蚯蚓体内超氧化物歧化酶分析

摘要

蚯蚓体内SOD含量甚高,35℃饲养的蚯蚓其SOD比活最高,因此,纯化前将蚯蚓在35℃养殖4周以上.采用硫酸铵分级沉淀和柱层析的方法,从蚯蚓体内分离得到纯的铜锌超氧化物歧化酶.每100g组织得到SOD制品总活力为17,190 U,比活7995 U/mg,回收率为35%.该酶呈淡蓝绿色,最大紫外吸收波长为270nm.该酶分子量为33,000,亚基分子量为16,500.该酶亚基含156个氨基酸残基,不含酪氨酸.N-末端为丙氨酸,等电聚焦为三条谱带,等电点分别为5.30 、5.59和6.22.%The superoxide dismutase activity (units/mg protein) was determined for earthworm (Eisenia foetida) acclimated from 10℃to 35℃at 5 degree intervals. Worms acclimated at 35℃gave a value of SOD units significantly higher than those acclimated at other temperatures . So the worms were acclimated for four weeks at 35℃prior to purification. Cupro-zinc superoxide dismutase extracted from the cell of earthworm had been purified by ammonium sulphate precipitation and special column chromatography . The purified enzyme obtained from 100 grams of tissue possessed a total activity of 17 190 units with specific activity of 7 995 units per mg protein .The yield was 35%. The enzyme isolated by this procedure showed pale blue-green color . It exhibited one absorption maximum in the ultraviolet at 270 nm .The molecular weight of the purified enzyme was 33 000 daltons as determined by gel filtration on Sephadex G-100 , and that of the enzyme subunits was 16 500 daltons as estimated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis .The enzyme subunits consisted of 156 amino acid residues , in which tyrosine was absent . The N-terminal of the enzyme was alanine as assayed by dansyl chloride . Three bands of the enzyme were seen in isoelectrofocusing. They focused at pH5.30, 5.59 and 6.22 , respectively .