意蜂蜂王浆超氧化物歧化酶的分离纯化及部分性质  

Isolation, purification and characterization of superoxide dismutase from royal jelly of the Italian worker bee, Apis mellifera

作　　者：闵丽娥[1] 李佳[1] 刘克武[1] 江琰[1] 刘晓雯[1] 刘鑫[1] 

机构地区：[1]四川大学生命科学学院,成都610064

出　　处：《昆虫学报》2004年第2期171-177,共7页Acta Entomologica Sinica

基　　金：国家自然科学基金项目 ( 3 9970 5 77)

摘　　要：以意蜂Apismellifera蜂王浆为材料 ,经过硫酸铵分段盐析 ,DEAE Sepharose柱层析和SephacrylS 2 0 0凝胶过滤 ,得到纯化的超氧化物歧化酶 (SOD) ,纯化倍数 10 4 0 0 ,比活力 5 3 0 5U mg。该SOD经SDS PAGE显示单一蛋白带。温度对该酶活力的影响较小。Cu、Zn、Fe和Mn等元素含量测定发现该酶只含有Cu和Zn。酶经圆二色谱测定后 ,其α螺旋、β折叠和无规则卷曲蛋白构型的含量分别为 2 6 1%、5 3 8%和 2 2 0 %。等电聚焦电泳测得酶的等电点为 4 6 9、4 85和 5 0 1。NR R单向和双向SDS PAGE表明该酶含有链内二硫键。氨基酸组成分析发现该酶由约 4 0 2个氨基酸残基组成 ,其中Asp、Gly、Leu、Ala、Glu和Val的含量较高。脲可抑制SOD活性 ,并使其紫外光谱发生变化 ,荧光发射峰强度变小。溴乙酸 (BrAc)抑制酶的活力 ,使其紫外光谱发生变化 ,荧光发射峰强度变小。二巯基苏糖醇 (DTT)使酶的活力发生变化 ,紫外吸收峰增大 。After salting out with ammonium sulfate, chromatography with DEAE-Sepharose and Sephacryl S-200, SOD was purified from royal jelly of the Italian worker bee, Apis mellifera with purification factor of 104.00, and the specific activity 53.05 U/mg proteins. The obtained enzyme showed a single band on SDS-PAGE. The effect of temperature on SOD activity was studied, and it was found that the enzyme was very stable. The contents of Cu, Zn, Fe, and Mn was also studied, and it was found that the enzyme contained Cu and Zn. Circular dichroism spectrum was investigated and it was found that the proportion of α-helix,β-sheet and random coiling form of proteins was 26.1%,53.8% and 22.0% respectively. The isoelectric focusing of SOD indicated that pI of the enzyme contained intrachain disulfate bond. The amino acid composition was investigated and it was found that the enzyme contained 402 amino acid residues. The enzyme had relatively high content of Asp, Gly, Leu, Ala, Glu and Val. Both urea and BrAc could inhibit enzyme activity, lead the change of ultraviolet absorption and induce the decrease of fluorescence emission. DTT could lead the change of enzyme activity, the increase of ultraviolet absorption and the decrease of fluorescence emission.

关 键 词：意大利蜜蜂 蜂王浆 超氧化物歧化酶 分离 纯化 生化性质 

分 类 号：Q55[生物学—生物化学]