Studies on low molecular mass phytocystatins purified from Phaseolus mungo (Urd)

In the present study two phytocystatins (thiol protease inhibitors) have been isolated and purified to homogeneity from Phaseolus mungo by a simple two-step procedure using ammonium sulfate fractionation and gel filtration on Sephacryl-100 HR. The latter procedure yielded two peaks of the inhibitors (PMC I and PMC II). The pH optimum of both phytocystatins was pH 7.0; the temperature optima for PMC I and PMC II were 65 and 70 degrees C, respectively. The molecular masses of the purified phytocystatins were 19 and 17 kD, respectively, as determined by SDS-PAGE and mass spectrometry. Antibodies raised against the purified cystatins gave a single precipitin line in Ouchterlony double immunodiffusion. Kinetics of inhibition showed that PMC I and PMC II strongly inhibit papain and ficin but not trypsin and chymotrypsin. Binding stoichiometry of PMC I and PMC II with both papain and ficin was 1 : 2. The effect of urea on PMC I and PMC II was analyzed by fluorescence and circular dichroism spectroscopy. The CD results suggest an unfolding of PMC I and PMC II accompanying a decrease in the amount of extended (hydrated) coil structure and an increase in sheet-like structure. FTIR results show that PMC I is structurally similar to PMC II. Hydrophobic interactions are observed over a long time scale (5-150 min). Furthermore, fluorescence spectroscopy results were found to be in accordance with CD results, by showing quenching of fluorescence intensity of PMC I and PMC II, although to different extents, due to perturbations of the environment of aromatic residues in the protein. Both cystatins showed strong inhibitory activity against Escherichia coli and Staphylococcus aureus.