PURIFICATION AND PARTIAL CHARACTERIZATION OF 2 PROTEINACEOUS ALPHA-AMYLASE INHIBITORS FROM TRITICALE

A total of six alpha-amylase inhibitory proteins (isoinhibitors) were extracted from triticale (Triticum x Secale) seeds and two of them wer e purified to homogeneity. The isoinhibitors were extracted by 70% eth anol and produced, by Sephadex G-100 chromatography, two peaks that ex hibited alpha-amylase inhibitory activity. Further purification of the most active peak by DEAE-cellulose chromatography resulted in six act ive fractions. Two of them designated as TAI-5 and TAI-6, considered t o be homogeneous by both acidic and alkaline electrophoresis, were par tially characterized. The isoelectric points were 4.80 and 4.70, and t he molecular weights 39,200 and 29,200, respectively. Under dissociati ng conditions the molecular weights were 13,500 and 13,000, suggesting that the isoinhibitors are composed of three and two subunits, respec tively. Both isoinhibitors were stable at different pHs, relatively st able at 98C, and resistant to proteolysis by trypsin, chymotrypsin and pepsin. The optimum interaction pH for both isoinhibitors with human salivary amylase was 7.9. They exhibited specificity to human salivary and porcine pancreatic alpha-amylases, but had no inhibitory activity on Bacillus subtillis, Aspergillus oryzae and endogenous triticale al pha-amylases.