Wheat Varietal Identification by Rapid Ion-Exchange Chromatography of Gliadins (1)
A rapid ion-exchange chromatography system for proteins has also become available. This system, known as ‘ Fast Protein Liquid Chromatography’ (FPLC) utilises cation or anion exchange columns , or a chromatofocusing column. The separation of proteins on these ion-exchange columns is claimed to occur solely on the basis of charge, and not to involve other interactions with the column support material.
The gliadin fraction was isolated from flour by aqueous ethanol (70%, v/v) extraction after preliminary extraction of the water- and salt-soluble components with 10% (w/v) sodium chloride. The gliadin samples were prepared for chromatography by dispersing them in starting buffer (5 mg gliadin/ml), The samples were stirred for 16 h at room temperature (c. 20-25°C) and centrifuged at 8000g.
For direct extraction of gliadins from straight-run (76% extraction) flour, samples were extracted by stirring for 30min at 20-25°C in the starting buffer, using 100mg flour/ml of buffer. The suspension was then centrifuged at 8000g. Immediately prior to application to the column, each gliadin or flour extract was filtered through a 0.22 urn filter.
The medium for chromatography was 1 M urea containing 0.01 M buffer with a linear gradient of 0-0.5 M Na acetate. Buffers used were as follows: pH 8.0, tris-(hydroxymethyI)-aminomethane (TRIS); pH9.0, triethanolamine; pH9.5, 2-(cyclohexylamino)-ethane-sulphonicacidand, pH 10.4, 3-(cyclohexylamino)-propane-sulphonic acid. In order to minimise corrosion of stainless steel fittings in the chromatographic system, acetate was chosen in preference to chloride as the counterion for the buffers and the salt gradient. Flow rate was 1.2 ml/min. The sample volume was generally 0.1-0.2 ml. After each analysis, the column was washed with 1 M Na acetate (2 ml) at the appropriate pH , followed by regeneration with a starting buffer.
Elution patterns of the Mono-Q column for four particular pH values are shown, and other buffers within the pH range 4.0-10.5 have been examined. Despite the large number of components shown by electrophoresis, chromatographic separation was poor unless buffers of quite high pH were used. Other varieties gave similar results, and pH 10.4 was chosen as the optimum pH for chromatography on Mono-Q.
Each variety could be differentiated readily. The varieties Egret and Condor are difficult to distinguish by electrophoresis, and failure to do so may cause problems for wheat users because they are wheats of different quality types.
Flour from the variety, Songlen, was extracted with a starting buffer and the proteins in this extract were subjected to chromatography on the Mono-Q column. The elution profile of the extract (a) differed from that of the isolated gliadins (d) mainly in the appearance of peaks that were eluted before and after those for isolated gliadins. Chromatography of an albumin and globulin preparation indicated that the material that was eluted early from the column was water- and/or salt-soluble protein. The broad peaks that were eluted after the gliadin peaks (c. 13-20 min) are presumed to be glutenin components that were extracted in the starting buffer.
