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Characterization and Quantification of Native Glutenin Aggregates by Multistacking Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Procedures¹

¹Published with the approval of the Director, Agricultural Experiment Station, North Dakota State University, Fargo, ND 58105. ²Postdoctoral fellow and professor, respectively. Department of Cereal Science, North Dakota State University, Fargo. ³Corresponding author. E-mail: kkhan@prairie.nodak.edu

Native glutenin aggregates of two different quality flours containing the same high molecular weight (HMW) glutenin subunit compositions were investigated by multistacking sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) procedures. Five stacking gels (4, 6, 8, 10, and 12%) with a separating (resolving) gel of 14% were used to separate nonreduced glutenin aggregates solubilized from flour by SDS sodium phosphate buffer. There were large differences in protein solubility of the two flours. It took 8 hr to extract 91% of total proteins from the strong flour (variety Len) while it took only 2 hr for the weak flour sample 205. Total glutenin proteins and the proportions of glutenins at the different origins (including the origin of the 14% separating gel) were quantified by high-resolution densitometry procedures. As the duration of extraction increased, both total glutenins and glutenins at the 4% origins increased. The good quality flour Len had a higher total glutenin protein (3% more) and higher proportion of glutenins with the largest molecular sizes (also 3% more) at the 4% origins than the poor quality flour sample 205. After glutenin aggregates from each origin were reduced and analyzed by SDS-PAGE, the largest glutenins at the 4% origin contained twice the amount of total HMW glutenin subunits when compared to the smaller aggregates at the 12% origin. Among the total HMW glutenin subunits, the proportion of subunit 5 (which published literature reports to be the largest molecular weight based on calculations of DNA-derived amino acid sequence analysis) was twice that at the 12% origin. A randomized structure of native glutenins is proposed based on the results of our investigations.