蚯蚓体内超氧化物歧化酶分析(英文)

SUPEROXIDE DISMUTASE FROM THE EARTHWORM (EISENIA FOETIDA)

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 1 0 0 g组织得到 SOD制品总活力为 1 7,1 90 U,比活 7995U/ mg,回收率为 35%。该酶呈淡蓝绿色 ,最大紫外吸收波长为 2 70 nm。该酶分子量为 33,0 0 0 ,亚基分子量为 1 6 ,50 0。该酶亚基含 1 56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 .2

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 .