Purification and Partial Characterization of Two Proteinaceous ?-Amylase Inhibitors From Triticale

Abstract

C. Postal 6001, 84051-970 -Londrina, PR -Brazil 2Dept. Alimentos e Nutricdo Experimental Faculdade de Ci6ncias Famc8uticas Universidade de Sdo Paul0 C. Postal 66083. 05389-970 -SaO Paulo, SP -Brazil ABSTRACTA total of six a-amylase inhibitory proteins (isoinhibitors) were extracted from triticale (Triticum x Secale) seeds and two of rhem were pur$ed to homogeneity. The isoinhibitors were extracted by 70% ethanol and produced, by Sephade-r G l 0 0 chromatography, two peaks that exhibited a-amylase inhibitory activity. Further purijkation ofthe most active peak by DEAE-cellulose chromatography resulted in six activefractions. Two of them designated as TAl-5 and TAl-6, considered to be homogeneous by both acidic and alkaline electrophoresis, were partially characterized. Ihe isoelectric points were 4.80 and 4.70, and the molecular weights 39,200 and 29,200, respectively. Under dissociating conditions the molecular weights were 13,500 and 13,000, suggesting that the isoinhibitors are composed of three and two subunits, respectively. Both isoinhibitors were stable at different pHs, relatively stable 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 specijkiry to human salivary and porcine pancreatic a-amylases, but had no inhibitory activiry on Bacillus subtillis, Aspergillus oryzae and endogenous tnticale a-amylases.