过氧化物酶的定性反应实验的改进

过氧化物酶(peroxidase)广泛存在于植物组织中,少数动物组织也有该酶的存在。过氧化物酶能催化过氧化氢对各种多元酚式芳香族胺的氧化,即 ZMH~++H_2O_2→ZM+H_2O,是生物细胞代谢中起氧化还原反应的重要酶类,在生物氧化中具有重要的位置。此外,它还能有效地防止过氧化氢在机体内的积累,促进组织代谢,对机体具有独特的保护作用。由于该酶在生物氧化、组织代谢等生物化学反应中的重要地位,人们对它的存在、分布、含量、理化性质、提取纯化及结构等方面颇为重视,在诸方面都进行了较深刻的     

过氧化物酶和PeroxiBase过氧化物酶数据库

过氧化物酶是存在于生物体中的一大类以过氧化物为电子受体催化底物氧化的酶,在生物体的生命活动中发挥着重要作用.过氧化物酶由多态性丰富的多基因家族所编码,其结构和功能具有多样性.近年来,随着研究和应用的深入,过氧化物酶领域迫切需要建立一个专业的信息交流平台.PeroxiBase数据库收集了大量的过氧化物酶数据,并进行生物信息学加工,为生命科学研究人员免费共享、最大化利用与综合开发过氧化物酶资源搭建了信息平台,在过氧化物酶的研究与应用中发挥着重要作用.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

植物谷胱甘肽过氧化物酶研究进展

氧化胁迫可诱导植物多种防御酶的产生,其中包括超氧化物歧化酶(SOD,EC1.15.L1)、抗坏血酸过氧化物酶(APX,EC1.11.1.11)、过氧化氢酶(CAT,E.C.1.11.1.6)和谷胱甘肽过氧化物酶(GPXs,EC1.11.1.9).它们在清除活性氧过程中起着不同的作用.GPXs是动物体内清除氧自由基的主要酶类,但它在植物中的功能报道甚少.最近几年研究表明,植物体内也存在类似于哺乳动物的GPXs家族,并对其功能研究已初见端倪.本文综述了有关GPXs的结构以及植物GPXs功能的研究进展.     

“探究pH对过氧化氢酶的影响”实验改进

\"探究pH对过氧化氢酶的影响\"是中学生物学实验教学的重要内容之一。为了简便实验操作过程,提高实验成功率,对浙科版高中生物学教材中的实验装置进行了改进。结果表明:改进后的实验装置容易操作,并且实验现象明显。在pH=5.0～8.0范围内,随着pH值的上升,过氧化氢酶催化H₂O₂溶液生成氧气的速率呈现先上升后下降的变化趋势,过氧化氢酶最适pH值在7.0左右。     

萝卜过氧化物酶对小鼠机体抗氧化作用的研究

目的 探索新的抗氧化剂.方法 研究萝卜过氧化物酶(POD)对小鼠肝、脾和肾脏超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)的影响.结果 用不同剂量的POD处理后,可以提高肝、脾和肾的SOD、GSH-Px的活性,减低丙二醛的含量.结论 萝卜过氧化物酶可以提高机体的抗氧化能力.     

谷子种质资源抗黑穗病鉴定与过氧化物酶研究

对全国2050份谷子种质资源进行了抗黑穗病鉴定,对不同抗、感黑穗病的谷子种质资源进行了过氧化物酶活性、过氧化物酶同工酶的比较研究。结果表明,谷子不同种质资源对黑穗病的抗性存在着明显的差异。谷子种质资源对黑穗病的抗性比较稳定。谷子感染黑穗病后,抗病品种的过氧化物酶活性明显高于感病品种。过氧化物酶同工酶可以作为一种遗传标记。     

植物抗坏血酸过氧化物酶

植物抗坏血酸过氧化物酶沈文飚黄丽琴徐朗莱（南京农业大学理学院应用化学系,南京２１００９５）关键词抗坏血酸过氧化物酶植物抗坏血酸过氧化物酶（ＡＰＸ,ＥＣ１．１１．１．１１）的发现至今已有２０多年了。Ｆｏｙｅｒ和Ｈａｌｉｗｅｌ［１］首先于１９７６年发现以...     

银离子对辣根过氧化物酶的影响

实验研究Ag 对HRP的影响对检测银的污染有重要意义。以ABTS[2,2-连氮-双-(3-乙基苯并噻唑-6-磺酸)]和H2O2为底物,在pH值5.0的条件下,用分光光度法考察了Ag 存在下的辣根过氧化物酶催化氧化反应。Ag 对辣根过氧化物酶的催化活性显示出抑制作用,并进一步分别探讨了对两种底物的抑制类型和对酶结构的影响。结果表明Ag 对底物H2O2而言,对酶的抑制效应属于反竞争性抑制类型,抑制常数Ki=14.83mmol/L;对底物ABTS而言,对酶的抑制效应属于非竞争性抑制,抑制常数Ki=16.139mmol/L。不同浓度Ag 分别与酶作用后,测定酶的内源荧光光谱。光谱结果表明Ag 影响酶活性的同时也影响酶的构象。     

YhjA - An Escherichia coli trihemic enzyme with quinol peroxidase activity

The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50?kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be ?170?mV, +133?mV and +210?mV, respectively, at pH?7.5. YhjA has quinol peroxidase activity in vitro with optimum activity at pH?7.0 and millimolar range K_M values using hydroquinone and menadiol (a menaquinol analogue) as electron donors (K_M?=?0.6?±?0.2 and 1.8?±?0.5?mM H₂O₂, respectively), with similar turnover numbers (k_cat?=?19?±?2 and 13?±?2?s^?1, respectively). YhjA does not require reductive activation for maximum activity, in opposition to classical bacterial peroxidases, as P heme is always high-spin 6-coordinated with a water-derived molecule as distal axial ligand but shares the need for the presence of calcium ions in the kinetic assays. Formation of a ferryl Fe(IV)?=?O species was observed upon incubation of fully oxidized YhjA with H₂O₂. The data reported improve our understanding of the biochemical properties and catalytic mechanism of YhjA, a three-heme peroxidase that uses the quinol pool to defend the cells against hydrogen peroxide during transient exposure to oxygenated environments.     

Amperometric detection of lignin-degrading peroxidase activities from Phanerochaete chrysosporium

An amperometric enzyme sensor for rapid and simultaneous detection of the lignin-degrading peroxidase activities secreted by Phanerochaete chrysosporium was developed, using H₂O₂, hydroquinone and veratryl alcohol as substrates. In the amperometric measurement, samples of culture filtrate with different lignin-degrading peroxidase activities measured by spectrophotometry were placed into electrochemical cells. The slope of the current increase (Δ_current/Δ_time) upon the addition of H₂O₂ into the culture filtrate solution containing hydroquinone was used as the index for total activity of lignin peroxidase and manganese peroxidase. Then a specific detection of lignin peroxidase was achieved by the addition of veratryl alcohol, which led to current decrease due to the redox competition between veratryl alcohol and hydroquinone. A good linear correlation was found between the electrochemical response and lignin peroxidase activity, manganese peroxidase activity in the range of 8.14–29.79 U l⁻¹ and 0.085–1.37 U l⁻¹, respectively. A regression model was established describing the relationship. The amperometric sensor described here is more rapid, sensitive and precise than conventional spectrophotometric assays, free from interference of turbidity and UV–vis-light-absorbing substances. In this paper, it was also applied in the detection of lignin-degrading peroxidases in compost bioremediation using P. chrysosporium, showing considerable advantages.     

Removal of pentachlorophenol by immobilized horseradish peroxidase

Researches on the polymerization of aqueous pentachlorophenol (PCP) by the catalysis of horseradish peroxidase (HRP) with the existence of hydrogen peroxide (H₂O₂) were conducted. Factors, such as acidity, temperature, enzyme activity, and initial concentration of PCP and H₂O₂ that could influence the degradation were studied. Results showed that the optimum pH value for free enzyme was 5–6; relative higher temperature could accelerate the reaction greatly; PCP removal increased with an increase of enzyme concentration, and PCP (initial concentration 12.6 mg/L) removal percentage could reach nearly 70% under the highest enzyme concentration (about 0.05 u/ml) adopted in the experiment; removal percentage increased slightly with an increase of initial concentration of PCP, and when initial PCP concentrations were 13.0 and 0.7 mg/L, the removal percentages were about 73.7% and 35.7%, respectively; the molar ratio of the reaction between PCP and H₂O₂ was about 1:2.Based on the above results, researches on the removal of PCP by the immobilized HRP were conducted. The free HRP was immobilized on the polyacrylamide gel prepared by gamma-ray radiation method; then the immobilized HRP was filled into a column, and PCP was successfully removed by the immobilized HRP column. The results were compared with results using free HRP enzyme, which showed that the optimum pH value for the immobilized HRP is similar to that for the free HRP, and when pH=5.15, the immobilized HRP could reduce PCP with initial concentration 13.4 mg/L to the concentration of 4.9 mg/L within 1 h, and the immobilized HRP column could be used to repeatedly.     

Purification,characterization and catalytic activity of anionic zucchini peroxidase

The isolation and purification, by preparative electrofocusing, of the major anionic (ZPOA) and cationic (ZPOC) isoenzymes, collected from young zucchini squash, are reported. The M _r and sugar content are similar to those found previously for the major isoenzymes from the ripe fruits and in the range commonly observed for plant peroxidases. The amount of the two cationic enzymes was very low compared with that of anionic ZPOA. The anionic enzyme has been characterized by electronic, circular dichroism, proton NMR and electron paramagnetic resonance spectroscopy. The spectra are qualitatively similar to those of the corresponding anionic horseradish peroxidase (HRPA) derivatives, with minor differences attributable to the particular protein environment around the heme. The kinetics of the enzymatic oxidation of a series of phenols by H₂O₂ have been studied. ZPOA shows a parallel behavior to HRPA, but it is systematically more active than HRPA, indicating that the zucchini enzymes have a marked tendency to carry out oxidation of this type of compounds.     

荔枝果皮过氧化酶的纯化与性质研究

荔枝果皮采后褐变是影响这一重要热带水果经济价值的主要问题,酚类物质的酶促氧化一直被认为是造成植物组织褐变的关键因素,其中多酚氧化酶被研究得最多.过氧化物酶在植物体中分布很广,能够氧化多种底物,在荔枝果皮中的含量也很高.非结合性过氧化物酶已经被证明在果实的采后成熟与老化过程中参与多种过程.在这项研究中,用磷酸缓冲液提取荔枝果皮的非结合性过氧化物酶,并通过硫酸铵沉淀,DEAFSephadex A-50离子交换柱层析以及Sephadex G-100凝胶过滤进行纯化.对得到的酶溶液进行了酶学性质的研究,发现荔枝果皮过氧化物酶具有较高的热稳定性和高的最适反应pH值(6.8),能够氧化许多底物尤其是单酚和各种多酚类物质,反应抑制剂专一性与其他植物来源的过氧化物酶略有不同显示了过氧化物酶参与荔枝果皮褐变过程的可能性,并为提高荔枝采后贮藏性提供了新的思路.     

Reactivation of Horseradish peroxidase in organic solvent using extraction

A novel extraction method was developed aiming at increasing the stability of enzymes in organic solvent media. Horseradish peroxidase (HRP), inactivated in a tetrahydrofuran (THF)/water (1:1, v/v), regained and maintained its activity when HRP was extracted by adding a THF/benzene mixture to the original solution. However, the HRP activity was drastically lowered in the enzyme-free blank solution that had been formed by employing the same extraction procedure. As a result, the reactivation after the extraction is believed to depend on enzyme history, and might be arisen from an irreversible structural change of the enzyme.     

Cyclooxygenase and peroxidase inactivation of prostaglandin-H-synthase during catalysis

Prostaglandin-H-synthase (PGHS) is a bifunctional enzyme catalyzing cyclooxygenase and peroxidase reactions and undergoing irreversible inactivation during catalysis. A new method for kinetic studies of both PGHS activities in the course of cyclooxygenase as well as peroxidase reactions and also preincubation with hydroperoxides is suggested. It is shown that peroxidase activity is retained after complete cyclooxygenase inactivation and cyclooxygenase activity is retained after complete peroxidase inactivation. Two-stage cyclooxygenase inactivation occurs on preincubation of PGHS with hydrogen peroxide. Studies on inactivation under various conditions indicate that chemical mechanisms of cyclooxygenase and peroxidase inactivation are different. The data allow development of kinetic models.     

Incorporation of glutathione peroxidase active site into polymer based on imprinting strategy

Glutathione peroxidase (GPx, EC 1.11.1.9) is a key enzyme involved in scavenging of reactive oxygen species in biological system. For developing an efficient GPx-like antioxidant, catalytically necessary amino acid derivatives which located near the GPx active center were prepared as functional monomers. Via predetermined imprinting with substrate glutathione (GSH), a polymer-based GPx mimic with a similar structure of catalytic center of natural GPx was developed, and it demonstrated high-catalytic efficiency and substrate specificity. The imprinting polymer (I-PEM) exhibits GPx-like activity about three times higher than that of 2-SeCD, a cyclodextrin-based GPx mimic. The detailed studies on kinetics revealed that not only the substrate binding but also positional arrangement of reacting groups contribute significantly to the catalytic efficiency of the peroxidase model.     

Identification of a copper-sensitive ascorbate peroxidase in the unicellular green alga Selenastrum capricornutum

Extracts from the unicellular green alga Selenastrum capricornutum exhibit high superoxide dismutase activity, but only traces of catalase activity. The excess hydrogen peroxide (HO) generated by the superoxide dismutase in S. capricornutum may be degraded by a unique peroxidase. This peroxidase has a high specificity for ascorbate as its electron donor. The enzyme has an optimum pH at 8, is insensitive to cyanide and is inhibited by oxine. Addition of low concentrations of copper to algal cultures stimulates the peroxidase activity threefold. This enzymatic system could be used as a sensitive bioindicator for copper in fresh water.