Two Kunitz-type inhibitors with activity against trypsin and papain from Pithecellobium dumosum seeds: purification, characterization, and activity towards pest insect digestive enzyme

• Published in: Protein and peptide letters Vol. 16; no. 12; p. 1526
• Main Authors: Oliveira, A S, Migliolo, L, Aquino, R O, Ribeiro, J K C, Macedo, L L P, Bemquerer, M P, Santos, E A, Kiyota, S, de Sales, M P
• Format: Journal Article
• Language: English
• Published: Netherlands 01.12.2009
• Subjects: Amino Acid Sequence; Animals; Bromelains - antagonists & inhibitors; Bromelains - metabolism; Cattle; Chymotrypsin - antagonists & inhibitors; Fabaceae - chemistry; Larva - drug effects; Larva - enzymology; Lepidoptera - drug effects; Lepidoptera - enzymology; Molecular Sequence Data; Pancreatic Elastase - antagonists & inhibitors; Papain - antagonists & inhibitors; Peptides - chemistry; Peptides - pharmacology; Plant Proteins - chemistry; Plant Proteins - pharmacology; Seeds - chemistry; Sequence Alignment; Sequence Analysis; Trypsin - metabolism
• ISSN: 1875-5305
• DOI: 10.2174/092986609789839403

Two trypsin inhibitors (called PdKI-3.1 and PdKI-3.2) were purified from the seeds of the Pithecellobium dumosum tree. Inhibitors were obtained by TCA precipitation, affinity chromatography on Trypsin-Sepharose and reversed-phase-HPLC. SDS-PAGE analysis with or without reducing agent showed that they are a single polypeptide chain, and MALDI-TOF analysis determined molecular masses of 19696.96 and 19696.36 Da, respectively. The N-terminal sequence of both inhibitors showed strong identity to the Kunitz family trypsin inhibitors. They were stable over a wide pH (2-9) and temperature (37 to 100 degrees C) range. These inhibitors reduced over 84% of trypsin activity with inhibition constant (Ki) of 4.20 x 10(-8) and 2.88 x 10(-8) M, and also moderately inhibited papain activity, a cysteine proteinase. PdKI-3.1 and PdKI-3.2 mainly inhibited digestive enzymes from Plodia interpunctella, Zabrotes subfasciatus and Ceratitis capitata guts. Results show that both inhibitors are members of the Kunitz-inhibitor family and that they affect the digestive enzyme larvae of diverse orders, indicating a potential insect antifeedant.