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SUDAKAR PATENT 1 - IDENTIFICATION AND PURIFICATION OF A NOVEL POTENT GALACTOSAMINE/GALACTOSE SPECIFIC, CA2+ DEPENDENT HEMOLYTIC LECTIN FROM FRESH WATER GASTROPOD VILLORITA CYPRYNOIDES
SUDAKAR PATENT 1 - IDENTIFICATION AND PURIFICATION OF A NOVEL POTENT GALACTOSAMINE/GALACTOSE SPECIFIC, CA2+ DEPENDENT HEMOLYTIC LECTIN FROM FRESH WATER GASTROPOD VILLORITA CYPRYNOIDES
The muscle of clam Villorita cyprinoides contains a galactose specific agglutinin. This has been isolated by a three step of purification procedure. DEAE- Cellulose, affinity chromatographical procedure using Fetuin sepharaose 4 Fast Flow, firstly immobilized fetuin followed by its binding to and elution from Sepharose and gel filtration chromatography using Sephadex G-75.. The hemolytic activity was higher in rabbit erythrocytes. The hemolytic activity was proved by erythrocyte agarose plate assay and spectrophotometer assay. Trypsinated rabbit erythrocytes found to be highest activity, but Papain treated erythrocytes did not produced more activity. The scanning experiment was conducted in lectin lysed erythrocytes and purified lectin, the results showed high absorption at 238, 264, 364, 444, 542, 578 nm respectively. Ca2+ -ions are required for ligand binding of the lectin; the pH-optimum for binding reactions is between pH 7.0-8.0. Heating to 50°C onwards reduces hemolytic activity which is completely destroyed at 62°C. Mgcl2 and BaCb gave higher activity at 25 mm and 12.5m mol. The FeCh and HgCl2 have no activity this means these ions were not involved during other hemolytic activity. MnSo4 and MnCb recorded very low activity. The requirement of divalent cations was very much involved in the hemolytic activity. EDTA and without CaCl2 contained TBS, the hemolytic activity was greatly reduced as compared with the presence of CaCb. This shows that this hemolytic lectin was highly calcium dependent activity. Kinetics of hemolysis was conducted in different concentration erythrocytes and lectin concentration, which indicated that 1.5% of erythrocytes produce highest activity. Hemolytic lectin and streptokinase conjugate, which well perform in clot lysis. The purified protein showed two subunits with approximately 42 KDa and 39 kDa in SDS-PAGE.
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