| 摘要: |
| 以40%～70%饱和度硫酸铵分级沉淀、DEAE SepharoseiCL-6B、Sephadex G-100、羟基磷灰石、Phenyl Sepharose CL-4B和制备电泳分离纯化了小麦的尿卟啉原Ⅲ合酶(UROS)。酶的亚基分子质量为54 ku,全酶分子质量为66 ku,酶的等电点6.3,在pH8.2时比活为257μmol/(mg·min),最适反应温度40℃,5mmol/L的巯基乙醇和Mg2+促进酶活,纯化的小麦UROS在-20℃下0.1mol/L,pH8.5的Tris-HCl,内含质量分数为50%的甘油,5mmol/L的巯基乙醇和MgCl2中贮藏7d酶活损失90%。 |
| 关键词: 尿卟啉原Ⅲ合酶 小麦 纯化 |
| DOI： |
| 分类号:S512.10; Q555 |
| 基金项目:国家自然科学基金资助项目(39570066) |
|
| Isolation and purification of uroporphyrinogen Ⅲ synthase from wheat leaf |
|
Fanjun and GuoAiguang,Fanjun and GuoAiguang
|
| Abstract: |
| Uroporphyrinogen Ⅲ synthase (UROS) is isolated and purified from wheat leaves by 40%-70% ammonium sulphate fractionation and followed by chromatography DEAE Sepharose CL-6B, Sephadex G-100, hydroxylatite, Phenyl Sepharose CL-4B and finally by electrophoresis.The purified enzyme has a specific activity of 257 μmol/(mg·min) protein at pH of 8.5. It shows homogeneity and has a subunit Mr of 54 ku by SDS-PAGE.The whole enzyme has about a relative Mr of 66 ku by Sephadex G-100 and thus appears to be monomeric. Its pI is about 6. 3. It was maximally active at a temperature of 40℃ and activated by 2-mercaptothanol and Mg2+.The purified enzyme after concentrated is stored with a loss of 90% activity for aweek at-20℃in the dark in 0.1mol/L Tris-HCl buffer(pH8.5), including 50% of glycerol, 5mmol/L 2-mercapoethanol and MgCl2. |
| Key words: uroporphyrinogen III synthase (UROS) purification wheat |
