Purification And Structure-Function Study Of Novel α-Amylase Inhibitors From Wild Amaranth (Amaranthus Paniculatus) Weeds

Two novel proteinaceous inhibitors, named WAI-1 and WAI-2 respectively, have been found in the wild amaranth(Amaranthus paniculatus) weeds. In the present study, WAI-1 and WAI-2 were purified by RP-HPLC (reverse-phase high performance liquid chromatography), and their properties, structures and biochemical activities were investigated. WAI-1 and WAI-2 have molecular weights of 986.5 Da and 1027.5 Da respectively, determined by MALDI-TOF-MS (matrix-assisted laser-desorption /ionization time- of-flight mass spectrometry). They are the smallest proteinaceous inhibitors of α-amylase found so far.WAI-1 could potently inhibit the α-amylase of Periplaneta americana digestive duct in a noncompetitive manner under mild acid conditions, with the optimal inhibitory activity at pH 6. 0. WAI-1 exhibited the highest inhibitory activity after preincubation with the enzyme at 37°C for about 30 min. When a fixed amount of α-amylase used, along with the increase of the inhibitory/ enzyme ratio the inhibition percentages of the α-amylase activity were linearly increased up to about 50%, and then increased slowly up to a maximum of about 65%. Afterpreincubation at 80°C for 120 rain WAI-1 could inhibit 58. 7% of a -amylase activity; after preincubation at 100°C for 10 min, WAI-1 could still lead to the inhibition rate of 23.08%, which indicated that WAI-1 has strong thermal stability. WAI-1 did not inhibit the human salivary a -amylase, hog pancreatic a-amylase, bacillus subilisa -amylase and aspergillus oryzae a -amylase. The inhibitory activity of WAI-1 to Periplaneta americana digestive duct a -amylase was higher than that to yellow mealworm digestive duct a-amylase. Amino acid composition analysis, Edman degradation and tandem mass spectrometry were used to determine the chemical structure of WAI-1, and the results indicated that WAI-1 was a cyclic nonapeptide with N-terminal pyroglutamate. Dilute hydrochloric acid hydrolysis of WAI-1 made pyroglutamic acid open side-chain loop, which had no significant effect on the inhibitory activity. Tryptic hydrolysis of WAI-1 made the molecule cleaved at c-terminal of arginine, which made WAI-1 lose its inhibitory activity.WAI-2 potently inhibited the a -amylase of the Periplaneta americana digestive duct in a competitive manner under mild acid conditions, with the optimal inhibitory activity at pH 5. 5. WAI-2 exhibited the highest inhibitory activity after preincubation with the enzyme at 37°C for about 35 min. When...