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

For the identification of the binding sites of glutathione (GS) in glutenin s, flour of the wheat cultivar "Canadian Western Red Spring" was mixed with water containing S-35-labelled reduced GS as a tracer. The resulting dough was washed in a Glutomatic, and, in order to remove gliadins, the gluten o btained was extracted with 70% aqueous ethanol adjusted to pH 5.5 with acet ic acid. The residual proteins (glutenins) were hydrolyzed with thermolysin , and the hydrolysate was separated by gel permeation chromatography on Sep hadex G25 and by several steps of reversed-phase HPLC on C-18 silica gel. T he major radioactive disulphide peptides identified by scintillation analys is were collected and analysed for their amino acid sequences. Twenty-five peptides linked to GS could be assigned to known sequences of gluten protei ns. Most peptides (16) were derived from low molecular weight (LMW) subunit s of glutenin. Among these, 13 peptides contained the cysteine residue C-b* , which is present in the repetitive sequence region of LMW subunits and wh ich has been postulated to form intermolecular disulphide bonds. This pepti de type represented 45% of the total radioactivity of isolated peptides. Th ree further peptides from LMW subunits representing 46% of radioactivity in cluded cysteine C-x, which has also been proposed to form intermolecular di sulphide bonds. Four peptides with 3.2% of radioactivity could be assigned to high molecular weight subunits (cysteines C-b, C-d, C-e, C-y) and four p eptides (3.0% of radioactivity) to glutenin-bound gamma -gliadins (C-b*, C- w, C-z). One peptide (3.3% of radioactivity) corresponded to cysteine C-c f rom gamma -gliadins or LMW subunits. Altogether the cysteine residues in gl utenins. which are usually linked by intermolecular disulphide bonds, contr ibuted up to 95% of total radioactivity. The results obtained are in accord ance 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.