辣根过氧化物酶模拟酶研究进展

辣根过氧化物酶 (HRP)是一种常用的工具酶 ,对其模拟酶的研究是近年来生物化学和有机化学的重要课题 ,具有重要的理论意义和应用价值。本文评述了近十年来HRP模拟酶的研究进展。     

中性辣根过氧化物酶制法新进展

中性辣根过氧化物酶制法新进展季钟煜,费锦鑫（上海普洛麦格生物产品有限公司,上海２００２３３）关键词中性辣根过氧化物酶辣根过氧化物酶（ＨＲＰ）是生物检测中用得非常多的工具酶,其应用和经济价值都很大。因此,制备ＨＲＰ的技术和方法也是相关行业的一个重要研究...     

甲醇酵母Pichia pastoris高水平表达有活性的辣根过氧化物酶

表达有活性的辣根过氧化物酶（ＨＲＰ） 不仅可以深入揭示ＨＲＰ 结构与功能及其生理作用规律, 而且为ＨＲＰ的广泛需要提供新的来源． 为了在甲醇酵母Ｐ． ｐａｓｔｏｒｉｓ 中成功表达, 将编码ＨＲＰＣ成熟肽的ｃＤＮＡ 构建到ｐＰＩＣ９ 上, 再转化到Ｐ． ｐａｓｔｏｒｉｓ 中, 筛选到了分泌表达非糖基化ＨＲＰ 和高糖基化ＨＲＰ（ 分子质量超过１００ ｋｕ） 两种主要产物的重组细胞株． 优化表达条件, 目标产物在摇瓶发酵液中高效表达, 可达４～６ ｇ／Ｌ． 并且直接从发酵液中可获得具有活性的高糖基化ＨＲＰ, 每毫升发酵液中酶活力约有２ Ｕ, 经初步的纯化ＨＲＰ具有最大吸收峰４０３ ｎｍ ．     

反相胶束体系对辣根过氧化物酶结构与功能的影响

在十六烷基三甲基溴化铵（ＣＴＡＢ）／异辛烷－正戊醇反相胶束中,研究了含水量（Ｗ０）和表面活性剂对辣根过氧化物酶（ＨＲＰ）和活力的影响机制。在测定不同含水量（Ｗ０）和ＣＴＡＢ不同浓度下的ＵＶ－Ｖｉｓ光谱（即Ｓｏｒｅｔ吸收光谱）及活力的变化的基础上,发现含水量不同时,反相胶束主要通过影响ＨＲＰ的活性中心而影响酶的活力,但ＣＴＡＢ对酶活性中心没有明显影响。此外通过反相胶束与水相中的ＨＲＰ与Ｈ２Ｏ２复合物     

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。     

辣根过氧化物酶产品的测定

辣根过氧化物酶产品的测定季钟煜,陈佩颖（上海普洛麦格生物产品有限公司,上海２００２３３）关键词辣根过氧化物酶（ＨＲＰ）,邻苯三酚辣根过氧化物酶（ＨＲＰ）是从辣根植物块根中提取制造的。它的实用价值很高,在临床检验上用作为酶指示剂和酶标记,藉以检验体液和...     

辣根过氧化物酶在水相胶束中的动力学

研究了辣根过氧化物酶在三种表面活性剂（ＳＤＳ,ＴｒｉｔｏｎＸ－１００及ＣＴＡＢ）的水相胶束中催化联苯胺聚合反应的动力学。结果表明水相胶束体系有利于反应的进行。辣根过氧化物酶在水相胶束体系中遵循米氏反应,Ｋ_ｍ在ＳＤＳ、ＴｒｉｔｏｎＸ－１００及ＣＴＡＢ三种体系中分别为３．０１４×１０~（－４）ｍｏｌ／Ｌ、１．７２８×１０~（－４）ｍｏｌ／Ｌ和５．６６４×１０~（－５）ｍｏｌ／Ｌ。由于微环境的不同,ＨＲＰ在三种体系中表现出不同的最适反应温度和最适ｐＨ。     

辣根过氧化物酶在一种新型有机介质中的催化反应

选择合适的酶反应介质体系,是酶应用于有机合成的一个重要环节。利用适宜分子量的聚乙二醇（ＰＥＧ）可以将辣根过氧化物酶（ＨＲＰ）分散在甲苯中,摸索了ＨＲＰ在聚乙二醇（ＰＥＧ）－甲苯互溶体系反应的适宜条件,即ＰＥＧ／甲苯的比例、含水量、ｐＨ值、底物浓度等对酶活性影响,结果发现ＰＥＧ含量越低,含水量越高,酶的活力越高;酶在此体系中的最适ｐＨ值为７．０,最适过氧化氢浓度为２０ｍｍｏｌ／Ｌ,愈创木酚的浓度为０     

Cysteine and Crocin Oxidation Catalyzed by Horseradish Peroxidase

The amino acid cysteine is oxidized by horseradish peroxidase, and the water-soluble carotenoid crocin is bleached by cooxidation. The rnonophenol p-hydroxyacetophenone stimulates oxygen uptake, cysteine oxidation and crocin bleaching, whereas its concentration does not change. Superoxide dismutase significantly enhances all these oxidative reactions. Addition of H₂O₂ is not required for these peroxidase-catalyzed oxidations.     

Cysteine and Crocin Oxidation Catalyzed by Horseradish Peroxidase

The amino acid cysteine is oxidized by horseradish peroxidase, and the water-soluble carotenoid crocin is bleached by cooxidation. The rnonophenol p-hydroxyacetophenone stimulates oxygen uptake, cysteine oxidation and crocin bleaching, whereas its concentration does not change. Superoxide dismutase significantly enhances all these oxidative reactions. Addition of H₂O₂ is not required for these peroxidase-catalyzed oxidations.     

MACROMOLECULAR ABSORPTION : Mechanism of Horseradish Peroxidase Uptake and Transport in Adult and Neonatal Rat Intestine

The immature small intestine of neonatal mammals is permeable to gamma globulins as a source of passive immunity. Allegedly, macromolecular absorption ceases when the epithelial cell membrane matures. However, some evidence exists that adult animals retain a limited capacity to transport antigenic and biologically active quantities of large molecules. In this study, the mechanism of absorption of the tracer protein, horseradish peroxidase (HRP), was tested in neonatal and adult rat gut sacs. Transport into serosal fluid was quantitated by enzymatic assay and monitored morphologically by histochemical techniques. A greater transport of HRP was noted in the adult jejunum compared to adult ileum and neonatal intestine. Morphologically, the uptake mechanism in adult intestine was similar to the endocytosis previously reported in neonatal animals Like other endocytotic processes, HRP uptake in adult rats is an energy-dependent process as determined by metabolic inhibitors and temperature-controlled studies. An understanding of the mechanism whereby macromolecules are bound to intestinal membranes and engulfed by them is necessary before the action of physiologic macromolecules such as enterotoxins can be appreciated.     

The Horseradish Peroxidase Catalysed Oxidation of Deoxyribose Sugars

The ability of horseradish peroxidase (E.C. 1.11.1.7. Donor: H₂O₂ oxidoreductase) to catalytically oxidize 2-deoxyribose sugars to a free radical species was investigated. The ESR spin-trapping technique was used to denionstrate that free radical species were formed. Results with the spin trap 3.5-dibronio-4-nitrosoben-zene sulphonic acid showed that horseradish peroxidase can catalyse the oxidation of 2-deoxyribose to produce an ESR spectrum characteristic of a nitroxide radical spectrum. This spectrum was shown to be a composite of spin adducts resulting from two carbon-centered species, one spin adduct being characterized by the hyperfine coupling constants a^N = 13.6Ganda^H_β = 11.0G, and the other by a^N = 13.4G and a^H = 5.8 G. When 2-deoxyribose-5-phosphate was used as the substrate, the spectrum produced was found to be primarily one species characterized by the hyperfine coupling constants a^N = 13.4G and a^H= 5.2. All the radical species produced were carbon-centered spin adducts with a β hydrogen, suggesting that oxidation occurred at the C(2) or C(5) moiety of the sugar. Interestingly, it was found that under the same experimental conditions, horseradish peroxidase apparently did not catalyze the oxidation of either 3-deoxyribose or D-ribose to a free radical since no spin adducts were found in these cases.

It can be readily seen that 2-deoxyribose and 2-deoxyribose-5-phosphate can be oxidized by HRP/H₂O₂ to form a free radical species that can be detected with the ESR spin-trapping technique. There are two probable sites for the formation of a CH type radical on the 2-deoxyribose sugar, these being the C(2) and the C(5) carbons. The fact that there is a species produced from 2-deoxy-ribose, but not 2-deoxy-ribose-5-phosphate, suggests that there is an involvement of the C(5) carbon in the species with the 1 1.0G β hydrogen. In the spectra formed from 2-deoxy-ribose, there is a big difference in the hyperfine splitting of the β hydrogens, suggesting that the radicals are formed at different carbon centers, while the addition of a phosphate group to the C(5) carbon seems to inhibit radical formation at one site. In related work, the chemiluminescence of monosaccharides in the presence of horseradish peroxidase was proposed to be the consequence of carbon-centered free radical formation (10).     

Stabilization of Diluted Aqueous Solutions of Horseradish Peroxidase

Effects of pH, enzyme concentration, and various supplements on the catalytic activity, temperature stability, and secondary structure of horseradish peroxidase (HRP) were studied in diluted aqueous solutions. In 5.0 mM citrate-phosphate buffer (pH 4.2) at 55°C and infinite dilution, HRP was inactivated with a rate constant of 2.86 × 10^–3 s^–1. CaCl₂, BSA, and glycerol caused protective effects, whereas KCl, LiCl, maltose, PEG-6000 (at a concentration above 3%), Triton X-100, ethanol, and Kathon CG had an opposite effect and altered the secondary structure of HRP. Two HRP-stabilizing media: the glycerol-based one containing 10% ethanol and 20% glycerol, or the protein-based one containing 0.1% Kathon CG and 0.2 mg/ml of BSA in 50.0 mM Tris-HCl buffer (pH 7.2) supplemented with 50 mM CaCl₂ were developed, and the stability of HRP (0.36 nM) and its immunoglobulin, cortisol, and progesterone conjugates were compared in these two media. The protein-based medium displayed a greater stabilizing effect particularly on HRP-steroid conjugates.     

The Horseradish Peroxidase Catalysed Oxidation of Deoxyribose Sugars

The ability of horseradish peroxidase (E.C. 1.11.1.7. Donor: H₂O₂ oxidoreductase) to catalytically oxidize 2-deoxyribose sugars to a free radical species was investigated. The ESR spin-trapping technique was used to denionstrate that free radical species were formed. Results with the spin trap 3.5-dibronio-4-nitrosoben-zene sulphonic acid showed that horseradish peroxidase can catalyse the oxidation of 2-deoxyribose to produce an ESR spectrum characteristic of a nitroxide radical spectrum. This spectrum was shown to be a composite of spin adducts resulting from two carbon-centered species, one spin adduct being characterized by the hyperfine coupling constants a^N = 13.6Ganda^H_β = 11.0G, and the other by a^N = 13.4G and a^H = 5.8 G. When 2-deoxyribose-5-phosphate was used as the substrate, the spectrum produced was found to be primarily one species characterized by the hyperfine coupling constants a^N = 13.4G and a^H= 5.2. All the radical species produced were carbon-centered spin adducts with a β hydrogen, suggesting that oxidation occurred at the C(2) or C(5) moiety of the sugar. Interestingly, it was found that under the same experimental conditions, horseradish peroxidase apparently did not catalyze the oxidation of either 3-deoxyribose or D-ribose to a free radical since no spin adducts were found in these cases.

It can be readily seen that 2-deoxyribose and 2-deoxyribose-5-phosphate can be oxidized by HRP/H₂O₂ to form a free radical species that can be detected with the ESR spin-trapping technique. There are two probable sites for the formation of a CH type radical on the 2-deoxyribose sugar, these being the C(2) and the C(5) carbons. The fact that there is a species produced from 2-deoxy-ribose, but not 2-deoxy-ribose-5-phosphate, suggests that there is an involvement of the C(5) carbon in the species with the 1 1.0G β hydrogen. In the spectra formed from 2-deoxy-ribose, there is a big difference in the hyperfine splitting of the β hydrogens, suggesting that the radicals are formed at different carbon centers, while the addition of a phosphate group to the C(5) carbon seems to inhibit radical formation at one site. In related work, the chemiluminescence of monosaccharides in the presence of horseradish peroxidase was proposed to be the consequence of carbon-centered free radical formation (10).     

In Vitro Slow Fluctuation in Horseradish Peroxidase Activity

In vitro slow fluctuations in the level of horseradish peroxidase activity were observed in long-range experiments (72–144 h). Besides random fluctuations, regular slow oscillatory patterns with period lengths ranging from 10.0 to 39.0 h were detected by statistical analysis. The possibility that these oscillations in enzyme activity could have reflected changes in the physical environment of the experimental setup has been thoroughly examined and ruled out. Periodic exposition of the enzyme solution, otherwise kept in darkness, to blue light illumination was shown to influence the period of the oscillations. The changes in enzyme activity were correlated with a modification of the Michaelis constant estimated using guaiacol as substrate. This result was confirmed by the action of chemical modifiers of the enzyme, such as ferulic acid and rutin. It is thought that the observed oscillations in horseradish peroxidase activity are due to spontaneous and specific changes in the tridimensional structure of the enzyme in the thermic reservoir.     

In Vitro Slow Fluctuation in Horseradish Peroxidase Activity

In vitro slow fluctuations in the level of horseradish peroxidase activity were observed in long-range experiments (72-144 h). Besides random fluctuations, regular slow oscillatory patterns with period lengths ranging from 10.0 to 39.0 h were detected by statistical analysis. The possibility that these oscillations in enzyme activity could have reflected changes in the physical environment of the experimental setup has been thoroughly examined and ruled out. Periodic exposition of the enzyme solution, otherwise kept in darkness, to blue light illumination was shown to influence the period of the oscillations. The changes in enzyme activity were correlated with a modification of the Michaelis constant estimated using guaiacol as substrate. This result was confirmed by the action of chemical modifiers of the enzyme, such as ferulic acid and rutin. It is thought that the observed oscillations in horseradish peroxidase activity are due to spontaneous and specific changes in the tridimensional structure of the enzyme in the thermic reservoir.