生物酶HRP催化H~2O~2氧化间苯二胺反应的研究

应用电化学分析,高效液相色谱(HPLC),紫外-可见光谱(UV-vis),红外光谱(IR)和核磁共振(NMR)等技术对辣根过氧化物酶(HRP)催化H~2O~2氧化间苯二胺(MPD)的反应进行了研究。伏安法和高效液相色谱实验说明,在所选择的酶催化反应条件下,酶催化反应生成一种产物。用化学方法制得了HRP酶催化H~2O~2氧化MPD的产物纯品。经UV-vis,IR和^1HNMR谱鉴定,产物为2,7-二氨基吩嗪。写出了酶催化反应过程,同时对酶催化反应产物的电极还原过程也进行了研究。     

Horseradish Peroxidase Enzyme Electrodes for Nadh and Nadph

Abstract

The aerobic oxidation of reduced pyridine phosphonucleotides catalyzed by horseradish peroxidase (E.C.1.11.1.7) (HRP) in combination with Mn²⁺-ions, phenolics and redox dyes was used to perform NADH and NADPH determinations with an amperometric HRP enzyme electrode. Optimal operational conditions have been elaborated for such a sensor and a linear relationship between derivative current (dI/dt) and NADH-concentration was obtained between 2.10^?5 and at least 2.10^?4 mol/l. The coefficient of variation was equal to or smaller than 4 %. The time for one measurement was less than 10 minutes including equilibration of the electrode.     

MAP-H~2O~2-HPR伏安酶联免疫分析新体系和光谱及电化学研究

提出了间氨基酸(MAP)-H~2O~2-辣根过氧化物酶(HRP)伏安酶联免疫分析新体系.本方法以线性扫描二阶导数伏安法检测HRP催化H~2O~2氧化MAP的产物,用于游离HRP和各种HRP标记物的测定,灵敏度均高于经典的ELISA显色光度法.测定游离HRP的线性范围为1.0x10^-^8-1.0x10-6/L,检测限达3.8x10^-^9g/L.制备出了HRP催化H~2O~2氧化MAP的产物纯品并应用电化学分析,高效液相色谱,元素分析,紫外-可见光谱,红外光谱,^1H核磁共振谱,^1^3C核磁共振谱及质谱等技术对体系酶促反应进行了深入的研究.在选择的酶促反应条件下,生成的产物为2-氨基-5-[(3-差苯基)]-2,5-环己烯基-1,4-二酮.提出了酶催化反应机理及其产物的电极还原过程。     

Enzymatic oxidation of cadmium and lead metals photodeposited on cadmium sulfide

Cadmium and lead metals deposited on CdS particles are shown to act as substrates--electron donors for enzymes, hydrogenase from Thiocapsa roseopersicina (HG), NAD-dependent hydrogenase from Alcaligenes eutrophus (NLH), and ferredoxin:NADP oxidoreductase (FNR) from Chlorella in the formation of hydrogen, NADH and NADPH, respectively. Adsorption of the enzyme on the surface of the metallized CdS particle is required for enzymatic oxidation of metal. The maximum rates for the formation of hydrogen and NADH catalyzed by hydrogenase and NAD-dependent hydrogenase with metals as electron donors are comparable with the rates obtained for these enzymes using soluble substrates. Kinetic analysis of the enzymatic oxidation of cadmium metal has revealed that the rate decreases mainly due to the formation of a solid product, which is supposed to be Cd(OH)2. The deceleration of lead oxidation catalyzed by hydrogenase proceeds at the expense of the inhibitory effect of the formed Pb2+. The enzymatic oxidation of electrochemically prepared cadmium metal is also shown. Based on these results, a new mechanism of action of the enzymes involved in anaerobic biocorrosion is proposed. By this mechanism, the enzyme accelerates the process of metal dissolution through a mediatorless catalysis of the reduction of the enzyme substrate.     

生物酶振荡器研究谷胱甘肽的抗氧化效应

组装了一种生物酶振荡器（Peroxidase-Oxidase Biochemical Oscillator） 的实验装置、建立了相应的实验方法,在此基础上得到单倍周期、四倍周期、混沌 等丰富的振荡图谱信息,并采用它作为“探针”,检测了谷胱甘肽（GSH）的抗氧 化效应,它可作为定量分析的基础,线性范围为9.38 * 10~(-7) ～ 7.50 * 10~(- 5) mol·L~(-1),探讨了某些可能的反应机理。     

Spectrophotometric quantification of lactose in solution with a peroxidase-based enzymatic cascade reaction system

A spectrophotometric assay was developed for the quantification of lactose in aqueous solution via a one-pot enzymatic cascade reaction at 25 °C and pH 7.2. Lactose (0.2-1.8 mM), E. coli β-galactosidase (β-Gal), Aspergillus niger glucose oxidase (GOD), horseradish peroxidase (HRP) and o-phenylenediamine (OPD) were incubated, and the increase in absorbance at 417 nm (A (417)) due to the formation of DAP (2,3-diaminophenazine), the dimeric oxidation product of OPD, was followed. The increase in A (417) was found to depend linearly on the initial lactose concentration via three consecutive but simultaneously occurring enzymatic reaction steps catalyzed by β-Gal, GOD, and HRP. No pre-incubation of lactose with β-Gal is needed with this simple lactose assay.     

PAP-H2O2-HRP酶促反应产物光谱及电化学研究

前文［１］首次提出了以对氨基酚（ＰＡＰ）为底物的ＰＡＰ－Ｈ２Ｏ２－辣根过氧化物酶（ＨＲＰ）伏安酶联免疫分析新体系,并成功地应用于烟草花叶等植物病毒的血清学检测．在此之前,我们也曾报道过一些伏安酶联免疫分析新体系［２,３］．迄今,还没有人对 ＨＲＰ催化 Ｈ２Ｏ２氧化 ＰＡＰ的酶促反应进行过探讨．Ｐｒａｔｉ等［４］报道了在铜催化作用下,Ｏ２氧化对氨基酚生成３－［（４－个羟苯基）氨基」－４－（２－氨基－５－羟苯基）－６－［（４－羟苯基）亚胺基］－２,４－环己二烯基－１－酮．本文的实验结果与此文献不符． 为…     

Horseradish peroxidase-mediated oxidation of carcinogenic non-aminoazo dyes

Fifteen non-aminoazo dyes were oxidized when catalyzed by horseradish peroxidase (HRP) in the presence of H2O2. Aromaticdiazonium ions, the ultimate carcinogen, were detected in the reaction with all of azo compounds as the substrates for HRP. The rates of oxidative cleavage fairly depended on pH. Relative enzymatic activities were compared among 15 non-aminoazo dyes, and the structural requirements of the substrates for HRP were discussed in detail.     

OSCILLATORY LOW-LEVEL CHEMILUMINESCENCE FROM A NONEQUILIBRIUM β-NICOTINAMIDE ADENINE DINUCLEOTIDE-PEROXIDASE SYSTEM: EXPERIMENTAL OBSERVATIONS and COMPUTER SIMULATIONS

Using a highly sensitive photon counting technique, oscillatory low-level chemiluminescence was observed from a non-equilibrium oxidation of β-nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase (HRP). Emergence and stability of the oscillation were affected by the concentration of HRP, 2,4-dichlorophenol and infusion rate of NADH. We also constructed a simultaneous rate equation to explain the oscillatory phenomena of this non-equilibrium system, in which the photon emission was assumed to be generated from NAD'-O₂ interaction.     

流动注射化学发光免疫分析研究II. 偶合反应测定HRP及其标记 物

本文详细考查了壳质胺,多孔玻璃和粗孔硅胶作为流动注射免疫分析免疫反应器基质的可行性,在此基础上将HRP催化H~2O~2氧化K~4Fe(CN)~6生成K~3Fe(CN)~6的反应,与H~2O~2和K~3Fe(CN)~6对luminol的共氧化化学发光反应相偶合首次提出了一种新的流动注射免疫分析的最终检测手段,由于酶促反应与化学发光反应在检测系统的不同位置进行,因而这种方法克服了已报道的流动注射化学发光免疫分析不能协调酶催化和化学发光反应的最佳pH,底物与酶不能充分接触及载体对光的散射等缺点,具有灵敏度高、精密度好等优点,该方法测定HRP及其标记物检测限均可达fmol级,测定时间为1-2min,相对标准偏差为3.9%。     

Studies on the oxidation reaction of tyrosine (Tyr) with H2O2 catalyzed by horseradish peroxidase (HRP) in alcohol-water medium by spectrofluorimetry and differential spectrophotometry

An oxidation reaction of tyrosine (Tyr) with H(2)O(2) catalyzed by horseradish peroxidase (HRP) was studied by spectrofluorimetry and differential spectrophotometry in the alcohol(methanol, ethanol, 1-propanol and isopropanol)-water mutual solubility system. Compared with the enzymatic-catalyzed reaction in the water medium, the fluorescence intensities of the product weakened, even extinguished. Because the addition of alcohols made the conformation of HRP change, the catalytic reaction shifted to the side of polymerization and the polymer (A(n)H(2), n>or=3) exhibited no fluorescence. The four alcohols cannot deactivate HRP. Moreover isopropanol activated HRP remarkably.     

Meldola’s Blue — doped titania sol-gel sensor for NADH determination

Titania layers obtained by a sol-gel technique doped with redox mediator, Meldola’s Blue, were employed for construction of a new NADH senor. Optimization of preparation process as well as experimental conditions affecting the response of the sensor were examined. Under optimal conditions NADH could be determined in the wide linear range from 90 to 2300 μM with detection limit 12 μM and a high sensitivity 12.5 nA μM⁻¹. The usefulness of developed sensor was preliminarily checked in determination of NADH forming during enzymatic oxidation of ethanol catalyzed by alcohol dehydrogenase (ADH).      

A Biofuel Cell Based on Biocatalytic Reactions of Glucose on Both Anode and Cathode Electrodes

Biofuel cells based on electrocatalytic oxidation of NADH and reduction of H₂O₂ have been prepared using carbon fiber electrodes functionalized with graphene nano‐flakes. The electrochemical oxidation of NADH was catalyzed by Meldola's blue (MB), while the reduction of H₂O₂ was catalyzed by hemin, both catalysts were adsorbed on the graphene flakes due to their π‐π staking. In the next set of experiments, the MB‐ and hemin‐electrodes were additionally modified with glucose dehydrogenase (GDH) and glucose oxidase (GOx), respectively. The enzyme catalyzed reactions in the presence of glucose, NAD⁺ and O₂ resulted in the production of NADH and H₂O₂ in situ. The produced NADH and H₂O₂ were oxidized and reduced, respectively, at the bioelectrocatalytic electrodes, thus producing voltage and current generated by the biofuel cell. The enzyme‐based biofuel cells operated in a human serum solution modelling an implantable device powered from the natural biofluid. Finally, two enzyme‐based biofuel cell connected in series and operating in the serum solution produced electrical power sufficient for activation of an electronic watch used as an example device.     

An improved ELISA for the determination of southern bean mosaic virus with linear sweep voltammetry detection based on new system of PAP-H_2O_2-HRP

An improved enzyme-linked immunosorbent assay (ELISA) for the determination of southern bean mosaic virus (SBMV) with linear sweep voltammetry based on a new system of p-aminophenol (PAP)-H2O2-horseradish peroxidase (HRP) has firstly been developed. The enzymatic product 3-[(4-hydrox-yphenyl) amino]-4-(2-amino-5-hydroxyphenyl)-6-[ (4-hydrox-yphenyl)imino]-2,4-cyclohexadiene-l-one, produced from the oxidation of PAP with H2O2 catalyzed by HRP, yielded a sensitive linear sweep voltammetric response at - 0.45 V ( vs. SCE) in Britton-Robinson (BR) buffer solution. Based on the voltammetric peak, HRP can be measured with a detection limit of 0.4 mU/L and a linear range of 1.0-1.0 × 102 mU/ L. The detection limit for the SBMV is 8.0 ng/mL and the highest dilution ratio for the detection of infected leaf sap is 1: 1.5×105.     

REGENERATION OF NAD+ AND NADP+ COFACTORS BY PHOTOSENSITIZED ELECTRON TRANSFER

Abstract The irradiation with visible light of photosensitizer dyes like methylene blue or N-methyl phenazonium methyl sulfate leads to the oxidation of reduced coenzymes such as pyridine nucleotides (NADH or NADPH). This photoredox reaction can be used to regenerate the oxidized form of these coenzymes in enzymatic reactions and total consumption of a substrate with catalytic amounts of enzyme, coenzyme and photosensitizer can be performed. The process has been studied on two common enzymatic reactions: ethanol oxidation by alcohol-NAD ⁺ -oxidoreductase and gluconate-6-phosphate oxidation by 6-phospho-D-gluconate-NADP⁺-2-oxidoreductase. In the first case, a turnover number of 1125 has been obtained for the photoregeneration of NAD ⁺ from NADH.     

Enzyme-catalyzed reaction of OPD-H_2O_2-HRP voltammetric enzyme-linked immunoassay system

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Immobilized horseradish peroxidase as a reusable catalyst for emulsion polymerization

The study on the adsorption of horseradish peroxidase (HRP) onto silicon wafers was carried out by means of in situ ellipsometry, atomic force microscopy (AFM) and contact angle measurements. A smooth HRP layer adsorbed onto Si wafers. The enzymatic activity of free or adsorbed HRP was determined by the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and by the emulsion polymerization of ethylene glycol dimethacrylate (EGDMA). Upon adsorbing, HRP molecules might have undergone some conformational changes, which caused a small reduction of enzymatic activity in comparison to that observed for HRP solution. However, it was possible to reuse the same HRP-covered Si wafer as catalyst in the polymerization of EGDMA three times.     

Reduction of an aldehyde by a NADH/Zn2+ -dependent redox active ribozyme

We report here the ability of an alcohol dehydrogenase (ADH) ribozyme to reduce a benzaldehyde. While the ribozyme was initially evolved in vitro based on the activity for the NAD+-dependent oxidation of the benzyl alcohol, we found that this ADH ribozyme is also capable of reducing the aldehyde in the presence of NADH and Zn2+. The rate acceleration gained by ribozyme catalysis was more than 6 orders of magnitude larger than the spontaneous reaction. Although the reversibility of phosphordiester and acyl transfer reactions catalyzed by ribozymes was known, that of other chemical reactions has not been well established. This study has demonstrated the reversibility of a hydride transfer chemistry catalyzed by the ADH ribozyme. Most interestingly, the ribozyme shares many features with the protein ADHs, e.g., reversibility and NADH/Zn2+ dependence.     

The influence of structure and composition of a reverse SDS microemulsion on enzymatic activities and electrical conductivities

The activity of the enzyme horse radish peroxidase (HRP) is studied in a series of reverse microemulsions composed of dodecane, aqueous buffer, sodium dodecylsufate (SDS) and alcohols of the homologous series 1-butanol to 1-octanol. The HRP catalyzed reaction is the oxidation of a classical water soluble substrate, the 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) by hydrogen peroxide. In parallel electrical conductivity measurements are performed on the same solutions. The structural changes in the microemulsions, as inferred by the conductivity measurements, correlate remarkably well with the changes in the enzymatic activities. In particular it is found that (a) the maximum activity of the enzyme is always related to its optimum hydration and that this hydration can be related to the microemulsion structures, (b) the enzyme inhibition caused by the alcohols in microemulsions is a consequence of both the solubility of the alcohols in the buffer and the rigidity of the interfacial film. Consequently, it can be concluded that enzymatic activity measurements are a valuable tool to study confined systems such as microemulsions and, in particular, the amount of available hydration water. Enzymatic activities can be finely tuned by small changes in microemulsion structures, probably in a predictive way.