鸭血超氧化物歧化酶(SOD)的纯化及性质研究

Purification and Characterization of Superoxide Dismutase from Duck Erythrocytes

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。

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.