摘要
In the present study two phytocystatins (thiol protease inhibitors) have been isolated and purified to homogeneity form Catharanthus roseusby a simple two step procedure using ammonium sulphate fractionation and gelfiltration chromatography on Sephacryl- 100HR.The two inhibitors were named as CRCI and CRCII (Catharanthus roseuscystatin I and II). CRCI and CRCII were purified with a fold purification of 1333.3, 1348.5 and percent yield of 18.18 and 16.35% respectively. The molecular weight of purified phytocystatins were 19.1 kDa and 16.9 kDa respectively, as determined by SDS-PAGE and mass spectrometry. Effect of denaturants like ureaon CRCI and II was analysed by Fluorescence spectroscopy. Results suggest an unfolding of CRCI and II. FTIR results show that structurally CRCI is different from CRCII. Hydrophobic interactions are observed over a longer timescale (5 - 150 min). Furthermore, fluorescence spectroscopy results show quenching of fluorescence intensity of CRC I and II, although to different extent, due to perturbations of the environment of aromatic residues in the protein. Both the cystatins showed strong inhibitory/antibacterial activity against E. coliand S.
In the present study two phytocystatins (thiol protease inhibitors) have been isolated and purified to homogeneity form Catharanthus roseusby a simple two step procedure using ammonium sulphate fractionation and gelfiltration chromatography on Sephacryl- 100HR.The two inhibitors were named as CRCI and CRCII (Catharanthus roseuscystatin I and II). CRCI and CRCII were purified with a fold purification of 1333.3, 1348.5 and percent yield of 18.18 and 16.35% respectively. The molecular weight of purified phytocystatins were 19.1 kDa and 16.9 kDa respectively, as determined by SDS-PAGE and mass spectrometry. Effect of denaturants like ureaon CRCI and II was analysed by Fluorescence spectroscopy. Results suggest an unfolding of CRCI and II. FTIR results show that structurally CRCI is different from CRCII. Hydrophobic interactions are observed over a longer timescale (5 - 150 min). Furthermore, fluorescence spectroscopy results show quenching of fluorescence intensity of CRC I and II, although to different extent, due to perturbations of the environment of aromatic residues in the protein. Both the cystatins showed strong inhibitory/antibacterial activity against E. coliand S. aureus
