High Performance Liquid Chromatography: How Proteins Look in Cereals

The fractionation and characterization of cereal proteins may be among the most difficult problems in biochemistry: these proteins are heterogeneous. have unusual solubility characteristics. and have marked tendencies to aggregate. both covalently and noncovalently. Recent reviews (Bietz and Huebner 1980. Kasarda et a11976. Khan and Bushuk 1979. Miflin and Shewry 1979. Wall 1979) demonstrate that progress has been made in our understanding of cereal proteins. particularly the wheat endosperm storage proteins. gliadin. and glutenin. Nevertheless. much remains to be known about these proteins. and there is a continuing need for improved methods of separation and analysis. Cereal protein classification still follows fairly closely that proposed by Osborne (1907). Albumins and globulins are extractable by water or dilute salt solutions. respectively. These classes include enzymes and other proteins necessary for normal physiological processes. Prolamins (gliadin in wheat. zein in corn) are those proteins soluble in aqueous solutions of alcohols (e.g.. 70C;i: ethanol or 559i: isopropanol). Their solubilities result primarily from their unusual amino acid compositions (rich in glutamine. proline. and hydrophobic amino acids). Most prolamins are monomers of molecular weight (iv1 W) 30.000-40.000. Prolamins have no known function other than storage and are very heterogeneous because of duplication and subsequent nonlethal mutation of genes: as a result. much homology exists among prolamins (Bietz et al 1977). In addition. wheat polyploidy further increases gliadin heterogeneity. If all albumins. globulins. and prolamins are extracted from a cereal. the remaining endosperm protein can be regarded as glutelins. Such a fractionation is. however. complicated by the tendency for cereal proteins to associate: for example. gliadin strongly binds to glutenin (Bietz and Wall 1975). the glutelin fraction of wheat. Glutelins are high-ivIW polymers consisting of subunits joined both through covalent (disulfide) and noncovalent (primarily hydrogen and hydrophobic) bonds. They are soluble. if at all. only in dilute acids. alkali. high concentrations of denaturants (such as urea or guanidine hydrochloride). or in solutions containing detergents (such as sodium dodecyl sulfate [SDS]). To fully solubilize glutelins. it is frequently necessary to cleave and stabilize disulfide bonds through reduction and alkylation and to disrupt noncovalent bonds with denaturants and detergents. Glutelin subunits are then soluble and more easily characterized: some are similar to prolamins. but in many cereals. such as wheat and corn. glutelin subunits and prolamins have uniquely different structures. Other glutelin subunits resemble albumins and globulins. whereas some. such as the high-M W subunits of wheat glutenin. occur in no other protein class. Numerous reasons exist to isolate and characterize cereal proteins. Amounts or types of proteins may directly influence functional properties: for example. native glutenin ivI W relates closely to wheat breadmaking quality (Orth and Bushuk 1972. Huebner and Wall 1976). as does the presence of specific high-tv! W glutenin subunits (Payne et al 1980). If the identity of these quality-