Large-scale preparation of gliadin proteins (1)
The column (22 x 10 cm), packed with CM 52 cellulose (1.7 kg), was equilibrated for 24 h with eluting buffer (0.005 M-sodium acetate, 1 M-dimethylformamide, pH 3.5 with acetic acid) prior to use. It was loaded with a solution of gliadin in the buffer. Further buffer (1.5 litres) was passed down the column, followed by buffer containing the following concentrations of sodium chloride: 10 to 25 mM (2 litres, linear gradient), 25 mM (4 litres), 45 mM (4 litres), 65 to 90 mM (4 litres, hear gradient), 100 to 500 mM (2 litres, linear gradient). This was followed by 8 M-urea (1 litre), after which the column was repacked and re-equilibrated. Pooled fractions were concentrated, were dialysed for 48 h against 0.1 M-acetic acid and were then freeze-dried.
Gel filtration was carried out on Sephadex GI00 (column: 200 x 5 cm) using 0.05 M-acetic acid as eluent. The required fractions were freeze-dried. The separation achieved during ion-exchange chromatography. Each of the four separations gave an identical “Uvicord” trace. The eluate was split into 13 sets of fractions which were freeze-dried after concentration and dialysis. The composition of each fraction was assessed by electrophoresis. The total amounts of the major gliadin fractions are given. Fractions (3), (5), (8) and (11) contained mixtures of the various groups of the gliadin proteins, while after fraction (11) the water-soluble proteins (albumins and globulins) were eluted. Fractions (2),(4), (6)/(7) and (9)/(10) contained the w-, g-, b- and a-gliadin mixtures, respectively, as judged by their relative mobilities in starch gel electrophoresis.
The s.g.e. pattern for the four protein groups so obtained. The purity of the w-, g- and b-fractions is high, as assessed by electrophoresis. However, the a-gliadin fraction is contaminated with material which makes the s.g.e. pattern streak. Accordingly, in order to prepare pure a-gliadin, the a-fraction was subjected to G100 gel filtration. The contaminants, low molecular weight glutenin and b-gliadin, can be removed in this way. A pure w-component can be obtained by the use of the CMC column alone. By running the first linear salt gradient from 10 to 22 mM and then eluting with 22 mM-NaCl in buffer, a pure w-gliadin is obtained.
