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

Storage proteins from three cereal species were reduced at acidpH in the presence or absence of urea. Reactions were usuallycarried out at 100°C and pH 3.2, using dithiothreitol asthe reductant. Changes in electrophoretic mobility of prolaminsin polyacrylamide gels were taken as evidence of protein reduction.One-dimensional polyacrylamide gel electrophoresis, densitometricanalyses and computerized evaluation of data were used to quantifyreactions. Parameters tested include the influence of time,heat, reductant concentration and the presence or absence ofurea. More than 45% of secalin, the alcohol-soluble proteinfrom rye, readily underwent reductive cleavage to yield bandsof substantially different mobilities. Results suggest thatat least 35% of the alcohol-soluble rye components contain intermoleculardisulphide bonds and that at least 12% consist of intramoleculardisulphides; other components appear to be already fully reducedor devoid of disulphide linkages. Analogous measurements forgliadins and hordeins showed that at least 10% of these prolaminsunderwent similar reductions; mobilities of the products suggestthat intramolecular linkages are involved. The method employedprovides a convenient measure of both the qualitative and quantitativechanges associated with the reduction of protein disulphidebonds and the results are important to an understanding of themolecular reactions responsible for the properties of glutenproteins. Key words: Rye proteins, Wheat proteins, Barley proteins, Reduction, Quantitative PAGE, Densitometry