Showing papers on "Substrate (chemistry) published in 2003"
TL;DR: This first demonstration of nanotube-based biosensors provides a new tool for enzymatic studies and opens the way to biomolecular diagnostics.
Abstract: We demonstrate the use of individual semiconducting single-wall carbon nanotubes as versatile biosensors. Controlled attachment of the redox enzyme glucose oxidase (GOx) to the nanotube sidewall is achieved through a linking molecule and is found to induce a clear change of the conductance. The enzyme-coated tube is found to act as a pH sensor with large and reversible changes in conductance upon changes in pH. Upon addition of glucose, the substrate of GOx, a steplike response can be monitored in real time, indicating that our sensor is also capable of measuring enzymatic activity at the level of a single nanotube. This first demonstration of nanotube-based biosensors provides a new tool for enzymatic studies and opens the way to biomolecular diagnostics.
1,242 citations
TL;DR: The structures of naphthalene dioxygenase are determined that show a molecular oxygen species bound to the mononuclear iron in a side-on fashion and provide the basis for a reaction mechanism and for the high stereospecificity of the reaction catalyzed by naphthaene diodesase.
Abstract: Binding of oxygen to iron is exploited in several biological and chemical processes. Although computational and spectroscopic results have suggested side-on binding, only end-on binding of oxygen to iron has been observed in crystal structures. We have determined structures of naphthalene dioxygenase that show a molecular oxygen species bound to the mononuclear iron in a side-on fashion. In a complex with substrate and dioxygen, the dioxygen molecule is lined up for an attack on the double bond of the aromatic substrate. The structures reported here provide the basis for a reaction mechanism and for the high stereospecificity of the reaction catalyzed by naphthalene dioxygenase.
473 citations
TL;DR: The results show that an enzymatic oxidation can diminish the toxicity of some polycyclic aromatic hydrocarbons, phenols, organophosphorus pesticides and azo dyes in laboratory and some field conditions.
Abstract: The ability of peroxidases and laccases enzymes to treat organic pollutants is reviewed. Enzymatic methods generally have low energy requirements, are easy to control, can operate over a wide range of conditions and have a minimal environmental impact. Peroxidases and laccases have broad substrate specificities and can catalyze the oxidation of a wide range of toxic organic compounds. The results show that an enzymatic oxidation can diminish the toxicity of some polycyclic aromatic hydrocarbons (PAHs), phenols, organophosphorus pesticides and azo dyes in laboratory and some field conditions. Due to the hydrophobicity and low aqueous solubility of these substrates, reactions are usually performed in the presence of organic solvents. However, it was detected that organic solvents can provoke enzyme denaturation, unfavorable substrate partition, inhibition or stabilization of enzyme–substrate complexes, depending on the enzyme, substrate and organic solvent used. Strategies to overcome these problems are proposed. Additionally, the low stability of heme-containing peroxidases to hydrogen peroxide, the low reaction rates of laccases, the mediators toxicity, the limited availability and high costs of these enzymes are other limitations detected for commercial applications. Due to field reaction conditions are more complex than laboratory conditions efforts have to be made to achieve the cheap overproduction of these biocatalysts in heterologous hosts and also their modification by chemical means or protein engineering to obtain more robust and active enzymes.
405 citations
TL;DR: The 1.7-Å structure of the reversible isatin–MAO-B complex has been determined; it forms a basis for the interpretation of the enzyme's structure when bound to either reversible or irreversible inhibitors.
Abstract: Monoamine oxidase B (MAO-B) is an outer mitochondrial membrane-bound enzyme that catalyzes the oxidative deamination of arylalkylamine neurotransmitters and has been a target for a number of clinically used drug inhibitors. The 1.7-A structure of the reversible isatin-MAO-B complex has been determined; it forms a basis for the interpretation of the enzyme's structure when bound to either reversible or irreversible inhibitors. 1,4-Diphenyl-2-butene is found to be a reversible MAO-B inhibitor, which occupies both the entrance and substrate cavity space in the enzyme. Comparison of these two structures identifies Ile-199 as a "gate" between the two cavities. Rotation of the side chain allows for either separation or fusion of the two cavities. Inhibition of the enzyme with N-(2-aminoethyl)-p-chlorobenzamide results in the formation of a covalent N(5) flavin adduct with the phenyl ring of the inhibitor occupying a position in the catalytic site overlapping that of isatin. Inhibition of MAO-B with the clinically used trans-2-phenylcyclopropylamine results in the formation of a covalent C(4a) flavin adduct with an opened cyclopropyl ring and the phenyl ring in a parallel orientation to the flavin. The peptide bond between the flavin-substituted Cys-397 and Tyr-398 is in a cis conformation, which allows the proper orientation of the phenolic ring of Tyr-398 in the active site. The flavin ring exists in a twisted nonplanar conformation, which is observed in the oxidized form as well as in both the N(5) and the C(4a) adducts. An immobile water molecule is H-bonded to Lys-296 and to the N(5) of the flavin as observed in other flavin-dependent amine oxidases. The active site cavities are highly apolar; however, hydrophilic areas exist near the flavin and direct the amine moiety of the substrate for binding and catalysis. Small conformational changes are observed on comparison of the different inhibitor-enzyme complexes. Future MAO-B drug design will need to consider "induced fit" contributions as an element in ligand-enzyme interactions.
371 citations
TL;DR: A phosphotriesterase with a very fast kcat (over 105 s−1), 63 times higher than the already very efficient wild‐type enzyme is selected using a novel strategy based on linking genotype and phenotype by in vitro compartmentalization using water‐in‐oil emulsions.
Abstract: We describe the selection of a phosphotriesterase with a very fast k(cat) (over 10(5) s(-1)), 63 times higher than the already very efficient wild-type enzyme. The enzyme was selected from a library of 3.4 x 10(7) mutated phosphotriesterase genes using a novel strategy based on linking genotype and phenotype by in vitro compartmentalization (IVC) using water-in-oil emulsions. First, microbeads, each displaying a single gene and multiple copies of the encoded protein, are formed by compartmentalized in vitro translation. These microbeads can then be selected for catalysis or binding. To select for catalysis the microbeads are re-emulsified in a reaction buffer of choice with a soluble substrate. The product and any unreacted substrate are coupled to the beads when the reaction is finished. Product-coated beads, displaying active enzymes and the genes that encode them, are detected with anti-product antibodies and selected using flow cytometry. This completely in vitro process selects for all enzymatic features simultaneously (substrate recognition, product formation, rate acceleration and turnover) and single enzyme molecules can be detected.
365 citations
TL;DR: Although the inhibitor did not directly coordinate to the molybdenum ion, numerous hydrogen bonds as well as hydrophobic interactions with the protein matrix were observed, most of which are also used in substrate recognition.
359 citations
TL;DR: The purpose of the research was to study the purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11, which was shown to have a relative low molecular weight and improve the cleansing power of various detergents.
Abstract: The purpose of the research was to study the purification and partial characterization of thermostable serine alkaline protease from a newly isolatedBacillus subtilis PE-11. The enzyme was purified in a 2-step procedure involving ammonium sulfate precipitation and Sephadex G-200 gel permeation chromatography. The enzyme was shown to have a relative low molecular weight of 15 kd by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and was purified 21-fold with a yield of 7.5%. It was most active at 60°C, pH 10, with casein as substrate. It was stable between pH 8 and 10. This enzyme was almost 100% stable at 60°C even after 350 minutes of incubation. It was strongly activated by metal ions such as Ca2+, Mg+2, and Mn+2. Enzyme activity was inhibited strongly by phenylmethyl sulphonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP) but was not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with iodoacetate,p-chloromercuric benzoate (pCMB), and β-mercaptoethanol (β-ME). The compatibility of the enzyme was studied with commercial and local detergents in the presence of 10mM CaCl2 and 1M glycine. The addition of 10mM CaCl2 and 1M glycine, individually and in combination, was found to be very effective in improving the enzyme stability where it retained 52% activity even after 3 hours. This enzyme improved the cleansing power of various detergents. It removed blood stains completely when used with detergents in the presence of 10mM CaCl2 and 1M glycine.
290 citations
TL;DR: This approach provides a new means for rapid determination of enzyme kinetics in microfluidic systems, which may be useful for clinical diagnostics, and drug discovery and screening.
Abstract: This paper describes a microanalytical method for determining enzyme kinetics using a continuous-flow microfluidic system. The analysis is carried out by immobilizing the enzyme on microbeads, packing the microbeads into a chip-based microreactor (volume ∼1.0 nL), and flowing the substrate over the packed bed. Data were analyzed using the Lilly−Hornby equation and compared to values obtained from conventional measurements based on the Michaelis−Menten equation. The two different enzyme-catalyzed reactions studied were chosen so that the substrate would be nonfluorescent and the product fluorescent. The first reaction involved the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and N-acetyl-3,7-dihydroxyphenoxazine (amplex red) to yield fluorescent resorufin, and the second the β-galactosidase-catalyzed reaction of nonfluorescent resorufin-β-d-galactopyranoside to yield d-galactose and fluorescent resorufin. In both cases. the microfluidics-based method yielded the same result obtained ...
220 citations
TL;DR: It is demonstrated that AZ1 acts on ODC to enhance the association of ODC with the proteasome, not the rate of its processing.
Abstract: Ornithine decarboxylase (ODC) is regulated by its metabolic products through a feedback loop that employs a second protein, antizyme 1 (AZ1). AZ1 accelerates the degradation of ODC by the proteasome. We used purified components to study the structural elements required for proteasomal recognition of this ubiquitin-independent substrate. Our results demonstrate that AZ1 acts on ODC to enhance the association of ODC with the proteasome, not the rate of its processing. Substrate-linked or free polyubiquitin chains compete for AZ1-stimulated degradation of ODC. ODC–AZ1 is therefore recognized by the same element(s) in the proteasome that mediate recognition of polyubiquitin chains. The 37 C-terminal amino acids of ODC harbor an AZ1-modulated recognition determinant. Within the ODC C terminus, three subsites are functionally distinguishable. The five terminal amino acids (ARINV, residues 457–461) collaborate with residue C441 to constitute one recognition element, and AZ1 collaborates with additional constituents of the ODC C terminus to generate a second recognition element.
212 citations
TL;DR: In this paper, a simple surface modification technique was developed to functionalize polymeric and cellulosic materials with bactericidal polycationic groups, such as pyridinium groups.
Abstract: A simple surface modification technique was developed to functionalize polymeric and cellulosic materials with bactericidal polycationic groups. The poly(ethylene terephthalate) (PET) film was first graft copolymerized with 4-vinylpyridine (4VP) and subsequently derivatized with hexyl bromide via the quaternization of the grafted pyridine groups into pyridinium groups. The amount of pyridinium groups on the film surface could be controlled by varying the 4VP monomer concentrations used for grafting. The pyridinium groups introduced on the surface of the substrate possess antibacterial properties, as shown by their effect on Escherichia coli (E. coli). The bacteria killing efficiency is very high when the concentration of pyridinium groups on surfaces is 15 nmol/cm2 or higher. E. coli adhered on the functionalized surfaces are no longer viable when released into an aqueous culture medium. Filter paper, as a typical cellulosic material, was also functionalized in the same manner to introduce the pyridinium ...
195 citations
TL;DR: A new DNAzyme-based biosensor for Pb(2+) was designed by introducing both inter- and intramolecular quenchers, and the background fluorescence was significantly suppressed, with 660% increase of fluorescence intensity as compared to 60% increase for the previous design.
Abstract: A previously reported DNAzyme-based biosensor for Pb(2+) has shown high sensitivity and selectivity at 4 degrees C. In the system, the substrate and the enzyme strand of the DNAzyme are labeled with a fluorophore and a quencher, respectively. In the presence of Pb(2+), the substrate strand is cleaved by the enzyme strand, and the release of the cleaved fragment results in significant fluorescence increase. However, the performance of the sensor decreases considerably if the temperature is raised to room temperature because of high background fluorescence. A careful analysis of the sensor system, including measurement of the melting curve and fluorescence resonance energy-transfer (FRET) study of the free substrate, suggests that a fraction of the fluorophore-labeled substrate strand is dissociated from the enzyme strand, resulting in elevated background fluorescence signals at room temperature. To overcome this problem, we designed a new sensor system by introducing both inter- and intramolecular quenchers. The design was aided by the FRET study that showed the dissociated substrate maintained a random coil conformation with an end-to-end distance of approximately 39 A, which is much shorter than that of the fully extended DNA. With this new design, the background fluorescence was significantly suppressed, with 660% increase of fluorescence intensity as compared to 60% increase for the previous design. This suppression of background fluorescence signals was achieved without losing selectivity of the sensor. The new design makes it possible to use the sensor for practical applications in a wide temperature range. The design principle presented here should be applicable to other nucleic acid-based biosensors to decrease background fluorescence.
Patent•
25 Jul 2003
TL;DR: In this article, an atomic layer deposition system is described that includes a deposition chamber, and a transport mechanism transports a substrate in a path through the first reaction chamber and through the second reaction chamber, thereby depositing a film on the substrate by atomic-layer deposition.
Abstract: An atomic layer deposition system is described that includes a deposition chamber. A first and second reaction chamber are positioned in the deposition chamber and contain a first and a second reactant species, respectively. A monolayer of the first reactant species is deposited on a substrate passing through the first reaction chamber. A monolayer of the second reactant species is deposited on a substrate passing through the second reaction chamber. A transport mechanism transports a substrate in a path through the first reaction chamber and through the second reaction chamber, thereby depositing a film on the substrate by atomic layer deposition. The shape of the first and the second reaction chambers are chosen to achieve a constant exposure of the substrate to reactant species when the transport mechanism transports the substrate in the path through the respective reaction chambers at the constant transport rate.
TL;DR: The structure of the complex of ascorbate peroxidase–ascorbate defines the ascorBate-binding interaction for the first time and provides new rationalization of the unusual functional features of the related cytochrome c peroxIDase enzyme, which has been a benchmark for per oxidase catalysis for more than 20 years.
Abstract: Heme peroxidases catalyze the H2O2-dependent oxidation of a variety of substrates, most of which are organic. Mechanistically, these enzymes are well characterized: they share a common catalytic cycle that involves formation of a two-electron, oxidized Compound I intermediate followed by two single-electron reduction steps by substrate. The substrate specificity is more diverse — most peroxidases oxidize small organic substrates, but there are prominent exceptions — and there is a notable absence of structural information for a representative peroxidase–substrate complex. Thus, the features that control substrate specificity remain undefined. We present the structure of the complex of ascorbate peroxidase–ascorbate. The structure defines the ascorbate-binding interaction for the first time and provides new rationalization of the unusual functional features of the related cytochrome c peroxidase enzyme, which has been a benchmark for peroxidase catalysis for more than 20 years. A new mechanism for electron transfer is proposed that challenges existing views of substrate oxidation in other peroxidases.
TL;DR: The difference between polystyrene and enzyme diffusivity suggests that protein-matrix and protein-protein interactions govern the distribution of the enzyme within the macroporous resin.
TL;DR: The structure exhibits conformational changes indicative of an adaptive fit to the substrate reflecting both the hydration and size of the substrate, indicating how structurally diverse substrates are recognized by drug-metabolizing P450 enzymes.
Abstract: The structure of the anti-inflammatory drug diclofenac bound in the active site of rabbit microsomal cytochrome P450 2C5/3LVdH was determined by X-ray crystallography to 2.1 A resolution. P450 2C5/3LVdH and the related enzyme 2C5dH catalyze the 4‘-hydroxylation of diclofenac with apparent Km values of 80 and 57 μM and kcat values of 13 and 16 min-1, respectively. Spectrally determined binding constants are similar to the Km values. The structure indicates that the π-electron system of the dichlorophenyl moiety faces the heme Fe with the 3‘- and 4‘-carbons located 4.4 and 4.7 A, respectively, from the Fe. The carboxyl moiety of the substrate is hydrogen bonded to a cluster of waters that are also hydrogen bonded to the side chains of N204, K241, S289, and D290 as well as the backbone of the protein. The proximity of the diclofenac carboxylate to the side chain of D290 together with an increased binding affinity at lower pH suggests that diclofenac is protonated when bound to the enzyme. The structure exhib...
TL;DR: In the course of searching for BACE1 (beta-secretase) inhibitors from natural products, the ethyl acetate soluble fraction of green tea, which was suspected to be rich in catechin content, showed potent inhibitory activity.
TL;DR: An alternative hypothesis for the development of filamentous structures in biological flocs has been formulated and it is hypothesized that bulking sludge originates from the presence of substrate gradients in sludge aggregates.
TL;DR: It is concluded that there is no accumulation of an activated form of O2 before C-H abstraction in the DbetaM and peptidylglycine-alpha-hydroxylating monooxygenase class of copper Monooxygenases, presenting a mechanism in which a diamagnetic Cu(II)-superoxo complex, formed initially at very low levels, abstracts a hydrogen atom from substrate to generate Cu( II)-hydroperoxo and substrate-free radical as
Patent•
18 Jun 2003
TL;DR: Ion-induced, UV-induced and electron-induced sequential chemical vapor deposition (CVD) processes are disclosed where an ion flux, a flux of ultra-violet radiation, or an electron flux, respectively, is used to induce the chemical reaction in the process as discussed by the authors.
Abstract: Ion-induced, UV-induced, and electron-induced sequential chemical vapor deposition (CVD) processes are disclosed where an ion flux, a flux of ultra-violet radiation, or an electron flux, respectively, is used to induce the chemical reaction in the process. The process for depositing a thin film on a substrate includes introducing a flow of a first reactant gas in vapor phase into a process chamber where the gas forms an adsorbed saturated layer on the substrate and exposing the substrate to a flux of ions, a flux of ultra-violet radiation, or a flux of electrons for inducing a chemical reaction of the adsorbed layer of the first reactant gas to form the thin film. A second reactant gas can be used to form a compound thin film. The ion-induced, UV-induced, and electron-induced sequential CVD process of the present invention can be repeated to form a thin film of the desired thickness.
Patent•
23 Jun 2003
TL;DR: In this paper, a method for energy-assisted atomic layer deposition and removal of a dielectric film is presented, where a substrate is placed into a reaction chamber and a gaseous precursor is introduced into the reaction chamber.
Abstract: A method for energy-assisted atomic layer deposition and removal of a dielectric film are provided. In one embodiment a substrate (14) is placed into a reaction chamber (10) and a gaseous precursor is introduced into the reaction chamber (10). Energy is provide by a pulse of electromagnetic radiation which forms radical species of the gaseous precursor. The radical species react with the surface of the substrate (14) to form a radical terminated surface on the substrate (14). The reaction chamber (10) is purged and a second gaseous precursor is introduced. A second electromagnetic radiation pulse is initiated and forms second radical species. The second radical species of the second gas react with the surface to form a film on the substrate (14). Alternately, the gaseous species can be chosen to produce radicals that result in the removal of material from the surface of the substrate (14).
TL;DR: In addition to analysis of nucleic acids and enzymes, immunoassays are the third group of analytical assays performed in microfluidic chips and utilize either affinity capillary electrophoresis as a homogeneous assay format, or immobilized antigens or antibodies in heterogeneous assays with serial supply of reagents and washing solutions.
Abstract: Microfluidic systems are capillary networks of varying complexity fabricated originally in silicon, but nowadays in glass and polymeric substrates. Flow of liquid is mainly controlled by use of electroosmotic effects, i.e. application of electric fields, in addition to pressurized flow, i.e. application of pressure or vacuum. Because electroosmotic flow rates depend on the charge densities on the walls of capillaries, they are influenced by substrate material, fabrication processes, surface pretreatment procedures, and buffer additives. Microfluidic systems combine the properties of capillary electrophoretic systems and flow-through analytical systems, and thus biochemical analytical assays have been developed utilizing and integrating both aspects. Proteins, peptides, and nucleic acids can be separated because of their different electrophoretic mobility; detection is achieved with fluorescence detectors. For protein analysis, in particular, interfaces between microfluidic chips and mass spectrometers were developed. Further levels of integration of required sample-treatment steps were achieved by integration of protein digestion by immobilized trypsin and amplification of nucleic acids by the polymerase chain reaction. Kinetic constants of enzyme reactions were determined by adjusting different degrees of dilution of enzyme substrates or inhibitors within a single chip utilizing mainly the properties of controlled dosing and mixing liquids within a chip. For analysis of kinase reactions, however, a combination of a reaction step (enzyme with substrate and inhibitor) and a separation step (enzyme substrate and reaction product) was required. Microfluidic chips also enable separation of analytes from sample matrix constituents, which can interfere with quantitative determination, if they have different electrophoretic mobilities. In addition to analysis of nucleic acids and enzymes, immunoassays are the third group of analytical assays performed in microfluidic chips. They utilize either affinity capillary electrophoresis as a homogeneous assay format, or immobilized antigens or antibodies in heterogeneous assays with serial supply of reagents and washing solutions.
TL;DR: The crystal structure of SULT1A1 provides the molecular basis for substrate inhibition and reveals the first clues as to how the enzyme sulfonates a wide variety of lipophilic compounds.
TL;DR: A wide range of fatty acid esters can be synthesised by esterification and transesterification reactions catalysed by lipases in non-aqueous systems, as lipases have been shown to have broad substrate specificities and can catalyse conversions with many other non-glycerol based compounds.
Abstract: A wide range of fatty acid esters can be synthesised by esterification and transesterification reactions catalysed by lipases in non-aqueous systems. As well as modifications to the natural substrate triacylglycerol, many other commercially important substances can be produced using this enzyme technology, as lipases have been shown to have broad substrate specificities and can thus catalyse conversions with many other non-glycerol based compounds. In mild reaction conditions, emollient esters for use in the personal care industry can be made with excellent quality. Development of economical processes was necessary to commercialise this technology for the manufacture of cosmetic ingredients. Enzymatic production methods had to be established which could compete against standard established chemical synthetic methods. Now relatively simple esters are being produced enzymatically. Extensive purification procedures to clean up products after high temperature reaction steps are not required. Product quality and specifications, such as the acid and hydroxy values of commercial ester products, could be improved. Examples of products manufactured with better purity and yields in economical and ecologically friendly processes are decyl oleate, cetyl ricinoleate, myristyl myristate and decyl cocoate.
TL;DR: The crystal structures have revealed a closed form in which a TDP ligand is bound at a donor substrate site in the interdomain cleft, thereby illustrating not only binding interactions, but the conformational changes in the enzyme that accompany substrate binding.
Abstract: During the biosynthesis of the vancomycin-class antibiotic chloroeremomycin, TDP-epi-vancosaminyltransferase GtfA catalyzes the attachment of 4-epi-vancosamine from a TDP donor to the beta-OHTyr-6 of the aglycone cosubstrate. Glycosyltransferases from this pathway are potential tools for the combinatorial design of new antibiotics that are effective against vancomycin-resistant bacterial strains. These enzymes are members of the GT-B glycosyltransferase superfamily, which share a homologous bidomain topology. We present the 2.8-A crystal structures of GtfA complexes with vancomycin and the natural monoglycosylated peptide substrate, representing the first direct observation of acceptor substrate binding among closely related glycosyltransferases. The acceptor substrates bind to the N-terminal domain such that the aglycone substrate's reactive hydroxyl group hydrogen bonds to the side chains of Ser-10 and Asp-13, thus identifying these as residues of potential catalytic importance. As well as an open form of the enzyme, the crystal structures have revealed a closed form in which a TDP ligand is bound at a donor substrate site in the interdomain cleft, thereby illustrating not only binding interactions, but the conformational changes in the enzyme that accompany substrate binding.
TL;DR: In this article, a wild strain of Penicillium sp in solid-state fermentation (SSF) was used as carbon and nitrogen source and solid matrix for synthesizing a serine protease.
TL;DR: Several examples illustrating these concepts in a variety of enzyme classes have appeared recently, including altering the reaction mechanism of the enzyme to catalyse new reactions, switching substrate specificity, expanding substrate Specificity, and improving substrate specificity.
TL;DR: The presented structure of apo-RADH provides plausible explanations for the metal dependence of the enzyme.
TL;DR: Here substrate permeation through the membrane and subsequent chemical transformation at the membrane surface are realized and these methods are expected to lead to development of complicated and sophisticated chemical systems involving membrane permeation and chemical reactions.
Abstract: Here we report a design and synthesis of a chemically functional polymer membrane by an interfacial polycondensation reaction and multilayer flow inside a microchannel. Single and parallel dual-membrane structures are successfully prepared by using organic/aqueous two-layer flow and organic/aqueous/organic three-layer flow inside the microchannel followed by an interfacial polycondensation reaction. By using the inner-channel membrane, permeation of ammonia species through the inner-channel membrane is successfully achieved. Furthermore, horseradish peroxidase is immobilized on one side of the membrane surface to integrate the chemical transform function onto the inner-channel membrane. Here substrate permeation through the membrane and subsequent chemical transformation at the membrane surface are realized. The polymer membrane prepared inside the microchannel has an important role in ensuring stable contact of different phases such as gas/liquid or liquid/liquid and the permeation of chemical species th...
TL;DR: Immunosensor using surface plasmon resonance (SPR) onto self-assembled protein G layer was developed for the detection of Legionella pneumophila and its detection limit could find up to 10(5) cells/ml.
TL;DR: An assay strategy for determining the substrate for denitrase was developed combining 2D-gel electrophoresis and an on-blot enzyme assay and the resulting substrate was Histone H1.2, an isoform protein of linker histone, which has only one tyrosine residue in the entire molecule.
Abstract: Several reports have described an activity that modifies nitrotyrosine-containing proteins and their immunoreactivity to nitrotyrosine Abs. Without knowing the product of the reaction, this new activity has been called a “denitrase.” In those studies, some nonspecific proteins, which have multiple tyrosine residues, e.g., albumin, were used as a substrate. Therefore, the studies were based on an unknown mechanism of reaction and potentially a high background. To solve these problems, one of the most important things is to find a more suitable substrate for assay of the enzyme. We developed an assay strategy for determining the substrate for denitrase combining 2D-gel electrophoresis and an on-blot enzyme assay. The resulting substrate from RAW 264.7 cells was Histone H1.2, an isoform protein of linker histone. Histone H1.2 has only one tyrosine residue in the entire molecule, which ensures the exact position of the substrate to be involved. It has been reported that Histones are the most prominent nitrated proteins in cancer tissues. It was also demonstrated that tyrosine nitration of Histone H1 occurs in vivo. These findings lead us to the idea that Histone H1.2 might be an intrinsic substrate for denitrase. We nitrated recombinant and purified Histone H1.2 chemically and subjected it to an on-blot enzyme assay to characterize the activity. Denitrase activity behaved as an enzymatic activity because the reaction was time dependent and was destroyed by heat or trypsin treatment. The activity was shown to be specific for Histone H1.2, to differ from proteasome activity, and to require no additional cofactors.