金属螯合亲和层析法纯化猪红细胞超氧化物歧化酶

本文报道以Sephadex G-200为基质的金属螯合亲和层析法纯化SOD、其比活为4508U/mg蛋白,收率为90.4％,经酸、碱、SDS-PAGE、考马斯亮兰染色均呈一条带,特异性的SOD活性染色呈阳性。分子量为34,000,氨基酸组成测定表明Tyr含量少,Gly含量高,紫外吸收光谱最大吸收在258nm处,园二色谱结果表明SOD空间结构为β—折迭及无规态,含极少量或几乎不含α—螺旋,等电聚焦电泳测定SOD有微不均一性,等电点分别为5.25,5.35和5.65。

Purification of the Superoxide Dismutase (SOD) From Porcine Red Cells by Metal Chelate Affinity Chromatography

A simple method--Metal Chelate Affinity Chromatography to purifySOD from porcine red cells and some preliminary physicochemical characteristics of the purified SOD were reported.SephadexG-200 was used as substrate for chromatography, epichlorohydrin as activator and iminodiacetic acid as ligands, then Cu was chelated to form Cu-metal chelate affinity chromatographic column. The SOD was purified to homogeneous in one step affinity chromatography. Its specific activity is 4508U/mg and recovery is 90.4%.The purified SOD showed a single band on the disc-PAGE (pH8.9 and pH4.3) and SDS-PAGE (pH7.0) . The activity stain of SOD is positive. The molecular weight is 34,000 daltons according to SDS-PAGE. The composition of amino acid indicates that SOD has a few Tyr. UV spectrum indicates that the UV maxium absorption of porcine SOD is at 258nm. The CD spectrum analysis indicates that the polypeptide chain has mainly β-pleated sheets, random coils and little α-helix. The purified SOD exhibited microheterogenity on the IEF, which showed three bands. The isoelectric points of SOD were pH5.25, pH5.35 and pH5.65, respectively.