Liberation of subunit polypeptides of glutenin by partial reduction at pH 6.0.

Abstract

Glutenin was reduced with various concentrations of 2-mercaptoethanol (2-ME) at pH 6.0 and the liberation of subunits was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The amount of sulfhydryl groups in protein (protein-SH) liberated by reduction increased steeply with the increase of 2-MEconcentration around 1 mM.But the increase of protein-SH declined at higher concentrations to give a plateau at 6 mM.The level of this plateau, which may correspond to the amount of reactive disulfide (SS) bonds, was 40%of the total SS bonds of glutenin. In order to investigate whether inter-polypeptide SS bonds are cleaved by the partial reduction, the liberation of subunit monomers was observed by SDS-PAGE.By the partial reduction, Band 1+1′ (MW 104Kd), Band 2 (MW 90 Kd), Band 3 (MW 81 Kd) and Band 7 (MW 35 Kd) appeared in the electrophoretic pattern even at low concentrations of 2-ME, indicating the liberation of these subunits. However, although all monomersof Band 7 subunits were almost liberated at low concentrations of 2-ME (0.5mM), Bands 1+1′, 2, and 3 did not show suffic ient amounts of monomerseven at 6 mM.Further, another group of band, which had molecular weights of 136 Kd, 132 Kd, and 1 10 Kd, were observed at low concentrations of2-ME but dissapeared at extremely high concentrations, indicating these bands must be oligomers of glutenin subunits. These observations from the SDS-PAGE patterns suggested that the reactivities of interpolypeptide SS bonds differ according to the kinds ofsubunits. Band 6 (MW42 Kd) appeared at the position of MW38 Kd by reduction with lower concentrations of2-ME, indicating the retention of intra-polypeptide SS bonds. Bands 1+1′ 2, 3, and 7 that always appeared at the same positions independent of the 2-MEconcentrations, were separated from partial reduced glutenin (0.5 mMof 2-ME) and their SHand SS determined, in order to knowwhether these subunits retain intrapo lypeptide SS bonds. While the amount of liberated SH was much less than 2 mol per mol protein, the amount of SS bands was near 2mol, indicating that the liberated subunits retain intrapolypeptide SS bonds.