Studies on distribution and binding of endogenous glutathione in wheat dough and gluten. I. Distribution of glutathione in Osborne fractions

Glutathion (GS) present in wheat flour or added to wheat flour has a large effect on the rheological properties of dough and gluten. To study the distribution of endogenous glutathione (GS) in Osborne fractions of dough and gluten,³⁵S-labelled reduced glutathione (GSH) was added as a tracer (about 0.1% of endogenous GSH) to the flours of three wheat cultivars (Canadian Western Red Spring, Dark Northern Spring, Maris Huntsman). After mixing of flours with water, the doughs obtained were fractionated into water-solubles, globulins, gliadins and glutenins. The radioactivity of the freeze-dried fractions was determined with a scintillation counter. The distribution of radioactivity was similar for the different cultivars and about 65% in the water-soluble fraction (mainly non-protein bound GS), 6–7 % in each of the globulin and gliadin fractions and 21% in the glutenin fraction. RP-HPLC of water-solubles, globulins and gliadins using simultaneous UV and radioactivity detection demonstrated that protein-bound labelled GS was distributed over the whole area of eluted protein. A dough resting time of 35 min did not influence distribution and³⁵S-labelled oxidized glutathione (GSSG) was distributed in the same proportions as GSH. In gluten washed from dough, radioactivity was increased in the gliadin (11%) and glutenin fractions (35%) and decreased in the water-soluble fraction. The addition of GSH to flour (100 nmol/g) led to an increase of radioactivity in the water-soluble fraction of dough and to a decrease in the other dough fractions; the effect of GSSG (50 nmol/g) was less pronounced. The influence of ascorbic acid (170 nmol/g) was dependent on the content of endogenous GSH in flour; at high levels of endogenous GSH, a strong shift of radioactivity from the water-soluble fraction to the glutenin fraction was observed. Amongst oxidants studied, CuSO₄(315 nmol/g) had only small effects. KBrO₃(300 nmol/g) increased radioactivity of the water-soluble fraction and decreased that of the glutenin fraction, whereas the effect of KIO₃(117 nmol/g) was dependent on the wheat cultivar.