Two Kunitz-Type Inhibitors with Activity Against Trypsin and Papain from Pithecellobium dumosum Seeds: Purification, Characterization, and Activity Towards Pest Insect Digestive Enzyme

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 oC) 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 Kunitzinhibitor family and that they affect the digestive enzyme larvae of diverse orders, indicating a potential insect antifeedant.