Separation and Characterization of Wheat Protein Fractions by High - Performance Capillary Electrophoresis 1

Cereal Chem. 72(6):527-532 Wheat protein fractions, separated by the Osborne Solvent Fractionaseparated by HPCE. Combined results of this study and our previous tion Procedure, were characterized by high-performance capillary elecstudies provide a catalog of individual gliadin information from HPCE, trophoresis (HPCE). Each fraction was separated on a 27-cm fused-glass HPLC, acid-polyacrylamide gel electrophoresis (A-PAGE) and sodium capillary (20 gm, i.d.) using 0.1M phosphate buffer (pH 2.5) containing dodecyl sulfate (SDS)-PAGE, relating class, relative molecular size, hyhydroxypropylmethylcellulose, a polymer additive, at 45 C and 22 kV drophobicity, relative charge, and separation times of each gliadin constant voltage. Albumins and globulins migrated in the first 4 min, subclass by HPLC and HPCE. The main advantages of HPCE are: 1) the whereas gliadins and glutenins migrated after 4 min. Individual x, 13, y, complementing of other electrophoretic and chromatographic protein and Xo gliadin proteins, which were collected from reversed-phase highseparation methods, and 2) safety because no toxic acrylamides and only performance liquid chromatography (RP-HPLC) separations, were also minute amounts of organic solvents and buffers are used. The baking quality of wheat flour is primarily dependent on the quantity and quality of the flour proteins (Finney 1943). As most of the proteins in flour are gluten, at least one gluten component should relate to breadmaking quality. In fact, two of the more important flour quality factors, mixing time and loaf volume, are related to one or more gluten components (Finney et al 1982). Gliadins comprise about half of the gluten and the endosperm storage proteins of wheat. They have been used to identify wheat varieties (Wrigley and Baxter 1974, Bushuk and Zillman 1978, Jones et al 1982, Lookhart et al 1982) and are known to correlate with breadmaking quality (Finney et al 1982). Glutenins are the other major component of gluten and endosperm storage proteins. Numerous studies (Bietz et al 1975; Payne et al 1981, 1984; Shewry et al 1989; Graybosch et al 1990; Dong et al 1991) over the past 20 years have established that allelic variation among high molecular weight glutenin subunits (HMW-GS) is a major contributor to the mixing and baking quality of bread wheats. Breadmaking quality has also been related to allelic variation among low molecular weight glutenins (Gupta and Shepherd 1987). High-performance capillary electrophoresis (HPCE) recently has been used to identify wheat cultivars by characterizing gliadin protein patterns (Werner et al 1994, Bietz and Schmalzreid 1995, Lookhart and Bean 1995a). Gliadins were previously divided into four classes: o, P, y, and w (Jones et al 1959), based on electrophoretic migration. Lookhart and Albers (1988) were the first to analyze each gliadin HPLC peak (25 peaks) of one cultivar, and relate each peak to acid polyacrylamide gel electrophoresis (APAGE) and sodium dodecyl sulfate (SDS)-PAGE patterns. Some individual proteins that migrate within the range of these classes have also been identified by reversed-phase high-performance liquid chromatography (RP-HPLC) of gliadins extracted from genetic stocks (Bietz and Burnouf 1985). The HPLC assignments 'Contribution 95-358-J of the Department of Grain Science and Industry, Kansas State University, Manhattan, KS. Cooperative investigation with the U.S. Dept. of Agriculture, Agricultural Research Service. Mention of firm names or trade products does not constitute endorsement by the U.S. Department of Agriculture over others not mentioned. 2 Research chemist, USDA/ARS, U.S. Grain Marketing Research Laboratory, 1515 College Ave., Manhattan, KS 66502. E-mail: george(crunch.usgmrl.ksu.edu 3 Research assistant, Department of Grain Science and Industry, Kansas State University, Manhattan. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. American Association of Cereal Chemists, Inc., 1995. as to gliadin class elution order by Lookhart and Albers (1988) and Bietz and Burnouf (1985) were both made by inference to the assignments of Jones et al (1959) and were in agreement. HPCE, which separates mainly by the same principles (combination of size and charge) as A-PAGE but has the automation and convenience of RP-HPLC, also provides rapid high-resolution separations of gliadins (Lookhart and Bean 1995a). To take full advantage of the high resolution of HPCE, knowing the order of migration for the respective proteins could prove useful in the study of gliadins and other components and their relationships to quality. Werner et al (1994) analyzed one representative protein from each gliadin class by HPCE using an aluminum-lactate buffer (pH 3.1) in conjunction with a charge reversal coating (MicroCoat) to determine the migration order of the respective gliadin classes. The MicroCoat system separated proteins against the electroosmotic flow (EOF), causing the fastest migrating proteins to appear last on the electrophoregram. The phosphate buffer separation system described for rapid differentiation of wheat cultivars (Lookhart and Bean 1995a) was much faster than that of Werner et al (1994). This article thus reports, for the first time, HPCE separations of the classical Osborne wheat protein fractions (albumins, globulins, gliadins, and glutenins) and of several proteins from each of the four classes of gliadins using a low pH phosphate buffer system. Results from the HPCE analyses of gliadins were related to RP-HPLC, A-PAGE, and SDS-PAGE analyses (Lookhart and Albers 1988) of the same proteins. MATERIALS AND METHODS Wheat Flour Samples The hard red winter (HRW) wheat Shawnee was obtained from the Hard Winter Wheat Quality Laboratory in Manhattan, KS. It was grown in 1981 at Manhattan, KS; it had a protein content of 18.1%. Flour was produced by a Brabender Senior experimental