PURIFICATION AND CHARACTERIZATION OF ?-AMYLASE INHIBITORS IN WHEAT (TRITICUM AESTIVUM VAR. ANZA)

Abstract

Three α-amylase inhibitors were purified to homogeneity from Anza wheat (Triticum aestivum var. Anza) by extraction with 70% ethanol, ammonium sulfate fractionation and column chromatography on DEAE-cellulose, CM-ceillulose and Sephadex G-50. Homogeneity was determined by disc gel and isoelectric focusing electrophoresis and by sedimentation equilibrium centrifugation. The inhibitors are designated 0.19, 0.28 and 0.55 on basis of their relative electrophoretic mobilities on polyacrylamide gels. The 0.19, 0.28 and 0.55 inhibitors had molecular weights of 24,000, 18,500 and 30,000 by polyacrylamide gel electrophoresis with different gel concentrations while the former two were 29,000 and 14,500 by sedimentation equilibrium centrifugation, respectively. The molecular weight of the 0.55 inhibitor was not determined by centrifugation. The isoelectric points were 5.9, 5.2 and 4.2 for the 0.19, 0.28 and 0.55 inhibitors, respectively. The three inhibitors had similar amino acid compositions but differed significantly in amounts of lysine, arginine, histidine, alanine, valine and phenylalanine. The 0.19 inhibitor was active against human salivary and hog pancreatic α-amylases but inactive against Bacillus subtilis and Aspergillus oryzae α-amylases. The 0.28 inhibitor had very weak activity against only human salivary α-amylase. The 0.55 inhibitor had activity against only human salivary α-amylase. The 0.55 inhibitor appears to differ from all previously reported wheat α-amylase inhibitors while the 0.28 inhibitor (protein) is unique in having essentially no inhibitory activity.