Determination of the number of cysteine residues in high molecular weight subunits of wheat glutenin

Abstract

A simple method allowing the determination of the number of cysteine residues of the high molecular weight glutenin subunits (HMW-GS) is presented. The method was adapted from that of Creighton (T. E. Creighton, Nature 1980, 2840, 487-489) with modified reagents for alkylation of the cysteine residues and the electrophoretic system. The acid-urea-polyacrylamide gel electrophoresis (PAGE) method, developed by Morel (M. H. Morel, Cereal Chem. 1994, 713, 238-242), was adopted and mixtures of iodoacetic acid and 4-vinylpyridine were used to alkylate the glutenin subunits. The accuracy of the method was checked with some HMW-GS whose number of cysteine residues was already known from molecular biology approaches. In subunits 5*, 2.2, 2.2* and 4.1 (of Dx types) from cultivars Ben and Fiorello, MG 7249, MG 315, and Kador, respectively, only 4 cysteine residues were demonstrated. Subunit 20 was found to exist as a regular Bx and By subunit pair, with the Bx subunit containing only 2 cysteine residues, which is unusual.

Sequence and properties of HMW subunit 1Bx20 from pasta wheat (Triticum durum) which is associated with poor end use properties

“…Since x-type subunits typically contain three cysteines in their N-terminal domain and one in their C-terminal domain (Shewry et al 1992), this suggested that subunit 1Bx20 contained only two cysteine residues. Indirect evidence in support of this conclusion came from comparison of the mobilities of "native" and reduced and alkylated subunits by acid-PAGE and reversed phase-HPLC (Morel and Bonicel 1996;Margiotta et al 1993).…”

Analysis of the relationship between bread-making quality and dough stress during the proofing process using near-isogenic lines of ‘Harunoakebono’

“…Basically, HMWGs, except for HMWGs encoded by Glu-A1 locus, are composed of two subunits of x-type and y-type encoded by two genes in each locus (Payne 1987). It is known that the y-type subunit has more cysteine residues related to the disulfide bond in its molecular than the x-type subunit (Megan and Skerritt 1999, Morel and Bonicel 1996). Models have also been proposed for glutenin polymers composed of HMWGs and LMWGs that are believed to form a gluten backbone.…”

“…However, only HMWGs 5 among the x-type HMWGs have three cysteine residues related to interchange disulfide bond and it is known that the contribution to glutenin polymerization is specifically large. On the other hand, x-type HMWGs 20 have only two cysteine residues in the molecule (The other x-type HMWGs except for HMWGs 5 usually have four cysteine residues), so it is believed that the contribution to glutenin polymerization is very small (Megan and Skerritt 1999, Morel and Bonicel 1996, Pirozi et al . 2008).…”