Hans-Dieter-Belitz-Institute for Cereal Grain Research

Gluten Research

To elucidate the functional properties of the gluten proteins of different cereal species the following topics have been investigated at the Hans-Dieter-Belitz-Institute for Cereal Research:

1. Quantitative Determination and Localisation of Protein-Bound Thiol Groups in Wheat Flour

2. Disulphide Bonds in Wheat Gluten

1. Quantitative Determination and Localisation of Protein-Bound Thiol Groups in Wheat Flour^a [Top of Page]

Susanne Antes and Herbert Wieser

Introduction: Disulphide bonds play a key role in determining the structure and properties of wheat gluten proteins. The well-known effects of oxidising or reducing agents on the rheological properties of dough and gluten are undoubtedly due to changes of the thiol/disulphide structure of gluten proteins. About 95 % of total cysteines in wheat flour are present in the disulphide (SS) form. Most α- and γ-type gliadins have only intramolecular disulphide bonds located in the C-terminal domains. LMW and HMW subunits of glutenin form both intra- and intermolecular disulphide bonds and occur in an aggregated state. About 5 % of total cysteines in flour are present in the thiol (SH) form. Only small amounts of free SH groups (0.5 %) are present in low-molecular-weight compounds, mainly in glutathione and cysteine, most beeing present in flour proteins (4.5 %).

Aims: The aim of the present work was, firstly, to optimise the classical method of Ellman for the quantitative determination of thiol groups in wheat flour. Secondly, the distribution of thiol groups on the Osborne fractions and their location in gluten protein were investigated using a fluorescent reagent as a marker.

Results: The determination of thiol groups with Ellman's reagent is strongly influenced by the extraction procedure and solvent. The accessibility of thiol groups can be increased by extraction with solvents containing SDS or urea. For the photometric determination of coloured extracts, measurement against a blank derived from the same amount of flour is recommended, because the flour extract itself is coloured slightly yellow. Using the optimum procedure with a solvent containing SDS the content of accessible thiol groups of freshly milled flour (cv. Rektor) was 1.21 µmol/g. Enzymic digestion with thermolysin, however, did not significantly influence the results.

Flour milled under nitrogen (REK-N₂) led to higher amounts of free thiol groups than flour milled under oxygen (REK-O₂). Gluten and dough prepared under nitrogen led to a lower maximum resistance and a greater extensibility than those prepared under air. Preparing dough and gluten from flour milled under nitrogen but under oxidising conditions, only dough led to differences concerning the maximum resistance whereas gluten corresponded to REK-O₂.


Positions of freee thiol groups in wheat gluten proteins after fluorescence labelling

In order to locate free thiol groups a method using a fluorescent reagent (DACM = N-(7-Dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide) was optimised according to methods described in the literature. Using a modified Osborne fractionation and labelling the free thiol groups with DACM led to amounts which were comparable with those obtained with Ellman's reagent. The highest amounts could be detected within the SDS fraction, which contained the glutenin proteins. Separation by HPLC led to three fluorescent peaks. N-terminal sequencing of these peaks allowed the assignment of the sequences to known sequences of gluten proteins. One peptide was derived from gluten proteins of the LMW s-type, the second from an α-type gliadin and the third from a γ-type gliadin. The position of the cysteine residues was determined by partial hydrolysis of the fluorescent peaks with thermolysin and sequencing of the fluorescent peptides.

Publications:
Antes S, Wieser H (2000) Untersuchungen über den Thiolgehalt in Weizenmehl. Lebensmittelchemie 54: 107
Antes S, Wieser H (2000) Quantifizierung und Lokalisierung proteingebundener Thiolgruppen in Weizenmehl. Getreide Mehl Brot 54: 290-294
Antes S, Wieser H (2000) Quantitative determination and localisation of thiol groups in wheat flour. In: Wheat Gluten. Proceedings of the 7th International Workshop Gluten (Shewry PR, Tatham AS, eds) Royal Society of Chemistry, Campridge, pp 211-214

^a Supported by the European Community (FAIR CT-97-3010)

2. Disulphide Bonds in Wheat Gluten^b [Top of Page]

Peter Köhler, Bettina Keck, Silvia Müller and Herbert Wieser

Introduction: Mixing of wheat flour with water gives a viscoelastic dough that is suitable for breadmaking. This viscoelasticity is due to the presence of gluten, that consists to 90 % of protein. The most important gluten proteins are the high-molecular weight glutenins that are responsible for the elasticity of wheat dough, and the low-molecular weight gliadins that are responsible for its viscosity. Gluten proteins have to be distinguished from the gluten protein components which can be considered as building blocks of gluten. Gluten protein components can be classified into the five groups high-molecular weight (HMW) subunits of glutenin, low molecular weight (LMW) subunits of glutenin, ω-, α-, and γ-gliadins. The HMW and LMW subunits of glutenin and a portion of the ω-gliadins build up the glutenin, gliadin consists of α-, γ-, and the rest of the ω-gliadins. Whereas amino acid sequences have been determined for several gluten protein components only a few information is available about disulphide bonds that link the protein components in glutenin.

Aims: The standard positions of the cysteine residues in gluten protein components are well-known. In gluten 95 % of the cysteine residues are connected via disulphide bonds. Only a few experimental proofs for the type and the positions in gluten are available up to now. Aim of the project was the identification of disulphide bonds in gluten proteins. Therefore, cystine containing peptides in enzymic partial hydrolysates of glutenin should be identified, isolated and sequenced. The sequences should be assigned to known sequences of gluten protein components and give insight into the linkages of cysteine residues in gluten.


Please click on the thumbnails to enlarge
Standard positions of the cysteine residues		Disulphide bonds of gliadins		Disulphide bonds of glutenins

Results: Gliadin and glutenin were used as starting material for the determination of disulphide bonds. For this, a dough was prepared from flour of the German wheat cultivar 'Rektor'. Gluten was isolated by washing the dough with water and subsequently extracted with 70 % aqueous ethanol to give two fractions, gliadin and residual glutenin. Then gliadin was separated by reversed-phase high-performance liquid chromatography (RP-HPLC) into individual gliadin components. These were partially hydrolysed with thermolysin, cystine containing peptides were identified by differential HPLC prior and after reduction, isolated, reduced with dithioerythritol, alkylated with 4-vinylpyridine and sequenced by automated Edman degradation. To remove low-molecular weight components glutenin was extracted with dilute acetic acid (0.1 mol/L) and partially hydrolysed with trypsin and thermolysin. Peptides were fractionated according to molecular weight by gel permeation chromatography on Sephadex G25. The identification, isolation and characterisation of cystine peptides was carried out as described above. The amino acid sequences of the peptides were then assigned to known sequences of gluten protein components.

On the basis of the results two-dimensional models of the disulphide structure of wheat gluten have been postulated. The three protein types of the LMW group (α- and γ-gliadins, LMW subunits of glutenin) have in common the linkage C^c/C^f1C^f2/C^y corresponding to a disulphide loop within domain III and a second loop between domains III and V. γ-gliadins and LMW subunits of glutenin are stabilised by an additional loop from cysteine residue C^d to C^e within domain III. A portion of the γ-gliadins was found in the glutenin fraction. This is due too cysteine residue C^b* which is able to form intermolecular disulphide bonds. This cysteine residue is present in only a minor portion of the γ-gliadins and might act as chain terminators during gluten formation. The same can be assumed for a minor portion of the α-gliadins with an odd number of cysteine residues.

The cysteine residue C^a of LMW subunits of glutenin is not a standard position. In contrast, C^b* seems to occur more frequently. Like C^x it is able to form intermolecular disulphide bonds. C^x was found to be linked with C^b* of LMW subunits, C^b* of γ-gliadins, and C^y of HMW subunits. C^x and C^b* are responsible for the aggregative nature of LMW subunits of glutenin. They act as chain extenders during gluten formation.

For HMW subunits of glutenin disulphide bonds have only been proven for the linkages C^a/C^b* in the x-type and C^c1C^c2 in the y-type. Furthermore, C^y of the y-type was found as part of a disulphide bond with an LMW subunit. For HMW subunits further investigations have to be performed. Especially the linkages involving C^d and C^z are of interest as they seem to play an important role in the polymerisation of HMW subunits of glutenin via head-to-tail disulphide bonds.

On the basis of the present results it can be assumed that disulphide bond formation in gluten doesn't occur randomly but strongly directed. Only a small number of cysteine residues are able to form intermolecular disulphide bonds. These cysteine residues are named C^b* and C^x and are the reason for the high aggregation tendency of LMW subunits of glutenin. Normally, these cysteine residues are not present in the monomeric α- and γ-gliadins.

Publications:
Köhler P, Belitz H-D, Wieser H (1991) Disulphide bonds in wheat gluten: isolation of a cystine peptide from glutenin. Z Lebensm Unters Forsch 192:234-239
Köhler P, Belitz H-D, Wieser H (1992) Disulphide bonds in wheat paste. Veroeff Arbeitsgem Getreideforsch 242:7-20
Köhler P, Belitz H-D, Wieser H (1993) Disulphide bonds in wheat gluten: further cystine peptides from high molecular weight (HMW) and low molecular weight (LMW) subunits of glutenin and from γ-gliadins. Z Lebensm Unters Forsch 196:339-247
Köhler P, Belitz H-D, Wieser H (1993) Disulphide Bonds in Wheat Gluten. In: Gluten proteins 1993 (Association of Cereal Research, Hrsg), Detmold, Germany, S. 79-89
Köhler P, Keck B, Müller S, Wieser H (1994) Disulphide Bonds in Wheat Gluten. In: Wheat Kernel Proteins (Universita Degli Studi Della Tuscia, Consiglio Nazionale Della Richerche, Hrsg), Viterbo, Italia, S. 45-53
Keck B, Köhler P, Wieser H (1995) Disulphide bonds in wheat gluten: cystine peptides derived from gluten proteins following peptic and thermolytic digestion. Z Lebensm Unters Forsch 200:432-439
Müller S, Wieser H (1995) The location of disulphide bonds in α-type gliadins. J Cereal Sci 22:21-27
Wieser H, Müller S (1996) Disulphide bonds of wheat gliadins. In: Gluten'96. Proceedings of the 6th International Gluten Workshop (Wrigley CW, ed) RACI, North Melbourne, Australien, pp 169-172
Köhler P, Keck-Gassenmeier B, Wieser H, Kasarda DD (1997) Molecular Modeling of the N-terminal Regions of High Molecular Weight Glutenin Subunits 7 and 5 in Relation to Intramolecular Disulphide Bond Formation. Cereal Chem 74:154-158
Müller S, Wieser H (1997) The location of disulphide bonds in monomeric γ-type gliadins. J Cereal Sci 26:169-176
Müller S, Vensel WH, Kasarda DD, Köhler P, Wieser H (1998) Disulphide bonds of adjacent cysteine residues in low molecular weight subunits of wheat glutenin. J Cereal Sci 27: 109-116
Müller S, Wieser H, Popineau Y (1998) Disulphide bonds of γ-46-gliadin. J Cereal Sci 27:23-25

^b AiF-Project No. 8684

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