【作者】 闵丽娥；

【导师】 刘克武；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2002， 硕士

【摘要】 对意大利工蜂及意大利工蜂蜂蛹、蜂王浆、蜂蜜、荞麦花粉和油菜花粉中超氧化物歧化酶（SOD）的分布进行了研究，发现这几种蜂产品中都含有SOD活性，这些蜂产品SOD活力顺序为：蜂蛹〉蜂王浆〉蜂蜜〉工蜂〉荞麦花粉〉油菜花粉。以蜂王浆为材料，经过硫酸铵分段盐析，DEAE-Sepahrose柱层析和Seohacryl S-200凝胶过滤，得到了纯化SOD。产率2.98％，纯化倍数104.0，比活53.05U／mg，该SOD经PAGE和SDS-PAGE中都显示单一蛋白区带。以邻苯三酚自氧化法为酶活力测定方法，发现该酶最适pH为8.3，研究了Cu²⁺、Ba²⁺、Mg²⁺、Mn²⁺等离子对酶活力的影响。发现Cu²⁺、Ba²⁺、Mg²⁺、Mn²⁺对SOD有不同程度的激活作用。其中，激活作用最强的是Cu²⁺。研究了温度对SOD活力的影响，发现该酶在55℃保温15分钟后，酶仍然保留62％的酶活力，在70℃保温15分钟后仍有20％的酶活力。还研究了Cu²⁺存在下温度对酶活的影响，发现在有Cu²⁺存在下，70℃保温15分钟后测得的酶活比25℃下所测酶活更高。酶敏感性实验发现该酶对H₂O₂敏感，对乙醇和氯仿稳定，表明该酶为Cu／Zn-SOD。用220FS Atomic Absorption Spectrometer测定了酶液中Cu、Zn、Fe、Mn的含量，发现该酶液中只含有Cu、Zn。经计算，每个亚基含1.6个Cu原子和3.3个Zn原子。用SDS-PAGE测得该Cu／Zn-SOD的相对分子质量为5.0×10⁴，考察了BrAC和DTT对酶侧链修饰的情况，发现BrAC明显抑制酶活力，抑制程度与BrAC浓度成正相关。DTT对酶活力的影响可以分为两个阶段，低浓度DTT抑制酶活力，而较高浓度DTT则对酶活有激活作用。脲的加入明显地抑制酶活力。脲的浓度为2mol／L时，酶完全失去活力。 用UV-VIS spectrophometer TU-1800测定了酶的紫外光谱，发现该酶的最大紫外吸收在260nm左右而不是280nm。BrAC对酶的修饰引起酶最大紫外吸 收不断变小。DTT对酶的修饰引起酶的最大紫外吸收不断变大，最大吸收波长 不断发生红移。向酶液中加入眠后，酶的最大紫外吸收变大，但随着眠浓度的 增大，紫外吸收变小。 用 FL4500－iuorence nctrophotometer测定了不同浓度的 BrAC和 D＊修 饰后酶的荧光光谱变化。BrAC和DTT修饰后均引起酶的荧光发射峰强度变小， 但并没有引起发射峰位置的显著改变。酶液中加入腺后引起酶的荧光发射峰强 度变小。 用从8＊0600C型自动记录分光偏振仪测定了酶的圆二色谱。经计算， 酶的口螺旋和p折叠和无规卷曲的含量分别为26．125％，53．83％和22．02％。 圆盘等电聚焦电泳测得酶的等电点为4．69，4．85和5刀1。NR／R－单向和双向 SDS．PAGE表明该酶含有链内二硫键。用日立835－50型高速氨基酸分析仪测 定了酶的氨基酸组成，发现该酶由约402个氨基酸残基组成，其中ASp、Gly、 LeU、AI＊、Gill、Val的含量较高。更多还原

【Abstract】 The paper studied the distribution of SOD (superoxide dismutase) activity in Italian worker bee, bee pupa, royal jelly, bee honey, pollen of buckwheat and pollen of cole. It was found that SOD was present in all of the six kinds of bee products and the order of activity was as the following: bee pupa >royal jelly >bee honey >worker bee >pollen of buckwheat >pollen of cole. After salting out with ammonium sulfate, chromatography with DEAE-Sepharose and Sephacryl S-200, SOD was purified from royal jelly of Italian worker bee with yield of 2.98%, purification factor of 104.0, and the specific activity 53.05 U/mg proteins. The obtained enzyme showed a single band on PAGE and SDS-PAGE. Its optimum pH was 8.3. The effect of Cu2, Ba2+, Mg2+, Mn2+ on enzyme activity was investigated and it was found thatCu2+, Ba2+, Mg2+, Mn2+ could activate enzyme activity. Among them, Cu2+ was the strongest activator. The effect of temperature on SOD activity was studied. It was found that the enzyme was very stable and its stability could be improved at the presence of Cu2+. The enzyme was sensitive to H2O2 and insensitive to ethanol/chloroform, which indicated that the enzyme was Cu/Zn-SOD. The content of Cu, Zn, Fe, Mn was studied by 220FS Atomic Absorption Spectrometer and it was found that the enzyme contained Cu and Zn. It was calculated that each subunit had 1.6 Cu atoms and 3.3 Zn atoms. SDS -PAGE showed that the molecular weight of SOD subunit was 5.0 X104.The residual chain modification by BrAC and DTT was studied. It was found that BrAC obviously inhibited enzyme activity and the inhibition degree was in direct correlation with the concentration of BrAC. The effect of DTT on enzymevaried when DTT concentration increased. DTT inhibited enzyme activity when its concentration was 1 and 3 mmol/L and activated enzyme activity when the concentration was 5 and 7 mmol/L. Urea could obviously inhibit enzyme activity. The enzyme lost its total activity at the urea concentration of 2 mol/L. Ultraviolet spectrum was investigated by UV-VIS spectrophometer TU-1800 and it was found that the maximal ultraviolet absorption was at 260nm around, and not 280nm. BrAC chemial modification led to the decrease of ultraviolet absorption. DTT chemical modification led to the increase of the maximal ultraviolet absorption and the maximal absorption wavelength. Urea led to the increase of the maximal ultraviolet absorption, but when the concentration of urea increased, the maximal ultraviolet absorption decreased.Effect of BrAC, DTT and Urea on the fluorescence spectrum was investigated by FL4500-fluorence spectrophotometer. It was found that BrAC and DTT incuced the decrease of fluorescence emission. Urea also induced the decrease of fluorescence emission.CD spectrum was investigated and it was found that a helix and P sheet was 6. 125% and 53. 83% respectively. IEF found that pI of the enzyme was 4.69, 4.85 and 5.01. NR/R-single and two dimension SDS-PAGE showed that 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, Val.更多还原