Purification and Properties of Phaseolamin, an Inhibitor of ar-Amylase, from the Kidney Bean, Phaseohs vulgaris

Kidney beans, Phaseolus vulgaris, contain a proteinaceous inhibitor of a-amylase, which we have named phaseolamin. The inhibitor has been purified to homogeneity by conventional protein fractionation methods involving heat treatment, dialysis, and chromatography on DEAE-cellulose, Sephadex G-100, and CM-cellulose. Phaseolamin is specific for animal a-amylases, having no activity towards the corresponding plant, bacterial, and fungal enzymes, or any other hydrolytic enzyme tested. Optimal inhibitory activity is expressed during preincubation of enzyme and inhibitor at pH 5.5 and 37” Substrate prevents inhibition. Measurement of the stoichiometry of inhibition showed that a 1:l complex of a-amylase and inhibitor is formed. Complex formation was demonstrated by chromatography on Sephadex G-100. The phaseolamin .amylase complex is dissociated at low pH values, apparently as a result of destruction of the enzyme; the complex cannot be dissociated by other conditions unfavorable for inhibition (low temperature or high pH). Phaseolamin inhibits hog pancreatic a-amylase in a noncompetitive manner.