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...     

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     

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...     

辣根过氧化物酶的结构与作用机制

辣根过氧化物酶是一种重要的酶制剂,它已经有一个多世纪的研究历史了。近几年,有关它的结构、催化中间体、催化机制以及特殊氨基酸残基功能等又有了新的发现。     

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。     

辣根过氧化物酶生物传感器催化与抑制动力学研究

为研究固定化的辣根过氧化物酶降解酚类有机污染物及检测环境有毒物质过程的动力学机制 ,利用伴刀豆蛋白A与糖蛋白的特异性吸附性质将辣根过氧化物酶层层固定在玻碳电极表面 ,制备了一种灵敏度高、性能稳定的辣根过氧化物酶多层膜生物传感器 ,并推导出了辣根过氧化物酶对过氧化氢、对苯二酚的催化反应以及苯肼对该反应的抑制作用的动力学模型。运用酶传感器实验数据 ,对推导出的动力学模型进行了拟合与参数估计。     

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     

反相胶束体系中辣根过氧化物酶的活力和动力学性质

本文系统研究辣根过氧化物酶在ＣＴＡＢ／Ｈ２Ｏ／ＣＨＣ．３－ｉｓｏｏｃｔａｎｅ（１∶１,Ｖ／Ｖ）反相胶束体系中的催化行为。在一定条件下酶反符合Ｍｉｃｈａｅｌｉｓ－Ｍｅｎｔｅｎ动力学。研究水含量、底物浓度、ＰＨ、温度、表面活性剂的浓度等对酶反应的影响,结果表明表面活性剂对酶表现非竞争性抑制作用,高浓度的过氧化氢抑制酶活,最适ＰＨ为７．０。在低水含量（Ｗ０＜５）的胶束体系中保温后,酶的活力发生不可逆的改     

Fe(Ⅱ)对漆酶催化活性的影响

以2,2-连氮-双（3-乙基苯并噻唑-6-磺酸）（ABTS）为底物,在pH4.5的条件下,用分光光度法考察了Fe^2 离子存在下的漆酶催化氧化反应,发现Fe^2 离子对漆酶的催化活性显示出抑制作用,并进一步探讨了其抑制特征,抑制位点和作用方式。结果表明,Fe^2 离子对漆酶催化活性抑制属竞争性抑制过程,抑制作用是通过还原ABTS来实现的。     

Inhibition of the Peroxidase-Catalyzed Oxidation of Chromogenic Substrates by Alkyl-Substituted Diatomic Phenols

A comparative kinetic study of the peroxidase oxidation of three chromogenic substrates--2,2-azino-bis(3-ethyl-2,3-dihydrobenzothiazoline-6-sulfonic acid), o-phenylenediamine (PDA), and 3,3,5,5-tetramethylbenzidine--inhibited by trimethylhydroquinone and six tert-butylated pyrocatechols (InH) was carried out at 20°C in 0.015 M phosphate–citrate buffer (pH 6.0) containing organic cosolvents (0–10% ethanol or DMF). The inhibitors were quantitatively characterized by the inhibition constants (K _i), the duration of the lag period in the oxidation product formation (), and the stoichiometric coefficient of inhibition that specifies the number of radicals terminated by one InH molecule (f). The inhibition could be competitive, noncompetitive, mixed, or uncompetitive, which depended on the nature and structure of the chromogenic substrate–diatomic phenol pair. Various substrate–diatomic phenol pairs exhibited K _i values within the range of 11–240 M andfvalues from 0.7 to 2.6. The absence of a lag period was characteristic of oxidation of the substituted o-phenylenediamine–substituted pyrocatechol. The total kinetic parameters and properties of the components allowed us to suggest six chromogenic substrate–substituted diatomic phenol pairs for use in test systems for the determination of antioxidant activity in human body fluids, natural biological preparations, and food.     

Effect of Enhancers on the Pyridopyridazine–Peroxide–HRP Reaction

4-Substituted phenyl boronic acids (e.g., 4-iodo, 4-bromo, 4-phenyl) are effective enhancers of the horseradish peroxidase (Type VIA) catalysed chemiluminescent oxidation of various pyrido[3,4-d]pyridazine-1,4(2H,3H)dione derivatives. The most effective combination was 4-biphenylboronic acid and 8-amino-5-chloro-7- phenylpyrido[3,4-d]- pyridazine-1,4(2H,3H)dione. Generally, the intensity of light emission in the presence of peroxidase was higher with the pyridopyridazines than with sodium luminol. However, the blank light emission was much lower with sodium luminol than with the pyridopyridazines. A synergistic enhancement phenomenon was demonstrated for the combination of a 4-iodophenol and a 4-biphenylboronic acid enhancer with 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione. The combination of these two enhancers produced a light emission intensity in an assay for 5 fmol of peroxidase that was 25% higher than expected from the sum of the individual light intensities.     

Noncompetitive Activation of the Peroxidase-Catalyzed Oxidation of o-Phenylenediamine by Melamine

Peroxidase-catalyzed oxidation of o-phenylenediamine (PDA) is greatly activated with melamine (MA) in 15 mM phosphate–citrate buffer at pH 6.0–7.4 in a noncompetitive manner: k _cat and K _m increase in direct proportion to the MA concentration. An extent of the activation is quantitatively characterized with a coefficient (in M^–1), which essentially increases along with the rise in pH from 6.0 to 7.4. MA acts as a nucleophilic catalyst in the oxidation process: it most likely affects the peroxidase active site from the distal position of heme. MA noncompetitively inhibits the peroxidase oxidation of PDA at pH 4.3, since it completely loses its nucleophilic properties in acidic medium. A rapid, highly accurate, and simple analytical test system based on the kinetics of melamine-activated oxidation of PDA is proposed for the quantitative determination of melamine within the concentration range of 10^–4–10^–3 M. This test system uses the spectrophotometric determination of the PDA oxidation product at 455 nm.     

A highly efficient and selective turn‐on fluorescent sensor for Hg2+, Ag+ and Ag nanoparticles based on a coumarin dithioate derivative

Based on chelation‐enhanced fluorescence, a new fluorescent coumarin derivative probe 3(1‐(7‐hydroxy‐4‐methylcoumarin)ethylidene)hydrazinecarbodithioate for Hg²⁺, Ag⁺ and Ag nanoparticles is reported. Fluorescent probe acts as a rapid and highly selective “off–on” fluorescent probe and fluorescence enhancement by factors 5 to12 times was observed upon selective complexation with Hg²⁺, Ag⁺ and Ag nanoparticles. The molar ratio plots indicated the formation of 1:1 complexes between Hg²⁺ and Ag⁺ with the probe. The linear response range covers a concentration range 0.1 × 10^–5–1.9 × 10^–5 mol/L, 0.1 × 10^–5–2.3 × 10^–5 mol/L and 0.146 × 10^–12–2.63 × 10^–12 mol/L for Hg²⁺, Ag⁺ and Ag nanoparticles, respectively. The interference effect of some anions and cations was also tested. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

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).     

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).