Natural plant enzyme inhibitors. Characterization of an unusual α-amylase/trypsin inhibitor from ragi (Eleusine coracana Geartn.)

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Abstract
An inhibitor I-1, capable of acting on both .alpha.-amylase and trypsin, was purified to homogeneity from ragi (finger-millet) grains. The factor was stable to heat treatment at 100.degree. C for 1 h in the presence of NaCl and was stable over the wide pH range 1-10. Pepsin and Pronase treatment of inhibitor I-1 resulted in a gradual loss of both the inhibitory activities. Formation of trypsin-inhibitor I-1 complex, amylase-inhibitor I-1 complex and trypsin-inhibitor I-1-amylase trimer complex was demonstrated by chromatography on a Bio-Gel P-200 column. The inhibitor may be double-headed in nature. The inhibitor was retained on a trypsin-Sepharose 4B column at pH 7.0. Elution at acidic pH resulted in almost complete recovery of amylase-inhibitory and trypsin-inhibitory activities. .alpha.-Amylase was retained on a trypsin-Sepharose column to which inhibitor I-1 was bound but not on trypsin-Sepharose alone. Modification of amino groups of the inhibitor with 2,4,6-trinitrobenzenesulfonic acid resulted in complete loss of amylase-inhibitory activity but only 40% loss in antitryptic activity. Modification of arginine residues by cyclohexane-1,2-dione led to 85% loss of antitryptic activity after 5 h but no effect on amylase-inhibitory activity. A single bifunctional protein factor is responsible for both amylase-inhibitory and trypsin-inhibitory activities with 2 different reactive sites.