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.     

The fractionation and purification of gliadins by hydrophobic interaction chromatography

The apolar nature of wheat gliadins has been studied. Apolar properties have been exploited to fractionate components by hydrophobic interaction chromatography and fractions have been analysed further by polyacrylamide gel electrophoresis. Experiments were carried out in pH 5.0 buffer in absence of additives such as urea or dimethylformamide. Gliadin was not retarded by ethyl or butyl agarose but showed a strong affinity for both the phenyl and octyl derivatives. The strength of the hydrophobic interactions was such that aqueous alcohol was required to achieve appreciable desorption. Even so, only two-thirds of the bound gliadin could be eluted with aqueous ethanol alone; complete desorption required the presence of small concentrations of tetramethylammonium chloride. The elution of protein from columns followed the general order ω-,β-, α-, and γ-gliadin, respectively. This procedure provides a simple system for the fractionation of wheat prolamines under mild conditions. Results suggest that a higher degree of resolution may be attainable by further modification of experimental conditions. Interaction of gliadin with nonpolar groups covalently bound to agarose apparently reflects the presence of hydrophobic patches on the surface of the gliadin molecule.     

Gliadin proteins from maris widgeon wheat

α₁-, α₂- and β₁-gliadin proteins have been separated from the English wheat, Maris Widgeon, by Sephadex SP C25 ion-exchange chromatography and G-100 gel-filtration. Minimal molecular weights from amino acid analyses of these proteins are 32 000, 22 000 and 28 000 respectively. α₁-Gliadin has a comparatively high sulphur amino acid content for a gliadin protein.     

Electrophoretic Characteristics of Gluten Proteins as Influenced by Crop Year and Variety

The glutenin and gliadin fractions in different wheat varieties produced during two different cropping years were determined through SDS-PAGE and Acid-PAGE. It was evident from the electropherograms that greater numbers of polypeptides were present in the region falling under low molecular weight glutenin subunits. The SDS-PAGE patterns of molecular weight of glutenin subunits of different wheat varieties showed the presence of glutenin subunits in the range of 28.23 to 110.89 kDa and 28.29 to 113.51 kDa during the cropping years 2010-11 and 2011-12, respectively. The highest molecular weight glutenin subunit (110.89 kDa) was observed in wheat variety Lassani-08, during the crop year 2010-11, while during the cropping season 2011-12, the highest molecular weight glutenin subunit (113.51 kDa) was found in wheat variety AARI-10. It was also evident from the results that the maximum numbers of gliadin electropherograms were found in wheat variety Lassani-08. The results regarding gliadin bands revealed that total gliadin electropherograms ranged from 31.03 to 89.61 kDa and 32.91 to 92.22 kDa among different wheat varieties, during the crop years 2010-11 and 2011-12, respectively. The polypeptides with molecular weight between 31.03 to 54.50 kDa and 32.91 to 55.21 kDa belongs to α-, β- and γ-subunits of gliadin, during the crop years 2010-11 and 2011-12 respectively. The group of polypeptides with a molecular weight of about 54.50 to 89.61 kDa and 55.21 to 92.22 kDa was composed of subunits of ω-gliadins during the crop years 2010-11 and 2011-12, respectively.     

Large-scale preparation of gliadin proteins

By a combination of carboxymethylcellulose ion-exchange chromatography and gel-filtration α, β and γ-gliadin proteins have been isolated in quantities large enough to be suitable for clinical feeding and baking tests. A pure ω-gliadin has been obtained by the use of ion-exchange chromatography alone.     

Sodium dodecyl sulphate electrophoresis of wheat gliadins

Polypeptide chain weights of α-, β- and γ-gliadin fractions have been estimated by polyacrylamide gel electrophoresis in the presence of mercaptoethanol and sodium dodecyl sulphate. Values lie in the range 32 000 to 44 000. When available data on the amino acid analyses were adjusted and compared, significant similarities emerged. It is suggested that these gliadins contain a minimum of 4 cystine residues/molecule. The results provide support for the hypothesis that glutenins and gliadins have evolved from a common precursor. The results of an amino acid analysis of a previously unreported β-gliadin component are included.     

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.     

The influence of gamma irradiation on the immunoreactivity of gliadin and wheat flour

The gliadin fraction of wheat flour gluten is potentially responsible for an allergic reaction in individuals with a genetic predisposition to allergy. In order to study the influence of ionising radiation on the immunogenicity of gliadin, samples of commercial gliadin powder and wheat flour were irradiated with doses between 2.2 and 12.8 kGy. Ethanolic extracts (40% ethanol/water, v/v) of treated samples were analysed by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies against ω-gliadin or polyclonal anti-gliadin antibodies and by immunoblotting after polyacrylamide gel electrophoresis. Irradiated gliadin samples show the increase in allergenicity measured by ELISA. A linear relationship between the immune response induced by irradiated gliadin and the applied radiation dose was observed. The increase in immunoreactivity was confirmed by immunoblot assays with sera containing anti-gliadin antibodies. Immunoreactivity of gliadin extracted from irradiated wheat flour was higher than the immune response of pure gliadin irradiated with the same dose. RP-HPLC was applied for the analysis of gliadin constituents after irradiation. The content of ω-gliadins in irradiated samples decreased with increasing radiation dose.     

Isolation of further components from wheat gliadins

Ion-exchange chromatography on CM-Sephadex C-50 has enabled a wheat protein, the β₃-gliadin, to be isolated in a pure state for the first time from two widely differing varieties. The two proteins were shown to be almost identical in amino acid composition. The preparation of an athin from gliadin, and its analysis, is also reported.     

A cappelle-desprez β-gliadin

A β-gliadin from Cappelle-Desprez wheat has been purified sufficiently to be analysed. Its amino acid composition is typical of the gliadin class. The molecule appears to be a single polypeptide chain with no carbohydrate or SH groups and a molecular weight of 38 000.     

Genetic analysis of detailed milk protein composition and coagulation properties in Simmental cattle

The objective of this study was to estimate genetic parameters for milk protein fraction contents, milk protein composition, and milk coagulation properties (MCP). Contents of α_S1-, α_S2-, β-, γ-, and κ-casein (CN), β-lactoglobulin (β-LG), and α-lactalbumin (α-LA) were measured by reversed-phase HPLC in individual milk samples of 2,167 Simmental cows. Milk protein composition was measured as percentage of each CN fraction in CN (α_S1-CN%, α_S2-CN%, β-CN%, γ-CN%, and κ-CN%) and as percentage of β-LG in whey protein (β-LG%). Rennet clotting time (RCT) and curd firmness (a₃₀) were measured by a computerized renneting meter. Heritabilities for contents of milk proteins ranged from 0.11 (α-LA) to 0.52 (κ-CN). Heritabilities for α_S1-CN%, κ-CN%, and β-CN% were similar and ranged from 0.63 to 0.69, whereas heritability of α_S2-CN%, γ-CN%, and β-LG% were 0.28, 0.18, and 0.34, respectively. Effects of CSN2-CSN3 haplotype and BLG genotype accounted for more than 80% of the genetic variance of α_S1-CN%, β-CN%, and κ-CN% and 50% of the genetic variance of β-LG%. The genetic correlations among the contents of CN fractions and between CN and whey protein fractions contents were generally low. When the data were adjusted for milk protein gene effects, the magnitude of the genetic correlations among the contents of milk protein fractions markedly increased, indicating that they undergo a common regulation. The proportion of β-CN in CN correlated negatively with κ-CN% (r = −0.44). The genetic relationships between CN and whey protein composition were trivial. Low milk pH correlated with favorable MCP. Genetically, contents and proportions of α_S1- and α_S2-CN in CN were positively correlated with RCT. The relative proportion of β-CN in CN exhibited a genetic correlation with RCT of −0.26. Both the content and the relative proportion of κ-CN in CN did not correlate with RCT. Weak curds were genetically associated with increased proportions in CN of α_S1- and α_S2-CN, decreased contents of β-CN and κ-CN, and decreased proportion of κ-CN in CN. Negligible effects on the estimated correlations between a₃₀ and κ-CN contents or proportion in CN were observed when the model accounted for milk protein gene effects. Increasing β-CN and κ-CN contents and relative proportions in CN and decreasing the content and proportions of α_S1-CN and α_S2-CN and milk pH through selective breeding exert favorable effects on MCP.     

Mechanism of gliadin–glutenin cross-linking during hydrothermal treatment

The gluten proteins, gliadin and glutenin, are important for wheat flour functionality and they undergo changes during heat treatment involving sulfhydryl (SH) groups. To change the level of SH-groups during hydrothermal treatment, the oxidant, potassium iodate (2.1 μmol/g protein) and the reducing agent dithiothreitol (DTT, 6.1 μmol/g protein) were added to 20% (w/w) gluten-in-water suspensions at room temperature, at 90 °C and after 15 min at 95 °C, and the viscosity was measured by the Rapid Visco Analyser (RVA). Protein extractabilities after hydrothermal treatment were determined by size-exclusion and reversed-phase HPLC. DTT decreased maximal RVA viscosity and the levels of extractable α- and γ-gliadin and this decrease was independent of the time of addition during hydrothermal treatment. In contrast, potassium iodate increased the levels of extractable α- and γ-gliadin. Its impact was less when added at later times during RVA analysis. A SH-blocking agent (N-ethylmaleimide, 8.0 μmol/g protein), added at room temperature to the gluten suspension, decreased RVA viscosity at 95 °C and increased the extractabilities of glutenin and α- and γ-gliadin after hydrothermal treatment. Subsequent addition, at 90 °C, of a reducing agent (glutathione, 3.1 and 6.2 μmol/g protein) recovered the control RVA profile and restored the control protein extractabilities after RVA analysis. This shows the importance of heat-induced gliadin–glutenin reactions for gluten viscosity and of the presence of free SH-groups for the polymerization of gluten proteins. A model explaining gliadin–glutenin polymerization through a sulfhydryl-disulfide exchange mechanism and demonstrating the effects of redox agents is put forward.     

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.     

Electrophoretic characterisation of low-molecular weight wheat protein of variable solubility

A low mol. wt protein (S protein) which has a high affinity for endogenous polar lipid was isolated from wheat gluten, partially purified and compared electrophoretically with low mol. wt components of several previously studied wheat protein preparations. Mobilities of S protein components in acidic polyacrylamide gel electrophoresis (PAGE) were very close to those of the chloroform/methanol soluble low mol. wt proteins (CM proteins). A fraction of S protein separated by gel filtration chromatography on Sephadex G-50 (S-III protein) was almost identical to CM proteins by two-dimensional electrophoresis. Bands with mobilities similar to those of S-III protein were present in PAGE patterns of gliadin, the low mol. wt fractions IV and V obtained from gliadin by gel filtration chromatography on Sephadex G-200, and the albumin/globulin fraction of flour prepared by the Osborne solubility fractionation. Because of its variable solubility S protein cannot be unambiguously classified according to the Osborne solubility classification of plant proteins.     

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.     

A fast-moving beta-gliadin from Cappelle-Desprez

A Cappelle-Desprez gliadin, in the fast β-region, has been isolated. The amino acid analysis is typical of gliadins in general. The protein has a single polypeptide chain of molecular weight 42 000 and appears to be free from carbohydrate and SH groups. The fact that its electrophoretic mobility is higher than that of a β-gliadin of lower mol. wt. is attributed to the presence of 2 His and 1 Arg extra.     

Aggregation states of alcohol-soluble storage proteins of barley, rye, wheat and maize

Two approaches were used to study the aggregation states of prolamins extracted from milled whole grain of wheat, barley, rye and maize with 500 ml litre^?1 aqueous propan-1-ol. In the first, a comparison was made of the electrophoretic patterns of the samples separated by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions. In the second, they were chromatographed on a column of controlled pore glass (CPG) using a modified acetic acid, urea, cetyltrimethyl ammonium bromide (AUC) solvent and aliquots of the major peaks were reduced and analysed by SDS-PAGE. When the CPG chromatography was repeated under reducing conditions some of the peaks were eliminated indicating that they were composed of disulphide-linked aggregates. The results from both approaches showed that in barley, rye and wheat the HMW prolamins and some of the S-rich prolamins (B hordein, some γ-secalins and subunits of high molecular weight gliadins) are present predominantly or partially in high molecular weight (above 1 × 10⁶ daltons) aggregates which appear to be stabilised by disulphide bonds. Other S-rich prolamins (including some γ-secalins, α, β and γ-gliadins) and all the S-poor prolamins (‘C’ hordein, ω-secalin, ω-gliadin) are present predominantly or only as monomers. These results are discussed in relation to the structural and genetic homology of the prolamins in the three species. Although prolamin aggregates are also present in maize, these are of lower molecular weight.     

Isolation of a Cappelle-Desprez gliadin.

A Cappelle-Desprez gliadin, previously unreported, has been isolated. It appears to be a single-chain protein with a molecular weight of only 18 000, considerably lower than that of any other gliadin so far isolated. Its amino acid composition, though broadly typical of the class, has surprising characteristics, such as 7 Met, 7 CySSCy, but less Gln and Pro and no Lys or His. Although at acid pH the mobility is less than that of the γ-gliadin group, at pH 8.9 the molecule is still positively charged. The high isoelectric point implies that almost all the carboxyl side-chains are amidated. The gliadan contains no SH groups and does not appear to be a glycoprotein. The amino acid analysis suggests that microheterogeneity is present.     

小麦α-醇溶蛋白体外克隆表达及脱酰胺改性对面条机械特性的影响

本研究以pUC57-α-gliadin重组质粒为对象进行同源性分析,构建表达质粒pET-22b-α-gliadin,利用大肠杆 菌（Escherichia coli）体外诱导表达α-醇溶蛋白,以体积分数70%乙醇溶液提纯,利用柠檬酸对α-醇溶蛋白进行脱 酰胺改性,通过傅里叶变换红外光谱、内源性荧光扫描、扫描电子显微镜表征改性后α-醇溶蛋白的结构变化,通过 质地剖面分析（texture profile analysis,TPA）实验探究其对面条质地的影响。结果表明：α-醇溶蛋白柠檬酸脱酰胺 改性后α-螺旋结构含量减少,β-折叠结构含量增加,α-螺旋/β-折叠由1.00降为0.64,说明α-醇溶蛋白的分子柔韧性增 加;α-醇溶蛋白的内源性荧光扫描结果显示λmax发生红移,说明α-醇溶蛋白的内部结构发生伸展,使内部的色氨酸 暴露;扫描电子显微镜观察结果表明α-醇溶蛋白表面呈现光滑均匀的块状;TPA实验发现脱酰胺改性的α-醇溶蛋白 使面条硬度降低,黏着性提高。     

Effect of parity, days in milk, and milk yield on detailed milk protein composition in Mediterranean water buffalo

The effects of some nongenetic factors on milk protein fraction contents and relative proportions were estimated in 606 individual milk samples of Mediterranean water buffalo. Content of α(S1)-casein (CN), α(S2)-CN, β-CN, γ-CN, κκ-CN, glycosylated κ-CN (glyco-κ-CN), α-lactalbumin, and β-lactoglobulin was measured by reversed-phase HPLC. Relative contents of α(S1)-CN%, α(S2)-CN%, β-CN%, and κ-CN% were, respectively, 32.1, 17.1, 34.5, and 15.7%, whereas γ-CN% accounted for 0.6% of total casein content. Increasing total casein content in milk would result in a greater proportion of β-CN% at the expense of all of the other major casein fractions, especially of κ-CN%. Values of α(S2)-CN%, β-CN%, and γ-CN% tended to decrease with parity, although their variations were not significant, whereas α(S1)-CN% and glyco-κ-CN% showed the opposite trend. Contents of most protein fractions showed the typical trends observed for milk components as lactation progressed, with high contents in early lactation, a minimum in midlactation, followed by a gradual increase toward the latter part of lactation. Values of α(S1)-CN% increased during lactation, whereas α(S2)-CN% decreased. The proportion of β-CN% had its maximum value between 60 and 160 d of lactation, followed by a decrease, whereas κ-CN% had its minimum value in early lactation (<60 d) and remained relatively constant in the period of mid and late lactation. Glyco-κ-CN% and β-lactoglobulin% decreased in the first part of lactation, to reach their minimum values in midlactation, followed by an increase. Milk of top-producing buffaloes, compared with that of low-producing ones, had a significantly greater value of β-CN% and glyco-κ-CN%, and lower proportion of α(S1)-CN%. The possible effect exerted by protein genetic variants in affecting variation of milk protein fraction contents and relative proportions should be further considered to better get insight into buffalo milk protein composition.