无花果叶超氧化物歧化酶的分离、纯化及性质研究

以我国山东无花果树(Brunswike, 原产法国Ficus carica)树叶为原料, 采用缓冲液抽提、硫酸铵分级、DEAE-Sepharose Fast Flow阴离子交换层析和Sephacryl S-100 HR分子筛层析分离纯化得到电泳纯CuZn-SOD, 并对其酶学性质进行研究.     

脱水糖苷化方法合成肝素酶底物寡糖

乙酰肝素酶（Heparanase,Hpa）是哺乳动物体内的内切葡萄糖醛酸苷水解酶,通过水解葡萄糖醛酸（GlcA）与己胺糖（GlcN）之间的β-糖苷键,选择性地降解肝素和硫酸肝素糖胺聚糖,从而释放多功能的肝素寡糖.本文报道一条高效的肝素酶底物寡糖的合成路线：采用苯甲酰基保护待硫酸化的羟基,苄基保护羧基和裸露的羟基,叠氮基保护氨基,应用脱水糖苷化方法高效地构建关键的a-GlcN-（1→4）-GlcA糖苷键.然后通过标准化的保护基脱除和硫酸化操作,获得肝素酶底物三糖和四糖1-4.最后五步反应的总收率超过52%.肝素酶底物寡糖的合成为研究肝素酶的底物选择性和活性检测打下了基础.     

临床肝素类药物酶解分析二糖组成

肝素是一类结构复杂的高分子糖类,以N-硫酸、6-O硫酸氨基葡萄糖和2-O硫酸艾杜糖醛酸为主要成分组成二糖。常见的肝素注射液是以未分级肝素为原料纯化灭菌制备而来,在临床上使用更为广泛的是将肝素降解得到的低分子片段,低分子肝素保留了肝素糖链基本结构,但是不同制备工艺导致其具有不同的还原及非还原端。本研究以肝素类药物为研究对象,使用肝素裂解酶玉,域及芋将肝素注射液、低分子肝素注射液(达肝素、那屈肝素、依诺肝素)、化学合成的类肝素(磺达肝素)进行酶催化降解产生二糖,结合强阴离子交换色谱( Strong anion exchange high performance liquid chromatography,SAX-HPLC)以及紫外检测器在线分析,使用市售肝素二糖标准品确定各种二糖结构。此外,使用反向离子对色谱和电喷雾质谱联用分析磺达肝素中的甲基二糖以及达肝素和那屈肝素中的脱氨二糖结构,为肝素以及类肝素药物的质量控制提供更为精确的结构信息。     

毛细管电泳法测定肝素和低分子量肝素平均硫酸化程度

肝素和低分子量肝素（LMWHs）作为临床上常用的抗凝血药物,其抗凝血活性与硫酸化程度（SD）密切相关。然而,肝素类药物的生产需经历一系列复杂的工艺过程,在制备和储存过程中,肝素的硫酸基团容易水解丢失,影响抗凝血活性,这将直接影响肝素药物的使用安全性。为保证产品质量,需要发展一种快速检测肝素硫酸化程度的技术,以监测原料质量和工艺条件的稳定性。虽然已有一些测定肝素硫酸化程度的报道,但这些方法均有局限性,不适用于肝素生产的质量控制。为此,开发了一种基于毛细管电泳技术（CE）检测肝素和低分子量肝素的平均硫酸化程度的方法。首先,用肝素酶混合液彻底消化未分级肝素（UHF）和低分子量肝素,然后用毛细管电泳分离酶解得到的所有寡糖和二糖构建模块,并对它们进行定性和定量分析。随后,根据每种寡糖和二糖的峰面积及其硫酸酯基团的数量,便可计算出每个构成肝素二糖单元硫酸化程度的平均值。使用该方法对来自两个生产商各4个批次依诺肝素（低分子量肝素）和5个批次肝素原料进行检测,并计算了各批次样品的相对标准偏差（RSD）,对不同厂家生产的依诺肝素平均硫酸化程度进行了比较,验证了该方法的实用性。该方法灵敏度高,准确可靠,分析速度快,在肝素类药物生产过程的质量控制中具有良好的应用潜力。     

含N-乙酰化肝素寡糖的制备及序列分析

建立了含N-乙酰化肝素寡糖的分离提纯及其序列结构分析方法.首先应用肝素酶Ⅰ深度酶解低分子量肝素来富集含N-乙酰化结构寡糖,通过Bio-Gel P10凝胶色谱法分离制备了包括二糖至十四糖的系列肝素寡糖粗样品,ProPac PA-1强阴离子高效液相色谱(SAX-HPLC)等方法对粗样品进一步分离,提纯得到4种六糖和3种八糖片段.其次应用肝素酶Ⅰ,Ⅱ和Ⅲ复合酶解与HPLC法分析各纯化寡糖的二糖组分,并结合肝素酶Ⅰ底物特异性,初步推断4种六糖和3种八糖的序列结构.在寡糖的糖链两端均含有N-硫酸化二糖,而N-乙酰化二糖分布在糖链当中.应用电喷雾离子阱-飞行时间质谱(ESI-IT-TOF-MS)在负离子模式下进一步表征寡糖并分析其裂解规律.结果表明,各寡糖中均出现大量因SO32-丢失形成的碎片离子峰,六糖中主要有双电荷和三电荷碎片离子峰;在八糖中出现了一系列从双电荷至五电荷的离子峰.各寡糖的双电荷离子峰质荷比进一步确定了上述寡糖的序列结构.六糖的裂解规律表明,裂解主要存在于糖苷键,N-乙酰葡糖胺和糖醛酸上的裂解方式分别为0,2X和0,2Z.本研究提供了切实有效的分离、分析未知结构肝素寡糖序列的新方法.     

肝素化丝素支架作为骨形态发生蛋白-2缓释载体的研究

骨形态发生蛋白-2(BMP-2)的缓释载体一直是骨组织工程中的研究热点.本研究通过化学改性制备了两种肝素化丝素支架,并浸渍吸附BMP-2,研究了BMP-2在不同丝素支架样品上的吸附能力、体外释放性能及其对人骨肉瘤细胞MG-63碱性磷酸酶活性(ALP)的影响.结果表明,肝素化丝素支架对BMP-2具有较强的吸附能力,并能保持其体外缓慢释放性能;MG-63细胞在肝素化支架上生长状态良好,并具有显著的增殖能力,负载BMP-2后的肝素化支架能显著促进MG-63细胞的分化.因此,肝素化丝素支架是一种较理想的BMP-2缓释载体.     

铜锌超氧化物歧化酶活性中心铜与组氨酸的相互作用

本工作应用电子自旋共振(ESR)和核磁共振(NMR)等手段深入研究了铜锌超氧化物歧化酶(CuZn-SOD)在水溶液中与组氨酸的相互作用。发现酶活性中心的Cu(Ⅱ)在水溶液中可以与外部物质进行交换相互作用,并不像固态那样稳定在络合位置上,Cu(Ⅱ)与组氨酸形成络合物并在活性中心与络合物间达成动态平衡。酶活性同时受到相应的影响。本文同时介绍了其它氨基酸与酶的作用情况。     

具有抗凝性能的肝素化ε-己内酯/L-丙交酯共聚酯的合成及电纺丝加工

以辛酸亚锡为催化剂,1,4-丁二醇为引发剂,制备出ε-己内酯/L-丙交酯共聚酯(PLCL).以1-(3-二甲氨基丙基)-3-乙基碳二亚胺/N-羟基琥珀酰亚胺为缩合剂将肝素连接在PLCL两侧端基上.采用1HNMR技术测定了共聚酯端基的肝素化率;用XPS分析了肝素化后聚酯中N和S含量,利用甲苯胺蓝紫外分光光度计法测定了表面肝素含量,并根据静态接触角测定结果讨论了材料表面的亲水性变化.凝血酶原时间、凝血酶时间和部分凝血活酶时间测试数据表明,肝素化后PLCL的抗凝血性能得到明显改善.探索了该共聚酯进行电纺丝加工的可行性.     

具有抗凝性能的肝素化ε-己内酯/L-丙交酯共聚酯的合成及电纺丝加工

以辛酸亚锡为催化剂, 1,4-丁二醇为引发剂, 制备出ε-己内酯/L-丙交酯共聚酯(PLCL). 以1-(3-二甲氨基丙基)-3-乙基碳二亚胺/N-羟基琥珀酰亚胺为缩合剂将肝素连接在PLCL两侧端基上. 采用 1H NMR技术测定了共聚酯端基的肝素化率; 用XPS分析了肝素化后聚酯中N和S含量, 利用甲苯胺蓝紫外分光光度计法测定了表面肝素含量, 并根据静态接触角测定结果讨论了材料表面的亲水性变化. 凝血酶原时间、凝血酶时间和部分凝血活酶时间测试数据表明, 肝素化后PLCL的抗凝血性能得到明显改善. 探索了该共聚酯进行电纺丝加工的可行性.     

肝素亲和柱分离纯化乳铁蛋白

1　实验部分1.1　主要仪器设备及试剂　　肝素亲和色谱柱(Illinois大学Hurley教授馈赠):美国BIO RAD公司,60mm×12mmi d ,填料为肝素与琼脂糖交联的产物,颗粒直径50μm、孔径100nm,洗脱液流速1～3mL/min;UV 120 02SHIMADZU紫外分光光度计:日本岛津公司;DYYⅢ 2型稳压稳流电泳仪和DYYⅢ 78型转移电泳仪:北京六一仪器厂;硝酸纤维膜:美国GelmanScience公司;DG3022A型酶联检测仪:华东电子管厂;96孔酶标板:丹麦NUNC公司。丙烯酰胺、牛血清白蛋白(BSA):进口分装;辣根过氧化物酶标记的羊抗兔免疫球蛋白G(IgG):华美生物技术公司(酶…     

RNA Preparation of Saccharomyces cerevisiae Using the Digestion Method may Give Misleading Results

Zymolyase (lyticase) is used for cell wall digestion in yeast experiments and is needed for incubation processes under moderate experimental conditions. This has been thought to cause unfavorable effects, and many researchers are aware that the enzyme method is unsuitable for RNA preparation following several reports of stress responses to the enzyme process. However, RNA preparation with enzyme digestion continues to be used. This may be because there have been insufficient data directly comparing RNA preparation conditions with previous studies. We investigated the influence of enzyme processes in RNA preparation using a DNA microarray, and compared superoxide dismutase (SOD) activities with a non-treated control and the results of previous research. Gene expressions were commonly changed by enzyme processes, and SOD activities increased only during short-term incubation. Meanwhile, both SOD gene expressions and SOD activity during RNA preparation indicated different results than gained under conditions of long-term incubation. These results suggest that zymolyase treatment surely influences gene expressions and enzyme activity, although the effect assumed by previous studies is not necessarily in agreement with that of RNA preparation.     

Chemical Modification of Tryptophan Residues in Superoxide Dismutase from Camellia Pollen and Its Fluorescence Spectrum

The amino acid composition of the superoxide dismutase（SOD） from camellia pollen was measured and the tryptophan（Trp） residues were modified by using N-bromosuccinimide（NBS）. The results show that there are 21 Trp residues in an SOD molecule and seven of which are located on the surface of the enzyme. By researching the fluorescence spectra of the native SOD and the modified SOD, we have found that the emission wavelength of Trp is at 335 nm and the fluorescence intensity will decrease when the enzyme is modified. The results also show that potassium iodide（KI） can significantly quench the fluorescence of the native SOD, but it has a less pronounced effect on the modified enzyme. Glycerin as a surface activation reagent can stabilize the fluorescence of the modified enzyme.     

Study on the interaction of copper-zinc superoxide dismutase with aluminum ions by electrochemical and fluorescent method

The interaction of superoxide dismutase (SOD) with aluminum (Al) ions was investigated by cyclic voltammetry, fluorescence spectroscopy and synchronous fluorescence spectroscopy. The electrochemical activity of the SOD enzyme electrode was inhibited irreversibly by the addition of Al. Meanwhile, the static fluorescence quenching mechanism further revealed the existing of molecular complex of SOD with Al(3+). The association constant was obtained from Lineweaver-Burk plot. The experimental results of voltammetry and fluorescence spectroscopy indicated that the conformation of SOD molecule was altered by the formation of Al-SOD complex. It may influence the activity of SOD enzyme since the optimum action of SOD depends upon a particular configuration of electrostatic charges in the enzyme molecule.     

Zinc deficient bovine erythrocyte superoxide dismutase has low specific activity.

Zinc deficient bovine superoxide dismutase (Cu2E2SOD (E = empty)) was prepared and purified by high performance liquid chromatography (HPLC). Each peak was characterized as to protein, copper content and specific activity. The Cu2E2SOD peak fractionated by HPLC has a low specific activity at pH 7.8 (about 10% of the native enzyme (Cu2Zn2SOD)). With the addition of zinc ions, the specific activity of Cu2E2SOD was quantitatively restored to that of the native enzyme. This behavior implies that the zinc ion is very important for the appearance of enzyme activity.     

Superoxide dismutase mimetic properties exhibited by vacancy engineered ceria nanoparticles

In this report ceria nanoparticles are shown to act as catalysts that mimic superoxide dismutase (SOD) with the catalytic rate constant exceeding that determined for the enzyme SOD.     

Purification and Characterization of a Cu,Zn-SOD from Garlic (Allium sativum L.). Antioxidant Effect on Tumoral Cell Lines

Crude garlic extract contains one Mn-superoxide dismutase designated as SOD1 and two Cu,Zn superoxide dismutases as SOD2 and SOD3. The major isoform SOD2 was purified to homogeneity by Sephacryl S200-HR gel filtration, DEAE Sepharose ion exchange chromatography, and chromatofocusing using PBE 94. SOD2 was purified 82-fold with a specific activity of 4,960 U/mg protein. This enzyme was stable in a broad pH range from 5.0 to 10.0 and at various temperatures from 25 to 60°C. The native molecular mass of SOD2 estimated by high performance liquid chromatography on TSK gel G2000SW column was 39 kDa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed a single band near 18 kDa, suggesting that native enzyme was homodimeric. The isoelectric point as determined by chromatofocusing was 5. Analysis of its N terminal amino acid sequence revealed high sequence homology with several other cytosolic Cu,Zn-SODs from plants. Exposure of cancer cell lines to garlic Cu,Zn-SOD2 led to a significant decrease in superoxide content with a concomitant rise in intracellular peroxides, indicating that the enzyme is active in mammalian cells and could, therefore, be used in pharmacological applications.     

外源铜增强天然铜锌超氧化物歧化酶断裂DNA的活性

铜锌超氧化物歧化酶（CuZnSOD）作为一种抗氧化酶, 最重要的功能是催化超氧阴离子歧化为过氧化氢和氧气。然而最近研究发现CuZnSOD具有过氧化物酶活性,能导致核酸、蛋白质和细胞膜的损伤。本工作采用光谱学和酶学方法研究外源Cu(Ⅱ)与CuZnSOD之间的相互作用,以及H₂O₂存在下外源Cu(Ⅱ)对 CuZnSOD断裂DNA活性的增强效应。比较CuZnSOD + nCu(Ⅱ) (n=0, 1, 2, 4, 6, 8)和单独Cu(Ⅱ)分别断裂DNA的活性,表明外源Cu(Ⅱ)的加入可显著增强CuZnSOD断裂DNA的活性。相对酶活力和稳态动力学的测定证实了这种增强效应。pH依赖性实验表明断裂DNA的最适pH范围为pH3.6-5.6和pH9.0-10,在不同的pH区域CuZnSOD + nCu(Ⅱ)断裂DNA途径可能不同。     

Comparative superoxide dismutase activity studies

Our results indicate that the various methods for measurement of superoxide dismutase (SOD) activity differ considerably as regards sensitivity. The activity difference may be particularly striking in the determination of the SOD activities of crude homogenates, because of the large number of disturbing factors. We consider the epinephrine-adrenochrome method to be the most suitable for measurement of the SOD activities of crude homogenates and purified enzyme preparations.     

Superoxide Dismutase Biosensors for Superoxide Radical Analysis

ABSTRACT

Some basic work has been performed on the development and optimisation of superoxide dismutase (SOD) biosensors for superoxide radical analysis. Initially we studied the possibility of obtaining a SOD biosensor using the Clark electrode as indicating sensor. However, the best results were obtained using as indicator a classical amperometric electrode for H₂O₂. In both cases the superoxide radical was generated in situ using the xanthine/xanthine oxidase (XOD) enzyme system, while the SOD was immobilised in kappa-carrageenan gel. The first application was realised by studying the effects in vitro on the superoxide radical of some molecules commonly accepted as radical scavengers.     

Development of a new biosensor for superoxide radicals

A superoxide dismutase (SOD) biosensor for determination of superoxide radicals has been developed by immobilization of superoxide dismutase within gelatin (G) on a Pt electrode surface. The properties of the biosensor have been investigated and optimum conditions–enzyme concentration, glutaraldehyde concentration, and pH–were determined. The response of the G-SOD biosensor was proportional to concentration and the detection limit was 0.01 mmol L⁻¹ at a signal-to-noise ratio of 3. The biosensor retained 89% and 60% of its sensitivity after use for three and four weeks, respectively. Immobilization of SOD on gelatin provides a biocompatible microenvironment around the enzyme and stabilizes the activity of the enzyme very efficiently. The superoxide dismutase biosensor was used to determine the antioxidant properties of acetylsalicylic acid-based drugs and the anti-radical activity of healthy and cancerous human brain tissues.