Peroxidase Substrate Research Articles

Spatiotemporal Proximity-Enhanced Biocatalytic Cascades Within Metal-Organic Frameworks for Wearable and Theranostic Applications.

Enzymatic catalysis, particularly multi-enzyme cascade catalytic, is often limited by the spatial and temporal separation of enzymes and their signal substrates. Herein, a facile method for producing a spatiotemporal proximity-enhanced biocatalytic cascade system is introduced by encasing enzymes within metal-organic frameworks (MOFs) that are modulated with sulfonic acid-functionalized signal substrates. The modulated behavior relies on the sulfonic acid groups coordinated with Zn2+. As a proof of concept, by utilizing 2,2'-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS), a widely-used signal substrate for horseradish peroxidase, two-enzyme/substrate, and three-enzyme/substrate MOFs, which demonstrated a 7.4- and 10.2-fold increase in biocatalytic efficiency over free systems are successfully synthesized. Incorporating the synthesized MOFs into homemade wearable patches and in vivo settings, noninvasive sweat glucose colorimetric detection and photoacoustic imaging-guided photothermal tumor therapy are enabled, respectively. This advancement stems from the newly established coordinative bonds between Zn2+ centers and substrates' sulfonic acid groups, which negates the need for additional signal substrates, thereby not only enhancing but also streamlining bioapplication processes.

Intrinsic Peroxidase-like Activity of Polystyrene Nanoplastics Mediates Oxidative Stress.

Nanoplastics represent a global environmental concern due to their ubiquitous presence and potential adverse impacts on public and environmental health. There is a growing need to advance the mechanistic understanding of their reactivity as they interact with biological and environmental systems. Herein, for the first time, we report that polystyrene nanoplastics (PSNPs) have intrinsic peroxidase-like activity and are able to mediate oxidative stress. The peroxidase-like activity is dependent on temperature and pH, with a maximum at pH 4.5 and 40 °C. The catalytic activity exhibits saturation kinetics, as described by the Michaelis-Menten model. The peroxidase-like activity of PSNPs is attributed to their ability to mediate electron transfer from peroxidase substrates to H2O2. Ozone-induced PSNP aging can introduce oxygen-containing groups and disrupt aromatic structures on the nanoplastic surface. While ozonation initially enhances peroxidase-like activity by increasing oxygen-containing groups without degrading many aromatic structures, extended ozonation destroys aromatic structures, significantly reducing this activity. The peroxidase-like activity of PSNPs can mediate oxidative stress, which is generally positively correlated with their aromatic structures, as suggested by the ascorbic acid assay. These results help explain the reported oxidative stress exerted by nanoplastics and provide novel insights into their environmental and public health implications.

Heterologous expression and characterization of a dye-decolorizing peroxidase from Ganoderma lucidum, and its application in decolorization and detoxifization of different types of dyes.

Dye-decolorizing peroxidases (DyPs) belong to a novel superfamily of heme peroxidases that can oxidize recalcitrant compounds. In the current study, the GlDyP2 gene from Ganoderma lucidum was heterologously expressed in Escherichia coli, and the enzymatic properties of the recombinant GlDyP2 protein were investigated. The GlDyP2 protein could oxidize not only the typical peroxidase substrate ABTS but also two lignin substrates, namely guaiacol and 2,6-dimethoxy phenol (DMP). For the ABTS substrate, the optimum pH and temperature of GlDyP2 were 4.0 and 35°C, respectively. The pH stability and thermal stability of GlDyP2 were also measured; the results showed that GlDyP2 could function normally in the acidic environment, with a T50 value of 51°C. Moreover, compared to untreated controls, the activity of GlDyP2 was inhibited by 1.60 mM of Mg2+, Ni2+, Mn2+, and ethanol; 0.16 mM of Cu2+, Zn2+, methanol, isopropyl alcohol, and Na2EDTA·2H2O; and 0.016 mM of Fe2+ and SDS. The kinetic constants of recombinant GlDyP2 for oxidizing ABTS, Reactive Blue 19, guaiacol, and DMP were determined; the results showed that the recombination GlDyP2 exhibited the strongest affinity and the most remarkable catalytic efficiency towards guaiacol in the selected substrates. GlDyP2 also exhibited decolorization and detoxification capabilities towards several dyes, including Reactive Blue 19, Reactive Brilliant Blue X-BR, Reactive Black 5, Methyl Orange, Trypan Blue, and Malachite Green. In conclusion, GlDyP2 has good application potential for treating dye wastewater.

Facile synthesis of copper carbonate analog with peroxidase-like activity for colorimetric detection of isoniazid

In this article, copper carbonate analog with good peroxidase-like activity was successfully synthesized for the first time via a simple co-precipitation of CuSO4▪5H2O and Na2CO3. The obtained copper carbonate analog exhibited excellent intrinsic peroxidase-like activity towards a classical peroxidase substrate of 3, 3′, 5, 5′ -tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2) under an acidic environment. The study of the catalytic mechanism confirmed that the hydroxyl radical produced from the decomposition of H2O2 is the main reactive oxygen species responsible for the catalytic oxidation of TMB to oxTMB. Moreover, results from kinetic parameter analysis indicated that H2O2 was more easily and/or likely to attach to the copper carbonate analog than TMB. Subsequently, the effects of experimental conditions (buffer pH, temperature, and incubation time) on the catalytic activity of the copper carbonate analog were also optimized. Finally, a copper carbonate analog-based colorimetric sensor was developed to determine isoniazid. Under the optimal conditions, the linear range for isoniazid was as broad as 0–178.6 μM, and the detection limit was as low as 8.47 μM. The spiked recoveries of isoniazid in normal human serum has been observed in the range of 94.8%–105.5 %. This strategy focuses on the development of a green, cost-efficient peroxidase mimic with high activity, good biocompatibility, and a simple synthesis process.

Optimization of metal–organic framework nanozyme activity via histidine modification for simultaneous pesticide detection

Biosensors provide simple, rapid and sensitive detection of a wide range of analytes. Recently, they have been widely used for the rapid detection of pesticides, which makes up for the shortcomings of traditional detection methods. However, the catalytic activity of some nanozymes is not satisfactory in practical applications, limiting the performance of biosensors. Further development of highly active and stable nanozymes is conducive to improving the sensitivity of the sensors and expanding their application in practical detection. Herein we simulated the amino acid microenvironment of natural enzymes and synthesized His-MIL-101(Fe)-X with different histidine (His) modification amounts (X = 0, 25%, 50%, 75%) by regulating the ratio of histidine and terephthalic acid ligands using a one-step solvothermal method. With increasing histidine doping, the peroxidase-like activity of His-MIL-101(Fe)-X was gradually enhanced, and the sensing performance of the sensor arrays constructed by combining His-MIL-101(Fe)-X with three peroxidase substrates was also gradually improved, with a 25-fold increase in detection limit. The His-MIL-101(Fe)-75% sensor array was not only able to accurately discriminate five pesticides in the range of 2-100 μM, but also to quantitatively identify different concentrations of pesticides. Moreover, the His-MIL-101(Fe)-75% sensor array showed good anti-interference capability and was able to discriminate five pesticides at only 2 μM in soil, lake water, seawater, and apple samples, as well as quantitatively detecting diafenthiuron, which has great potential for practical application.

Accelerated and precise identification of antioxidants and pesticides using a smartphone-based colorimetric sensor array

The integration of smartphones with conventional analytical approaches plays a crucial role in enhancing on-site detection platforms for point-of-care testing. Here, we developed a simple, rapid, and efficient three-channel colorimetric sensor array, leveraging the peroxidase (POD)-like activity of polydopamine-decorated FeNi foam (PDFeNi foam), to identify antioxidants using both microplate readers and smartphones for signal readouts. The exceptional catalytic capacity of PDFeNi foam enabled the quick catalytic oxidation of three typical peroxidase substrates (TMB, OPD and 4-AT) within 3 min. Consequently, we constructed a colorimetric sensor array with cross-reactive responses, which was successfully applied to differentiate five antioxidants (i.e., glycine (GLY), glutathione (GSH), citric acid (CA), ascorbic acid (AA), and tannic acid (TAN)) within the concentration range of 0.1–10 μM, quantitatively analyze individual antioxidants (with AA and CA as model analytes), and assess binary mixtures of AA and GSH. The practical application was further validated by discriminating antioxidants in serum samples with a smartphone for signal readout. In addition, since pesticides could be absorbed on the surface of PDFeNi foam through π-π stacking and hydrogen bonding, the active sites were differentially masked, leading to featured modulation on POD-like activity of PDFeNi foam, thereby forming the basis for pesticides discrimination on the sensor array. The nanozyme-based sensor array provides a simple, rapid, visual and high-throughput strategy for precise identification of various analytes with a versatile platform, highlighting its potential application in point-care-of diagnostic, food safety and environmental surveillance.

Nuclear factor erythroid 2-related factor 2 promotes radioresistance by regulating glutamate-cysteine ligase modifier subunit and its unique immunoinvasive pattern.

The enzyme glutamate-cysteine ligase modifier subunit (GCLM) serves as the initial rate-limiting factor in glutathione (GSH) synthesis. GSH is the preferred substrate for glutathione peroxidase 4 (GPX4), directly impacting its activity and stability. This study aims to elucidate the expression of GCLM and its correlation with the nuclear factor erythroid 2-related factor 2 (NFE2L2), commonly referred to as NRF2, in esophageal squamous cell carcinoma (ESCC) and further investigate the potential signaling axis of radiotherapy resistance caused by NRF2-mediated regulation of ferroptosis in ESCC. The expression of NRF2, GCLM, and GPX4 in ESCC was analyzed by bioinformatics, and their relationship with ferroptosis was verified through cell function experiments. Their role in radioresistance was then investigated through multiple validation steps. Bioinformatics analysis was employed to determine the immune infiltration pattern of NRF2 in ESCC. Furthermore, the effect of NRF2-mediated massive macrophage M2 infiltration on radiotherapy and ferroptosis was validated through in vivo experiments. In vitro assays demonstrated that overactivated NRF2 promotes radioresistance by directly binding to the promoter region of GCLM. The Tumor Immune Estimation Resource (TIMER) and quanTIseq analyses revealed NRF2 enrichment in M2 macrophages with a positive correlation. Co-culturing KYSE450 cells with M2 macrophages demonstrated that a significant infiltration of macrophages M2 can render ESCC cells resistant to radiotherapy but restore their sensitivity to ferroptosis in the process. Our study elucidates a link between the NRF2-GCLM-GSH-GPX4 signaling axis in ESCC, highlighting its potential as a therapeutic target for antagonistic biomarkers of resistance in the future. Additionally, it provides a novel treatment avenue for ESCC metastasis and radioresistance.

Facile Synthesis of Copper Telluride Nanoparticles as a Robust Nanoenzymatic Catalyst for Intrinsic Peroxidase‐Like Activity

AbstractCopper telluride nanoparticles are classified as the family of metal chalcogenides and were synthesized using a simple solvothermal method using a new source of telluride, ditelluride diphenyl. The characterized techniques revealed that the atomic composition of copper tellurides is Cu2Te and particles are spherical. This study is greatly examined by different designed experiments for the proof of the intrinsic peroxidase‐like activity of copper telluride nanoparticles and its effectiveness in a variety of similar structures. Cu2Te nanoparticles can convert peroxidase substrates (TMB and OPD) to colored products in the presence of H2O2. In addition, the as‐prepared nanoparticles have the potential to oxidize DPPH and ABTS substrates. Michaelis–Menten constant KM value for TMB and H2O2 was found to be 12 mM and 0.60 mM for H2O2 and the Vmax value of the Cu2Te was found to be TMB and H2O2 was 2.50×10−7 and 0.82×10−8 MS−1. These data are found to be almost comparable with natural peroxidase HRP and other artificial enzymes reported in the literature. The natural peroxidase‐like activities of Cu2Te nanoparticles originated from the catalytic decomposition of H2O2 into ⋅OH radical, which was confirmed from the ESR spin‐trapping technique and using a fluorescence probe, terephthalic acid.

Development of a lateral flow immunoassay with HRP enhancement for spiked SARS-CoV-2 protein N detection in human saliva

The Coronavirus (SARS-CoV-2) is an enveloped RNA virus that caused a dangerous COVID-19 pandemic following an outbreak in Wuhan, China in 2019. In response to the pandemic, the development of lateral flow assays (LFAs) has been crucial for the detection of viruses, commonly targeting the spike (S) or nucleocapsid (N) protein in nasopharyngeal swab (NS) specimens. COVID-19, caused by the SARS-CoV-2 virus, predominantly manifests as a respiratory tract infection-like illness, characterized by symptoms such as fever, dry cough, upper airway congestion, runny nose, sore throat, myalgia, headache, and exhaustion. This study presents the development of an LFA targeting the N protein to detect the coronavirus in saliva specimens, using a sandwich format. The use of 80 nm gold nanoparticles (AuNPs) has been investigated, and the application of horseradish peroxidase (HRP) and TMB substrate has been employed to enhance the limit of detection (LOD). The results demonstrate a significant 200-fold improvement in LOD, from 10 to 50 pg/ml, for N protein spiked in saliva samples after the application of HRP-TMB. This finding highlights an important advancement towards the utilization of saliva samples in diagnostic applications.

Borophene Quantum Dots as Novel Peroxidase-Mimicking Nanozyme: A Dual-Mode Assay for the Detection of Oxytetracycline and Tetracycline Antibiotics.

The greater advantages and wide applications of zero-dimensional nanodots inspire researchers to develop new materials. Therefore, novel borophene quantum dots (QDs) were prepared by a hydrothermal liquid exfoliation technique using water medium. The borophene QDs proved to be highly stable in water medium for more than 120 days. The synthesized borophene QDs revealed intrinsic peroxidase mimetic activity using two chromogenic substrates, 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS). The excellent intrinsic peroxidase activity toward TMB and ABTS substrates was executed using optimal reaction conditions (pH, borophene QDs' concentration, incubation time, and temperature). The formation of hydroxyl radicals in the presence of H2O2 upon TMB and ABTS oxidation played a significant role in the peroxidase reaction. The borophene QDs further proved to be successful for the colorimetric detection of antibiotics (oxytetracycline and tetracycline) using both TMB and ABTS peroxidase substrates. The limit of detection (LOD) for oxytetracycline and tetracycline was found to be 1.10 and 1.02 μM using TMB and 1.03 and 1.02 μM using ABTS chromogenic substrates, respectively. In addition, the fluorescence sensing of oxytetracycline and tetracycline over borophene QDs was also examined. The high fluorescence of borophene QDs (turn ON) was quenched (turn OFF) by oxytetracycline and tetracycline through the inner filter effect mechanism. The LOD of the fluorescence sensing of oxytetracycline and tetracycline was 1.14 and 1.08 μM, respectively. Interestingly, the borophene QDs could be used for the sensitive and selective colorimetric and fluorometric sensing of oxytetracycline and tetracycline after 120 days of storage. The synthesized borophene QDs with long-term stability and real sample analysis provide new insight as nanozymes with higher peroxidase mimetic and fluorescence performance and can be further exploited for medical diagnosis and environmental toxicants' detection.

High Peroxidase-like Activity of Single Iron Atoms Anchored on N-doped Porous Carbon for the Colorimetric Detection of Biothiols

In this report, single iron atoms anchored on nitrogen-doped porous carbon (Fe-ISAs/CN) was prepared and shown to possess high peroxidase-like activity. Fe-ISAs/CN could effi ciently catalyze the oxidation of the peroxidase substrate 3,3?,5,5?-tetramethylbenzidine (TMB) in the presence of H2O2, producing a blue color. In terms of the excellent peroxidase-like activity, single iron atoms anchored on nitrogen-doped porous carbon playing a critical role. Besides, an inhibition effect on the oxidation of TMB catalyzed by Fe-ISAs/CN was induced by biothiols in the presence of H2O2. Taking these advantages together, a simple colorimetric method for rapid and sensitive detection of biothiols was established.

Recent Progress on Peroxidase Modification and Application.

Peroxdiase is one of the member of oxireductase super family, which has a broad substrate range and a variety of reaction types, including hydroxylation, epoxidation or halogenation of unactivated C-H bonds, and aromatic group or biophenol compounds. Here, we summarized the recently discovered enzymes with peroxidation activity, and focused on the special structures, sites, and corresponding strategies that can change the peroxidase catalytic activity, stability, and substrate range. The comparison of the structural differences between these natural enzymes and the mimic enzymes of binding nanomaterials and polymer materials is helpful to expand the application of peroxidase in industry. In addition, we also reviewed the catalytic application of peroxidase in the synthesis of important organic molecules and the degradation of pollutants.

A peroxidase-derived ligand that induces Fusarium graminearum Ste2 receptor-dependent chemotropism.

The fungal G protein-coupled receptors Ste2 and Ste3 are vital in mediating directional hyphal growth of the agricultural pathogen Fusarium graminearum towards wheat plants. This chemotropism is induced by a catalytic product of peroxidases secreted by the wheat. Currently, the identity of this product, and the substrate it is generated from, are not known. We provide evidence that a peroxidase substrate is derived from F. graminearum conidia and report a simple method to extract and purify the FgSte2-activating ligand for analyses by mass spectrometry. The mass spectra arising from t he ligand extract are characteristic of a 400 Da carbohydrate moiety. Consistent with this type of molecule, glycosidase treatment of F. graminearum conidia prior to peroxidase treatment significantly reduced the amount of ligand extracted. Interestingly, availability of the peroxidase substrate appears to depend on the presence of both FgSte2 and FgSte3, as knockout of one or the other reduces the chemotropism-inducing effect of the extracts. While further characterization is necessary, identification of the F. graminearum-derived peroxidase substrate and the FgSte2-activating ligand will unearth deeper insights into the intricate mechanisms that underlie fungal pathogenesis in cereal crops, unveiling novel avenues for inhibitory interventions.

Oxidase-like manganese oxide nanoparticles: a mechanism of organic acids/aldehydes as electron acceptors and potential application in cancer therapy.

Identifying the underlying catalytic mechanisms of synthetic nanocatalysts or nanozymes is important in directing their design and applications. Herein, we revisited the oxidation process of 4,4'-diamino-3,3',5,5'-tetramethylbiphenyl (TMB) by Mn3O4 nanoparticles and revealed that it adopted an organic acid/aldehyde-triggered catalytic mechanism at a weakly acidic or neutral pH, which is O2-independent and inhibited by the pre-addition of H2O2. Importantly, similar organic acid/aldehyde-mediated oxidation was applied to other substrates of peroxidase in the presence of nanoparticulate or commercially available MnO2 and Mn2O3 but not MnO. The selective oxidation of TMB by Mn3O4 over MnO was further supported by density functional theory calculations. Moreover, Mn3O4 nanoparticles enabled the oxidation of indole 3-acetic acid, a substrate that can generate cytotoxic singlet oxygen upon single-electron transfer oxidation, displaying potential in nanocatalytic tumor therapy. Overall, we revealed a general catalytic mechanism of manganese oxides towards the oxidation of peroxidase substrates, which could boost the design and various applications of these manganese-based nanoparticles.

Self-assembly of a unique triangle-like tungstovanadate containing pentagonal {(WO7)W3(SnR)2} cluster.

In aqueous solution, a novel triangle-like tungstovanadate estertin derivative K10H10.5[(W4O15(H2O)2){(SnCH2CH2COO)2(V0.75W10.75/V0.25O39)}{{(SnCH2CH2COO)2(μ-OH)}2(SnCH2CH2COO)(VW10O37)}2]·31H2O ((SnR)8-V3W35, R = CH2CH2COO) was assembled by a conventional synthetic method. (SnR)8-V3W35 is composed of one [VW11O39]7- ({VW11}) and two [VW10O37]9- ({VW10}) units connected by eight [Sn(CH2)2COO]2+ groups and a {W4O19} cluster. Interestingly, there exists a pentagonal bipyramid WO7 polyhedral center surrounded by two SnCO5 and three WO6 octahedra, forming a pentagonal {(WO7)W3(SnR)2} cluster in this polyoxometalate (POM), which is also the first example of a pentagonal structure formed by transition metals (TMs) and main group organometals in the POM family. Furthermore, the structure of this organic-inorganic hybrid POM also exhibits the largest number of organotin groups introduced into the POM system. It was characterized with various physico-chemical and spectroscopic methods, including X-ray single crystal and powder diffraction analysis, 119Sn and 51V NMR, IR, thermal gravimetric analysis (TGA), etc. In addition, the catalytic activity of (SnR)8-V3W35 as a mimic of peroxidase was evaluated using o-phenylenediamine (OPD) as a peroxidase substrate. The major factors influencing the oxidation reaction such as pH, the dosage of (SnR)8-V3W35, and concentrations of OPD and H2O2 were mainly studied. (SnR)8-V3W35 exhibits good peroxidase-like catalytic activity. From another perspective, the successful acquisition of (SnR)8-V3W35 further proves the instability and easy reassembly characteristics of TM-sandwich-type tungstovanadates, which also provides a new assembly strategy for synthesizing POM-estertin derivatives.

Gamma Irradiation Promotes the Growth Rate of Thai Pigmented Rice As Well As Inducing the Accumulation of Bioactive Compounds and Carbohydrate Hydrolyzing Enzymes Inhibitors (α-Glucosidase and α-Amylase) under Salt Conditions.

Rice contains many bioactive compounds that perform various biological activities. Some of these compounds have been identified as α-glucosidase and α-amylase inhibitors, including guaiacol, vanillin, methyl vanillate, vanillic acid, syringic acid, and 2-pentyl furan. In this study, we assessed the growth rate, photosynthetic pigment content, phenolic content, and flavonoid content of gamma-irradiated Thai pigmented rice. Bioactive components of gamma-irradiated rice that had been subjected to salt treatment were also investigated. The findings showed that production of photosynthetic pigments, which are associated with plant growth, was induced by low gamma exposure. Phenolic and flavonoid content of rice was increased after gamma irradiation at 5 to 1,000 Gy. Both gamma irradiation and the salt conditions changed the quantity of vanillin, methyl vanillate, and vanillic acid in the rice. However, at a salt concentration of 40 mM, the salt stress had more of an effect than the gamma dosage. However, the high concentrations of methyl vanillate and vanillic acid detected in the rice under salt conditions were ameliorated by gamma irradiation. Guaiacol served as the substrate of guaiacol peroxidase for catalyzed reactive oxygen species, as evidenced by the observation that the guaiacol content of rice decreased between increased gamma dosages. A gamma dose of 40 to 1,000 Gy resulted in the production of syringic acid. Under salt stress, syringic acid buildup was also seen to be ameliorated by gamma irradiation. In comparison to salt conditions, particularly for 20 mM salt, gamma irradiation had less of an impact on the 2-pentyl furan in rice.

Naked-Eye Sensing of SARS-CoV-2 Utilizing Nanozymatic Nanoassays

The first clinical diagnosis of the new infectious disease, COVID-19 was reported on December 31, 2019. The origin of this new infectious disease is a new generation of coronavirus, i.e., SARS-CoV-2. Since, the exploration of this concept, several methods have been developed for the diagnosis of COVID-19 via the detection of SARS-CoV-2. Among different methods, the nanozyme-based colorimetric sensors have attracted good attention due to the naked-eye response and simple procedure. Hence, the aim of this review is a quick overview of the nanozyme-based sensing and detection methods for early diagnosis of COVID-19. The main basis of these sensors is the detection of color variation of a nanozyme-mediated oxidation reaction in the presence and the absence of antigens of COVID-19. In this review article, we aimed to review the recent nanozyme-based colorimetric sensors for the detection of SARS-CoV-2 to provide a brief comprehensive insight into naked-eye sensing of SARS-CoV-2. The colorimetric nanozyme-based sensing of SARS-CoV-2 is based on the interaction of nanozymes with SARS-CoV-2 nucleocapsid protein, spike (S1) protein of SARS-CoV-2, SARS-CoV-2 phosphoprotein, SARSCoV-2 RNA, or receptor of CD147 and then probing the color change of the nanozyme-mediated oxidation of peroxidase substrates. The progress or inhibition of color intensity of the reaction system is then assigned to the presence of SARSCoV-2 and consequently positive result of the COVID-19 test.

Peroxidasin Inhibition by Phloroglucinol and Other Peroxidase Inhibitors.

Human peroxidasin (PXDN) is a ubiquitous peroxidase enzyme expressed in most tissues in the body. PXDN represents an interesting therapeutic target for inhibition, as it plays a role in numerous pathologies, including cardiovascular disease, cancer and fibrosis. Like other peroxidases, PXDN generates hypohalous acids and free radical species, thereby facilitating oxidative modifications of numerous biomolecules. We have studied the inhibition of PXDN halogenation and peroxidase activity by phloroglucinol and 14 other peroxidase inhibitors. Although a number of compounds on their own potently inhibited PXDN halogenation activity, only five were effective in the presence of a peroxidase substrate with IC50 values in the low μM range. Using sequential stopped-flow spectrophotometry, we examined the mechanisms of inhibition for several compounds. Phloroglucinol was the most potent inhibitor with a nanomolar IC50 for purified PXDN and IC50 values of 0.95 μM and 1.6 μM for the inhibition of hypobromous acid (HOBr)-mediated collagen IV cross-linking in a decellularized extracellular matrix and a cell culture model. Other compounds were less effective in these models. Most interestingly, phloroglucinol was identified to irreversibly inhibit PXDN, either by mechanism-based inhibition or tight binding. Our work has highlighted phloroglucinol as a promising lead compound for the design of highly specific PXDN inhibitors and the assays used in this study provide a suitable approach for high-throughput screening of PXDN inhibitors.

Biochemical characterization of enzyme-like silver nanoparticles toward nanozyme-catalysed oxidation reactions

In this contribution, the biochemical characterization of enzyme-like nanosilvers was performed toward nanozyme-catalyzed oxidation reactions. In this regard, silver nanoparticles were synthesized via a simple chemical reduction method and then characterized by the TEM imaging method. Afterward, their enzyme-like activity was investigated toward catalysis of the oxidation reaction of 3,3’,5,5’-tetramethyl-benzidine (TMB) as one of the most popular peroxidase substrates. The results exhibited a specific nanozymatic activity as high as 5400 nM min−1 for the as-synthesized nanosilvers toward TMB oxidation. Due to the high enzyme-like activity ofthe as-prepared nanosilvers, their biochemical properties including pH, thermal, light, and shelf stability were characterized to explore more precisely describing their nanozymatic behavior. The results ofthermal and pH stability studies showed that the as-prepared nanosilvers reveal their maximal enzyme-like activity at a wide temperature range of 25℃–35℃ and a pH range of 3.5–4.5, in order. Regarding the light stability and shelf-life studies, the results exhibited that 75% and 96% of the enzyme-like activity of the as-prepared nanozymes was saved after 7 days exposing visible light and 10 days of storage at 4℃ under dark conditions, in order.

Experimental Evaluation of Kinetic Characteristics of SiO2@AuNPs Nanocomposite and BSA-stabilized gold Nanoparticles toward Peroxidase-Mediated Reactions

In this study, BSA-stabilized gold nanoparticles and SiO2 @AuNPs nanocomposite were synthesized and then characterized by the TEM imaging method. The average size of BSA-stabilized gold nanoparticles was found to be as small as 13 nm while the SiO2 @AuNPs nanocomposite showed a mean size of 204 nm. The experimental studies revealed that both BSA-stabilized gold nanoparticles and SiO2 @AuNPs nanocomposite exhibit intrinsic peroxidase-like activity. Hence, to explore more precise no their catalytic efficiency and substrate affinity of them, the kinetic characteristics of both nanozymes were quantified using the Menten kinetic model, and the provided results were compared. Upon using TMB as peroxidase substrate, Vmax of 0.022 µM min-1 and a Km of 0.06 mM for the SiO2 @AuNPs nanocomposite was achieved while for the BSA-stabilized gold nanoparticles, a Vmax and Km at about 0.263 μM min-1and 0.03 mM, in order, was estimated. The Vmax of BSA-stabilized gold nanoparticles was 12.0-fold higher than that of SiO2 @AuNPs nanocomposite, revealing that the catalytic efficiency of BSA-stabilized gold nanoparticles is 12.0-fold higher than SiO2 @AuNPs nanocomposite. Besides, the Km value of SiO2 @AuNPs nanocomposite was 2-order higher than that of BSA-stabilized gold nanoparticles, indicating that the substrate affinity toward BSA-stabilized gold nanoparticles is 2.0-order higher than the SiO2 @AuNPs nanocomposite. Since, the active nodes of both nanozymes are same (i.e., gold), the difference between their catalytic efficiency and affinity can be assigned to their different sizes and the ability of the active nodes to bind the substrate. Based on the results of this work, small-size BSA-stabilized gold nanoparticles are characteristically more efficient peroxidase mimic materials than the SiO2 @AuNPs nanocomposite.