Vol. 74, No. 3, 1997 229 PROTEINS Characterization and Quantification of Native Glutenin Aggregates by Multistacking Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Procedures 1 DONG YIN HUANG 2 and KHALIL KHAN 2,3 ABSTRACT Cereal Chem. 74(3):229–234 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 polyacryla- mide 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 solubil- ity 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 sam- ple 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 ana- lyzed 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 ran- domized structure of native glutenins is proposed based on the results of our investigations. Native glutenin molecules have a molecular weight ranging from a few hundred thousand to millions estimated by gel-filtration chromatography (Huebner and Wall 1976, Payne and Corfield 1979, Graveland et al 1982). Huebner and Wall (1976) separated glutenin proteins into two fractions by using Sepharose 4B and 2B: a very high molecular weight fraction (>20 million) eluted at void volume and a lower molecular weight fraction with a broad peak. The ratio of the first glutenin peak to the second peak was greater for strong than for weak flours. Payne and Corfield (1979) found that glutenin proteins exclud- ed from Sepharose CL-4B consisted of three groups of subunits by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): group A with apparent molecular weights from 136,000 to 95,000 (high molecular weight [HMW] glutenin subunits); group B from 42,000 to 51,000; and group C from 35,500 to 31,500. Both groups B and C were referred to as low molecular weight (LMW) glutenin subunits. Glutenins of different molecular weights, eluted from an agarose gel column, had similar subunit composition but dif- ferent proportions of the three groups of glutenin subunits. As the size of the glutenin increased, the proportion of group A subunits (HMW) became larger relative to both the group B and C subunits (LMW), and the ratio of group B to group C also increased. Graveland et (1985) fractionated glutenin proteins into three fractions by differential solubility in SDS and 70% ethanol: an SDS-insoluble glutenin I fraction (MW >10 million), an SDS- soluble but ethanol-insoluble glutenin II fraction (MW 700,000– 10 million), and both SDS and ethanol soluble fraction III (MW 120,000–600,000). But they found that both glutenins I and II, which consisted of groups A, B, and C subunits, had the same ratio of the three groups of subunits (A-B-C = 3: 3.4:1.7). Because gel filtration offers limited resolutions for separation of large native glutenins, it is difficult to investigate the differences in the sizes of glutenin aggregates among wheat and flours with differ- ent baking quality. This could be seen from the similar profiles ob- tained by gel-filtration chromatography of glutenin proteins of wheat lines with varying numbers of HMW glutenin subunits (from 0 to 5 HMW subunits) (Gao and Bushuk 1993). Glutenin aggregates that contained all five HMW glutenin subunits had gel-filtration profiles very similar to those aggregates that contained only LMW glutenin subunits. Size-exclusion high-performance liquid chromatography (SE- HPLC) was also used to characterize native glutenin proteins (Singh et al 1991) by separating total glutenin proteins from nonglutenin proteins. To solubilize glutenins, the procedure involved the use of sonication which might cleave disulfide bonds of large glutenin aggregates to smaller aggregates (Khan et al 1994). The SE-HPLC procedure is limited in characterizing the molecular distributions of native glutenin proteins. A multistacking gel electrophoresis procedure (Khan and Huckle 1992) was devised to characterize native glutenin proteins by fractionating the glutenin aggregates into six fractions based on their sizes and mobilities on the multistacking gel. In this proce- dure, five stacking gels with various acrylamide concentrations (4, 6, 8, 10, and 12%) on top of a 14% separating gel were used to separate the different glutenin aggregates at the various origins of the stacking gels. The larger the size of the aggregates, the more difficult for the aggregates to move into the higher concentration stacking gels. We used this procedure to investigate the composi- tions of native glutenin proteins of two hard red spring wheats differing in breadmaking quality. MATERIALS AND METHODS Wheat and Flour Samples Two genotypes of hard red spring wheat were used for this study: Len, which has a good breadmaking quality, and the ex- perimental line 205, which has a weak dough mixing quality. Len is commonly used as a good quality reference variety in evalua- tions of hard red spring wheat in the wheat breeding program at 1 Published with the approval of the Director, Agricultural Experiment Station, North Dakota State University, Fargo, ND 58105. 2 Postdoctoral fellow and professor, respectively. Department of Cereal Science, North Dakota State University, Fargo. 3 Corresponding author. E-mail: kkhan@prairie.nodak.edu Publication no. C-1997-0404-01R. © 1997 by the American Association of Cereal Chemists, Inc.