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Methods to sequentially extract and fractionate wheat flour proteins were evaluated to reliably quantify gliadins, glutenins, and albumins/globulins in single flour samples. Compositions of the resulting protein fractions were analyzed by RP-HPLC combined with SDS-PAGE. Unknown proteins were identified by mass spectrometry or N-terminal sequencing. The best separation and recovery of discrete albumin/globulin, gliadin, and glutenin fractions from the same flour sample was achieved by extraction with 0.3 M NaI in 7.5% 1-propanol followed by 2% SDS, 25 mM DTT in 25 mM TRIS, pH 8.0, and precipitation of the solubilized proteins with ammonium acetate/methanol followed by acetone. Average flour composition for the variety Butte86 was 10% albumin/globulin, 40% gliadin, and 48% glutenin. This method should be useful for determining flour composition in diverse samples and evaluating relationships between proteins and end-use functionality.

Cereal Chem. 77(5):607-614 ω-Gliadins were purified from wheat (Triticum aestivum L. 'Butte') flour and characterized. Although reversed-phase HPLC (RP-HPLC) separated the 1B-encoded ω-gliadins into two fractions, 1B1 and 1B2, these fractions had nearly identical amino acid compositions, three similar protein bands in SDS-PAGE, 10 similar spots in two-dimensional PAGE, and two similar N-terminal amino acid sequences. The main components had a range in mass of 48,900-51,500 when estimated by mass spectrometry, significantly less than the mass estimated by SDS-PAGE. The 1B fractions were digested with thermolysin, the peptides were separated by RP-HPLC, the peptide mass was determined, and nine peptides from each fraction were sequenced with nearly identical results for the 1B1 and 1B2 digests. A possible consensus sequence of the 1B-encoded ω-gliadin internal repeat was QQQXP, where X was F, I, or L in descending order of occurrence. The 1D-encoded ω-gliadins were purified by RP-HPLC as a single fraction that had one band in SDS-PAGE, two spots in two-dimensional PAGE, two components with mass of 41,923 and 42,770 detected by mass spectrometry, and two N-terminal sequences. Circular dichroism (CD) spectra for the 1B and 1D ω-gliadins were similar and were suggestive of mainly flexible random structure with a significant amount of the left-handed polyproline II helical conformation in the 1D components.The ω-gliadin components of wheat flour are sulfur-poor prolamins with no cysteine. Closely related components that contain cysteine are referred to as D-glutenin subunits (Masci et al 1998).The ω-gliadins are composed almost entirely of high-glutamine, high-proline repeats and are related to the rye ω-secalins and the barley C-hordeins (Tatham and Shewry 1995). Several distinct ω-gliadins were identified in wheat based on electrophoretic patterns and N-terminal sequences, and their genes were localized to the Gli-B1 and Gli-D1 loci on the short arms of chromosome 1B and 1D, respectively (Kasarda et al 1983, Lafiandra et al 1984, Tatham and Shewry 1995, Zhao et al 1999. Additional ω-gliadin-like proteins, with genes located at the Gli-A1 locus on the short arm of chromosome 1A are less well characterized. Wheat was estimated to have at least 15-18 copies of ω-gliadin genes (Sabelli and Shewry 1991). In this article, we refer to the Gli-A1, Gli-B1, and Gli-D1 encoded ω-gliadins as the 1A, 1B, and 1D ω-gliadins.Sulfur availability and the ratio of sulfur to nitrogen in the soil affect dough elasticity and resistance and loaf volume, probably because of the effect of sulfur-to-nitrogen ratios on the gluten protein composition of wheat flour (Zhao et al 1999). The relative abundance of the sulfur-poor prolamins, particularly the ω-gliadins, and the intermediate-sulfur high molecular weight glutenin subunits (HMW-GS), increased when wheat plants were depleted of sulfur (Wrigley et al 1984, Zhao et al 1999 but the significance of changes in ω-gliadin amounts to dough quality is unknown. Because the ω-gliadins have no cyste...