Enhanced Peroxidase Activity Research Articles

Bioinspired design of an artificial peroxidase: introducing key residues of native peroxidases into F43Y myoglobin with a Tyr-heme cross-link.

Inspired by the structural features of native peroxidases, an artificial peroxidase was rationally designed using F43Y myoglobin with a Tyr-heme cross-link by further introduction of key residues, including both a distal Arg and a Trp close to the heme group, which exhibits an enhanced peroxidase activity similar to the most efficient native horseradish peroxidase. This study provides a simple approach for design of artificial heme enzymes by the combination of catalytic elements of native enzymes with the post-translational modifications of heme proteins.

ATP induced alteration in the peroxidase-like properties of hollow Prussian blue nanocubes: a platform for alkaline phosphatase detection.

Breaking the pH limitation of the enzyme-like activity of nanomaterials is of great importance for extending their applications in environmental and biomedical fields. Herein, to mimic the role of histidine residues in horseradish peroxidase (HRP), adenosine 5'-triphosphate (ATP) is reported to improve the peroxidase-like activity of hollow Prussian blue nanocubes (hPBNCs). Due to the inherited porous structures, hPBNCs can expose all the binding sites as far as possible to ATP to significantly amplify their catalytic activity and broaden their applicable pH range up to pH 12. Introduction of ATP provides the possibility of realizing efficient catalytic reactions under alkaline conditions. Upon binding with hPBNCs, ATP can enhance the stability of hPBNCs, increase the affinities of the catalysts towards substrates and improve the conductivity of hPBNCs as well as change the decomposed product from H2O2. Moreover, on the basis of the different catalytic activities of hPBNCs towards ATP, adenosine 5'-diphosphate and adenosine 5'-monophosphate, hPBNCs-ATP is utilized to construct a novel colorimetric sensor for the detection of alkaline phosphatase (ALP) activity in biological fluids, which is significantly important for the clinical diagnosis of ALP-related diseases.

Increasing the activity of DNAzyme based on the telomeric sequence: 2’-OMe-RNA and LNA modifications

Abstract2’-OMe-RNA analogues and LNA point modifications of DNA oligonucleotides were applied for the modulation of the G-quadruplex topology and enhancement of peroxidase activity of the resulting DNAzymes. The effect of the 2’-OMe-RNA analogue was studied for full length modified oligonucleotides with various sequences. In the case of LNA-point modification, we have chosen a telomeric DNA sequence and investigated various numbers of modifications. Our main goal was to prove that the application of these modifications can influence the activity of DNAzyme, especially those, which normally form poor DNAzymes. As an example, we have chosen the telomeric HT22 sequence which is known to form DNAzyme characterized by low activity. In all cases, the DNAzymes formed by a telomeric sequence with the application of the 2’-OMe-RNA analogue as well as LNA-point modification, showed significantly higher peroxidase activity. We were also able to shift the formation of hybrid or antiparallel topology to parallel topology. These results are important for the development of probes for biological applications as well as for the design of probes based on DNA sequences that normally form DNAzymes with low activity. This paper also provides information on how the application of nucleotide analogues can transform the topology of G-quadruplexes.

Precise engineering of ultra-thin Fe2O3 decorated Pt-based nanozymes via atomic layer deposition to switch off undesired activity for enhanced sensing performance

Unlike natural enzymes, nanozymes possess often multiple enzyme-mimic activities, which can be the impediment for their applications. Current strategies in activity engineering are based mainly on extensive cut-and-try screening and lack accurate engineering methods. It has not been reported through rational design to exaggerate the desired activity and suppress unwanted ones. In this study, inspired by catalytic theories in traditional nanocatalysis, ultra-thin Fe2O3 decorated Pt in inverse catalyst model (Fe2O3/Pt/CNTs) with high ratio of Pt0/Pt2+, new Pt-O-Fe3+ bonds and more interface sites was designed for excellent peroxidase activity and inhibited oxidase activity. Atomic layer deposition (ALD), as a novel approach for nanozyme preparation, enables the fine tailoring of surface microstructure of Fe2O3/Pt/CNTs. Activity analysis verified our design and the final Fe2O3/Pt/CNTs possess greatly enhanced peroxidase activity while the oxidase activity was eliminated. The detection limit of a colorimetric glucose sensor based on Fe2O3/Pt/CNTs was almost 10-fold lower over Pt/CNTs through the elimination of false-positive signal caused by the oxidase activity. These results demonstrated that, in virtue of the meticulous engineering method like ALD, rational design is feasible and powerful to engineer nanozymes with desired activity.

3,4:9,10-perylene tetracarboxylic acid-modified zinc ferrite with the enhanced peroxidase activity for sensing of ascorbic acid

3,4:9,10-perylene tetracarboxylic acid (PTCA) modified litchi-like zinc ferrite (ZnFe2O4) was prepared by a simple hydrothermal method and was found to possess the enhanced peroxidase-like activity. Under the catalysis of PTCA-ZnFe2O4, the colorless colorimetric substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was rapidly oxidized by H2O2 and fast turned into blue color. Comparatively, the peroxidase-like activity of PTCA-ZnFe2O4 nanocomposites is higher than that of pure ZnFe2O4. In addition, it is hydroxyl radical that plays the key role in the process of catalysis reaction, which verified by fluorescent probe. On the basis of the higher peroxidase-like activity of PTCA-ZnFe2O4, a simple colorimetric biosensor platform has been proposed for detection of ascorbic acid (AA) in the linear range of 1−10 μM with the detection limit of 0.834 μM.

Enhanced plant antioxidant capacity and biodegradation of phenol by immobilizing peroxidase on amphoteric nitrogen-doped carbon dots

A green method of immobilizing enzyme on biocompatible nitrogen-doped carbon dots (N-CDs) is proposed in this research. The N-CDs that were prepared in an environmentally friendly manner can improve plant antioxidant capacity to alleviate growth inhibition under stress. The fluorescence signal emitted by N-CDs present in vivo in plants indicates that N-CDs can immobilize peroxidase without a crosslinker. After immobilization, N-CDs-supported horseradish peroxidase (N-CDs@HRP) can significantly enhance peroxidase activity, stability, and phenol removal capacity when compared with free HRP. The N-CDs-immobilized enzyme with high activity and stability can be broadly applied to different environments and under harsh conditions.

Silicon increases cell wall thickening and lignification in rice (Oryza sativa) root tip under excess Fe nutrition

Iron (Fe) as a micronutrients and silicon (Si) as a cell wall element are important in plant cell wall extension and integrity. While the interaction of exogenous Si and excess Fe on root cell wall modifications is known, the effects of these nutritional parameters on the spatial changes in the activities of genes and/or enzymes involved in the lignification of root cell walls are not well studied. Thus, these parameters were investigated in the root apical part (AP) and basal part (BP) of rice (Oryza sativa L.) plants supplied with and without Si (1.5 mM) under normal (10 mg/L) and excess Fe (150 mg/L) nutrition for 7 days. Beside growth retardation, excess Fe increased the activities of phenylalanine ammonia lyase (PAL), superoxide dismutase and NADPH–oxidase and PAL and cell wall peroxidase (POD) genes expression, along with the increased phenols and H2O2 contents in the root AP. Furthermore, the increased thickening of endodermal, exodermal and metaxylem cell walls in the root AP by excess Fe was attributed to the enhanced POD activity. POD expression, endodermal and exodermal cell wall thickenings were not affected by excess Fe in the root BP. Si application under excess Fe exaggerated the effects of excess Fe on root cell wall thickening, increased POD activity but reduced H2O2 content in the root AP. Thus, Si application under excess Fe nutrition promotes earlier initiation of lignin polymerization closer to and toward the root tip and hence restricts the entry of excess Fe into the plant.

Naturally Occurring A51V Variant of Human Cytochrome c Destabilizes the Native State and Enhances Peroxidase Activity.

The A51V variant of human cytochrome c is linked to thrombocytopenia 4 (THC4), a condition that causes decreased blood platelet counts. A 1.82 Å structure of the A51V variant shows only minor changes in tertiary structure relative to the wild-type (WT) protein. Guanidine hydrochloride denaturation demonstrates that the global stability of the A51V variant is 1.3 kcal/mol less than that of the WT protein. The midpoint pH, pH1/2, of the alkaline transition of the A51V variant is 1 unit less than that of the WT protein. Stopped-flow pH jump experiments show that the A51V substitution affects the triggering ionization for one of two kinetically distinguishable alkaline conformers and enhances the accessibility of a high-spin heme transient. The pH1/2 for acid unfolding of the A51V variant is 0.7 units higher than for that of the WT protein. Consistent with the greater accessibility of non-native conformers for the A51V variant, the kcat values for its peroxidase activity increase by 6- to 15-fold in the pH range of 5-8 versus those of the WT protein. These data along with previously reported data for the other THC4-linked variants, G41S and Y48H, underscore the role of Ω-loop C (residues 40-57) in modulating the peroxidase activity of cytochrome c early in apoptosis.

Enzyme Mimic Based on a Self-Assembled Chitosan/DNA Hybrid Exhibits Superior Activity and Tolerance.

Nature has evolved enzymes with exquisite active sites that catalyze biotransformations with high efficiency. However, the exploitation of natural enzymes is often hampered by poor stability, and natural enzyme production and purification are costly. Supramolecular self-assembly allows the construction of biomimetic active sites, although it is challenging to produce such artificial enzymes with catalytic activity and stability that rival those of natural enzymes. We report herein a strategy to produce a horseradish peroxidase (HRP) mimic based on the assembly of chitosan with a G-quadruplex DNA (G-DNA)/hemin complex. A network-like morphology of the assembled nanomaterial was observed together with a remarkable enhancement of peroxidase activity induced by the chitosan and G-DNA components. The turnover frequency and catalytic efficiency of the enzyme-mimicking material reached or even surpassed those of HRP. Moreover, the catalytic complex exhibited higher tolerance than HRP to harsh environments, such as extremely low pH or high temperatures. In accord with the experimental and simulated results, it is concluded that the spatial distribution of the G-DNA and chitosan components and the exposure of the catalytic center may facilitate the coordination of substrates by the hemin iron, leading to the superior activity of the material. Our work provides a simple and affordable avenue to produce highly active and robust enzyme-mimicking catalytic nanomaterials.

In Vitro Selection of DNA Aptamers for a Small-Molecule Porphyrin by Gold Nanoparticle-Based SELEX.

In this study, a new strategy for the selection of aptamers against small-molecule target was established using gold nanoparticles (AuNPs) as the separation matrix and Zinc(II)-Protoporphyrin IX (ZnPPIX) as the target molecule without the immobilization step due to the absorption of ssDNA on AuNPs. The progress of the selection process was monitored by the recovery rate and the fluorescence enhancement of N-methyl mesoporphyrin IX (NMM) after reacting with each selected pool. After 11 rounds of selection, a truncated aptamer ZnP1.2 with a low-micromolar dissociation constant was obtained, and it also showed good fluorescence enhancement for NMM and the enhanced peroxidase activity after binding with hemin, indicating this functional aptamer has potential to be a light-up fluorescent probe and a DNAzyme which could be used as an alternative to peroxidases for many colorimetric or chemiluminescent detections in biosensing events. The experimental results show that the simple and convenient AuNP-based SELEX is very conducive to the selection of aptamers for small-molecule targets.

Identification of Intermediates in Peroxidase Catalytic Cycle of a DNAzyme Possessing Heme

Abstract Heme in the ferric state (heme(Fe3+)) binds to G-quadruplex DNAs to form stable complexes that exhibit enhanced peroxidase activities. The complexes are considered DNAzymes possessing heme as a prosthetic group (heme-DNAzymes), and have been extensively investigated as promising catalysts for a variety of applications. On ESR and stopped-flow measurements, an iron(IV)oxo porphyrin π-cation radical known as Compound I was detected in reaction mixtures of heme-DNAzymes and hydrogen peroxide. This finding not only resolved the long-standing issue of the mechanism underlying the enhancement of the peroxidase activity of heme(Fe3+) in the scaffold of a G-quadruplex DNA, but also provided new insights as to the design of novel heme-DNAzymes.

Investigating the role of ATP towards amplified peroxidase activity of Iron oxide nanoparticles in different biologically relevant buffers

Abstract Evaluating the colloidal stability and enzyme mimetic nature of nanozymes at different buffer conditions is necessary to develop novel biosensing, biomedical and environmental applications. It is established that Fe3O4 nanoparticles show optimum peroxidase activity at pH 4 and it has also been shown that nucleotides like ATP can exhibit synergistic effect to enhance peroxidase activity at neutral pH. In this study we show the effect of buffers, pH and presence of ATP on peroxidase activity of Fe3O4 NPs and elucidate the mechanism involved in enhanced activity at neutral pH. Fe3O4 NPs colloidal stability was assessed over a period of 72 h from acidic to neutral pH. It was explained that ˙OH radical generation from the synergistic combination of nucleotides and Fe3O4 NPs resulted in peroxidase activity at neutral pH and found that buffer concentration has a major effect on this activity. These findings challenge the existing theory of peroxidase activity demonstrated by Fe3O4 NPs at acidic pH. Moreover, the posibility can overcome the pH lacuna in designing novel biosensors and can also extend the heterogeneous Fenton reaction of Fe3O4 NPs over a wide range of pH.

Effect of V83G and I81A Substitutions to Human Cytochrome c on Acid Unfolding and Peroxidase Activity below a Neutral pH.

Mitochondrial cytochrome c is a highly conserved protein in eukaryotes. Certain functions of cytochrome c have been tuned during evolution. For instance, the intrinsic peroxidase activity of human cytochrome c is much lower than that of the yeast counterpart. Structural studies on K72A yeast iso-1-cytochrome c [McClelland, L. J., et al. (2014) Proc. Natl. Acad. Sci. USA, 111, 6648-6653] revealed that residues 81 and 83 in Ω-loop D (residues 70-85) may be gatekeeper residues for the peroxidase activity linked to intrinsic apoptosis. Amino acids at both positions evolve to more sterically demanding amino acids. We hypothesized that residues 81 and 83 evolved such that steric constraints at these positions tune down the peroxidase activity of human cytochrome c. To test this hypothesis, I81A and V83G variants of human cytochrome c were prepared. Our results show that the I81A substitution significantly influences the thermodynamics and kinetics of access to alternate conformers of human cytochrome c, while the V83G substitution has a more modest effect on these properties. The I81A variant also shows a significant enhancement in peroxidase activity, particularly below pH 7, whereas the V83G variant has a similar peroxidase activity to wild-type human cytochrome c. However, neither variant increases the peroxidase activity of human cytochrome c to the level of yeast iso-1-cytochrome c, indicating that other substructures of cytochrome c are also involved in tuning the intrinsic peroxidase activity of mitochondrial cytochrome c.

Self-Assembling Peptide Artificial Enzyme as an Efficient Detection Prober and Inhibitor for Cancer Cells.

A new hybrid nanoparticle (NP; fluorenylmethoxycarbonyl-arginine-glycine-aspartate and hemin, Fmoc-RGD/hemin NP) was developed for the simultaneous detection and inhibition of breast cancer cells. Hemin can regulate the reactive oxygen species (ROS), while Fmoc-RGD acts as a scaffold for hemin nanocrystallization. Fmoc groups interact with the porphyrin groups of hemin through hydrophobic and π-π interactions to form a hydrophobic core of the NPs. The hydrophilic RGD chains surround the core to maintain the stability of the nanoparticles in an aqueous medium. The RGD groups of Fmoc-RGD are also selective for tumor cells. This interaction can be exploited to enhance the selectivity of tumor detection. Based on enhanced peroxidase activity, Fmoc-RGD/hemin NPs were developed as signal transducers in a facile and fast point-of-care cancer diagnosis platform. This platform is sensitive to breast cancer cells and hydrogen peroxide (H2O2), a biomarker for breast cancer. In addition, these Fmoc-RGD/hemin NPs can be used as nanoscavengers for ROS and for regulating the redox status of cancer cells. They also exhibit a targeted inhibitory effect on the epithelial-mesenchymal transition (EMT). The peptide-tuned self-assembly of Fmoc-RGD/hemin NPs as functional artificial enzymes boasts simple preparation, biofriendliness, and the versatility required for on-demand therapeutics and diagnostics for metastatic cancer cells. These NPs can therefore be used as effective tools for potential applications in medicine and biotechnology.

One-pot synthesis of a composite consisting of the enzyme ficin and a zinc(II)-2-methylimidazole metal organic framework with enhanced peroxidase activity for colorimetric detection for glucose.

An enzyme-metal organic framework (MOF) composite with saucer-like structure was prepared via one-pot synthesis from ficin (a cysteine proteolytic enzyme with POx activity), zinc(II) ions and 2-methylimidazole. The composites exhibit a 2.5-fold higher catalytic activity and stronger affinity for substrates compared to free ficin. This was exploited to design a colorimetric assay for the determination of glucose. The addition of glucose oxidase causes the formation of H2O2 which is catalytically oxidized by ficin to form a blue coloration that can be measured at 652nm. The assay has a 0.12μM detection limit and excellent selectivity. It was successfully applied to the determination of glucose in diluted serum samples. Graphical abstract Schematic presentation of the synthesis process and the enhanced peroxidase activity of ficin@MOF composites. Ficin was immobilized in a new saucer-like shape of Ficin@MOF composites, which showed higher peroxidase activity than free ficin. Scheme and graphical abstract contains poor quality of text.We have attach a new picture with 600 dpi as scheme and graphical abstract.

New Insights into the Effects of Surface Functionalization on the Peroxidase Activity of Cytochrome c Adsorbed on Silica Nanoparticles.

The immobilization of proteins on inorganic supports is attracting increasing interest since the realization of active surfaces finds application in enzyme-assisted catalysis, environmental sciences, and medical fields. In the present study, cytochrome c (cyt c) is adsorbed on silica nanoparticles (SNPs) and amino-functionalized silica nanoparticles (SNPs-APTES), which are prepared for this purpose and having a diameter of about 50 nm. The peroxidase activity of the protein is investigated under different experimental conditions, to evaluate the impact of differently charged surfaces on the catalytic activity of the biomolecule. The peroxidase activity of cyt c increases upon adsorption on SNPs, and it shows a linear behavior with nanoparticles concentration; on the other hand, the contact with increasing amounts of SNPs-APTES does not affect the catalytic activity of the protein. The kinetic profile of the oxidation reaction is altered for cyt c-SNPs sample, suggesting that upon adsorption, changes in the catalytic process take place. Moreover, we observe that the enhancement of peroxidase activity of cyt c-SNPs is almost completely inhibited in high-ionic-strength buffer: this indicates that the protein establishes electrostatic interactions with SNP. The spectroscopic properties of the adsorbed protein on the two different matrices are investigated by using fluorescence and Raman spectroscopies to account for the enzymatic activity of the hybrid materials. The fluorescence spectra of cyt c-silica bio-composites reveal that the adsorption on silica modifies the microenvironments of the emitting amino acid residues of the protein. Indeed, their fluorescence gains intensity and appears blue-shifted compared to that of the native protein; these modifications are more evident when cyt c is adsorbed on SNPs. Raman spectra suggest that both oxidation and spin state of heme iron change when cyt c is adsorbed on SNPs but not on SNPs-APTES. The spectroscopic data of biocomposite materials are discussed in terms of structural changes to account for the increment of peroxidase activity upon adsorption on the negatively charged surface of SNPs.

Colorimetric Detection of MPT64 Antibody Based on an Aptamer Adsorbed Magnetic Nanoparticles for Diagnosis of Tuberculosis.

We have developed a colorimetric biosensing system for the detection of antibody against MPT64, a protein secreted by Mycobacterium tuberculosis, using aptamer DNA adsorbed Fe₃O₄ magnetic nanoparticles (MNPs) for diagnosis of tuberculosis (TB). In this system, MNPs were first incubated with single stranded (ss) DNA-type aptamer having a high affinity toward target antibody against MPT64 (anti-MPT64), resulting in quick inhibition of the peroxidase-like activity of MNPs via the adsorption of aptamer on the surface of MNPs. By the addition of sample solutions containing anti-MPT64, aptamer bound on the surface of MNPs would strongly interact with free anti-MPT64 and be detached from the MNPs, thereby increasing the available surface area of the MNPs and consequently yielding enhanced peroxidase activity. Using this strategy, target anti-MPT64 was successfully detected by displaying increased colorimetric intensities from the higher oxidation of employed peroxidase substrate, 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) (ABTS). Based on these results, we anticipate that aptamer adsorbed MNPs can serve as a potent probe system for the detection of clinically important target molecules.

A microwave-assisted approach to N-(2-nitrophenyl)benzenesulfonamides that enhanced peroxidase activity in response to excess cadmium

A facile and efficient approach to N-(2-nitrophenyl) benzenesulfonamides was developed under microwave irradiation. A series of pyrabactin analogues containing nitrophenyl scaffold was obtained in excellent yields. In addition, the method was pretty suitable to prepare flusulfamide. Significantly, the 3ae could enhance POD activity in response to heavy metal stress.

Effects of methyl jasmonate on growth, antioxidants, and carbon and nitrogen metabolism of Glycyrrhiza uralensis under salt stress

We investigated the effects of 0.025 or 0.05 mM methyl jasmonate (MeJA) on the growth characteristics, antioxidant enzyme activities, non-enzymatic antioxidant content, and carbon and nitrogen metabolizing enzyme activities in Glycyrrhiza uralensis exposed to 100 mM NaCl. Results showed that salt stress decreased the stem length and lateral root number and the treatment with 0.025 or 0.05 mM MeJA increased the root length of salt-stressed G. uralensis seedlings but decreased root diameter, stem length, and stem diameter. MeJA application modulated oxidative stress in salt-stressed G. uralensis seedlings. It decreased the catalase activity but enhanced peroxidase activity and ascorbate content. However, treatment with 0.05 mM MeJA significantly increased the malondialdehyde content of salt-stressed seedlings. Salt stress inhibited carbon and nitrogen metabolism. The application of MeJA enhanced the activities of sucrose synthase, and sucrose phosphate synthase, and nitrate reductase in salt-stressed seedlings.

Cytochrome c-poly(acrylic acid) conjugates with improved peroxidase turnover number.

Cytochrome c-poly(acrylic acid) (cyt c-PAA) conjugates with 34-fold enchancement in peroxidase turnover number (kcat) are reported. Cyt c-PAA conjugates were prepared by carbodiimide coupling. PAA with molecular weight (Mw) ranging from 1.8k to 250k g mol-1 were employed, and the effect of PAA Mw on peroxiodase kinetics was assessed. The kcat value increased with increased Mw of PAA, ranging from 0.077(±0.002) s-1 in the absence of PAA to 2.66(±0.08) s-1 for the conjugate of cyt c with 250k PAA. Enzymatic activity studies over pH 6-8 indicated improved activity for cyt c-PAA conjugates at neutral or slightly alkaline pH. Examination of the cyt c heme spectroscopy in the presence of H2O2 revealed that formation of compound III, a reactive intermediate that leads to enzyme inactivation, was supressed in cyt c-PAA conjugates. Thus, we suggest the kcat enhancement can be attributed to acidification of the pH microenvironment and inhibition of the formation of a reactive intermediate that deactivates cyt c during the catalytic cycle.