Protease Inhibitors from Coccinia Grandis (l.) Voigt. Leaves: Purification, Characterization and Kinetic Properties

Cucurbits are rich source of protease inhibitors. A protein of 14.3 kDa designated as Coccinia grandis protease inhibitors (CGPI) was isolated from leaves of Coccinia grandis and purified to homogeneity by ammonium sulfate precipitation, Sephadex G-75 column, DEAE Sepharose column and TrypsinSepharose affinity chromatography. The purity was checked by reversed phase chromatography. The molecular mass estimated by size exclusion chromatography was agreed well with the SDS-PAGE results. Both Native-PAGE as well as isoelectric focusing showed four isoinhibitors (pI values of 4.5, 5.0, 5.4, and 6.0). Inhibitory activity of CGPI remained unchanged over a broad range of temperatures (0 – 80 oC) and pH amplitude (2–12). CGPI exhibited a non-competitive-type inhibitory activity against both bovine pancreatic trypsin (Ki of 322.5 nM) and chymotrypsin (Ki of 11.6 μM). The protein inhibited trypsin in the stoichiometric molar ratio of 1:2, but lacked similar stoichiometry against chymotrypsin. The temperature-induced conformational changes in secondary structure are reversed when CGPI was cooled from 90 to 25oC. Further, upon reduction with dithiothreitol, CGPI lost both its inhibitory activity as well as secondary structural conformation. Lysine residue(s) present in the reactive site of CGPI are essential for the action of inhibiting the trypsin activity.