| Polysialic acid (PSA) is a homopolymer of sialic acid withα-2,8 or/andα-2,9 ketosidic linkages. It has strong hydrophilicity, biodegradability and good biocompatibility. Polysialic acid is considered an ideal alternative material of the polyethylene glycol (PEG) in controlled-drug release application due to its more effective safety in therapeutics. Modification with PSA, also known as polysialylation, means coupling long-chain PSA to the protein or peptide drugs by chemical cross-linking method, in order to improve stability and pharmacokinetics of modified enzyme.Purification process of PSA was studied based on the previous work to satisfy the PSA purity for medical application. The total processes were as follows: filter by ethanol precipitation, alkaline precipitation and complexation with cetyl pidinium chloride (CPC), and the final purity was about 90%. Then the product was further purified by ultra filtration (100kDa), DEAE F F column chromatography and Sephacryl S-200 HR column chromatography. As a result, the cumulative recovery was about 9.59%. The high-purity PSA contained protein 2.12×10-4 mg/mg PSA, and the removal of bacterial endotoxins was above 99.8%. the HPGFC test shows the purity is 99.71% and Mp is 16.2kDa. All these parameters meet the medical pure level, indicating that the obtained PSA can be used for polysialylation.This research used CuZn-Superoxide dismutase (CuZn-SOD) as the target protein for polysialylation. SDS-PAGE electrophoresis revealed that the CuZn-SOD was electrophoretically pure, with molecular weight of 32kDa and residual enzyme activity of 17.6×104 NU/mg protein.By using PSA as crosslinking agent and CuZn-SOD as the target protein, this research has established a convenient and efficient polysialylation process. The activated PSA was coupled to the free Lys in the surface of CuZn-SOD using the method of–NH2 covalent coupling, then the modified enzyme was isolated by TOSOH Toyopearl HW-55F column chromatography. In order to optimize crosslinking ratio and residual activity, the double-factors L9 (34) experimental method was used and the optimal conditions for the modifying reaction were as follows: PSA:SOD mole ratios of 40:1, 25℃and pH 7.4. It was shown that 3.86±0.17 PSA molecules linked to per SOD molecule in average, with the average residual activity of 77.8±1.6 % in these conditions.SDS-PAGE electrophoresis revealed that the molecular weight of polysialylated SOD was increased from 32kDa to 90～100kDa. Atomic force microscopy (AFM) showed that the average size of the polysialylated SOD was 10～15nm, about four-fold of native enzyme size. Besides, AFM showed two kinds of microscopic spherical shapes: coated single particle or coated multiple particles. TNBS method measured the average surface amino-modified rate was 24.4%, and surmised that the modified sites were Lys (9,23,73,89,151) by the analysis of protein residue accessibility. Further experiments indicated that the stability of modified enzyme against heat, acid, alkali and proteinases such as pepsin and trypsin was improved significantly as compared to native enzyme.
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