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

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Abstract:

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.





Keywords: Kunitz inhibitor; Leguminosae; Pest proteases; Pithecellobium dumosum

Document Type: Research Article

DOI: https://doi.org/10.2174/092986609789839403

Publication date: 2009-12-01

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  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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