Peroxide Substrate Research Articles

A facile preparation of FePt-loaded few-layer MoS2 nanosheets nanocomposites (F-MoS2-FePt NCs) and their application for colorimetric detection of H2O2 in living cells

BackgroundRapid and sensitive detection of H2O2 especially endogenous H2O2 is of great importance for series of industries including disease diagnosis and therapy. In this work, uniform FePt nanoparticles are successfully anchored onto Few-layer molybdenum disulfide nanosheets (F-MoS2 NSs). The powder X-ray diffraction, transmission electron microscopy, UV–Vis spectra and atomic force microscopy were employed to confirm the structure of the obtained nanocomposites (F-MoS2-FePt NCs). The prepared nanocomposites show efficient peroxidase-like catalytic activities verified by catalyzing the peroxidation substrate 4,4′-diamino-3,3′,5,5′-tetramethylbiphenyl (TMB) with the existence of H2O2.ResultsThe optimal conditions of the constructed colorimetric sensing platform is proved as 35 °C and pH 4.2. Under optimal catalytic conditions, the detection limit for H2O2 detection reaches 2.24 μM and the linear ranger is 8 μM to 300 μM. Furthermore, the proposed colorimetric sensing platform was successfully utilized to detect the intracellular H2O2 of cancer cells (MCF-7).ConclusionsThese findings indicated that the F-MoS2-FePt-TMB-H2O2 system provides a potential sensing platform for hydrogen peroxide monitoring in living cells.

Radioprotective Role of Peroxiredoxin 6.

Peroxiredoxin 6 (Prdx6) is a member of an evolutionary ancient family of peroxidase enzymes with diverse functions in the cell. Prdx6 is an important enzymatic antioxidant. It reduces a wide range of peroxide substrates in the cell, thus playing a leading role in the maintenance of the redox homeostasis in mammalian cells. Beside peroxidase activity, Prdx6 has been shown to possess an activity of phospholipase A2, an enzyme playing an important role in membrane phospholipid metabolism. Moreover, Prdx6 takes part in intercellular and intracellular signal transduction due to its peroxidase and phospholipase activity, thus facilitating the initiation of regenerative processes in the cell, suppression of apoptosis, and activation of cell proliferation. Being an effective and important antioxidant enzyme, Prdx6 plays an essential role in neutralizing oxidative stress caused by various factors, including action of ionizing radiation. Endogenous Prdx6 has been shown to possess a significant radioprotective potential in cellular and animal models. Moreover, intravenous infusion of recombinant Prdx6 to animals before irradiation at lethal or sublethal doses has shown its high radioprotective effect. Exogenous Prdx6 effectively alleviates the severeness of radiation lesions, providing normalization of the functional state of radiosensitive organs and tissues, and leads to a significant elevation of the survival rate of animals. Prdx6 can be considered as a potent and promising radioprotective agent for reducing the pathological effect of ionizing radiation on mammalian organisms. The radioprotective properties and mechanisms of radioprotective action of Prdx6 are discussed in the current review.

Nonequilibrium Spatiotemporal Sensing within Acoustically Patterned Two-Dimensional Protocell Arrays.

Acoustically trapped periodic arrays of horseradish peroxidase (HRP)-loaded poly(diallydimethylammonium chloride) / adenosine 5′-triphosphate coacervate microdroplet-based protocells exhibit a spatiotemporal biochemical response when exposed to a codiffusing mixture of substrate molecules (o-phenylenediamine (o-PD) and hydrogen peroxide (H2O2)) under nonequilibrium conditions. Unidirectional propagation of the chemical concentration gradients gives rise to time- and position-dependent fluorescence signal outputs from individual coacervate microdroplets, indicating that the organized protocell assembly can dynamically sense encoded information in the advancing reaction-diffusion front. The methodology is extended to arrays comprising spatially separated binary populations of HRP- or glucose oxidase-containing coacervate microdroplets to internally generate a H2O2 signal that chemically connects the two protocell communities via a concerted biochemical cascade reaction. Our results provide a step toward establishing a systematic approach to study dynamic interactions between organized protocell consortia and propagating reaction-diffusion gradients, and offer a new methodology for exploring the complexity of protocellular communication networks operating under nonequilibrium conditions.

Structural properties of the peroxiredoxin AhpC2 from the hyperthermophilic eubacterium Aquifex aeolicus

Peroxiredoxins (Prxs) are thiol peroxidases that scavenge various peroxide substrates such as hydrogen peroxide (H2O2), alkyl hydroperoxides and peroxinitrite. They also function as chaperones and are involved in signal transduction by H2O2 in eukaryotic cells. The genome of Aquifex aeolicus, a microaerophilic, hyperthermophilic eubacterium, encodes four Prxs, among them an alkyl hydroperoxide reductase AhpC2 which was found to be closely related to archaeal 1-Cys peroxiredoxins. We determined the crystal structure of AhpC2 at 1.8 Å resolution and investigated its oligomeric state in solution by electron microscopy. AhpC2 is arranged as a toroid-shaped dodecamer instead of the typically observed decamer. The basic folding topology and the active site structure are conserved and possess a high structural similarity to other 1-Cys Prxs. However, the C-terminal region adopts an opposite orientation. AhpC2 contains three cysteines, Cys49, Cys212, and Cys218. The peroxidatic cysteine CP49 was found to be hyperoxidized to the sulfonic acid (SO3H) form, while Cys212 forms an intra-monomer disulfide bond with Cys218. Mutagenesis experiments indicate that Cys212 and Cys218 play important roles in the oligomerization of AhpC2. Based on these structural characteristics, we proposed the catalytic mechanism of AhpC2. This study provides novel insights into the structure and reaction mechanism of 1-Cys peroxiredoxins.

Prognostic assessment of cryosensitivity level for umbilical cord blood hemopoietic tissue with the help of markers of prooxidant activity processes

Aim. To investigate the connection between the levels of human umbilical cord blood hemopoietic tissue (UCB HT) cryosensitivity (using of the granulocyte-macrophage progenitors (GMPs) percentage loss) and activity of prooxidant processes in whole blood before the beginning of its cryopreservation.Methods of research. Cryopreservation of UCB nuclear cell fraction was carried out by slow freezing under protection of the dimethyl sulfoxide in 5% final concentration (v/v). The loss of GMPs was determined by the difference in the total content of colonies and clusters prior to cryopreservation and after thawing the sample in a short-term tissue culture. The activity of prooxidant processes in UCB was studied by biochemical markers of lipid peroxidation products (LPO) using a spectrophotometric method for determining the concentrations of such products as substrates for lipid peroxidation (isolated double bonds - IDB), intermediate (conjugated dienes (CD), conjugated trienes (CT), conjugated oxodienes (COD)) and final LPO products such as Schiff bases (ShB) for neutral lipids and phospholipids. Data analysis performed on models of analytical groups, regression analysis, mutual conjugation.Results. It has been demonstrated that the level of UCB HT cryosensitivity (due to loss of GMPs) has direct correlation with LPO activity indices (from medium to high significance level). The relative risk (RR) of loss of GMPs is significantly increased under the condition of high levels of LPO phospholipid peroxidation in UCB before beginning cryopreservation. In particular, in the case of phospholipid peroxidation, it is: for IDB - RR = 5.29; 95% CI: 2.69-10.38; p <0.001; CD - RR = 5.73; 95% CI: 2.88-11.40; p <0.001; CT and COD - RR = 2.81; 95% CI: 1.72-4.60; p <0.001; ShB - RR = 1, 92, 95% CI: 1.16-3.18, p <0.01, respectively.Conclusions. Evaluation of the activity level of prooxidant processes in UCB using the biochemical markers before the start of the freezing procedure is valuable because of the possibility of creating an early prediction of the HT cryosensitivity, which can be useful in choosing cryopreservation tactics

P-276 - Vitamin E metabolism and its putative role as fat-soluble antioxidant in the redox-lipidomics era

Lipidomics is going to leave its role of “Cinderella” of omics technologies. Innovative mass spectrometry and bioinformatics technology is bursting into the omics scene and this is leading lipid science to gain renewed attention and interest by the scientific community. New lipidomics challenges have already launched including that of extensive (high-throughput) exploration of oxidized lipids in biological matrices (that is “redox lipidomics”). Alpha-tocopherol, the main form of vitamin E in human tissues, is a H donor with exclusive pertinence to lipid peroxide substrates of cellular membranes and lipoprotein particles. The direct or enzyme-mediated interaction of this vitamin with lipid oxidation pathways generates specific metabolites and functional responses. This area of redox lipidomics is now gaining increasing interest for its role in PUFA oxidation and lipid signaling, and more in general in long-chain lipid metabolism under both physiological and pathological conditions. Targeted and untargeted lipidomics protocols of investigation are now available to characterize the functional lipidome of vitamin E that is discussed in this paper along with other emerging aspects of redox lipidomics.

Selenoprotein L-inspired nano-vesicular peroxidase mimics based on amphiphilic diselenides

In this study, we developed selenoprotein L-inspired nano-vesicular peroxidase mimics based on amphiphilic diselenides. Selenocystine (SeCyst) was used as the starting material for the synthesis of four liposomal membrane-compatible diselenide derivatives (R−Se−Se−R’) with two hydrophobic tails and a polar part. The diselenide derivatives were successfully incorporated into the phosphatidylcholine (PC)-based nano-vesicular scaffold. The results of the particle diameter and zeta-potential measurements suggested that the functional diselenide moiety was placed around the outer surface, not in the hydrophobic interior, of the liposomal membrane structures. The GPx-like catalytic activity of the diselenide/PC liposomes was determined by the conventional NADPH method using glutathione as the reducing substrate. For three peroxide substrates, i.e., hydrogen peroxide, organic tert-butyl hydroperoxide and cummen hydroperoxide, the cationic property-possessing diselenide derivatives in the PC-based liposomes resulted in a higher catalytic activity in comparison to electrically neutral and anionic derivatives. Overall, the diselenide derivatives at the surface of a liposomal colloidal scaffold could exert a GPx-like catalytic activity in physiological aqueous media.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway.

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFβ-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

A continuous single organic phase process for the lipase catalyzed synthesis of peroxy acids increases productivity.

The design of an optimal process is particularly crucial when the reactants deactivate the biocatalyst. The reaction cascades of the chemo-enzymatic epoxidation where the intermediate peroxy acid is produced by an enzyme are still limited by enzyme inhibition and deactivation by hydrogen peroxide. To avoid additional effects caused by interfaces (aq/org) and to reduce the process limiting deactivation by the substrate hydrogen peroxide, a single-phase concept was applied in a fed-batch and a continuous process (stirred tank), without the commonly applied addition of a carrier solvent. The synthesis of peroxyoctanoic acid catalyzed by Candida antarctica lipase B was chosen as the model reaction. Here, the feasibility of this biocatalytic reaction in a single-phase system was shown for the first time. The work shows the economic superiority of the continuous process compared to the fed-batch process. Employing the fed-batch process reaction rates up to 36 mmolh-1 per gramcat, and a maximum yield of 96% was achieved, but activity dropped quickly. In contrast, continuous operation can maintain long-term enzyme activity. For the first time, the continuous enzymatic reaction could be performed for 55h without any loss of activity and with a space-time yield of 154mmolL-1h-1, which is three times higher than in the fed-batch process. The higher catalytic productivity compared to the fed-batch process (34 vs. 18 gProdg-1 cat) shows the increased enzyme stability in the continuous process.

Reusable, 3D-printed, peroxidase mimic–incorporating multi-well plate for high-throughput glucose determination

Three-dimensional (3D) printing technologies enable rapid laboratory-scale customization of experimental devices to fit the user’s requirements. To demonstrate how 3D-printed objects can be functionalized through incorporation of reactive substances into the raw materials prior to printing, this paper describes a multi-well plate fabricated using polylactic acid (PLA) filaments that had been treated in advance with iron oxide (Fe2O3, Fe3O4) nanoparticles that possess intrinsic peroxidase activities. The fabricated multi-well plate efficiently catalyzed the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by the peroxidase substrate hydrogen peroxide and allowed measurements of the resulting absorbance through direct loading of the plate into a plate reader. The applicability of this 3D-printed peroxidase mimic–incorporating multi-well plate has been examined in terms of the device’s reusability and analytical performance, analyses of reaction kinetics, and the high-throughput determination of glucose concentrations in clinical and food samples. The results suggest that this functionalization scheme can diversify the utility of current 3D printing technologies in the fabrication of experimental devices with properties comparable with or superior to those of conventional systems.

Amorphophallus paeoniifolius corm: A potential source of peroxidase for wide applications

ABSTRACTPeroxidases have wide applications in different areas including chemical synthesis, medicine, food industry, bioremediation of wastewater, biosensing, and biotechnology. Amorphophallus paeoniifolius (elephant foot yam) is an attractive source of enzymes; however, no effort has been made to assess peroxidase enzyme from this source. Therefore, the objectives of this study were to evaluate and compare peroxidases from corms of elephant foot yam and roots of Dacus carota (carrot) and Aramoracia rusticana (horseradish). Elephant foot yam corm peroxidase (ECP) demonstrated 4.5 times higher specific activity compared with carrot root peroxidase (CRP). ECP showed retention of high activity over a broad pH range and had higher temperature optima and thermal stability compared with CRP and horseradish peroxidase (HRP). The calculated KM values of ECP, CRP, and HRP for the substrates guaiacol and hydrogen peroxide were 4.5 mM and 307.8 µM, 4.6 mM and 55.5 µM, and 3.5 mM and 413.0 µM, respectively. Peroxidases are used for the bioremediation of wastewater contaminated with hazardous aromatic compounds. Heavy metals such as cadmium (Cd2+), lead (Pb2+), and toxic compounds such as sodium azide (NaN3) are often present in wastewater along with aromatic compounds. Results showed activation of ECP and inhibition of HRP at 250 and 500 µM concentrations of Cd2+ and Pb2+. Treatment with 1 mM NaN3 led to 8.4%, 55.5%, and 11.0% inhibition in the activities of ECP, CRP, and HRP, respectively. Overall, our results suggest that elephant foot yam can be used as a rich and convenient source of peroxidase for various applications including wastewater remediation.

Condition of the prooxidant-antioxidant system of some strains of Basidiomycetes

The article deals with the calculation and comparison indications of the condition of the prooxidant-antioxidant system (PAS) of strains of Basidiomycetes under periodic surface cultivation on a glucose-peptone medium. The research material consisted of the mycelium and culture filtrate (CF) from 57 strains, 52 of them belonging to 7 species of the order Agaricales and 5 belonging to 5 species of the order Polyporales. The intensity of the processes of lipid peroxidation was determined by a modified spectrophotometric method for contents of active products to thiobarbituric acid. Total antioxidant activity (АОА) of the mycological material was evaluated by intensity of inhibition from accumulated products of lipid peroxide oxidation (LPO) in a model reaction of oxidation by Twin-80 oxygen of the air. From the data obtained, indicators of prooxidant activity (POA), indicators of reserve of substrate peroxidation (SPO) and the balance coefficient of the prooxidant-antioxidant system (CbPАS) were calculated. It was established that strains of Basidiomycetes are characterized by significant predominance of prooxidant activity characteristic of PAS in the culture filtrate in comparison with the mycelium indicator. The highest values of POA in the Culture Filtrate were observed on the 12-th day of cultivation for the strain Р-089 genus Pleurotus and strain Gl-2 genus Ganoderma, and for the mycelium on the 9-th day of cultivation for the strains Р-сіtr, Р-089, Р-er and Р-082 of the genus Pleurotus. There is a direct dependence between the indicators of POA in the CF and mycelium for each strain, this dependence and level of indication do not reflect their systematic placement. We distinguished a more significant prevalence of indicators of reserve of substrates peroxidation of mycelium for most strains, than for such indicators with CF The highest value of reserve SPO of mycelium was recorded for strains Р-447, Р-998, Р-039, Р-94, Р-2175, Р-сіtr, Р-er, D-140 of the genus Pleurotus and strains F-107, F-610 of the genus Flammulina. For the majority of the strains there was a clear prevalence of the values of the balance coefficients for PAS of mycelium over those of CF. Maximum values of the balance coefficient PAS of mycelium on the 9-th day were recorded for strains Т-10 (F. fomentarius) and D-140, Р-035, Р-01, Р-105 (P. ostreatus) and on the 12-th day for strains Sc-10 (S. commune), Р-kl, Р-14 (P. ostreatus), Р-er (P. eryngii) і 960 (A. cylindracea). The abovementioned strains are distinguished by the with high level of AOA in the mycelium on certain days of cultivation and can be recommended as producers of antioxidants of fungal origin.

Catalytic activity of catalase-silica nanoparticle hybrids: from ensemble to individual entity activity.

We demonstrate the electrochemical detection and characterization of individual nanoparticle-enzyme hybrids. Silica nanoparticles were functionalized with catalase enzyme and investigated spectroscopically and electrochemically. The catalytic activity of the hybrids towards hydrogen peroxide decomposition was comparable to the activity of a freely diffusing enzyme in solution, exhibiting a Michaelis-Menten constant of KM = 74 mM and a turnover number of kcat = 8 × 107 s-1 per NP. The fast turnover number of the hybrid further enabled the electrochemical detection of individual nanoparticle-enzyme hybrid via a novel method: the hydrogen peroxide substrate was generated at a microelectrode which enabled enzymatic activity exclusively within the diffusion layer of the electrode. The method is the first electrochemical approach for measuring hybrid nanoparticles, at the single entity level.

The Peroxiredoxin Family: An Unfolding Story.

Peroxiredoxins (Prxs) are a large and conserved family of peroxidases that are considered to be the primary cellular guardians against oxidative stress in all living organisms. Prxs share a thioredoxin fold and contain a highly-reactive peroxidatic cysteine in a specialised active-site environment that is able to reduce their peroxide substrates. The minimal functional unit for Prxs are either monomers or dimers, but many dimers assemble into decameric rings. Ring structures can further form a variety of high molecular weight complexes. Many eukaryotic Prxs contain a conserved GGLG and C-terminal YF motif that confer sensitivity to elevated levels of peroxide, leading to hyperoxidation and inactivation. Inactive forms of Prxs can be re-reduced by the enzyme sulfiredoxin, in an ATP-dependent reaction. Cycles of hyperoxidation and reactivation are considered to play an integral role in a variety of H2O2-mediated cell signalling pathways in both stress and non-stress conditions. Prxs are also considered to exhibit chaperone-like properties when cells are under oxidative or thermal stress. The roles of various types of covalent modifications, e.g. acetylation and phosphorylation are also discussed. The ability of Prxs to assemble into ordered arrays such as nanotubes is currently being exploited in nanotechnology.

Facile synthesis of iridium nanoparticles with superior peroxidase-like activity for colorimetric determination of H2O2 and xanthine

A facile and efficient method was developed for the preparation of iridium nanoparticles (Ir NPs) with superior peroxidase-like activity. Under the catalytic action of the Ir NPs, the peroxidase substrate 3,3,5,5-tetramathylbenzidine (TMB) can be oxidized by H2O2 to form a blue-colored production oxTMB. Kinetic analysis indicated that the catalytic behavior was in accordance with the typical Michaelis-Menten kinetics and the catalytic reaction followed the ping-pang mechanism. Electron Spin Resonance (ESR) experiment showed that no hydroxyl radical formed in the reaction process. Peroxide substrates with different electron withdrawing ability (benzoyl peroxide, H2O2 and artemisinin) were used to study the catalytic reactions and results indicated that H2O2 can oxidize TMB with much faster rate than artemisinin and slower rate than benzoyl peroxide. These results suggest an electron transfer mechanism was involved in the system of TMB-H2O2-Ir NPs. By using TMB as the colorimetric substrate, H2O2 can be rapidly determined and the method was extended for the determination of xanthine based on its production of H2O2 in the presence of xanthine oxidase. The prepared Ir NPs exhibit good stability in wide range of pH and temperature. The Ir NPs retained at least 90% of their initial catalytic activity after stored at ambient temperature for three months. The high specificity for H2O2 and the excellent stability of the peroxidase-like Ir NPs showed the great application potential in biotechnology field.

Structural insights into the effects of charge-reversal substitutions at the surface of horseradish peroxidase.

Horseradish peroxidase (HRP), has gained significant interests in biotechnology, especially in biosensor field and diagnostic test kits. Hence, its solvent-exposed lysine residues 174, 232, and 241 have been frequently modified with the aim of improving its stability and catalytic efficiency. In this computational study, we investigated the effects of Lys-to-Glu substitutions on HRP structure to model charge-reversal manipulations at the enzyme surface. Simulation results implied that upon these substitutions, the number of stable hydrogen bonds and α-helical content of HRP are increased and the proximal Ca2+ binding pocket becomes more integrated. The results revealed that although Glu174-heme hydrogen bond is lost after mutation, formation of a new hydrogen bonding network contributes to the stability of heme-protein linkage. Together, it may be concluded that these substitutions enhance the stability of the protein moiety as well as the heme-protein non-covalent interactions. In the enzyme active site, we observed increased accessibility of peroxide binding site and heme prosthetic group to the peroxide and aromatic substrates, respectively. Results also demonstrated that the bottleneck entry of the peroxide-binding site has become wider and more flexible upon substitutions. Moreover, the hydrophobic patch functioning as a binding site or trap for reducing aromatic substrates is more extended in mutated enzyme. These observations suggest that the reactivity of the enzyme to its substrates has increased. Together, the results of this simulation study could provide possible structural clues to explain those experimental observations in which the protein stability achieved upon manipulation of charge distribution on protein surface.

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples.

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 µl makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments. Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes. Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

2’-OMe-RNA Analogues of Peroxidase-mimicking DNAzymes

In presented research we report the thorough investigation on 2’O-Me-RNA analogues of DNAzymes. DNAzyme with peroxidase-mimicking activity catalyses the reaction between hydrogen peroxide and adequate organic substrate. This system can be successfully used in development of new bioassays. The application of 2’-OMe-RNA analogues can enhance the activity of formed ribozyme as well as allows on the employment of probes in-vivo. Our results suggest that application of 2’-OMe-RNA analogues enhance the catalytic activity, even of ribozymes based on sequences characterized by low activity of DNAzyme. These results confirms that application of 2’-OMe-RNA ribozymes improve the catalytic behaviour as well as expand potential application.

Direct measurement of catalase activity in living cells and tissue biopsies

Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings.