Isolation and Characterization of Gliadin-Like Subunits from Glutenin

The polypeptide subunits of wheat glutenin obtained after reductive cleavage of disulfide bonds have been separated into two fractions by a modified Osborne technique. Approximately 62% of the subunits by weight are soluble, as is gliadin, in neutral 70% ethanol; the ethanol-soluble subunits are mainly of 44,000 molecular weight (MW). The ethanol-insoluble glutenin subunit fraction is markedly different in amino acid composition, and consists of high-MW subunits, and some of lower MW. Sodium dodecyl sulfate electrophoresis, starch-gel electrophoresis, and amino acid analyses suggested that some ethanol-soluble subunits of reduced glu tenin may be equivalent to subunits of high-MW gliadin. To test this possibility, both fractions were reduced and alkylated and 44,000-MW subunits were isolated and partially characterized. Wheat gluten is formed by the interaction of endosperm proteins with other cellular components when flour is hydrated and becomes the elastic cohesive mass known as dough. The major proteins responsible for the properties of gluten may be fractionated by Osborne's (1) procedure into gliadin, which is soluble in neu tral 70% ethanol, and glutenin, the alcohol-insoluble component. Gliadin consists mainly of many different single-chained profeins of relatively low molecular weigh t (MW), whose conformations are stabilized by intramolecular disulfide bonds (see 2, 3, and 4 for recent studies and reviews of earlier work). A small amount of high-MW protein has also been separated from gliadin by gel-filtration chromatography (4). Glutenin, however, contains subunits of many sizes joined through disulfide bonds into proteins with MWs ranging into the millions (see 5 for review). Glutenin's subunits have been partially separated and characterized after reduction of disulfide bonds (6-10). We also recently studied the number and MW of gluten proteins and their subunits by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) (I 1). Similarities between glutenin's subunits and gliadin molecules (see 12-13 for review) have led to theories that glutenin consisted, at least in part, of gliadin-like subunits joined by intermolecular disulfide bonds; other studies, however, emphasized differences between gliadin and glutenin. This apparent discrepancy was partially resolved by SDS-PAGE (Il), which showed that many glutenin subunits are unique, but at least three correspond in MW to proteins of gliadin. Prolamin-like subunits have also been observed in glu telins of corn (14-18), barley (19), and sorghum (20); for these cereals, fractionation of glutelin subunits was achieved by alcohol extraction in the presence of mercaptoethanol. Since, to our knowledge, no attempt had been made to fractionate wheat glutenin subunits by differential solubility in solvents of different polarity, we undertook the present study. This investigation demonstrated that certain glutenin subunits are soluble in neutral dilute ethanol; furthermore, these subunits are very similar to the subunits of high-MW gliadin. 1 Agricultural Research Service, U.S. Department of Agriculture. Mention of firm names or trade products does not constitute endorsement by the Department over others of a similar nature not mention~d. Copyright © 1973 American Association of Cereal Chemists, Inc., 3340 Pilot Knob Road, St. PaUl, Minnesota 55121. All r" hts reserved. \turchased by U. S. Dep · )Agriculture for Official USI 538 ISOLATIO or GLIADI -LIKE SUBU ITS MATERIALS AND METHODS VoL 50