Studies on the distribution and binding of endogenous glutathione in wheat dough and gluten. II. Binding sites of endogenous glutathione in glutenins

Abstract

For the identification of the binding sites of glutathione (GS) in glutenins, flour of the wheat cultivar " Canadian Western Red Spring " was mixed with water containing 35S-labelled reduced GS as a tracer. The resulting dough was washed in a Glutomatic, and, in order to remove gliadins, the gluten obtained was extracted with 70% aqueous ethanol adjusted to pH 5.5 with acetic acid. The residual proteins (glutenins) were hydrolyzed with thermolysin, and the hydrolysate was separated by gel permeation chromatography on Sephadex G25 and by several steps of reversed-phase HPLC on C18 silica gel. The major radioactive disulphide peptides identified by scintillation analysis were collected and analysed for their amino acid sequences. Twenty-five peptides linked to GS could be assigned to known sequences of gluten proteins. Most peptides (16) were derived from low molecular weight (LMW) subunits of glutenin. Among these, 13 peptides contained the cysteine residue Cb*, which is present in the repetitive sequence region of LMW subunits and which has been postulated to form intermolecular disulphide bonds. This peptide type represented 45% of the total radioactivity of isolated peptides. Three further peptides from LMW subunits representing 46% of radioactivity included cysteine Cx, which has also been proposed to form intermolecular disulphide bonds. Four peptides with 3.2% of radioactivity could be assigned to high molecular weight subunits (cysteines Cb, Cd, Ce, Cy) and four peptides (3.0% of radioactivity) to glutenin-bound γ-gliadins (Cb*, Cw, Cz). One peptide (3.3% of radioactivity) corresponded to cysteine Cc from γ-gliadins or LMW subunits. Altogether the cysteine residues in glutenins, which are usually linked by intermolecular disulphide bonds, contributed up to 95% of total radioactivity. The results obtained are in accordance with the effect of reduced GS on the rheological properties of dough, namely the weakening of dough by depolymerization of glutenin polymers via specific cleavage of intermolecular disulphide bonds.