重组人超氧化物歧化酶化学修饰的初步研究

在高效表达重组人铜锌超氧化物歧化酶(rh Cu/Zn SOD),并纯化得到比活大于4000单位的 rh Cu/Zn SOD 纯品的基础上,采用活化酯法将聚乙二醇(PEG)与 rhCu/Zn SOD 交联,获得分子量约6万的 PEG-SOD 交联物.经 PEG 修饰的酶稳定性增强,表现为对酸、碱和热的耐受力均较未交联酶高.修饰酶的生物半衰期为15h,是天然酶的90倍,酶活性保留80%以上.还实验观察了修饰剂用量与修饰酶保留活性之间的关系.     

长半衰期重期人超氧化物歧化酶的研制及性质

一种双亲有机化合物聚苯乙烯马来酸丁酯（ＳＭＡ）经酰胺键与重组人铜锌超氧化物歧化酶（ｒｈＣｕ／ＺｎＳＯＤ）共价交联,制得修饰酶,当４２％游离氨基被修饰时,保留酶活力为８８％。酶蛋白主链结构在修饰前后变化不大。与天然酶相比,修饰酶的生物半衰期延长了２２倍,抗蛋白水解酶能力亦有所增强。     

人超氧化物歧化酶分子生物学研究进展

人超氧化物歧化酶是一类极有开发前景的药用蛋白。hCu/Zn-SOD为二聚体,主要存在于细胞质,hMn-SOD为四聚体,主要存在于线粒体基质,hEC-SOD也为四聚体,主要存在于分泌液。各种同工酶的分子量、氨基酸序列、基因结构已经研究清楚,并进行了基因克隆和表达。细菌表达产物以包含体形式存在。复性工作复杂而困难。利用真核细胞进行克隆和表达是该酶今后的发展方向。     

棕榈酰化超氧化物歧化酶的制备及性质研究

为了增强超氧化物歧化酶的稳定性,用棕榈酸对其进行了修饰,在修饰条件下,酶分子表面氨基修饰率为55％时,酶的活力回收为63％。修饰后的酶在耐热、耐酸、耐碱、抗有机溶剂变性和抗蛋白水解能力上均高于天然超氧化物歧化酶,为将超氧化物歧化酶作成实用药物和进一步扩大其应用范围创造了条件。     

酰化人红细胞超氧化物歧化酶稳定性的研究

用月桂酸对人红细胞超氧化物歧化酶（ｈ－ＳＯＤ）进行化学修饰得到酰化ｈ－ＳＯＤ（Ａｃ－ｈＳＯＤ）,并对Ａｃ－ｈＳＯＤ和ｈ－ＳＯＤ的稳定性进行了比较。结果表明：Ａｃ－ｈＳＯＤ活力为ｈ－ＳＯＤ的７２％。比活力为４０００Ｕ／ｍｇ,Ａｃ－ｈＳＯＤ的热稳定性、酸碱稳定性及抗蛋白酶水解能力均比天然酶提高。     

月桂酸修饰超氧化物歧化酶的制备及其性质研究

用月桂酸对超氧化物歧化酶进行化学修饰以改善其稳定性.活化的月桂酸与SOD在40℃碱性条件下反应1h,再用Sephacryl S-200柱进一步纯化.修饰SOD活性回收率93%,比活为6000U / mg.具有较强的稳定性,基本上消除了免疫原性并明显地延长了在血液中的半衰期;该产品适用于化妆品和食品.     

硬脂酸修饰超氧化物歧化酶的制备及其性质研究

报道了一种修饰ＳＯＤ的方法。所得硬脂酸修饰ＳＯＤ比活力为每毫克蛋白１００００单位,经鉴定已达均一程度。测得其分子量为３５０００．修饰ＳＯＤ和天然ＳＯＤ在紫外光区的最大吸收均在２６５ｎｍ。修饰ＳＯＤ对温度、ｐＨ、蛋白酶水解的稳定性比天然ＳＯＤ增强,且免疫原性消除。在低浓度的某些有机介质中活性比在水中高。     

超氧化物歧化酶的功能,性质及临床意义

自从1969年（MeCord）和（Fridovich）发现了超氧化物歧化酶（EC．1．15．1．1,superoxidedismutase,以下简称SOD）以来,国内外学者对SOD的研究日益广泛而深人。现已证明SOD存在于所有需氧生物的细胞中。SOD是一类金属酶,根据其所含金属辅基的不同,可将其分为3类：即Cu．Zn－SOD,Mn－SOD,和Fe－SOD。Cu、Zn－SOD主要存在于真核细胞的胞浆和线粒体,Mn－SOD主要存在于真核细胞的线粒体和原核细胞中,也存在于灵长类的胞浆。Fe－S（）D只存在于原核细胞。研究表明SOD与机体的衰老、炎症、肿瘤、抗细胞毒性等方面关系…     

静电作用与修饰铜锌超氧化物歧化酶的稳定性

铜锌超氧化物歧化酶(Cu, Zn-SOD)表面的赖氨酸经化学修饰后, 酶的稳定性显著提高. 赖氨酸被修饰后, 酶的电荷结构遂发生变化, 从而影响到酶分子电场. 使用FDPB方法(有限差分法求解Poission-Boltzman方程)计算了酶修饰前后的静电场变化, 以及对维持酶的结构稳定起重要作用的Cu, Zn配位结构的影响.结果表明, Cu, Zn配位体的两级离解常数在酶修饰后分别约下降10³, 10⁶.     

牛血清白蛋白对超氧化物歧化酶的化学修饰

目的：通过化学修饰提高超氧化歧化酶（SOD）的稳定性,考察金属离子在不同浓度下对SOD活性的影响。方法：用戊二醛作为交联剂,用牛血清白蛋白（BSA）将牛红细胞超氧化物歧化酶进行化学修饰,得到SOD的修饰酶。对比研究三种SOD：修饰酶,混合酶及天然酶的理化性质。结果：修饰酶等电点降低,对温度、pH的稳定性较天然酶有很大提高,对胰蛋白酶和胃蛋白酶也表现出很强的耐水解性。二价离子Mg^2 、Mn^2 对天种SOD活力均有不同程度的抵制作用,Ca^2 、Zn^2 、Cu^2 对修饰酶活力有激活作用,一价离子K^ 对三种OSD活力均无明显影响.结论:修饰酶较天然酶的稳定性有很大的提高,加入Ca^2 、Zn^2 、Cu^2 可提高修饰酶的活力。     

人铜锌超氧化物歧化酶基因在乳酸乳球菌中的食品级表达

以lacF基因为食品级选择标记,构建了乳酸乳球菌食品级基因表达系统,并进而实现了人铜锌超氧化物歧化酶基因在乳酸乳球菌中的食品级表达。首先构建了含有lacF基因两侧同源DNA序列(0.5kb)的整合型质粒pUCEmDE,通过pUCEmDE与乳酸乳球菌MG5267染色体上单拷贝的乳糖操纵子之间的同源双交换,构建了lacF基因缺失突变的食品级受体菌WZ103 (Lac-),并经PCR及Lac表型检测所验证。然后构建了互补质粒pMG36eF,其lacF基因的表达受组成型的强启动子P32的控制。将其电转化导入WZ103后,Lac+表型得到恢复,表明WZ103中lacF基因的功能可被互补质粒pMG36eF上的lacF基因互补。随后,以互补质粒pMG36eF为基础,构建了不含任何抗生素抗性选择标记的人铜锌超氧化物歧化酶基因的食品级表达质粒pWZ104。通过非变性聚丙烯酰胺凝胶电泳和SOD活性凝胶染色分析,检测到WZ103(pWZ104)中Cu/Zn SOD的表达,并且具有生物活性。     

Effects of Liposomal Superoxide Dismutase on Human Neutrophil Activity

Study of the effects of liposomal bovine copper superoxide dismutase on human polymorphonuclear neutrophils with respect to production of active oxygen species, chemotaxis and random migration, or bacterial killing show that no significant interference with neutrophil function is observed at levels far exceeding the clinical doses used in the treatment of various pathologies.     

转铜/锌超氧化物歧化酶和抗坏血酸过氧化物酶基因甘薯的耐旱性

水分胁迫使转铜／锌超氧化物歧化酶基因（Cu／Zn SOD）和抗坏血酸过氧化物酶基因（APX）甘薯及未转基因甘薯中超氧阴离子（O2^-）、过氧化氢（H2O2）、丙二醛（MDA）含量和细胞膜相对透性增加,在相同条件下以上指标均为转基因甘薯低于未转基因甘薯;而叶片含水量、净光合速率（Pn）和气孔导度（Gs）均下降,SOD和APX酶活性随胁迫程度的加重先增大后减小,胞间CO2浓度（Ci）则先减小后增大,在相同条件下转基因甘薯中以上指标均高于未转基因甘薯。这些结果表明：转入Cu／Zn SOD和APX基因使转基因甘薯清除活性氧的能力增强,在水分胁迫下能保持较高的叶片含水量和Pn,耐旱性得到提高。     

Bisphenol-A Can Inhibit the Enzymatic Activity of Human Superoxide Dismutase

Bisphenol-A (BPA), a synthetic xenoestrogen, is currently being used to produce a wide variety of consumer products. Humans as well as animals are exposed to this ubiquitous compound through ingestion, inhalation, and dermal exposure. The effect of this compound on superoxide dismutase (SOD), an antioxidant enzyme, isolated from human blood was studied using an enzyme inhibition assay. The mode of interaction of BPA on SOD was investigated using modeling and docking studies. Purified human SOD from erythrocytes was used to study the enzyme inhibition assay of BPA. Molecular level interactions of BPA on SOD were also analyzed by modeling and docking studies. Our study demonstrates that BPA has an inhibitory effect on SOD. The docking results showed that it could bind to the active site residues of SOD and could interfere with the catalytic activity of the enzyme. Our study reveals for the first time that BPA can directly inhibit the enzymatic activity of human SOD and thus impairs the free radical scavenging mechanism.     

Chemically-Synthesized Gene Encoding Modified Human Superoxpde Dismutase: Its Construction,Expression and Properties of the Product

The gene encoding modified human superoxide dismutax (h-SOD) with 153 amino acid residues was constructed by chemical synthesis using the phosphoramidite method. The gene was designed so as to use bacterial codons for expression in prokaryotes and to introduce several unique restriction sites for further mutagenesis by the cassette exchange method. The distance between Shine-Dalgarno sequence and initiation codon was adjusted to maximum expression by using synthesized oligonucleotide. In addition, Cys 6 of h-SOD was changed to Ala to improve instability of native h-SOD.

Synthesized structural gene of h-SOD was expressed in E. coli after induction of isopropyl β-D-thiogalactoside by inserting the gene into the expression vector pKK223–3 having tac promoter. The gene that has 10 base pairs between Shine-Dalgarno sequence and initiation codøn showed the most efficient expression. The gene produced three active SOD isomers as revealed by chromatofocusing.

The main isomer was purified to homogeneity and characterized. The h-SOD-Ala⁶ showed similar properties to those of native h-SOD with respect to molecular weight, subunit structure, absorption spectrum. but the modified SOD was more resistant to heat denaturation than was native h-SOD; half-denaturing temperature was shifted by 10°C. Thus. the exchange of Cys 6 to Ala of h-SOD increased a stability of the enzyme.