Isolation, Purification And Bioactivities Of The Proteins From Two Chinese Marine Sponges, Hymeniacidon Perleve And Reniochalina Sp

The isolation and purification of proteins from the marine sponges, Hymeniacidon perleve and Reniochalina sp collected at the coast of the China Yellow Sea close to Dalian city, were studied in this thesis. The properties and antitumour activities of the purified proteins were characterized.Based on the establishment of the best conditions on the Bradford method, the optimal operating conditions were established for the high efficient isolation of proteins from marine sponges in terms of the extraction solvent systems, the concentration of (NH4)2SO4 for the saturated precipitation of proteins, techniques for protein concentration.Two extraction solvent systems were studied for the differences betweenproteins from two systems.The extraction solvent of "System I" was distilledwater, and the PBS buffer ( pH7.5,5.0 mM ) was the solvent of "System II "The crude protein extracts from Hymeniacidon perleve were further purified by column chromatography on Sephadex G-100 and DEAE cellulose. Three mainhomogeneous protein fractions were obtained and denoted asHI-1 'HH-1 'HII-2 respectively. They were identified to be homogeneous proteins by PAGE, and the relative molecular masses of H I -1, H H -1 and H E -2 were 23 kDa,52 kDa and 57kDa respectively. The H I -1 protein showed a single band on the SDS-PAGE, which implied only one subunit. The H H -1 protein and HE -2 protein both showedtwo bands on the SDS-PAGE, indicating each of them having two subunits. Further characterization showed that H I -1 and HII-1 contained about 36.1% and 21.5%total sugars detected by the phenol-sulfate method, and no sugar detected in H n -2protein.The proteins from Reniochalina sp contained too low protein content to characterize.H I -1,H H -1,H n -2 and other purified proteins were assayed for theantitumour activities in vitro. HII-1 protein exhibited strong antirumour activities, and IC50 of this protein against QGY-7701 and Raji rumour cells is approximately 28.6μ g/ml and 29.9μg/ml respectively .The protein HII-1 was heat stable at lower and higher temperature,but lost all activities.at room temperature. Stable activities were maintained at pH5 ~ 8. H II -2 protein and the protein fractions fromReniochalina sp showed weak antitumour activites. The protein H I -1 didn'tshowed antitumour activities, but it promoted the cell aggregation of marine sponge Hymeniacidon perleve.