Cereal Chem 56:505 - 512. | VIEW
Studies of Glutenin. XIII. Gel Filtration, Isoelectric Focusing, and Amino Acid Composition Studies.
K. Khan and W. Bushuk. Copyright 1979 by the American Association of Cereal Chemists, Inc.
Glutenin, isolated from the hard red spring wheat cultivar Manitou, was reduced with beta-mercaptoethanol and alkylated with 4-vinylpyridine. Gel-filtration chromatography on Sephadex G-200 was done with four different adqueous solvents (acetic acid, acetic acid-urea [AU], acetic acid-urea- hexadecyltrimethylammonium bromide, and tris-HCl-sodium dodecyl sulfate). Each solvent produced an elution profile with three peaks; the amount of protein represented by each peak depended on the solvent. In all cases, peak I eluted with the void volume (mol wt greater than 200,000). The three peaks obtained with each solvent were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed qualitative and quantitative differences among the peaks. The three obtained with the AU solvent were, however, chosen for further detailed characterization since SDS-PAGE showed that each peak obtained with this solvent comprised subunits of different molecular weights. Peak I protein comprised subunits with molecular weights ranging from 12,000 to 68,000. Peak II contained the highest molecular weight subunits ranging from 68,000 to 134,000. Peak III proteins contained predominantly the 35,000 and 45,000 mol wt subunits. Amino acid compositions of the three peaks were markedly different especially in the proportions of basic, acidic, and hydrophobic amino acids. Peak II proteins were examined by isoelectric focusing on a 6-8 pH gradient of Amphyline carrier ampholytes in 6M urea. Elution of the focused proteins gave a profile with 12 peaks (fractions). These 12 fractions contained 20 different protein species, detected by SDS-PAGE. Two homogeneous subunits, 134,000 and 90,000 mol wt, were isolated by isoelectric focusing. The two subunits were partially characterized; they differed in amino acid composition and N-terminal amino acid.