarity to 2S storage albumins of cotton and Brazil nut and sweet protein, Mabinlin. To identify the corresponding cDNA of the putative cocoa 2S albumin, 18 randomly chosen clones from the cDNA library of immature Theobroma cacao seed mRNA were sequenced, and a full-length cDNA clone encoding a protein harboring the N-terminal sequence of the novel polypeptide was selected. The open reading frame of the clone encodes a polypeptide of M(r) 17125. Comparison of the translated amino acid sequence of the precursor protein or the mature polypeptide against the Swiss-Prot and TrEMBL databases shows high sequence similarity (>52%) and identity (>38%) to many plant 2S albumins. Tryptic peptide mass fingerprinting of the purified polypeptide by high-performance liquid chromatography-electrospray ionization mass spectrometry shows 10 masses that match the expected tryptic peptides of the deduced sequence. Together with the published work on plant 2S albumin processing, the results presented here suggest that post-translational processing yields a 73-residue polypeptide (residue positions 78-150) corresponding to the 9 kDa subunit of the mature cocoa 2S albumin protein.     

Maillard reaction induced lactose attachment to bovine beta-lactoglobulin: electrospray ionization and matrix-assisted laser desorption/ionization examination

Nonenzymatic attachment of lactose to beta-lactoglobulin (beta-Lg) was investigated under different conditions. Solubilized conditions, dry environment, and a combination of dry and solubilized environments, were examined for their effects on lactosylation. Temperatures ranging from 50 to 65 degrees C and time intervals between 1 h and 4 days were used. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry were implemented to examine the reaction products. Maximum attachment efficiency occurred at 65 degrees C held for 3 h in dry-way conditions. Incubations held for long periods of time under dry-way conditions suggest possible denaturation. Both ESI and MALDI data suggest beta-Lg removal in the solubilized samples held for long periods of time. A combination of solubilized and dry environments led to very similar mass spectrogram results over time.     

Leaching and plant offtake of N in field pea/cereal cropping sequences with incorporation of 15N-labelled pea harvest residues

Abstract. Field peas (Pisum sativum L.) were grown in sequence with winter wheat (Triticum aestivum L.) or spring barley (Hordeum vulgare L.) in large outdoor lysimeters. The pea crop was harvested either in a green immature state or at physiological maturity and residues returned to the lysimeters after pea harvest. After harvest of the pea crop in 1993, pea crop residues (pods and straw) were replaced with corresponding amounts of ¹⁵N‐labelled pea residues grown in an adjacent field plot. Reference lysimeters grew sequences of cereals (spring barley/spring barley and spring barley/winter wheat) with the straw removed. Leaching and crop offtake of ¹⁵N and total N were measured for the following two years. These treatments were tested on two soils: a coarse sand and a sandy loam. Nitrate concentrations were greatest in percolate from lysimeters with immature peas. Peas harvested at maturity also raised the nitrate concentrations above those recorded for continuous cereal growing. The cumulative nitrate loss was 9–12 g NO₃‐N m^–2 after immature peas and 5–7 g NO₃‐N m^–2 after mature peas. Autumn sown winter wheat did not significantly reduce leaching losses after field peas compared with spring sown barley. ¹⁵N derived from above‐ground pea residues accounted for 18–25% of the total nitrate leaching losses after immature peas and 12–17% after mature peas. When compared with leaching losses from the cereals, the extra leaching loss of N from roots and rhizodeposits of mature peas were estimated to be similar to losses of ¹⁵N from the above‐ground pea residues. Only winter wheat yield on the coarse sand was increased by a previous crop of peas compared to wheat following barley. Differences between barley grown after peas and after barley were not statistically significant. ¹⁵N lost by leaching in the first winter after incorporation accounted for 11–19% of ¹⁵N applied in immature pea residues and 10–15% of ¹⁵N in mature residues. Another 2–5% were lost in the second winter. The ¹⁵N recovery in the two crops succeeding the peas was 3–6% in the first crop and 1–3% in the second crop. The winter wheat did not significantly improve the utilization of ¹⁵N from the pea residues compared with spring barley.     

Functionality of beta-casein peptides: importance of amphipathicity for emulsion-stabilizing properties.

To investigate structure-function relationships with regard to emulsion-stabilizing properties, peptides from bovine beta-casein (betaCN), obtained by plasmin hydrolysis and fractionation of the hydrolysate, were isolated and identified on the basis of their masses determined by electrospray ionization mass spectrometry, the primary structure of the intact protein, and the known specificity of the enzyme. An amphipathic peptide fraction was fractionated further by ion-exchange chromatography and subsequent hydrophobic interaction chromatography resulting in the components betaCN[f 1-105/107] and betaCN[f 29-105/107]. The latter peptides had poor emulsion-stabilizing properties compared to the former ones, and the stability of an emulsion formed with betaCN[f 29-105/107] was also more sensitive to hydrophobic impurities than that of an emulsion formed with betaCN[f 1-105/107]. The highly charged N-terminal part appeared to be important for the emulsion-stabilizing properties of these peptides. A hypothesis for the structure-function relationship is given.     

Comparison of the α-amylase inhibitor-1 from common bean (Phaseolus vulgaris) varieties and transgenic expression in other legumes--post-translational modifications and immunogenicity

The seeds of peas (Pisum sativum) and chickpeas (Cicer arietinum) expressing a gene for α-amylase inhibitor-1 (αAI) from the common bean (Phaseolus vulgaris) are protected from damage by old world bruchids (pea and cowpea weevils). Here, we used electrospray ionization time-of-flight mass spectrometry to compare the post-translational modifications of αAI from transgenic sources with the processed forms of the protein from several bean varieties. All sources showed microheterogeneity with differences in the relative abundance of particular variants due to differences in the frequency of addition of glycans, variable processing of glycans, and differences of C-terminal exopeptidase activity. The structural variation among the transgenics was generally within the range of the bean varieties. Previously, mice showed allergic reactions following ingestion of transgenic pea αAI but not bean αAI. Here, only minor differences were observed following intraperitoneal sensitization. Both of the transgenic pea and bean forms of αAI elicited Th1 and Th2 antibody isotype responses, suggesting that both proteins are immunogenic and could potentially be allergenic.     

Identification of an N-terminal formylated, two-peptide bacteriocin from Enterococcus faecalis 710C

Enterococcus faecalis 710C, isolated from beef product, has a broad antimicrobial activity spectrum against foodborne pathogens. Two bacteriocins, enterocin 7A (Ent7A) and enterocin 7B (Ent7B), were purified from the culture supernatant of E. faecalis 710C and characterized using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and electrospray infusion tandem mass spectrometry analyses. These data and subsequent genetic analysis showed that Ent7A and Ent7B are produced without N-terminal leader sequences and have amino acid sequences that are identical to those of enterocins MR10A and MR10B, respectively. However, the observed masses for Ent7A and Ent7B are 5200.80 and 5206.65 Da (monoisotopic mass), respectively, which are higher than the theoretical molecular masses of MR10A and MR10B, respectively. This study provides evidence that both Ent7A and Ent7B are formylated on the N-terminal methionine residue. Purified Ent7A and Ent7B are active against spoilage microorganisms and foodborne pathogens, including Clostridium sporogenes , Listeria monocytogenes , and Staphylococcus aureus as well as Brevundimonas diminuta , which has been associated with infections among immune-suppressed cancer patients.     

Studies of Partial Amino Acid Sequences of γ‐40k Secalins of Rye

Though γ‐40k secalins are a major protein type within rye storage proteins, total amino acid sequences are not as well known as the gluten proteins of wheat. Well‐reputed structural features such as amino acid compositions and molecular masses indicated a close relationship between γ‐40k secalins and γ‐gliadins of wheat, but the degree of homology of amino acid sequences and the positions of intramolecular disulfide bonds are unknown. Therefore, two major components of γ‐40k secalins (R1, R2) were analyzed for partial amino acid sequences. The R1 and R2, derivatized with 4‐vinylpyridine, were isolated from the prolamin fraction of rye cultivar Danko by means of a two‐step RP‐HPLC on C₁₈ silica gel. The proteins were digested in parallel with trypsin and thermolysin, and the partial hydrolyzates were separated by RP‐HPLC. Simultaneous measurement of UV absorbance at 210 and 254 nm allowed the detection of all peptides eluted as well as the specific detection of pyridylethylated cysteine peptides. Isolated peptides were characterized by sequence analysis, and in parts by mass spectrometry, and assigned to known sequences of γ‐gliadins. The results demonstrated that the N‐terminal domain of R1 and R2 remained undigested after tryptic hydrolysis; they were in agreement with the N‐terminal domain of γ‐gliadins in their molecular masses and in the absence of cysteine residues. Most of the isolated peptides originated from the C‐terminal domains, they covered 83% (R1) and 77% (R2), respectively, of the C‐terminal domain of a known γ‐gliadin (clone pW1020). Comparison of R1 and R2 revealed differences only in a few sequence positions. The degree of homology between the C‐terminal domains present in γ‐40k secalins and γ‐gliadins was ≈85%. All eight cysteine residues of γ‐gliadins were found in R1 and R2 sequences. Remarkably, sequences close to corresponding cysteine residues were identical for γ‐40k secalins and γ‐gliadins. Therefore, it can be assumed that the positions of intramolecular disulfide links are homologous.