| A rapid method was established for the isolation and purification of quantities of the native porcine hemoglobin. Native porcine hemoglobin with super-purity was extracted, isolated and purified from the fresh porcine whole blood. Red blood cell(RBC) was throughly washed with saline to remove plasma and white cell, then was lysed on low penetrative pressure solution at 4 ℃. Cell debris and cell membrane were removed by high-speed centriflgation at 4 ℃ to obtain rough products of porcine hemoglobin. Further purification was made by different ion exchange chromatographic systems to obtain super-purity porcine hemoglobin. Finally qualitative and quantitative determinations for the purity, concentration, yield and biological activity of porcine hemoglobin were performed by a series of experimental methods including protein electrophoresis, scanning spectrum, SEC-FPLC and Western Blotting, and a complete standard was formed for the identification of pure hemoglobin product. The purity of porcine hemoglobin was above 99.9%,the concentration was between 8.9g/dl and 12.5g/dl.Purified hemoglobin was sterile , contained no pyrogen and single spectrum was displayed by SEC-FPLC. The content of Met-hemoglobin determined by blood analytical instrument was below 5% in purified porcine hemoglobin. SDSPAGE electrophoresis of 13.5% separated gel showed that the product distributed evenly, of which 86.5% was hemoglobin monomer with a molecular weight of 16.5kD and 10.2% was hemoglobin dimer with a molecular weight of 32.5kD. The subunit constitutes , molecular weights and distribution of hemoglobin were confirmed by SDS-PAGE silver staining-Western blotting . The purity of hemoglobin was indirectly confirmed by the above method as well through different sampling between hemoglobin and standard protein(BSA and OVA).High-concentration,super-purity and superior-quality porcine hemoglobin could be prepared by the above experimental results. |
PDF Full Text Request