Biochemical and Functional Properties of Wheat Gliadins: A Review

Gliadins account for 40–50% of the total storage proteins of wheat and are classified into four subcategories, α-, β-, γ-, and ω-gliadins. They have also been classified as ω5-, ω1, 2-, α/β-, and γ-gliadins on the basis of their primary structure and molecular weight. Cysteine residues of gliadins mainly form intramolecular disulfide bonds, although α-gliadins with odd numbers of cysteine residues have also been reported. Gliadins are generally regarded to possess globular protein structure, though recent studies report that the α/β-gliadins have compact globular structures and γ- and ω-gliadins have extended rod-like structures. Newer techniques such as Mass Spectrometry with the development of matrix-assisted laser desorption/ionization (MALDI) in combination with time-of-flight mass spectrometry (TOFMS) have been employed to determine the molecular weight of purified ω- gliadins and to carry out the direct analysis of bread and durum wheat gliadins. Few gliadin alleles and components, such as Gli-B1b, Gli-B2c and Gli-A2b in bread wheat cultivars, γ-45 in pasta, γ-gliadins in cookies, lower gliadin content for chapatti and alteration in Gli 2 loci in tortillas have been reported to improve the product quality, respectively. Further studies are needed in order to elucidate the precise role of gliadin subgroups in dough strength and product quality.     

Influence of pH and salt concentration on the emulsifying properties of native wheat gliadins and of their chymotryptic hydrolysates

Chymotryptic hydrolysates with degrees of hydrolysis around 1% were prepared from purified β- and γ-gliadins of wheat. Some functional properties were studied at pH 4.0 and 6.5 in the absence or in the presence of 1% and 2% NaCl. The hydrolysates were highly soluble in all the conditions of pH and salt concentration tested, whereas the solubility of native gliadins did not exceed 20%, except at pH 4.0, 0% NaCl. The differences in emulsifying properties between hydrolysates and native proteins were great in the presence of salt. Phase separation was almost complete immediately after emulsification with the native gliadins, but creaming was slowed down and limited with the hydrolysates. Furthermore, emulsions stabilized with hydrolysates showed a very strong resistance to coalescence. Although some differences were observed between hydrolysates of β- and γ-gliadins, the peptides adsorbed at the alcane/water interface were in both cases the more hydrophobic and charged, corresponding to the non-repetitive domain of the gliadin sequences.     

The influence of nitrogen fertilisation on quantities and proportions of different protein types in wheat flour

The flours of 13 wheat varieties grown at different levels of nitrogen fertilisation were characterised by the quantitative determination of flour protein groups and gluten protein types using a combined extraction/HPLC procedure. The results demonstrate that the quantities of albumins and globulins were scarcely influenced by different nitrogen fertilisation, whereas those of gluten proteins (gliadins, glutenins) were strongly influenced. The effect on gliadins was more pronounced than on glutenins, as well as the effect on major protein types (α-gliadins, γ-gliadins, LMW subunits of glutenin) in comparison with minor types (ω-gliadins, HMW subunits of glutenin). The proportions of hydrophilic proteins (ω-gliadins, HMW subunits of glutenin) were increased by high levels of nitrogen and those of hydrophobic proteins (γ-gliadin, LMW subunits of glutenin) were decreased. The degree of the effects on both quantities and proportions of flour protein groups and gluten protein types was strongly dependent on the variety. © 1998 SCI.     

Large scale isolation of gliadin fractions

The ω,- γ-, and α-gliadin groups were isolated by chromatography of 10 g of crude gliadin on Sulfopropyl Sephadex C50, followed by chromatographies of the SPS-C50 fractions on Sephadex G100. The alcohol-soluble glutenins separated from the SPS-C50 fractions containing the γ- and β-gliadins had lower contents of Asx, Ala and Tyr but higher contents of Thr, Ser and Met than the gliadins. On the basis of weights of the isolated fractions, the proportions of ω-, γ-, β- and α-gladin groups in Cappelle wheat were estimated to be about 15, 30, 30 and 25% of the total gliadin, respectively. This method of isolation of the four gliadin groups is suitable for all wheat varieties, especially for the isolation of α-gliadin.     

β-gliadin proteins from Maris Widgeon wheat

Four β-gliadins have been isolated from the English wheat, Maris Widgeon by Sephadex SP C25 ion-exchange chromatography of the gliadin mixture followed by G-75 or G-100 gel-filtration of either the native or reduced and alkylated proteins. Amino acid analyses of the proteins indicate similar compositions of β₂- and β₄-gliadins and also of β₃- and β₅-gliadins. Minimal molecular weights from amino acid analysis for all four proteins lie within the range 28000 to 30000.     

Comparative investigations of gluten proteins from different wheat species I. Qualitative and quantitative composition of gluten protein types

In contrast to the hexaploid common (bread) wheat, little information is available on the qualitative and quantitative compositions of gluten proteins from other cultivated wheat species. Therefore, representatives of hexaploid spelt, tetraploid durum wheat and emmer, and diploid einkorn were compared with three classes of common wheat (winter wheat, spring wheat, wheat rye hybrid). The flours were extracted to yield total endosperm proteins and the gluten protein fractions (gliadins and glutenin subunits). The extracts were characterised using sodium dodecyl sulfate polyacrylamide gel electrophoresis and reversed-phase HPLC; both methods revealed that gluten protein groups and types known from common wheat (ω-, α-, γ-gliadins, HMW and LMW subunits of glutenin) were present in all species. The HPLC platterns of gliadins and glutenin subunits from species with the same genome composition (common wheat/spelt or durum wheat/emmer) were related, and those of einkorn quite different. According to the quantities determined by reversed-phase HPLC, α-gliadins were predominant in most cases, followed by γ-gliadins and LMW subunits; ω-gliadins and HMW subunits were generally minor components. Common wheats were characterised by the highest proportions of total glutenins and HMW subunits, which are known to be important for breadmaking quality. Moreover, the lower ratio of gliadins to glutenins was typical. Emmer had the lowest proportions of total glutenins and of HMW and LMW subunits, together with einkorn the highest proportion of α-gliadins, and, by far, the highest ratio of gliadins to glutenins. The values for spelt and durum wheat were mostly in a medium range between common wheats, emmer, and einkorn, respectively. Amongst common wheats, spring wheat was characterised by more balanced quantities of α- and γ-gliadins, and wheat rye hybrid by the highest proportions of ω-gliadins. Received: 26 November 1999     

A third β-Gliadin from Cappelle-Desprez wheat

A third β-gliadin has been isolated from Cappelle-Desprez wheat. It is the middle member of a triplet of almost equally spaced bands that appears on starch-gel electro-phoresis. Surprisingly, it has no very close affinity with the fastest of these three bands. Although conforming to the amino acid composition expected in gliadins, the content of (His + Arg) is four less than in the fastest component, but the small molecular weight of only 27 000 compensates for this, so that the mobility at acid pH is only a little less than that of its faster companion. The protein appears to be single-chain and free from SH groups and carbohydrate. Of the (Glx+Asx) content, 96% is amidated. Published analyses of β-gliadins have been calculated to the same basis and compared.     

Detection and quantification of adulteration of durum wheat flour by flour from common wheat using reverse phase HPLC

When common wheat (Triticum aestivum L) gliadins were separated by RP-HPLC, a major doublet peak eluted at 47.20 and 47.94 min. This peak was consistently found to be absent in Durum wheat (Triticum durum Desf) gliadins separated under identical conditions. In Durum wheat gliadins a characteristic small peak eluted at 48.30 min followed at 50.47, 510.37, 52.80 min by larger peaks. The peak area ratio of the peaks eluting at 50.47 and 51.37 min was found to be 2.18 (±0.14). This ratio was found to decrease proportionally on contamination of Durum wheat flour with flour from some common wheat varieties. This ratio alone was not enough to detect and quantify adulteration by all varieties of common wheat. An alternative method was found whereby the peak emerging between 47 and 49 min in the Durum wheat gliadin elution profile was expressed as a ratio of the total protein applied. This ratio was shown to increase when Durum wheat flour was adulterated with flour from common wheat thus enabling quantitative estimation of the level of adulteration. A third method of detecting adulteration of Durum wheat flour is also proposed in which the peak emerging between 47 and 49 min is collected and the protein separated by PAGE. The presence of more than one band of γ/β-gliadins is indicative of adulteration.     

Comparative investigations of gluten proteins from different wheat species II. Characterization of ω-gliadins

 Flours of different wheat species (common wheats including winter wheat, spring wheat, and wheat rye hybrid, spelt, durum wheat, emmer, and einkorn) were successively extracted with a salt solution and 60% (v/v) aqueous ethanol. The alcohol extracts (gliadins) were separated by reversed-phase HPLC. Six to nine different ω-gliadins were obtained for each wheat sample and were characterized by their relative amounts, the amino acid compositions, the N-terminal amino acid sequences, and the molecular masses. The wheats investigated showed typical differences in the qualitative and quantitative HPLC patterns. The amino acid compositions of all ω-type gliadins revealed significantly higher proportions of glutamine, proline, and phenylalanine compared with other gluten proteins. These three amino acids accounted for 70 to 86% of the total composition. Typical differences in amino acid compositions, N-terminal sequences, and molecular masses allowed a clear differentiation of the proteins into ω5- and ω1,2-type gliadins; the gliadin fractions of emmer and einkorn contained only the ω5-type, but not the ω1,2-type. ω5-Gliadins were characterized by extremely high proportions of glutamine (52–57 mol %) and relatively high proportions of proline (18–21 mol %) and phenylalanine (9–10 mol %). ω1,2-Gliadins had less glutamine (39–45 mol %) and phenylalanine (6–8 mol %), but much more proline (22–31 mol %). ω5-Gliadins of all wheats except emmer could be assigned to the N-terminal sequence variants SRQLSP or SRLLSP; the typical sequence of emmer ω5-gliadins was SMELQT. The N-terminal sequences of ω1,2-gliadins were characterized by two basic variants beginning with KELQSP or ARQLNP. The wheat rye hybrid had additionally components with the sequence RQLNPS known from ω-secalins of rye. The determination of molecular masses by MALDI-TOF mass spectrometry revealed a range of 44,000–55,000 for the ω5-type and a range of 36,000–44,000 for the ω1,2-type. Thus, the actual masses were by far lower than the values derived from SDS-PAGE mobility. Received: 25 May 2000     

The α-amylase, α-amylase inhibitor and proteinase inhibitor activities of proteins extracted from wheat varieties and their influence on the baking quality

The grain of six winter and spring wheat varieties, harvested in 1987, differing in baking quality were included in the study. A number of technological analysis were performed. Albumins, globulins and gliadins were washed out. The location of inhibitory activities in extracted proteins, against α-amylases of various origin. were tested and compared to the baking quality of the wheat grain. Also, the location of antitryptic activity and endogenous amylase activity in extractable proteins was compared to baking quality factors. Among the studied inhibitory activities against mammalian α-amylases some correlation were found between the inhibition activity of hog pancreas α-amylase and sedimentation test (r = 0.88). Furthermore inhibitory activities of wheat proteins towards insects α-amylase shown significant correlation between the inhibitors of Anagasta kuehniella α-amylase and sedimentation test (r = 0.90). as well as crude protein content (r = 0.86). Also the inhibition activity against Sitophilus granarius α-amylase versus sedimentation test presented some correlation (r = 0.80). Endogenous amylase activities (α- and β-amylase) have an inversely proportional correlation to crude protein content (r = ?0.82) and sedimentation test (r = ?0.85).     

Slow triplet β-gliadin from cappelle-desprez

The third and slowest member of a triplet of β-gliadins seen in the electrophoretic pattern of Cappelle-Desprez gliadin has been isolated. It appears to be a single-chain protein that is free from carbohydrate and SH groups and has a molecular weight of 41000. The amino acid analysis is typical of many gliadins, with high contents of Glx and Pro, 93% of the side-chain COOH amidated, and 23 (Tyr + Phe) per molecule. Microheterogeneity may be present.     

Hydrolysis of Cyclodextrin by Aspergillus oryzae α-Amylase

The hydrolysis of α-, β- and γ-cyclodextrins by Aspergillus oryzae α-amylase was studied at pH 5.2 and 37°C. The kinetic parameters were determined and it was found that the V _max value increased markedly in the order α-, β- and γ-cyclodextrin, but no significant difference was observed in the K_m values. The qualitative and quantitative distribution of the hydrolysis products were determined by HPLC. In the case of γ-cyclodextrin the time course of the kinetic parameters was compared to the qualitative and quantitative distribution of the hydrolysis products.     

Three α-gliadins of Cappelle-Desprez

The strong α-doublet running behind the fastest major α-gliadin of Cappelle-Desprez on gel electrophoresis has been separated into three proteins. They were named: slow, middle and fast (S, M and F) from their mobilities on alkaline gels. The amino acid compositions were typical of gliadins, with no free thiol groups. Molecular weights and % amidation were: S31000, 96; M32 000, 94; F29000, 91. No convincing evidence for a glycoprotein nature was found. At acid pH the order seems to be, in decreasing mobility, F, S, M.     

Comparative investigations of gluten proteins from different wheat species

The only commercially available immunoassay for gliadin determination in gluten-free food which has been ring-tested and in use for many years, is a test kit based on monoclonal antibodies against -gliadins. Various studies of the literature have shown that different gliadin standards resulted in different calibration curves, and it has been proposed that the affinity of -gliadins to the monoclonal antibodies varied among wheat varieties. To clarify this fundamental problem, total gliadins and the -gliadins from a winter wheat ("Rektor"), a spring wheat ("CWRS"), a wheat rye hybrid ("Herzog") and varieties of spelt, durum wheat, emmer and einkorn, were isolated and analyzed by means of an enzyme-linked immunoabsorbent assay (ELISA) kit based on antibodies against -gliadins. Additionally, single - and -gliadins of Rektor wheat were studied. The results demonstrated that the calibration curves derived for total gliadins differed, in parts, strongly from that of the kit gliadin standard; only the curves for the durum wheat and spelt gliadins were in congruence with the kit gliadin. Single -gliadins revealed strong differences between and within wheat species and ELISA equivalents had a range from 10 to 220% according to kit gliadin. The affinity of -gliadins was not correlated with the calibration curves of total gliadins. Some of the -gliadins of Rektor wheat showed ELISA equivalents similar to those of -gliadins. Because the proportions of -gliadins in total gliadins were significantly higher than those of -gliadins, the unspecific binding of -gliadins contributed much more to the total affinity of gliadins than the specific binding of -gliadins. -Gliadins, however, did not show any detectable affinity.     

α3-gliadin from Timmo wheat. Isolation and partial structure determination

Ion-exchange chromatography, followed by gel-filtration has been used to separate α₃-gliadin from the hard English wheat, Timmo. Amino acid sequence determination on the protein has revealed 10 N-terminal residues and six C-terminal residues. The gliadin has been cleaved by N-bromosuccinimide into two peptides and another two peptides have been obtained by treatment with cyanogen bromide. The amino acid analyses of the peptides and α₃gliadin are reported.     

Dinkel und Z:oliakie

Spelt wheat (Triticum spelta L.) has not been investigated for the toxicity on coeliac disease patients until now. Because clinical studies are out of considerations for ethical reasons, spelt wheat and coeliac-active bread wheat (Triticum aestivum L.) were compared by the analysis of N-terminal sequences of α-gliadins, which have been proposed to be responsible for the toxic effect. The gliadin fractions of the spelt wheats ‘Roquin’ and ‘Schwabenkorn’ and of the bread wheat ‘Rektor’ were preparatively separated by RP-HPLC and major α-gliadin components were then compared by N-terminal sequence analysis. The results did not reveal any significant difference between spelt and bread wheats within the first 25 positions. For the determination of sequences further from the N-terminus, the gliadin fractions of the spelt wheats were hydrolyzed with pepsin and trypsin. The resulting peptides were successively separated by gel permeation chromatography and RP-HPLC. Those peptides derived from the N-terminal part of α-gliadins were identified by reference peptides isolated previously from bread wheat [this journal 194: 229 (1992)]. Retention times upon RP-HPLC and amino acid compositions of corresponding peptides confirmed the identity of spelt and bread wheat concerning the N-terminal sequences of α-gliadins from position 3 to 56. For these reasons, it can be concluded that spelt wheat is a coeliac-toxic cereal and has to be avoided by coeliac patients.     

EVIDENCE FOR THE PRESENCE OF MALTASE AND α-GLUCOSIDASE ISOENZYMES IN BARLEY

An examination of the development of α-glucosidase and maltase activities (as measured by the hydrolysis of p-nitrophenyl α-D-glucoside and maltose respectively) indicated that two genotypes of Glacier barley had the same general pattern of enzyme development. However, the development of α-glucosidase activity followed a different course from that of maltase activity suggesting that separate enzyme proteins are involved in hydrolysing these substrates. Further evidence that separate enzyme proteins were responsible for hydrolysis of maltose and p-nitrophenyl α-glucoside was obtained by column chromatography of extracts of germinated barley which indicated the presence of two maltases and two α-glucosidases. The maltases and the α-glucosidases differed in molecular weight, pH of optimum activity and in thermostability. When isomaltose was used as a substrate the optimum pH and behaviour on gel chromatography were coincident with that of maltase activity but different from the α-glucosidases.     

The effects of α-amylase inhibitors on insect storage pests: Inhibition of α-amylase in vitro and effects on development in vivo

Protein α-amylase inhibitors were prepared from wheat and their effects tested against insect storage pests both in vitro against the insect α-amylases and in vivo in insect feeding trials. Inhibitor fraction A was found to inhibit porcine pancreatic α-amylase but not insect α-amylases, whereas fractions B, C and D (0.28) did not inhibit porcine pancreatic α-amylase but were strong inhibitors of digestive α-amylases from larvae of Tribolium confusum, a storage pest of wheat products, and Callosobruchus maculatus, a storage pest of legume seeds. Fraction D, which was a single polypeptide of M_r 13 000 was the most effective inhibitor in vitro. It would appear that the degree of inhibition by the wheat α-amylase inhibitor preparations can be correlated with the presence of the M_r 13 000 (0.28) polypeptide since the purer this polypeptide the stronger was the inhibition; fraction A which contained two polypeptides of M_r 60 000 and 58 000 caused no inhibition. The effects of fractions B and C on larval development were determined in insect feeding trials. With C. maculatus both fractions were toxic, their relative effectiveness being directly paralleled by their effectiveness observed in vitro. Only fraction C was tested against T. confusum in feeding trials. Despite this fraction being equally effective against both pests in vitro it had very little effect upon larval development of T. confusum in vivo, thus suggesting that this organism is able to detoxify the wheat α-amylase inhibitors. As far as the authors are aware, this is the first time that the effects of identified inhibitor fractions have been monitored both in vitro and in vivo. The results, in contrast to previous proposals, suggest that selecting wheat varieties for high α-amylase inhibitory activity may not be a very reliable criterion in selecting for insect resistance.     

Investigations on the extractability of gluten proteins from wheat bread in comparison with flour

 Immunochemical methods are recommended for the quantitation of small amounts of gluten in food produced for those with coeliac disease. A major problem, however, is the reduced extractability of gliadin, the toxic factor of gluten, with aqueous alcohol, when foods have been heat-processed. A combined extraction/HPLC procedure was used to study the extractability of all gluten protein types from wheat flour and bread under both non-reducing and reducing conditions. Gliadin isolated from wheat flour was used as a reference protein for quantitation. The results indicate that the extractability of gliadin from bread with 60% ethanol under non-reducing conditions is strongly reduced. α- and γ-gliadins are much more affected than ω-gliadins, and less gliadin was extracted from the crust than from the crumb. For a complete extraction of gliadins from bread, reducing conditions and increased temperature are required. However, glutenin subunits are coextracted with the gliadins. This extract can be used for the quantitation of total gluten proteins by RP-HPLC. The recovery of gliadin added to flour before dough-mixing and bread-making is 98%. Received: 16 February 1998     

Investigations on the extractability of gluten proteins from wheat bread in comparison with flour

 Immunochemical methods are recommended for the quantitation of small amounts of gluten in food produced for those with coeliac disease. A major problem, however, is the reduced extractability of gliadin, the toxic factor of gluten, with aqueous alcohol, when foods have been heat-processed. A combined extraction/HPLC procedure was used to study the extractability of all gluten protein types from wheat flour and bread under both non-reducing and reducing conditions. Gliadin isolated from wheat flour was used as a reference protein for quantitation. The results indicate that the extractability of gliadin from bread with 60% ethanol under non-reducing conditions is strongly reduced. α- and γ-gliadins are much more affected than ω-gliadins, and less gliadin was extracted from the crust than from the crumb. For a complete extraction of gliadins from bread, reducing conditions and increased temperature are required. However, glutenin subunits are coextracted with the gliadins. This extract can be used for the quantitation of total gluten proteins by RP-HPLC. The recovery of gliadin added to flour before dough-mixing and bread-making is 98%. Received: 16 February 1998