【作者】 顾洪雁；

【导师】 袁均林；

【作者基本信息】 华中师范大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 本文采用有机溶剂(乙醇-氯仿)的方法进行猪血SOD粗酶液的制备，然后分别用两种不同的方法对粗酶液进行过柱纯化：第一种方法是用葡聚糖G100，G50和阴离子交换树脂依次先后进行SOD纯化，所用猪血60ml，可得SOD纯品约2.44mg，比活力达4420.7U／mg左右，纯化倍数约28.6倍左右，总活力回收率约48％；第二种方法是只用葡聚糖G100-G50进行串连柱层析，所用猪血5000ml，可得SOD纯品约275.6mg，比活力达4700U／mg左右，纯化倍数约31倍左右，总活力回收率约70％。纯化后的两种样品经聚丙烯酰胺凝胶电泳鉴定和酶活性测定，结果表明：两种方法分离纯化的SOD纯度高，活力也高，说明后者在前者的基础上发展起来后，更趋于合理。省去了阴离子交换不说，还极大的提高了总产量和活力得率，而且由三次操作简化成一次过柱即可，节省了药品及缓冲液用量，降低了劳动强度和电力消耗，减少了操作过程中的人为浪费，缩短了生产周期，与生产极为有利。因而不管从第一次的纯化来看，还是从文献上报道的来看，本实验的串联柱层析是较为合理有效的一种生产方法，值得推广。 用不同的温度与不同的时间对猪血SOD粗酶液进行处理，结果表明：猪血粗酶液的处理以变性温度55-65℃，变性时间为15～26分钟最好，此时杂蛋白去除量较大而酶的损失却相对较小，比活力较高。 本文采用聚丙烯酰胺凝胶电泳，巯基已醇-SDS-聚丙烯酰胺凝胶电泳，等电聚焦电泳以及各种凝胶层析的方法对猪血铜／锌超氧化物歧化酶进行了研究。结果表明：猪血只含一种Cu，Zn-SOD类型，其分子量为32112，有两个相同的亚基构成，本文所测得的亚基约为16293，约为分子量的一半。在非变性聚丙烯酰胺凝胶电泳中，酶谱带数为最多时达4条，在等电聚焦电泳中，最多时酶谱带数亦达4条，此酶在电泳条件下出现的多谱带现象可能是由亚基的解聚和构象异构体两种原因造成的。由此排除了其是由不同基因编码的同工酶或在转录后加工及分离纯化过程中形成肽链长短不一和酶分子侧链基团差异等微不均一性所造成的可能性。更多还原

【Abstract】 The isolation of crude Cu, Zn-SOD can be started with organic solvents (ethanol-chloroform) in the classical manner. Then we take two different methods:the first one is that chromatography is carried out on Sephadex G100, G50 at room temperature with PH7. 6, 0. 02M phosphate buffer, respectively, Following this, ion-exchange chromatography is carried at pH7. 6 with 0. 02M and 0. 4M phosphate buffer;the second is that the crude enzyme runs on Sephadex G100-G50 column under the same experimental conditions(only with Sephadex G100, G50).At last polyacrylamide gel electrophoresis and SOD assay tell us both of products are highly purified SOD. These SOD activities are very high, too.Crude hog superoxide dismutase treated with different temperatures and for different time, the result.shows as following: The treatment is most appropriate at 55-65 C and for 15-26 minutes for thermal denatured extracting SOD from hog blood and specific activi is very high.In this thesis, we have done some research on the characterization of Cu, Zn-SOD from hog blood by polyacrylamide gel electrophoresis, electric focusing electrophoresis and dealing with some different reagents. The result shows as follows: one is that hog Cu, Zn-SOD has no isozymes and it contains the same two subunits , with molecular masses of 32112 daltons . the other is that the polymorphism of the Cu, Zn-SOD from hog blood is caused by polymerization and conformational isomers. So it has been found that this polymorphism of SOD was changed under different experimental conditions, which showed that the polymorphism was not caused by the difference of genes or the difference of length or side groups of the peptide chains which was caused during the processing after transcript or the purification. According to thereferences about the protein research, we estimated that the possible reasons of the polymorphism were polymerization, conformational isomers and charge isomers. There was some research on the SOD charge isomers, but not on this polymorphism.更多还原