J. Agric. Food Chem. 1994, 42, 2460-2465
2460
Fractionation of Wheat, Barley, and Rye Prolamins by Cation Exchange FPLC Fernando G. Chirdo,t,t,§Carlos A. Fossati,*J'and Maria C. Afion*ttJ1 Centro de Investigacion y Desarrollo en Criotecnologia de Alimentos (CIDCA) and Catedra de Inmunologia, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 116 (1900) La Plata, Argentina
Wheat, rye, and barley prolamins were separated using optimized elution conditions by cation exchange FPLC on an analytical Mono-S column. Wheat gliadins were resolved into a larger number of peaks than has been obtained hitherto. Most fractions contained a mixture of components as determined by SDS-PAGE and A-PAGE. At least one fraction was obtained for each group of gliadins that contains only one major component with minimal contamination by other components. Only w-gliadins occurred in five of the fractions. A preparative S-Sepharose Fast Flow column, run under similar chromatographic conditions, was used to isolate w-gliadins. This procedure could be used as the first step for the purification of individual gliadin components. Chromatography on the Mono-S column was highly reproducible and showed promise as a means of differentiating wheat cultivars. Furthermore, FPLC was applied t o fractionate prolamins from barley and rye.
Keywords: FPLC; gliadin fractionation; prolamin fractionation INTR 0DUCTION The major wheat storage protein groups are gliadin and glutenin, which are differentiated on the basis of their extractability. Up to 112 components can be found using high-resolution 2-D electrophoresis separation (Tkachuk and Mellish, 1987). The gliadin fraction is classified into a-, p-, y-, and w-gliadins, according to their relative mobilities by electrophoresis a t pH 3 (Bushuk and Zillman, 1978). These proteins have a characteristic amino acid composition, notable features being the presence of large proportions of glutamine and proline residues. Their sequences contain extensive regions of tandem repeats of five to nine amino acid consensus sequences (Kasarda et al., 1983, 1984; Shewry et al., 1986). Although there are some differences, these repetitive sequences are found in all groups of gliadins, hordeins, and secalins (the last two being the prolamin fractions of barley and rye, respectively) (Kreis et al., 1985; Shewry and Tatham, 1990). These highly polymorphic proteins contain many structurally related components with some similar biochemical properties. They have unusual extractability properties and have a marked tendency to aggregate. Such properties present many difficulties for the fractionation and characterization of the polypeptides. The structural homology among prolamins is also responsible for their known high degree of immunological cross-reactivity, thus making their purification difficult, even by immunochemical methods (Skerritt and Underwood, 1986; Festenstein et al., 1987). Gliadin polypeptide have been separated traditionally by conventional chromatographic methods, such as gel filtration followed by ionic exchange on sulfoethylcellulose (Huebner and Rothfus, 19681, (carboxymethyl)*Author to whom correspondence should be addressed (fax 54-21-25-4853). ' CIDCA. Catedra de Immunogia. § F.G.C. is a fellow of CONICET. "Member of the Carrera del Investigador of the National Council Research (CONICET).
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0021-8561/94/1442-2460$04.50/0
cellulose (Patey and Evans, 1973) or sulfopropylSephadex (Charbonnier and MOSSB,1980). The best analytical separation has been obtained by employing reversed-phase high-performance liquid chromatography (RP-HPLC) (Bietz, 19851, but only small amounts of proteins could be recovered. Higher resolution and shorter analysis times than those of low-pressure chromatography have been reported recently using fast protein liquid chromatography (FPLC) on anion exchanger (Mono-&) (Batey, 1984) or cation exchanger (Mono-S) (Larre et al., 1991). In the study presented here we have developed improved conditions for the analytical and preparative cation exchange FPLC of gliadin proteins and have explored the usefulness of the procedure for the differentiations of wheat cultivars. Furthermore, this is the first study in which cation exchange FPLC was applied to fractionate hordein and secalin proteins. MATERIALS AND METHODS Cereal Cultivars. Wheat cultivars included Triticum aestiuum L. (hard red spring wheat) INTA La Paz, Staparka, Buck Bagual, Buck Pucara, INTA Marcos Juarez, Buck Fogon, Buck Catriel, ProINTA Pigue, and ProINTA Oasis. The barley cultivar used was Hordeum vulgare L. Linea 371; the rye cultivar used was Secale cereale L. Insave FH. Prolamin Preparation. The gliadin fraction was extracted from ground grain using 70% (v/v) aqueous ethanol extraction (10 m u g , 2 h at room temperature) after prior extraction of the albumin-globulin fraction using 0.15 M NaCl (10 m u g , 4 x 1h at room temperature). After each extraction step, a clear supernatant was obtained by centrifugation at lOOOOg for 15 min at 8 "C. The whole gliadin fraction was lyophilized, dissolved in and dialyzed against the FPLC starting buffer, and finally filtered through a 0.22 pm Nucleopore membrane (Millipore). The protein concentration was determined according to the Lowry method (Lowry et al.,1951) using a gliadin solution as standard. Hordein and secalin fractions were obtained by the same procedure. Cation Exchange FPLC. FPLC separations were carried out on a Pharmacia FPLC system. Two columns were used:
0 1994 American Chemical Society
J. Agric. food Chem., Vol. 42, No. 11, 1994 2461
Wheat, Barley, and Rye Prolamins
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