Stepwise elution chromatographic fractionation on carboxymethylcellulose of S-zone proteins from gluten

Abstract

A simple method for the chemical preparation of enriched gluten is presented. The S-zone and Base proteins in gluten have been isolated in pure form and good yield by a combination of chemical fractionation with subsequent stepwise elution chromatography on CMC. The optimum experimental CMC chromatographic conditions were determined and found to be: stepwise elution; 300 ml eluent for each step; column size, 6.5 × 15 cm; sample loading, 2 gm; 0.005 M sodium acetate buffer, pH 3.5; 1 M dimethylformamide; flow rate, 250 ml/hr. Gradient elution experiments were conducted to determine the concentration of NaCl required to elute each protein class, and from this information the stepwise elution schedule was established. About 95% of the protein that was applied to the column was recovered. The chemically prepared enriched gluten contained at least 70% of S-zone proteins, and 14% Base proteins. The spread zone proteins were further fractionated into three distinct groups (S 1, S 2, and S 3). These three groups were also observed as separate SGUE bands. The S 1 component is predominant, constituting 58% of the enriched gluten. The S 1 component was further fractionated into four components (S 1.1, S 1.2, S 1.3, and S 1.4). These component bands were separated by SGUE. Although the four components of the S 1 group were separated by SGUE, their absorbance peaks on CMC chromatography did overlap. These four S 1 component proteins are quite similar, perhaps differing slightly in net charge. It is considered that they represent reversibly associating species. The bands observed for the S 1, S 2, and S 3 protein groups were designated in the decreasing order of their mobility (net charge) as S 1.1, S 1.2, S 1.3, S 1.4, S 2.1, S 2.2, S 3.1, and S 3.2. The dye-binding capacity of the S 3 proteins is weak resulting in faint SGUE bands.