Peroxidase-mediated oxidative coupling of 1-naphthol: characterization of polymerization products

The oxidative polymerization of 1-naphthol was investigated in the presence of horseradish peroxidase (HRP). Naphthol polymerization products (NPP) were characterized for their relative polarity using octanol--water partitioning experiments and reverse-phase high pressure liquid chromatography, for structure using size exclusion chromatography and liquid chromatography-mass spectrometry (LC/MS), and for ecotoxicity using inhibition of bacterial bioluminescence. Peroxidase addition resulted in the production of soluble and insoluble NPP. Soluble NPP was predominantly more polar than the parent naphthol and comprised of trimers and tetramers. Insoluble NPP oligomers included dimers, trimers and tetramers. The net aqueous-phase toxicity was significantly reduced due to polymer formation and subsequent precipitation. A reaction model deduced from the LC/MS fragmentation patterns of trimeric naphthol was proposed for NPP formation. Results from this study suggest that HRP-mediated treatment of naphthol contaminated soils can achieve risk reduction through (i) the formation of large hydrophobic oligomers that are immobilized on the soil matrix; and (ii) reduction in aqueous-phase toxicity due to polymer precipitation.     

Synaptic circuitry identified by intracellular labeling with horseradish peroxidase

During the past two decades new techniques have been developed to directly test the dogma that neuronal structure is correlated with neuronal function. In the earliest experiments, Procion yellow was injected into neurons after they had been characterized physiologically; these neurons were then viewed through the light microscope. Recent advances in the method generally employ horseradish peroxidase as the dye which is injected since it diffuses quite readily throughout the injected neuron and produces a stable reaction product for both light and electron microscopic studies. This review explores the utility of examining synaptic circuitry after physiologically recording from axons or neurons and then injecting horseradish peroxidase into them. As a model system, we studied the cat lateral geniculate nucleus and investigated, at the electron microscopic level, the synaptic contribution to this nucleus from retinogeniculate axons, from interneurons, and from the axon collaterals of neurons that project to visual cortex.     

HRP biosensor based on sugar-lectin biospecific interactions for the determination of phenolic compounds

A layer-by-layer self-assembly of concanavalin A (Con A) and glycoprotein horseradish peroxidase (HRP) afforded multilayer thin films on the surface of a thiol-modifed gold electrode, through biospecific complexation of Con A and sugar residues in the glycoenzymes. The performance of the HRP biosensor is reported for the amperometric detection of phenolic compounds. The concentration of hydrogen peroxide and assembly conditions of the precursor film, such as pH, the ionic strength of the polyelectrolyte solutions and the number of assembled bilayers were investigated using catechol. With optimized conditions, the biosensor presented a linear response for catechol from 6.0 to 48.0 μmol l⁻¹, with a high sensitivity of 160 μmol⁻¹ l nA and a detection limit of 0.6 μmol l⁻¹. The response time of the biosensor for phenolic compounds was very short, reaching 95% of its maximum response in about 2 s. The differences in sensitivity observed for a series of phenolic substrates were discussed in terms of the stability of the oxidized phenolic compounds and the properties of substituents.     

高压脉冲电场对辣根过氧化物酶活性及构象的影响效果

本实验旨在研究高压脉冲电场(PEF)对辣根过氧化物酶(HRP)的活性及酶构象的影响效果。结果显示:HRP的活性随电场强度的增强和脉冲处理数的增加而降低。在25kV/cm、207个脉冲和22kV/cm、1214个脉冲的时候,HRP的活性分别降低了14.3%和33.2%。PEF处理后的酶液在4℃下贮藏24h和48h后,HRP的活性呈缓慢下降。圆二色谱和荧光光谱分析表明PEF处理后HRP的蛋白构象发生了改变。PEF和热处理后HRP蛋白二级结构的平均摩尔椭圆率都发生了变化,α-螺旋含量分别下降了29.3%和57.7%。荧光光谱分析表明HRP蛋白的荧光强度在PEF处理后也发生了改变。     

超高压处理对辣根过氧化物酶二级结构及其活力的影响

本文研究了超高压处理对辣根过氧化物酶活力的影响，测定了其CD谱，并分析了酶的二级结构与酶活力的关系。试验压力为0.1～500MPa，温度为20～60℃，保压时间为10min，酶溶液pH7．0。试验结果表明：(1)在处理温度为40℃、保压时间为10min和酶溶液pH7．0的条件下，压力对酶活力有显著影响；在100MPa附近的低压处理时，酶活力会反常升高；大于400MPa处理时，酶活力下降趋势缓慢。(2)在处理压力为500MPa、保压时间为10min、酶溶液pH7．0条件下，在40℃以下的温度范围内，酶的活力下降趋势缓慢：40℃以后，酶活力随温度升高下降迅速。(3)辣根过氧化物酶的活力与β-折叠的含量密切相关；超高压处理将降低酶构象中的β-折叠构象比例，从而使酶失活；温度协同超高压处理将加剧β-折叠的含量的下降，从而加速辣根过氧化物酶活力的降低。     

Immobilization of horseradish peroxidase and nile blue into the ormosil nanocomposite for the fabrication of hydrogen peroxide biosensor based on MWCNT modified glassy carbon electrode

A novel approach to construct a second-generation amperometric biosensor is described. The classical redox dye nile blue (NB) as mediator and horseradish peroxidase as a base enzyme were coimmobilized into the multiwalled carbon nanotubes (MWCNTs) modified ormosil matrix. Nafion was dispersed into the matrix to enhance the rate of the electron transfer and prevent the cracking of the ormosil film. The surface morphology of MWCNT/NB/NAF/HRP nanocomposite was characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). Cyclic voltammetry and amperometry measurements were used to study and optimize the performance of the resulting peroxide biosensor. The apparent Michaelis–Menten constant was determined to be 1.1 mM. The effect of pH, applied potential and amount of the HRP enzyme on the electrochemical biosensor has been systematically studied. The fabricated biosensor demonstrated significant electrocatalytic activity for the reduction of hydrogen peroxide with wide linear range from 2 × 10⁻⁷ to 3.8 × 10⁻⁴ M, and low detection limit 1 × 10⁻⁷ M (S/N = 3) with fast response time <3 s. The facile procedure of immobilizing HRP and MWCNTs into the ormosil used in the present work can promote the development of electrochemical research for enzymes, proteins, biosensors, biofuel cells and other bioelectrochemical devices.     

Lipidic Cubic Phases as a Versatile Platform for the Rapid Detection of Biomarkers,Viruses, Bacteria,and Parasites

Rapid and affordable detection of analytes is critical in diagnostic technologies, but current methods are typically expensive and unsuitable for field detection. Lipidic cubic phases are optically isotropic, transparent lyotropic liquid crystals (LC), containing highly confined water nanochannels in‐between percolating lipid bilayers following defined space groups. Due to this nanoconfinement, the water in these systems provides a unique environment for chemical and enzymatic reactions. Here, it is shown that during the in meso peroxidase enzymatic reaction, the converted product crystallizes within the mesophase domains, generating a detectable birefringence signal and a new general assay principle is presented for the detection of an unprecedented vast class of analytes using such birefringence as sole optical output signal. By exploiting bienzymatic cascade reactions or introducing an enzyme‐linked immunosorbent assay based on birefringence (Birefringent‐ELISA), this approach is used for real‐time detection of exemplary analytes, such as glucose and cholesterol, model pathogenic microorganisms, Escherichia coli, and viruses such as Ebola and HIV. It is also shown how the same technology enables the rapid, naked‐eye screening of malaria infection via in meso detection of hemozoin crystallites. This new technology is general and readily adaptable to the rapid detection of virtually any type of analyte, such as disease biomarkers, viruses, bacteria, and parasites.     

辣根过氧化物酶催化合成水溶性导电聚苯胺的研究

以辣根过氧化物酶(HRP)为催化剂、十二烷基硫酸钠(SDS)为模板研究了生物催化水溶性导电聚苯胺(PANI)的合成.探讨了反应体系的pH值、过氧化氢浓度和SDS浓度对聚合反应的影响,并研究了HRP催化苯胺聚合过程中性质的变化.紫外可见扫描光谱分析和电导率测定的结果表明,在以SDS为模板的条件下,HRP催化水溶性导电聚苯...