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Chemistry of gluten proteins

Herbert Wieser
German Research Centre of Food Chemistry and Hans-Dieter-Belitz-Institute for Cereal Grain Research, D-85748 Garching, Germany

Abstract Gluten proteins play a key role in determining the unique baking quality of wheat by conferring water absorption capacity, cohesivity, viscosity and elasticity on dough. Gluten proteins can be divided into two main fractions according to their solubility in aqueous alcohols: the soluble gliadins and the insoluble glutenins. Both fractions consist of numerous, partially closely related protein components characterized by high glutamine and proline contents. Gliadins are mainly monomeric proteins with molecular weights (MWs) around 28,00055,000 and can be classied according to their different primary structures into the a/b-, g- and o-type. Disulphide bonds are either absent or present as intrachain crosslinks. The glutenin fraction comprises aggregated proteins linked by interchain disulphide bonds; they have a varying size ranging from about 500,000 to more than 10 million. After reduction of disulphide bonds, the resulting glutenin subunits show a solubility in aqueous alcohols similar to gliadins. Based on primary structure, glutenin subunits have been divided into the high-molecular-weight (HMW) subunits (MW 67,00088,000) and low-molecular-weight (LMW) subunits (MW 32,00035,000). Each gluten protein type consists or two or three different structural domains; one of them contains unique repetitive sequences rich in glutamine and proline. Native glutenins are composed of a backbone formed by HMW subunit polymers and of LMW subunit polymers branched off from HMW subunits. Non-covalent bonds such as hydrogen bonds, ionic bonds and hydrophobic bonds are important for the aggregation of gliadins and glutenins and implicate structure and physical properties of dough.

Keywords: Gluten; Gliadins; Glutenins; Proline and glutamine

1. Gluten Gluten can be dened as the rubbery mass that remains when wheat dough is washed to remove starch granules and water-soluble constituents. Depending on the thoroughness of washing, the dry solid contain 7585% protein and 510% lipids; most of the remainder is starch and nonstarch carbohydrates. In practice, the term gluten refers to the proteins, because they play a key role in determining the unique baking quality of wheat by conferring water absorption capacity, cohesivity, viscosity and elasticity on dough. Gluten contains hundreds of protein components which are present either as monomers or, linked by interchain disulphide bonds, as oligo- and polymers (Wrigley and Bietz, 1988). They are unique in terms of their amino acid compositions, which are characterized by high contents of glutamine and proline and by low contents of amino acids with charged side groups. The molecular
E-mail address: h.wieser@lrz.tum.de.

weights (MWs) of native proteins range from around 30,000 to more than 10 million. Traditionally, gluten proteins have been divided into roughly equal fractions according to their solubility in alcoholwater solutions of gluten (e.g. 60% ethanol): the solubles gliadins and the insoluble glutenins. Both fractions are important contributors to the rheological properties of dough, but their functions are divergent. Hydrated gliadins have little elasticity and are less cohesive than glutenins; they contribute mainly to the viscosity and extensibility of the dough system. In contrast, hydrated glutenins are both cohesive and elastic and are responsible for dough strength and elasticity. To simplify matters, gluten is a twocomponent glue, in which gliadins can be understood as a plasticizer or solvent for glutenins. A proper mixture of both fractions is essential to impart the viscoelastic properties of dough and the quality of the end product. Though cysteine belongs to the minor amino acids of gluten proteins (E2%), it is extremely important for the structure and functionality of gluten (Grosch and Wieser,

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1999; Wieser, 2003). Most cysteines are present in an oxidized state and form either intrachain disulphide bonds within a protein or interchain disulphide bonds between proteins. These bonds are the main target for most redox reactions that occur during kernel maturation, milling, dough preparation and baking (Wieser, 2003). Additional covalent bonds formed during breakmaking are tyrosine tyrosine crosslinks between gluten proteins (Tilley et al., 2001) and tyrosinedehydroferulic acid crosslinks between gluten proteins and arabinoxylans (Piber and Koehler, 2005). The covalent structure of the gluten network is superimposed by non-covalent bonds (hydrogen bonds, ionic bonds, hydrophobic bonds). Though this class of chemical bonds is less energetic than covalent bonds, they are clearly implicated in gluten protein aggregation and dough structure (Wieser et al., 2006). Evidence for the presence of hydrogen bonds in gluten proteins are the dough weakening effect of hydrogen bond breaking agents (e.g. urea) and the dough strengthening effect of heavy water compared with that of ordinary water. The importance of ionic bonds can be demonstrated by the strengthening effect of NaCl or of bipolar ions such as amino acids or dicarboxylic acids. Hydrophobic bonds contribute signicantly to the stabilization of gluten structure. They are different from other bonds, because their energy increases with increasing temperature; this can provide additional stability during the baking process. 2. Gliadins Most gliadins are present as monomers; they were initially classied into four groups on the basis of mobility at low pH in gel electrophoresis (a-, b-, g-, o-gliadins in order of decreasing mobility). Later studies on amino acid sequences, however, have shown that the electrophoretic mobility does not always reect the protein relationships and that a- and b-gliadins fall into one group (a/b-type). Modern methods such as two-dimensional electrophoresis or reversed-phase high-performance liquid chromatography (RP-HPLC) allow the separation of the gliadin fraction into more than hundred components. Based on the analysis of complete or partial amino acid sequences, amino acid compositions and MWs, they can be grouped
Table 1 Characterisation of gluten protein types Type MW 103 Proportionsa (%) Partial amino acid composition (%) Gln o5-Gliadins o1,2-Gliadins a/b-Gliadins g-Gliadins x-HMW-GS y-HMW-GS LMW-GS
a

into four different types: o5-, o1,2-, a/b- and g-gliadins (Table 1) (Wieser, 1996). Within each type, structural differences are small due to substitution, deletion and insertion of single amino acid residues. o-Gliadins are characterized by the highest contents of glutamine, proline and phenylalanine which together account for around 80% of the total composition. o5-Gliadins have higher MWs (E50,000) than o1,2-gliadins (E40,000). Most o-gliadins lack cysteine, so that there is no possibility of disulphide crosslinks. These proteins consist almost entirely of repetitive sequences rich in glutamine and proline (e.g. PQQPFPQQ). a/b- and g-gliadins have overlapping MWs (E28,00035,000) and proportions of glutamine and proline much lower than those of o-gliadins (Table 1). They differ signicantly in the contents of a few amino acids, e.g. tyrosine. Each of both types has two clearly different Nand C-terminal domains. The N-terminal domain (4050% of total proteins) consists mostly of repetitive sequences rich in glutamine, proline, phenylalanine and tyrosine and is unique for each type (sequence Sections I and II, Fig. 1). The repetitive units of a/b-gliadins are dodecapeptides such as QPQPFPQQPYP which are usually repeated ve times and modied by the substitution of single residues. The typical unit of g-gliadins is QPQQPFP, which is repeated up to 16 times and interspersed by additional residues. Within the C-terminal domains, a/b- and g-gliadins are homologous (sequence Sections IIIV, Fig. 1). They present sequences which are non-repetitive, have less glutamine and proline than the N-terminal domain and possess are more usual composition. With a few exceptions, a/b-gliadins contain six and g-gliadins eight cysteines located in the C-termional domain; they form three and four homologous intrachain crosslinks, respectively (Grosch and Wieser, 1999) (Fig. 1). Studies on the secondary structure have indicated that the N-terminal domains of a/b- and g-gliadins are characterized by b-turn conformation, similar to o-gliadins (Tatham and Shewry, 1985). The non-repetitive C-terminal domain contains considerable proportions of a-helix and b-sheet structures. Though the distribution of total gliadins among the different types is strongly dependent on wheat variety (genotype) and growing conditions (soil, climate, fertilization), it can be generalized that a/b- and g-gliadins are

Pro 20 26 16 17 13 11 13

Phe 9 8 4 5 0 0 4

Tyr 1 1 3 1 6 5 1

Gly 1 1 2 3 19 18 3

4955 3944 2835 3135 8388 6774 3239

36 47 2833 2331 49 34 1925

56 44 37 35 37 36 38

According to total gluten proteins.

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(GMP) make the greatest contribution to dough properties and their amount in wheat our (E2040 mg/g) is strongly correlated with dough strength and loaf volume. After reduction of disulphide bonds, the resulting glutenin subunits show a solubility in aqueous alcohols similar to gliadins. The predominant protein type are LMW glutenin subunits (LMW-GS), their proportion amounts to E20% according to total gluten proteins (Wieser and Kieffer, 2001). LMW-GS are related to a/b- and g-gliadins in MW and amino acid composition (Table 1). Similarly, they contain two different domains: The N-terminal domain (sequence Section I, Fig. 1) consists of glutamine- and proline-rich repetitive units such as QQQPPFS and the C-terminal domain (sequence Sections IIIV, Fig. 1) is homologous to that of a/b- and ggliadins within Sections III and V. LMW-GS contain eight cysteines (Grosch and Wieser, 1999; Wieser, 2003); six residues are in positions homologous to a/b- and g-gliadins, and therefore, are proposed to be linked by intrachain disulphide bonds (Fig. 1). Two additional cysteine residues unique to LMW-GS are located in Sections I and IV. They are not able to form an intrachain bond, probably for steric reasons. Consequently, interchain disulphide bonds with cysteines of different gluten proteins are generated. HMW glutenin subunits (HMW-GS) belong to the minor components within the gluten protein family (E10%). Each wheat variety contains three to ve HMW-GS which can be grouped into two different types, the x- and the y-type, with MWs from 83,00088,000 and 67,00074,000, respectively (Table 1). The nomenclature on single HMW-GS is based on the coding genome (A, B, D), the type (x, y) and the mobility of SDS-PAGE (nos. 112). HMW-GS consist of three structural domains (Fig. 1): a non-repetitive Nterminal domain (A) comprising about 80105 residues, a repetitive central domain (B) of about 480700 residues and a C-terminal domain (C) of 42 residues (Shewry et al., 1992). Domains A and C are characterized by the frequent occurrence of charged residues and by the presence of most or all of cysteines. Domain B contains repetitive hexapeptides (unit QQPGQG) as a backbone with inserted hexapeptides (e.g. YYPTSP) and tripeptides (e.g. QQP or QPG). The most important difference between the x- and the y-type lies within the A and B domains. For example, the y-type has an insertion of 18 residues including two neighbouring cysteines in domain A, and typical repetitive units are less frequently repeated and more frequently modied in domain B of the y-type. Because HMW-GS does not occur in our and dough as monomers, it is generally assumed that they form interchain disulphide bonds. The xtype except subunit Dx5 has four cysteines, three in domain A and one in domain C (Shewry and Tatham, 1997) (Fig. 1). Two residues of domain A are linked by an intrachain, the other two by interchain disulphide bonds. Subunit Dx5 has an additional cysteine at the beginning of domain B and it has been suggested that this might form another interchain bond. The y-type has ve cysteines in domain A and one in each of domains B and C. At present, interchain bonds have

Fig. 1. Schematic architecture and disulphide structures of gluten proteins (adapted from Grosch and Wieser, 1999).

major components, whereas the o-gliadins occur in much lower proportions (Wieser and Kieffer, 2001) (Table 1). A minor portion of gliadins have an odd number of cysteines due to point mutations and are linked together or to glutenins. They appear either in alcohol-soluble oligomers in the gliadin fraction or in the alcohol-insoluble glutenin polymers. This form of gliadins is proposed to act as terminator of glutenin polymerization (see below). The oligomeric fraction has been called high-molecular-weight (HMW) gliadin, aggregated gliadin or ethanol-soluble glutenin (Shewry et al., 1983; Huebner and Bietz, 1993). It contains a/b-, g-gliadins and low-molecular-weight (LMW) subunits linked by interchain disulphide bonds; the MWs range from around 100,000500,000. 3. Glutenins The glutenin fraction comprises aggregated proteins linked by interchain disulphide bonds; they have varying size ranging from about 500,000 to more than 10 million (Wieser et al., 2006). Thus, a part of glutenins belongs to the largest proteins in nature. The MW distribution of glutenins has been recognized as one of the main determinants of dough properties and baking performance. The largest polymers termed glutenin macropolymer

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only be found for the adjacent cysteines of domain A which are connected in parallel with the corresponding residue of another y-type, and for cysteine of domain B which is linked to a cysteine of LMW-GS. Various approaches have been combined to give details of the secondary structure of HMW-GS. Studies of the repetitive domain B indicated the presence of b-reverse turns (Shewry et al., 1992). These were predicted to be overlapping and form a loose spiral which was considered to contribute decisively to glutens elasticity. The nonrepetitive domains A and C were proposed to have globular structures containing a-helices. Numerous studies demonstrated that dough properties are strongly inuenced by the quantities of HMW-GS. Among HMW-GS the contribution of the x-type to dough properties has been found to be more important than that of the y-type (Wieser and Kieffer, 2001). Considering single subunits, the presence of subunit Dx5 (which has an additional cysteine for an interchain crosslink) and subunit Bx7 (which occurs in the greatest amounts) has been proposed to be particularly important for dough quality and loaf volume (Wieser and Zimmermann, 2000). 4. Disulphide bonds Disulphide bonds play an important role in determining the structure and properties of gluten proteins. Monomeric a/b- and g-gliadins present three and four intrachain disulpide bonds, respectively, whereas polymeric LMWand HMW-GS include both intra- and interchain bonds (Fig. 1) (Shewry and Tatham, 1997). The disulphide structure of native glutenins is not in a stable state, but undergoes a continuous change from the maturing grain to the end product (e.g. bread) (Wieser, 2003; Wieser et al., 2006). The MW distribution of glutenins has been recognized as one of the main determinants of dough quality and is governed by the state of disulphide structure that depends on genetic factors (e.g. presence of subunit Dx5, ratio of HMW-GS to LMW-GS, amount of chain terminators), environmental factors (e.g. sulphur deciency, heat or water stress), and the redox state (e.g. presence of reducing or oxidizing agents) (Wieser et al., 2006). Disulphide formation starts rapidly after synthesis of proteins within the lumen of endoplasmatic reticulum as an integral part of protein folding. It has been proposed that intrachain links form more rapidly than interchain links (Kasarda, 1999). To participate in a growing polymer, proteins need at least two cysteines forming interchain disulphide bonds. HMW- and LMW-GS usually full this requirement; they act as chain extender. The only crosslink found between HMW- and LMW-GS until now is a disulphide bond between a cysteine residue in domain B of y-HMW-GS and a cysteine residue in the C-terminal domain of LMW-GS. The backbone of HMW-GS

Fig. 2. A model double unit for the interchain disulphide structures of LMW-GS () and HMW-GS (&) of gluten polymers (adapted from Wieser et al., 2006).

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polymers is established by end-to-end, probably head-totail linkages. At least, four different cysteines lead to branching of the backbone. LMW-GS form also linear polymers via cysteines of the N-terminal and C-terminal domain. Terminators of polymerization were found to be glutathione or gliadins with an odd number of cysteines. With respect to quantitative data on the subunits composition of glutenins (ratio of HMW-GS to LMWGS E1:2 and of x- to y-type E2.5:1) and the MW of subunits, the basis molecular double unit of glutenin polymers might consist of 2 y-type HMW-GS, 4 x-type HMW-GS and around 30 LMW-GS covalently linked by interchain disulphide bonds (Wieser et al., 2006) (Fig. 2). The MW of this double unit amounts to about 1.5 million. The largest of the glutenins (GMP) might include more than ten of these double units in which the x/y-type ratio and the HMW/LMW-GS ratio increase. The overriding importance of disulphide bonds can be demonstrated by the addition of reducing agents weakening dough and of thiol-blocking or oxidizing agents strengthening dough (Wieser, 2003). During the whole process of breadmaking, the state of large glutenin polymers is characterized by three competitive redox reactions: (1) the oxidation of free SH groups which support polymerization; (2) the presence of terminators that stop polymerization and (3) SH/SS interchange reactions between glutenins and thiol compounds such as glutathione that depolymerize polymers. Oxygen is known to be essential for the formation of large glutenin polymers during dough mixing. Oxidizing agents such as potassium bromate, potassium iodate and L-ascorbic acid (after oxidation to dehydrascorbid acid) show the same effect as atmospheric oxygen. The baking process produces dramatic changes in the glutenin structure and functionality. For example, extractability in urea or SDS is strongly reduced and most of cysteine containing a/b- and g-gliadins are covalently bound to glutenin polymers. Disulphide interchange reactions between gliadins and glutenins have been postulated to be involved in the heat-induced effects (Wieser, 1998). 5. Conclusions Gluten proteins are among the most complex protein networks in nature due to numerous different components and different size, and due to variability caused by genotype, growing conditions and technological processes. They play a key role in determining the unique rheological dough properties and baking quality of wheat. In spite of the large number of excellent studies dedicated to characterizing structure and functionality of gluten proteins, important gaps of knowledge has to be lled by future research. One of the priorities should be to get more insight into changes of the disulphide structure beginning from the synthesis of proteins in the growing plant and ending in the baked products. Another point should be a better understanding of the relations between structure and functionality of gluten proteins, particularly in combination with additives such as dough improvers. Heterologous expression and protein engineering could be a valuable aid in structurefunction studies (Tamas and Shewry, 2006).

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