Studies On The Technology Of SS-PEG Modification And The New Medicine Of SOD Modified With PEG

In this article, a method was established, which can isolate crude SOD simply, quickly and effectively. Then two PEGs, PEG-ALD and SS-PEG, are compared on their modification results of SOD with different reaction conditions. Meanwhile, the optimized reaction condition of SS-PEG modification was acquired. At last, we investigated the stability of PEG-SOD by experiments of heat, acid, alkaliand and proteinase. The detailed contents of research were described as the following:1 Purification of crude SODA new approach of isolating crude Superoxide Dismutase (SOD), was established by anion exchange chromatography. In this way, using the Q XL column of anion exchange chromatography, 300mg of crude SOD was isolated to 98.8mg pure SOD after dialyzation and freeze-drying in about 1h. The purified protein appears as a single band on the SDS-PAGE electrophoretogram, whose position is similar to that of standard SOD. The activity experiment showed the enzymatic activity improved from 1774.812NU/mg to 4294.76NU/mg, with a recovery of 79.7%.2 Pegylation of SODPEG-ALD and SS-PEG were selected for the SOD modification. These two reagents were compared with each other on their modification results of SOD with different reaction conditions. With different reaction temperature, or the molar ratio of SS-PEG to SOD, degrees of modification of SOD with SS-PEG are all changed. Reaction conditions of preparation of SS-PEG-SOD conjugation were investigated (reaction time 0.5h, pH7.4, 4℃, the mole ratio of PEG: SOD is 12:1). Under these conditions the enzyme activity of PEG-SOD is 89.3%.3 Purification, identification and property study of PEG-SODThe mixture of SS-PEG-SOD conjugate was isolated by Sephadex G100 gel filtration column. The conjugate solution was eluted with 0.02M phosphate buffer and the flow speed is 0.5ml/min. The protein peaks were detected at 280nm. Purified PEG-SOD was identified with SDS-PAGE and the modified ratio of SOD was determined according to a TNBS method. The possible modification sites of amino residues were prognosticated by using accessibility data. The activity and stability of PEG-SOD were also detected. Compare native bovine SOD with PEG-SOD in stability by experiments of heat, acid, alkaliand and proteinase. The stability of PEG-SOD...