鸭血超氧化物歧化酶(SOD)的纯化及性质研究

超氧化物歧化酶(E、C、1.15.1.1)是催化超氧阴离子起歧化反应的金属酶类,血红细胞中的SOD属Cu Zn—SOD。本文详细报道了从鸭血分离纯化SOD的改进方法(同时用猪血作对比研究)。设计了热变性、(NH_4)_2SO_4分级监析、低浓乙醇—氯仿短时去除残留血红蛋白、凝胶层析四步纯化方案,获得高产率电泳纯SOD。用紫外扫描,PAGE电泳后考马斯亮蓝和SOD活性杂色,SDS—电泳,HPLC分离和各电泳带组分的N—端测定等技术检测纯度,并进行性质研究。证明所得SOD是均一的;N—端为Ala;分子量和亚分子量各为32,359和16,500dt;并与猪血SOD对比研究其某些性质;对HPLC出现的多个活力峰及PAGE电泳显现的多条活性带进行了讨论。

Purification and Characterization of Superoxide Dismutase from Duck Erythrocytes

In this paper, an improved method for purification of superoxide dismutase (SOD) from duck (also from pig for comparation) erythrocytes is preset-ed.The procedures designed are:1.Heat denaturation; 2.(NH4)2SO4 fractional salting out; 3. Short time treatment with Alc-CHCl3 at low concentration to remove residual hemoglobin, and 4. Sephadex G column chromatography. Using this method very high yield of Cu-Zn SOD has been obtained from duck and pig erythrocytesThe purified SOD exhibits an unique UV-spectrum with a maximun at 265nm. SOD activity staining and coomassie brilliant blue staining of PAGE pattern of purified SOD exhibit same profile of the multiple bands.The purified SOD obtaind from sephadex G chromatofraphy also studied by H-PIC and DEAE-cellulose chromatography separation, N-terminal amino acid determination, molecular weight and subuhit molecular weight determination and other properties examinations. All these results proved that the purified SOD obtaind from above method is homogeneous.Its N-terminal amino acid is alanine. Its M.W. and its subunit M.W.are 32, 500 and 16,000 dt.respectively.Some properties of this homogeneous SOD and its profile of the multiple band on PAGE-pattern are also discussed.