Characterization of Two α‐Amylase Inhibitors from Black Bean (Phaseolus vulgaris)

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Abstract
Two inhibitors of α‐amylase, I‐1 and I‐2, were purified from the black bean (Phaseolus vulgaris). The rate of complexation with porcine pancreatic α‐amylase was very slow. I‐l required 12 hr and I‐2 6 hr for maximum inhibition at 30°C. The stoichiometry of complexation of α‐amylase with I‐1 was 2:1 and 1:1 with I‐2. Optimum pH for complexation with I‐1 ranged from 4.5 to 5.0 and 5.0 to 5.5 for I‐2 and both were most stable at pH 3.0 to 4.0. An increase in ionic strength of the preincubation buffer from 0.106 to 0.906 enhanced the rate of inhibition 3 fold for both inhibitors. Maltose, a competitive inhibitor of α‐amylase, added to the preincubation solution prevented complex formation for both inhibitors; however, it did not reverse the inhibition of the preformed complex. The dissociation constant for I‐1 was 2.2 × 10−9M and 3.8 × 10−9M for I‐2 at 30°C and pH 6.9. Both inhibitors functioned via a noncompetitive mechanism. I‐2 was also stable at pH 2.0. I‐2 was more stable to proteolytic digestion by trypsin than I‐1 and was also stable to pepsin digestion.