Reduction of Disulphide Bonds at Acid pH and Quantitative Estimation of Prolamins from Rye, Wheat and Barley following Separation by Electrophoresis

Abstract

Storage proteins from three cereal species were reduced at acid pH in the presence or absence of urea. Reactions were usually carried out at 100 °C and pH 3-2, using dithiothreitol as the reductant. Changes in electrophoretic mobility of prolamins in polyacrylamide gels were taken as evidence of protein reduction. One-dimensional polyacrylamide gel electrophoresis, densitometric analyses and computerized evaluation of data were used to quantify reactions. Parameters tested include the influence of time, heat, reductant concentration and the presence or absence of urea. More than 45% of secalin, the alcohol-soluble protein from rye, readily underwent reductive cleavage to yield bands of substantially different mobilities. Results suggest that at least 35% of the alcohol-soluble rye components contain intermolecular disulphide bonds and that at least 12% consist of intramolecular disulphides; other components appear to be already fully reduced or devoid of disulphide linkages. Analogous measurements for gliadins and hordeins showed that at least 10% of these prolamins underwent similar reductions; mobilities of the products suggest that intramolecular linkages are involved. The method employed provides a convenient measure of both the qualitative and quantitative changes associated with the reduction of protein disulphide bonds and the results are important to an understanding of the molecular reactions responsible for the properties of gluten proteins.