Purification and Characterization of α-Amylase Inhibitors from the Seeds of Three Cultivars of the Genus Phaseolus

Abstract

Two kinds of proteinaceous α-amylase inhibitors were purified from the respective seeds of three cultivars of Phaseolus beans (Phaseolus vulgaris L. cv Otebou, Phaseolus coccineus L. cv Murasakihanamame, and Phaseolus coccineus L. cv Shirohanamame) by water extraction, ammonium sulfate fractionation, DEAE-Sepharose ion-exchange chromatography and Sephacryl S-200HR gel filtration. Among 6 inhibitors (OAI-1, OAI-2 from Otebou, MAI-1, MAI-2 from Murasakihanamame, and SAI-1, SAI-2 from Shirohanamame) purified, 4 inhibitors (OAI-1, OAI-2, MAI-2, and SAI-2) were shown homogeneous by disc-PAGE. Isoelectrofocusing indicated that the isoelectric points of these inhibitors were 4.6 for OAI-1, 4.5 for OAI-2, and 4.9 for MAI-2 and SAI-2. Each inhibitor was a glycoprotein with a molecular weight of about 45000. SDS-PAGE showed that these inhibitors were composed of subunits with molecular weights between 14000 and 30000. Amino acid composition of these inhibitors was similar to each other and characterized by high contents of aspartic acid and serine, low contents of methionine and histidine, and no cysteine. OAI-2, MAI-2, and SAI-2 showed almost the same inhibitory activity against porcine pancreatic α-amylase at pH 6.9, while OAI-1 showed weaker inhibitory activity than the above inhibitors. Heat treatment at 70°C indicated that OAI-2, MAI-2, and SAI-2 were thermostable, while OAI-1, MAI-1, and SAI-1 were thermolabile.