Tris¬HCl, pH 8.0,¬was used for bovine trypsin and chymotrypsin. Enzyme and various amounts of kalata B1, kalata B2, or Nicotiana alata proteinase inhibitor were mixed and preincubated in a 60-¬µl volume for 30¬min at 30¬C before the addition of substrate (40¬¬µl). The reactions were continued for 30¬min at 30¬C before release of p-nitroanilide was recorded at 405¬nm on a SpectraMax 250¬microtiter plate reader (Molecular Devices). Trypsin activity was determined by using 4¬¬µg (1.67¬¬µM) bovine trypsin [Type XIII 1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) treated, Sigma] or 3.2¬¬µg of gut protein. Chymotrypsin assays used 0.1¬¬µg (0.04¬¬µM) of bovine chymotrypsin (Type VII TLCK treated, Sigma) or 2.6¬¬µg of gut protein. Amylase assays were performed at 30¬C in 200 ¬µl of reaction mixture containing 26 ¬µg of gut protein and 0.5% (wt/vol) starch (Sigma) in CAPS buffer. Samples (20 ¬µl) were removed at 10, 20, and 30 min for reducing sugar assay by using dinitrosalicyclic acid reagent. Potential inhibitory activity of kalata B1 and B2 was measured by mixing 20 ¬µg with gut protein (26 ¬µg) and preincubating at 30¬C for 1 h in CAPS buffer before the enzyme assay was initiated by the addition of an equal volume of 1% (wt/vol) starch in the same buffer. The -amylase inhibitor from wheat seeds (Sigma A1520) was used as a positive control.