- Building three-dimensional nanostructures with active enzymes by surface templated layer-by-layer assembly
-
We show that well-defined three-dimensional nanostructures of functional enzymes can be controllably fabricated by layer-by-layer assembly of avidin and biotinylated horseradish peroxidase on micro-contact printing patterned surface templates. The Royal S
- Rauf, Sakandar,Zhou, Dejian,Abell, Chris,Klenerman, David,Kang, Dae-Joon
-
-
Read Online
- A Fluorogenic Trehalose Probe for Tracking Phagocytosed Mycobacterium tuberculosis
-
Tuberculosis (TB) disease is a global epidemic caused by the pathogenic Mycobacterium tuberculosis (Mtb). Tools that can track the replication status of viable Mtb cells within macrophages are vital for the elucidation of host-pathogen interactions. Here, we present a cephalosphorinase-dependent green trehalose (CDG-Tre) fluorogenic probe that enables fluorescence labeling of single live Bacille Calmette-Guérin (BCG) cells within macrophages at concentrations as low as 2 μM. CDG-Tre fluoresces upon activation by BlaC, the β-lactamase uniquely expressed by Mtb, and the fluorescent product is subsequently incorporated within the bacterial cell wall via trehalose metabolic pathway. CDG-Tre showed high selectivity for mycobacteria over other clinically prevalent species in the Corynebacterineae suborder. The unique labeling strategy of BCG by CDG-Tre provides a versatile tool for tracking Mtb in both pre- and postphagocytosis and elucidating fundamental physiological and pathological processes related to the mycomembrane.
- Bertozzi, Carolyn R.,Dai, Tingting,Jiang, Ke,Kamariza, Mireille,Rao, Jianghong,Xie, Jinghang,Zhu, Qihua
-
-
Read Online
- Increased potassium conductance of brain mitochondria induces resistance to permeability transition by enhancing matrix volume
-
Modulation of K+ conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K+ channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca2+ and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K+ or H+ conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoKATP channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K+ conductance did not result in augmented ΔpH. The beneficial effect of valinomycin on CRC was not mediated by H2O2-induced protein kinase C∈ activation. Rather, increased K+ conductance reduced H2O2 generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges.
- Hansson, Magnus J.,Morota, Saori,Teilum, Maria,Mattiasson, Gustav,Uchino, Hiroyuki,Elmer, Eskil
-
-
Read Online
- Target-Selective Fluorescence Imaging and Photocytotoxicity against H2O2 High-Expressing Cancer Cells Using a Photoactivatable Theranostic Agent
-
A purpose-designed and synthesized H2O2-reactive and photoactivatable theranostic agent 1 consisting of 1) an arylboronic acid moiety, 2) pro-fluorophore moiety, and 3) photoactivatable moiety (photosensitizer), selectively and effectively reacted with H2O2 while simultaneously releasing resorufin for fluorescence detection under neutral aqueous conditions. In addition, 1 was cell-permeable, and exhibited effective photocytotoxicity against fluorescently visualized cells only upon photoirradiation. The results also showed that 1 produced a selective fluorescence response to H2O2, even in living cultured cells.
- Takagi, Ryoma,Takeda, Ayano,Takahashi, Daisuke,Toshima, Kazunobu
-
-
Read Online
- Cu2+-Modified Metal–Organic Framework Nanoparticles: A Peroxidase-Mimicking Nanoenzyme
-
The synthesis and characterization of UiO-type metal–organic framework nanoparticles (NMOFs) composed of Zr4+ ions bridged by 2,2′-bipyridine-5,5′-dicarboxylic acid ligands and the postmodification of the NMOFs with Cu2+ ions are described. The resulting Cu2+-modified NMOFs, Cu2+-NMOFs, exhibit peroxidase-like catalytic activities reflected by the catalyzed oxidation of Amplex-Red to the fluorescent Resorufin by H2O2, the catalyzed oxidation of dopamine to aminochrome by H2O2, and the catalyzed generation of chemiluminescence in the presence of luminol/H2O2. Also, the Cu2+-NMOFs mimic NADH peroxidase functions and catalyze the oxidation of dihydronicotinamide adenine dinucleotide, NADH, to nicotinamide adenine dinucleotide, NAD+, in the presence of H2O2. The Cu2+-NMOFs-catalyzed generation of chemiluminescence in the presence of luminol/H2O2 is used to develop a glucose sensor by monitoring the H2O2 formed by the aerobic oxidation of glucose to gluconic acid in the presence of glucose oxidase. Furthermore, loading the Cu2+-NMOFs with fluorescein and activating the catalyzed generation of chemiluminescence in the presence of luminol/H2O2 yield an efficient chemiluminescence resonance energy transfer (CRET) process to the fluorescein reflected by the activation of the fluorescence of the dye (λ = 520 nm, CRET efficiency 35%).
- Chen, Wei-Hai,Vázquez-González, Margarita,Kozell, Anna,Cecconello, Alessandro,Willner, Itamar
-
-
Read Online
- Superresolution fluorescence mapping of single-nanoparticle catalysts reveals spatiotemporal variations in surface reactivity
-
For the practical application of nanocatalysts, it is desirable to understand the spatiotemporal fluctuations of nanocatalytic activity at the single-nanoparticle level. Here we use time-lapsed superresolution mapping of single-molecule catalysis events on individual nanoparticles to observe time-varying changes in the spatial distribution of catalysis events on Sb-doped TiO2 nanorods and Au triangle nanoplates. Compared with the active sites on well-defined surface facets, the defects of the nanoparticle catalysts possess higher intrinsic reactivity but lower stability. Corners and ends are more reactive but also less stable than flat surfaces. Averaged over time, the most stable sites dominate the total apparent activity of single nanocatalysts. However, the active sites with higher intrinsic activity but lower stability show activity at earlier time points before deactivating. Unexpectedly, some active sites are found to recover their activity ("self-healing") after deactivation, which is probably due to desorption of the adsorbate. Our superresolution measurement of different types of active catalytic sites, over both space and time, leads to a more comprehensive understanding of reactivity patterns and may enable the design of new and more productive heterogeneous catalysts.
- Zhang, Yuwei,Lucas, J. Matthew,Song, Ping,Beberwyck, Brandon,Fu, Qiang,Xu, Weilin,Alivisatos, A. Paul
-
-
Read Online
- A 3,7-Dihydroxyphenoxazine-based Fluorescent Probe for Selective Detection of Intracellular Hydrogen Peroxide
-
A novel N-borylbenzyloxycarbonyl-3,7-dihydroxyphenoxazine fluorescent probe (NBCD) for detecting H2O2 in living cells is described. The probe could achieve high selectivity for detecting H2O2 over other biological reactive oxygen species (ROS). In addition, upon addition of H2O2, NBCD exhibited color change from colorless to pink, which makes it a "naked-eye" probe for H2O2 detection. NBCD could not only be used to detect enzymatically generated H2O2 but also to detect H2O2 in living systems by using fluorescence spectroscopy, with a detection limit of 2 μm. Importantly, NBCD enabled the visualization of epidermal growth factor (EGF)-stimulated H2O2 generation inside the cells.
- Han, Zhiqiang,Liang, Xiao,Ren, Xuejiao,Shang, Luqing,Yin, Zheng
-
-
Read Online
- Polyphenol-Mediated Assembly of Proteins for Engineering Functional Materials
-
Functional materials composed of proteins have attracted much interest owing to the inherent and diverse functionality of proteins. However, establishing general techniques for assembling proteins into nanomaterials is challenging owing to the complex physicochemical nature and potential denaturation of proteins. Here, a simple, versatile strategy is introduced to fabricate functional protein assemblies through the interfacial assembly of proteins and polyphenols (e.g., tannic acid) on various substrates (organic, inorganic, and biological). The dominant interactions (hydrogen-bonding, hydrophobic, and ionic) between the proteins and tannic acid were elucidated; most proteins undergo multiple noncovalent stabilizing interactions with polyphenols, which can be used to engineer responsiveness into the assemblies. The proteins retain their structure and function within the assemblies, thereby enabling their use in various applications (e.g., catalysis, fluorescence imaging, and cell targeting).
- Caruso, Frank,Guo, Rui,Han, Yiyuan,Lin, Zhixing,Richardson, Joseph J.,Yun, Gyeongwon,Zhou, Jiajing
-
-
Read Online
- Development of an improved Amplex Red peroxidation activity assay for screening cytochrome P450 variants and identification of a novel mutant of the thermophilic CYP119
-
Abstract: Biocatalysts are increasingly utilized in the synthesis of drugs and agrochemicals as an alternative to chemical catalysis. They are preferred in the synthesis of enantiopure products due to their high regioselectivity and enantioselectivity. Cytochrome P450 (P450) oxygenases are valuable biocatalysts, since they catalyze the oxidation of carbon–hydrogen bonds with high efficiency and selectivity. However, practical use of P450s is limited due to their need for expensive cofactors and electron transport partners. P450s can employ hydrogen peroxide (H2O2) as an oxygen and electron donor, but the reaction with H2O2 is inefficient. The development of P450s that can use H2O2 will expand their applications. Here, an assay that utilizes Amplex Red peroxidation, to rapidly screen H2O2-dependent activity of P450 mutants in cell lysate was developed. This assay was employed to identify mutants of CYP119, a thermophilic P450 from Sulfolobus acidocaldarius, with increased peroxidation activity. A mutant library of CYP119 containing substitutions in the heme active site was constructed via combinatorial active-site saturation test and screened for improved activity. Screening of 158 colonies led to five mutants with higher activity. Among improved variants, T213R/T214I was characterized. T213R/T214I exhibited fivefold higher kcat for Amplex Red peroxidation and twofold higher kcat for styrene epoxidation. T213R/T214I showed higher stability towards heme degradation by H2O2. While the Km for H2O2 and styrene were not altered by the mutation, a fourfold decrease in the affinity for another substrate, lauric acid, was observed. In conclusion, Amplex Red peroxidation screening of CYP119 mutants yielded enzymes with increased peroxide-dependent activity. Graphic abstract: [Figure not available: see fulltext.].
- Ba?lar, M. Semih,Sakall?, Tu??e,Güralp, Gülce,Kestevur Do?ru, Ekin,Hakl?, Emre,Surmeli, Nur Basak
-
-
Read Online
- A Reaction-Based Fluorescent Probe for Imaging of Native Hypochlorous Acid
-
Hypochlorous acid (HOCl), one of the reactive oxygen species (ROS), is highly reactive and short-lived. It is a challenge to dynamic monitor HOCl activity in living systems. Hence, we synthesized a new fluoresce nt probe RF1 based on protection of the hydroxyl group by N,N-dimethylthiocarbamate recognition group, which reached a low fluorescence background signal and highly sensitive property. On account of the electrophilic addition of Cl+ to the sulfide of thiocarbamate moiety, probe RF1 was converted to resorufin and triggered emitting bright. RF1 showed not only the highly sensitive and selective response to HOCl in vitro, but also can be applied in environmental water samples and detected HOCl by test strips. Besides, the ability of RF1 monitoring HOCl in HeLa cells by exogenous simulation and tracing native HOCl in macrophages cells were also explored.
- Wang, Jiamin,Men, Yuhui,Niu, Linqiang,Luo, Yang,Zhang, Jian,Zhao, Weili,Wang, Jianhong
-
-
Read Online
- Covalent hemin-DNA adducts for generating a novel class of artificial heme enzymes
-
(Chemical Equation Presented) Well-defined conjugates between DNA and heme enzymes are accessible by the reconstitution of apo-enzymes with covalent hemin-DNA adducts (see picture). This concept allows access to novel redox catalysts with programmable binding properties that may be applied as biocatalysts, sensors, and biomaterials.
- Fruk, Ljiljana,Niemeyer, Christof M.
-
-
Read Online
- Protein encapsulation: A new approach for improving the capability of small-molecule fluorogenic probes
-
Herein, we report a protein-based hybridization strategy that exploits the host-guest chemistry of HSA (human serum albumin) to solubilize the otherwise cell impermeable ONOO- fluorescent probe Pinkment-OAc. Formation of a HSA/Pinkment-OAc supramolecular hybrid was confirmed by SAXS and solution-state analyses. This HSA/Pinkment-OAc hybrid provided an enhanced fluorescence response towards ONOO-versusPinkment-OAc alone, as determined by in vitro experiments. The HSA/Pinkment-OAc hybrid was also evaluated in RAW 264.7 macrophages and HeLa cancer cell lines, which displayed an enhanced cell permeability enabling the detection of SIN-1 and LPS generated ONOO- and the in vivo imaging of acute inflammation in LPS-treated mice. A remarkable 5.6 fold (RAW 264.7), 8.7-fold (HeLa) and 2.7-fold increased response was seen relative to Pinkment-OAc alone at the cellular level and in vivo, respectively. We anticipate that HSA/fluorescent probe hybrids will soon become ubiquitous and routinely applied to overcome solubility issues associated with hydrophobic fluorescent imaging agents designed to detect disease related biomarkers.
- Han, Hai-Hao,Sedgwick, Adam C.,Shang, Ying,Li, Na,Liu, Tingting,Li, Bo-Han,Yu, Kunqian,Zang, Yi,Brewster, James T.,Odyniec, Maria L.,Weber, Maria,Bull, Steven D.,Li, Jia,Sessler, Jonathan L.,James, Tony D.,He, Xiao-Peng,Tian, He
-
-
Read Online
- Heterolytic reduction of fatty acid hydroperoxides by cytochrome c/cardiolipin complexes: Antioxidant function in mitochondria
-
(Chemical Equation Presented) Cytochrome c (cyt c), a mitochondrial intermembrane electron shuttle between complexes III and IV, can, upon binding with an anionic phospholipid, cardiolipin (CL), act as a peroxidase that catalyzes cardiolipin oxidation. H
- Belikova, Natalia A.,Tyurina, Yulia Y.,Borisenko, Grigory,Tyurin, Vladimir,Samhan Arias, Alejandro K.,Yanamala, Naveena,Furtmueller, Paul Georg,Klein-Seetharaman, Judith,Obinger, Christian,Kagan, Valerian E.
-
-
Read Online
- Intracellular implantation of enzymes in hollow silica Nanospheres for protein therapy: Cascade System of Superoxide Dismutase and Catalase
-
An approach for enzyme therapeutics is elaborated with cell-implanted nanoreactors that are based on multiple enzymes encapsulated in hollow silica nanospheres (HSNs). The synthesis of HSNs is carried out by silica sol-gel templating of water-in-oil micro
- Chang, Feng-Peng,Chen, Yi-Ping,Mou, Chung-Yuan
-
-
Read Online
- Monitoring of Heparin Activity in Live Rats Using Metal-Organic Framework Nanosheets as Peroxidase Mimics
-
Metal-organic framework (MOF) nanosheets are a class of two-dimensional (2D) porous and crystalline materials that hold promise for catalysis and biodetection. Although 2D MOF nanosheets have been utilized for in vitro assays, ways of engineering them into diagnostic tools for live animals are much less explored. In this work, a series of MOF nanosheets are successfully engineered into a highly sensitive and selective diagnostic platform for in vivo monitoring of heparin (Hep) activity. The iron-porphyrin derivative is selected as a ligand to synthesize a series of archetypical MOF nanosheets with intrinsic heme-like catalytic sites, mimicking peroxidase. Hep-specific AG73 peptides as recognition motifs are physically adsorbed onto MOF nanosheets, blocking active sites from nonspecific substrate-catalyst interaction. Because of the highly specific interaction between Hep and AG73, the activity of AG73-MOF nanosheets is restored upon the binding of Hep, but not Hep analogues and other endogenous biomolecules. Furthermore, by taking advantages of biocompatibility and diagnostic property enabled by AG73-MOF nanosheets, the elimination process of Hep in live rats is quantitatively monitored by coupling with microdialysis technology. This work expands the biomedical applications of 2D MOF nanomaterials and provides access to a promising in vivo diagnostic platform.
- Cheng, Hanjun,Liu, Yufeng,Hu, Yihui,Ding, Yubin,Lin, Shichao,Cao, Wen,Wang, Qian,Wu, Jiangjiexing,Muhammad, Faheem,Zhao, Xiaozhi,Zhao, Dan,Li, Zhe,Xing, Hang,Wei, Hui
-
-
Read Online
- A hemin/G-quadruplex acts as an NADH oxidase and NADH peroxidase mimicking DNAzyme
-
Enzyme impressionist: A DNAzyme consisting of hemin combined with a G-quadruplex was shown to mimic the activity of NADH oxidase under aerobic conditions and of NADH peroxidase under anaerobic conditions. Along with these new biocatalytic activities, the hemin/G-quadruplex complex is also capable of regenerating the biologically important NAD+ cofactor in both cycles (see picture). Copyright
- Golub, Eyal,Freeman, Ronit,Willner, Itamar
-
-
Read Online
- Single Molecule Investigation of Nanoconfinement Hydrophobicity in Heterogeneous Catalysis
-
Nanoconfinement imposes physical constraints and chemical effects on reactivity in nanoporous catalyst systems. In the present study, we lay the groundwork for quantitative single-molecule measurements of the effects of chemical environment on heterogeneous catalysis in nanoconfinement. Choosing hydrophobicity as an exemplary chemical environmental factor, we compared a range of essential parameters for an oxidation reaction on platinum nanoparticles (NPs) confined in hydrophilic and hydrophobic nanopores. Single-molecule experimental measurements at the single particle level showed higher catalytic activity, stronger adsorption strength, and higher activation energy in hydrophobic nanopores than those in hydrophilic nanopores. Interestingly, different dissociation kinetic behaviors of the product molecules in the two types of nanopores were deduced from the single-molecule imaging data.
- Chen, Minda,Cheng, Xiaodong,Dong, Bin,Fang, Ning,Filbrun, Seth L.,Huang, Tengxiang,Huang, Wenyu,Mansour, Nourhan,Pei, Yuchen,Pruski, Marek,Wang, Zhuoran
-
-
Read Online
- Super-resolution mapping of photogenerated electron and hole separation in single metal-semiconductor nanocatalysts
-
Metal-semiconductor heterostructures are promising visible light photocatalysts for many chemical reactions. Here, we use high-resolution superlocalization imaging to reveal the nature and photocatalytic properties of the surface reactive sites on single Au-CdS hybrid nanocatalysts. We experimentally reveal two distinct, incident energy-dependent charge separation mechanisms that result in completely opposite photogenerated reactive sites (e- and h+) and divergent energy flows on the hybrid nanocatalysts. We find that plasmon-induced hot electrons in Au are injected into the conduction band of the CdS semiconductor nanorod. The specifically designed Au-tipped CdS heterostructures with a unique geometry (two Au nanoparticles at both ends of each CdS nanorod) provide more convincing high-resolution single-turnover mapping results and clearly prove the two charge separation mechanisms. Engineering the direction of energy flow at the nanoscale can provide an efficient way to overcome important challenges in photocatalysis, such as controlling catalytic activity and selectivity. These results bear enormous potential impact on the development of better visible light photocatalysts for solar-to-chemical energy conversion.
- Ha, Ji Won,Ruberu, T. Purnima A.,Han, Rui,Dong, Bin,Vela, Javier,Fang, Ning
-
-
Read Online
- Major involvement of rabbit liver cytochrome P4501A in thiabendazole 5-hydroxylation
-
1. Thiabendazole is a widely used food preservative and anthelmintic drug for breeding animal species. In order to characterize precisely the cytochrome P450 isozyme(s) involved in its major route of metabolism, a rapid and sensitive spectrofluorimetric method was developed for the simultaneous determination of thiabendazole and its main hepatic metabolite 5-hydroxythiabendazole. 2. The kinetics of thiabendazole 5-hydroxylation were determined in microsomal preparations from control rabbits or animals previously treated with either β-naphthoflavone, isosafrole, phenobarbital, rifampicin or clofibrate. These treatments led to specific induction of CYP1A1, 1A2, 2B4, 3A6 and 4A1 respectively. 3. By considering this panel of characterised microsomal preparations, only those obtained from BNF-treated rabbits exhibited an increase in thiabendazole 5-hydroxylase activity. Ethoxyresorufin O-deethylation in these microsomes was solely inhibited by thiabendazole. These argue for a specific involvement of the CYP1A, subfamily. 4. In the CYP1A subfamily, CYP1A2 appears to be responsible for basal 5-hydroxylation and further unidentified metabolism of thiabendazole in control livers. However, the major involvement of CYP1A1 is supported by the following characteristics of 5-hydroxylation of thiabendazole: (1) the correlation with CYP1A1 expression and (2) the inhibition by ellipticine and not by furafylline, inhibitors of CYP1A1 and CYP1A2 respectively. 5. All these data demonstrated that the rabbit cytochrome P4501A is predominantly involved in thiabendazole 5-hydroxylation which has been suspected to be critical in terms of safety of the parent drug.
- Rey-Grobellet,Eeckhoutte,Sutra,Alvinerie,Galtier
-
-
Read Online
- Cytochrome P4501A (CYP1A) induction in rat and man by the benzodioxino derivative, fluparoxan
-
1. Fluparoxan is an α2-adrenoceptor antagonist that has a relatively planar, tricyclic structure and was considered a potential substrate and inducer of cytochrome P4501A (CYP1A) enzymes. 2. Structure-activity analysis indicated some potential for CYP1A interaction, although its greater log P and molecular depth, compared with many CYP1A inducers, suggested fluparoxan would be a weak ligand for the aryl hydrocarbon (Ah) receptor and only a weak inducer. 3. In vitro, fluparoxan showed little affinity for the CYP1A enzymes. The compound was not metabolized by human CYP1A1 or 1A2 heterologously expressed in yeast and its rate of metabolism in rat and human microsomes was unaffected by the addition of the 1A inhibitor α-naphthoflavone. Furthermore K(i)'s for fluparoxan against EROD activity were > 4000-fold higher than those of α-naphthoflavone. 4. In vivo, however, fluparoxan did show some capacity for CYP1A induction. In rat, hepatic EROD activity increased approximately 40-fold with seven once-daily oral doses of fluparoxan (50 mg/kg, solution), and immunoblotting studies confirmed induction of CYP1A2, though not of 1A1. In man, administration of 11 twice-daily oral doses of fluparoxan (8 mg tablet) produced some reduction in plasma levels of orally administered phenacetin and in the ratio of phenacetin AUC/urinary paracetamol, consistent with increased O-deethylation.
- Beresford,Ellis,Ayrton,Johnson,Lewis
-
-
Read Online
- Antioxidant capacity of two novel bioactive Fe(III)-cyclophane complexes
-
The cyclophanes 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7, 10, 24,27,30,33-octaaza-17,40-dioxa[10.1.10.1]paracyclophane (PO) and 2,9,25,32-tetraoxo- 4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33- octaaza[10.1.10.1]paracyclophane
- Salazar-Medina, Alex J.,Sugich-Miranda, Rocio,Teran-Cabanillas, Eli,Hernandez, Jesus,Gonzalez-Aguilar, Gustavo A.,Rudino-Pinera, Enrique,Sotelo-Mundo, Rogerio R.,Velazquez-Contreras, Enrique F.
-
-
Read Online
- Silica nanoparticle-based microfluidic immunosensor with laser-induced fluorescence detection for the quantification of immunoreactive trypsin
-
The purpose of this study was to develop a silica nanoparticle-based immunosensor with laser-induced fluorescence (LIF) as a detection system. The proposed device was applied to quantify the immunoreactive trypsin (IRT) in cystic fibrosis (CF) newborn screening. A new ultrasonic procedure was used to extract the IRT from blood spot samples collected on filter papers. After extraction, the IRT reacted immunologically with anti-IRT monoclonal antibodies immobilized on a microfluidic glass chip modified with 3-aminopropyl functionalized silica nanoparticles (APSN-APTES-modified glass chips). The bounded IRT was quantified by horseradish peroxidase (HRP)-conjugated anti-IRT antibody (anti-IRT-Ab) using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as enzymatic mediator. The HRP catalyzed the oxidation of nonfluorescent ADHP to highly fluorescent resorufin, which was measured by LIF detector, using excitation lambda at 561 nm and emission at 585 nm. The detection limits (LODs) calculated for LIF detection and for a commercial enzyme-linked immunosorbent assay (ELISA) test kit were 0.87 and 4.2 ng ml-1, respectively. The within- and between-assay variation coefficients for the LIF detection procedure were below 6.5%. The blood spot samples collected on filter papers were analyzed with the proposed method, and the results were compared with those of the reference ELISA method, demonstrating a potential usefulness for the clinical assessment of IRT during the early neonatal period.
- Seia, Marco A.,Stege, Patricia W.,Pereira, Sirley V.,De Vito, Irma E.,Raba, Julio,Messina, Germn A.
-
-
Read Online
- Measurement of enzyme kinetics using a continuous-flow microfluidic system
-
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 from the standard Michaelis-Menten treatment. The continuous-flow method required ～10μL of substrate solution and 109 enzyme molecules. 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.
- Seong, Gi Hun,Heo, Jinseok,Crooks, Richard M.
-
-
Read Online
- A resorufin-based red-emitting fluorescent probe with high selectivity for tracking endogenous peroxynitrite in living cells and inflammatory mice
-
Peroxynitrite (ONOO?) plays essential roles on various physiological and pathological processes of living systems as a short-lived and highly reactive nitrogen (RNS) specie. The construction of novel long-wavelength fluorescent probes with high specificity towards ONOO? for imaging in vivo is still demand urgently. About this work, a novel resorufin-based red-emitting fluorescent probe for tracking ONOO? has been constructed. The probe RFP exhibited high selectivity towards ONOO? anion over other analytes. Utilizing the probe, ONOO? could be directly observed by the naked eye. Furthermore, RFP was successfully applied for imaging endogenous ONOO– in RAW264.7 cells and inflammatory mice. This work offers a convenient method for monitoring the intercellur ONOO– that be expected to be applied for explaining the bio-functional roles of ONOO? in living system.
- Su, Huihui,Wang, Nannan,Wang, Jiamin,Wang, Han,Zhang, Jian,Zhao, Weili
-
-
Read Online
- Inhibition of human cytochrome P450 enzymes by 1,2-dithiole-3-thione, oltipraz and its derivatives, and sulforaphane
-
Recent studies have demonstrated that two chemoprotective agents, oltipraz (OPZ), a synthetic derivative of the natural compound 1,2-dithiole- 3-thione (D3T), and sulforaphane (SF), an isothiocyanate, are not only inducers of glutathione S-transferases but also inhibitors of some major cytochrome P450 enzymes (P450s) involved in xenobiotic metabolism. We examined P450 inhibition by the two compounds and compared two OPZ metabolites (OPZ M3 and M8) and D3T using human P450s expressed in Escherichia coli membranes. OPZ was a more potent inhibitor than D3T or SF, in the following order of inhibition: P450 1A2 > 3A4 > 1A1 ~ 1B1 > 2E1. OPZ M3 also inhibited P450s 1A2, 1A1, 1B1, and 3A4 but not more effectively than OPZ. OPZ Ms was not inhibitory. OPZ behaved as a competitive inhibitor of P450 1A2, with a K(i) of 1.5 μM. Incubation of P450 1A2 with OPZ and NADPH led to a first-order loss of the P450 spectrum, and the loss was not blocked by glutathione. No such time-dependent loss of P450 was seen with P450 1A2 and D3T, P450 1A2 and OPZ M3, P450 1A2 and SF, P450 3A4 and OPZ, P450 3A4 and D3T, P450 2E1 and OPZ, or P450 2E1 and D3T. The time- and concentration- dependent loss of P450 1A2 activity in the presence of OPZ was characterized with a K(i) of 9 μM and a k(inactivation) of 0.19 min-1. The activation of 2-amino-3,5- dimethylimidazo[4,5-f]quinoline (MeIQ) in an E. coli lac-based mutagenicity tester system containing functional human P450 1A2 was inhibited by OPZ (IC50 1 μM) but not appreciably by 40 μM D3T. Our results indicate that OPZ is a competitive and mechanism-based inhibitor of P450 1A2, and the extent of this inhibition is significantly greater than that of other chemoprotective chemicals with P450 1A2 or other human P450s.
- Langouet, Sophie,Furge, Laura Lowe,Kerriguy, Nathalie,Nakamura, Katsunori,Guillouzo, Andre,Guengerich, F. Peter
-
-
Read Online
- Resazurin as an electron acceptor in glucose oxidase-catalyzed oxidation of glucose
-
The behavior of resazurin (1) as an electron acceptor in glucose oxidase (GOD)-catalyzed oxidation of glucose under anaerobic conditions is described. When a mixture of 1, glucose, and GOD in phosphate buffer (pH 7.4, 0.1 M) was incubated at 25°C, the resulting solution turned purple to fluorescent pink due to the deoxygenated product, resorufin (2). On incubation of 1 with GOD alone or with H2O2 under essentially the same conditions, no color change was seen, indicating that generation of 2 in the enzymatic reaction is brought about through reduction of 1 by the reduced form (GODred) of GOD, which was also supported by the voltammetric behavior of 1. However, it was found that the enzymatic transformation of I to 2 is of no practical use as an indicator reaction for glucose determination using only GOD due to a slow reaction of 1 with GODred. Based on a ping-pong type mechanism with a steady-state approximation, KM and kcat for 1 as an electron acceptor from GODred were estimated to be 15±1.3 μM and (5.0±0.5)×10-2s-1, respectively.
- Maeda,Matsu-Ura,Yamauchi,Ohmori
-
-
Read Online
- Facet Effects of Ag3PO4 on Charge-Carrier Dynamics: Trade-Off Between Photocatalytic Activity and Charge-Carrier Lifetime
-
Silver phosphate (Ag3PO4) is a promising visible-light-driven photocatalyst with a strong oxidation power and exceptionally high apparent quantum yield of O2 evolution. Although engineering Ag3PO4 facets is widely known to enhance its photocatalytic activity, most studies have explained its facet effect by calculating surface energies. Herein, the charge carrier dynamics in three kinds of Ag3PO4 crystals (mixed facets, cubic, and tetrahedral structures) were first investigated using single-particle photoluminescence microscopy and femtosecond time-resolved diffuse reflectance spectroscopy. As a result, we clarified that the disagreement between the photocatalytic activities (dye degradation and O2 evolution) of different Ag3PO4 facets are the consequence of trade-off between catalytic activity and lifetime of photogenerated charge carriers in addition to surface energy.
- Kim, Sooyeon,Wang, Yue,Zhu, Mingshan,Fujitsuka, Mamoru,Majima, Tetsuro
-
-
Read Online
- Single-Molecular Catalysis Identifying Activation Energy of the Intermediate Product and Rate-Limiting Step in Plasmonic Photocatalysis
-
Plasmon-mediated photocatalysis provides a novel strategy for harvesting solar energy. Identification of the rate-determining step and its activation energy in plasmon-mediated photocatalysis plays critical roles for understanding the contribution of hot carriers, which facilitates rational designation of catalysts with integrated high photochemical conversion efficiency and catalytic performance. However, it remains a challenge due to a lack of research tools with spatiotemporal resolution that are capable of capturing intermediates. In this work, we used a single-molecule fluorescence approach to investigate a localized surface plasmon resonance (LSPR)-enhanced photocatalytic reaction with subturnover resolution. By introducing variable temperature as an independent parameter in plasmonic photocatalysis, the activation energies of tandem reaction steps, including intermediate generation, product generation, and product desorption, were clearly differentiated, and intermediate generation was found to be the rate-limiting step. Remarkably, the cause of the plasmon-enhanced catalysis performance was found to be its ability of lowering the activation energy of intermediate generation. This study gives new insight into the photochemical energy conversion pathways in plasmon-enhanced photocatalysis and sheds light on designing high-performance plasmonic catalysts.
- Li, Di,Li, Kun,Li, Wei,Lv, Hui,Miao, Junjian,Peng, Tianhuan,Wang, Jun-Gang,Zhu, Ying
-
-
Read Online
- Hydrogel-Immobilized Coacervate Droplets as Modular Microreactor Assemblies
-
Immobilization of compartmentalized microscale objects in 3D hydrogels provides a step towards the modular assembly of soft functional materials with tunable architectures and distributed functionalities. Herein, we report the use of a combination of micro-compartmentalization, immobilization, and modularization to fabricate and assemble hydrogel-based microreactor assemblies comprising millions of functionalized polysaccharide–polynucleotide coacervate droplets. The heterogeneous hydrogels can be structurally fused by interfacial crosslinking and coupled as input and output modules to implement a UV-induced photocatalytic/peroxidation nanoparticle/DNAzyme reaction cascade that generates a spatiotemporal fluorescence read-out depending on the droplet number density, intensity of photoenergization, and chemical flux. Our approach offers a route to heterogeneous hydrogels with endogenous reactivity and reconfigurable architecture, and provides a step towards the development of soft modular materials with programmable functionality.
- Li, Mei,Liu, Jianbo,Mann, Stephen,Patil, Avinash J.,Qiao, Yan,Tian, Liangfei,Wang, Kemin,Zhou, Shaohong
-
-
Read Online
- Rapid analysis of human fecal bile acids
-
A rapid, accurate, precise method for determining human fecal bile acids is reported. Feces are homogenized and then briefly extracted with boiling absolute ethanol. A portion of the extract is evaporated to dryness and the residue heated with mild alkali to hydrolyze bile acid 3α-hydroxyl esters. Aliquots of hydrolyzed crude extract are treated with resazurin reagent which effects a series of enzyme catalyzed reactions in which bile acid free 3α-hydroxyls are first oxidized to 3-oxo-groups in a reaction catalyzed by 3α-hydroxysteroid dehydrogenase. Resulting protons are transferred to β-nicotinamide adenine dinucleotide, yielding reduced β-nicotinamide adenine dinucleotide (β-NADH). β-NADH then reduces nonfluorescent resazurin to fluorescent resorufin in a reaction catalyzed by diaphorase. Developed fluorescence, which is proportional to the extract aliquots bile acid content, is excited at 565 nm and read at 580 nm, wavelengths which lie in a spectral region in which there is minimal fecal pigment absorption. 3-Oxo-bile acids and bile acid 3α-sulfates are extracted in the procedure but reduction and/or solvolysis is necessary before quantification.
- Beher,Stradnieks,Lin,Sanfield
-
-
Read Online
- Biologically relevant chemical properties of peroxymonophosphate ({double bond, long}O3POOH)
-
It has been suggested that peroxymonophosphate could serve as an endogenous hydrogen peroxide-derived regulator of cellular protein tyrosine phosphatase activity under physiological or pathophysiological conditions. To facilitate further consideration of
- LaButti, Jason N.,Gates, Kent S.
-
-
Read Online
- Hydroxyl radical and free and shallowly trapped electron generation and electron/hole recombination rates in TiO2 photocatalysis using different combinations of anatase and rutile
-
Hydroxyl radical (OH[rad]) and electron (e?) generation in aqueous suspensions during TiO2 photocatalysis was studied, varying the properties of the photocatalyst and using the molecular probes terephthalic acid (TA) for OH[rad] and resazurin dye (Rz) for electrons. Overall photocatalytic activity was calculated from rhodamine B dye degradation. The percentage of e? recombination was found as the difference between the results for aqueous Rz in the presence and absence of a hole scavenger (glycerol). The findings show that the properties of the TiO2 particles affect e? and OH[rad] generation rates differently. The generation rates of these active species and their overall photocatalytic activity were observed to be heavily dependent upon the physical properties of the TiO2 used and the recombination percentage.
- Jimenez-Relinque, Eva,Castellote, Marta
-
-
Read Online
- The excited-state interaction of resazurin and resorufin with amines in aqueous solutions. Photophysics and photochemical reactions.
-
The photophysics and photochemical behavior of the phenoxazin-3-one dyes, resazurin and resorufin, have been studied in aqueous solutions. The irradiation of resazurin in the presence of amines leads to deoxygenation of the N-oxide group, giving resorufin. This photoreaction is highly dependent on the amine structure and is efficient only in the presence of tertiary aliphatic amines. The absorption and fluorescence properties of these dyes are dependent on pH. At pH above 7.5 both dyes are in their anionic form. For resorufin this form is highly fluorescent (phiF = 0.75). At lower pH the fluorescence is strongly reduced. The N-oxide dye presents a very weak fluorescence quantum yield (0.11), which also is reduced at low pH. Flash photolysis experiments allowed characterization of the triplet state and the transients formed after irradiation of these dyes in the absence and presence of amines. The triplet quantum yields are 0.08 for resazurin and 0.04 for resorufin. The photodeoxygenation of N-oxide in the presence of amines occurs from the triplet state.
- Bueno,Villegas,Bertolotti,Previtali,Neumann,Encinas
-
-
Read Online
- Effects of molecular confinement and crowding on horseradish peroxidase kinetics using a nanofluidic gradient mixer
-
The effects of molecular confinement and crowding on enzyme kinetics were studied at length scales and under conditions similar to those found in biological cells. These experiments were carried out using a nanofluidic network of channels constituting a nanofluidic gradient mixer, providing the basis for measuring multiple experimental conditions simultaneously. The 100 nm × 40 μm nanochannels were wet etched directly into borosilicate glass, then annealed and characterized with fluorescein emission prior to kinetic measurements. The nanofluidic gradient mixer was then used to measure the kinetics of the conversion of the horseradish peroxidase (HRP)-catalyzed conversion of non-fluorescent Amplex Red (AR) to the fluorescent product resorufin in the presence of hydrogen peroxide (H2O2). The design of the gradient mixer allows reaction kinetics to be studied under multiple (five) unique solution compositions in a single experiment. To characterize the efficiency of the device the effects of confinement on HRP-catalyzed AR conversion kinetics were studied by varying the starting ratio of AR:H2O2. Equimolar concentrations of Amplex Red and H2O2 yielded the highest reaction rates followed by 2:1, 1:2, 5:1, and finally 1:5 [AR]:[H2O2]. Under all conditions, initial reaction velocities were decreased by excess H2O2. Crowding effects on kinetics were studied by increasing solution viscosity in the nanochannels in the range 1.0-1.6 cP with sucrose. Increasing the solution viscosities in these confined geometries decreases the initial reaction velocity at the highest concentration from 3.79 μM min-1 at 1.00 cP to 0.192 μM min-1 at 1.59 cP. Variations in reaction velocity are interpreted in the context of models for HRP catalysis and for molecular crowding.
- Wichert, William R. A.,Han, Donghoon,Bohn, Paul W.
-
-
Read Online
- A GGCT fluorogenic probe: Design, synthesis and application to cancer-related cells
-
Cancer-related γ-glutamyl cyclotransferase (GGCT) specifically converts γ-glutamyl amino acids (γ-Glu-Xaa) into pyroglutamate and the corresponding amino acids (Xaa). Here we report a novel GGCT fluorogenic probe LISA-101 containing a masked O-acylated fluorophore resorufin on the side chain of the P′1 amino acid (Xaa). Upon GGCT treatment, the P′1 amino acid was liberated and spontaneously released the intact fluorophore. Thus, the fluorescence was regained. LISA-101 will expand the strategies for cancer studies.
- Yoshiya, Taku,Ii, Hiromi,Tsuda, Shugo,Kageyama, Susumu,Yoshiki, Tatsuhiro,Nishiuchi, Yuji
-
-
Read Online
- Design of fluorogenic probes and fluorescent-tagged inhibitors for γ-glutamyl cyclotransferase
-
A tumor-related protein, human chromosome 7 ORF 24 (C7orf24), is involved in regulation of the glutathione homeostasis cycle as a γ-glutamyl cyclotransferase (GGCT). The singular substrate preference of this enzyme had long hampered its chemical probe development. That is, substrate of GGCT is definitely ‘γ-Glu-Xaa’, where Xaa is an L-α-amino acid. Based on the structure of substrates and GGCT fluorogenic probes, LISA-4/101, we succesfully developed a fluorescent-tagged inhibitor, gKFA. These chemical tools will assist cancer-related researches in the future.
- Yoshiya, Taku,Ii, Hiromi,Tsuda, Shugo,Mochizuki, Masayoshi,Kageyama, Susumu,Yoshiki, Tatsuhiro
-
-
Read Online
- Multiplex analysis of enzyme kinetics and inhibition by droplet microfluidics using picoinjectors
-
Enzyme kinetics and inhibition is important for a wide range of disciplines including pharmacology, medicine and industrial bioprocess technology. We present a novel microdroplet-based device for extensive characterization of the reaction kinetics of enzyme substrate inhibitor systems in a single experiment utilizing an integrated droplet picoinjector for bioanalysis. This device enables the scanning of multiple fluorescently-barcoded inhibitor concentrations and substrate conditions in a single, highly time-resolved experiment yielding the Michaelis constant (Km), the turnover number (kcat) and the enzyme inhibitor dissociation constants (ki, k i′). Using this device we determine Km and k cat for β-galactosidase and the fluorogenic substrate Resorufin β-d-galactopyranoside (RBG) to be 442 μM and 1070 s-1, respectively. Furthermore, we examine the inhibitory effects of isopropyl-β-d-thiogalactopyranoside (IPTG) on β-galactosidase. This system has a number of potential applications, for example it could be used to screen inhibitors to pharmaceutically relevant enzymes and to characterize engineered enzyme variants for biofuels production, in both cases acquiring detailed information about the enzyme catalysis and enzyme inhibitor interaction at high throughput and low cost.
- Sjostrom, Staffan L.,Joensson, Haakan N.,Svahn, Helene Andersson
-
-
Read Online
- PVP stabilized Pt nano particles catalyzed de-oxygenation of phenoxazine group by hydrazine in physiological buffer media: Surfactant competes with reactants for the same surface sites
-
PVP capped platinum nano particles (PNP) of 5 nm diameter were prepared and characterized as homogeneous and of spherical nature. At physiological pH range (6.0-8.0), these PNP catalyze the deoxygenation of phenoxazine group containing resazurin (1) by hydrazine. The observed rate constants (ko), increase linearly with [PNP] at constant [1] and [Hydrazine]; but first increase and then after reaching a maximum it decrease with increase in [1] as well as in [Hydrazine]. The ko values increase linearly with 1/[H +] indicating N2H4 as the reducing species that generates from the PNP assisted deprotonation of N2H 5+. The kinetic observations suggest Langmuir-Hinshelwood type surface reaction mechanism where both 1 and hydrazine are adsorbed on nano particles surface and compete for the same sites. Interestingly, the surfactant molecules, polyvinylpyrrolidone (PVP), though do not take part into reduction reaction but having same type of functional groups as reactants, competes with them for the same surface sites. Adsorption on PNP with same type of functional group is further supported by the FTIR spectra of Pt-PVP and Pt-1. Thus on increasing [PVP], ko decreases linearly and only when [PVP] is held constant, the plot of kovs. [PNP] passes through the origin indicating the insignificance of uncatalyzed reaction. The plot of ln k ovs. [1] or [Hydrazine] shows two different linear zones with different exponent values with respect to [1] and [Hydrazine]. This indicates that along with the complex heterogeneous surface adsorption processes, the mutual interactions between the reactants are also changing with the relative concentrations of reactants or, in general, with the molar ratio ([Hydrazine]/[1]).
- Das, Ranendu Sekhar,Singh, Bula,Banerjee, Rupendranath,Mukhopadhyay, Subrata
-
-
Read Online
- Laser-induced mixing in microfluidic channels
-
We demonstrate a novel strategy for mixing solutions and initiating chemical reactions in microfluidic systems. This method utilizes highly focused nanosecond laser pulses from a Q-switched Nd:YAG laser at λ = 532 nm to generate cavitation bubbles within 100- and 200-μm-wide microfluidic channels containing the parallel laminar flow of two fluids. The bubble expansion and subsequent collapse within the channel disrupts the laminar flow of the parallel fluid streams and produces a localized region of mixed fluid. We use time-resolved imaging and fluorescence detection methods to visualize the mixing process and to estimate both the volume of mixed fluid and the time scale for the re-establishment of laminar flow. The results show that mixing is initiated by liquid jets that form upon cavitation bubble collapse and occurs ～20 μs following the delivery of the laser pulse. The images also reveal that mixing occurs on the millisecond time scale and that laminar flow is re-established on a 50-ms time scale. This process results in a locally mixed fluid volume in the range of 0.5-1.5 nL that is convected downstream with the main flow in the microchannel. We demonstrate the use of this mixing technique by initiating the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and nonfluorescent N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) to yield fluorescent resorufin. This approach to generate the mixing of adjacent fluids may prove advantageous in many microfluidic applications as it requires neither tailored channel geometries nor the fabrication of specialized on-chip instrumentation.
- Hellman, Amy N.,Rau, Kaustubh R.,Yoon, Helen H.,Bae, Stephanie,Palmer, James F.,Phillips, K. Scott,Allbritton, Nancy L.,Venugopalan, Vasan
-
-
Read Online
- Modification of the heme active site to increase the peroxidase activity of thermophilic cytochrome P450: A rational approach
-
The site specific mutants of the thermophilic P450 (P450 175A1 or CYP175A1) were designed to introduce residues that could act as acid-base catalysts near the active site to enhance the peroxidases activity. The Leu80 in the distal heme pocket of CYP175A1 was located at a position almost equivalent to the Glu183 that is involved in stabilization of the ferryl heme intermediate in chloroperoxidase (CPO). The Leu80 residue of CYP175A1 was mutated with histidine (L80H) and glutamine (L80Q) that could potentially form hydrogen bond with hydrogen peroxide and facilitate formation and stabilization of the putative redox intermediate of the peroxidase cycle. The mutants L80H and L80Q of CYP175A1 showed higher peroxidase activity compared to that of the wild type (WT) CYP175A1 enzyme at 25°C. The activity constants (kcat) for the L80H and L80Q mutants of CYP175A1 were higher than those of myoglobin and wild type cytochrome b562 at 25°C. The optimum temperature for the peroxidase activity of the WT and mutants of CYP175A1 was ~70°C. The rate of catalysis at temperatures above ~70°C was higher for L80Q mutant of CYP175A1 compared to that of the well known natural peroxidase, horseradish peroxidase (HRP) that denatures at such high temperature. The peroxidase activities of the mutants of CYP175A1 were maximum at pH 9, unlike that of HRP which is at pH ~5. The results have been discussed in the light of understanding the structure-function relationship of the peroxidase properties of these thermostable heme proteins.
- Behera, Rabindra Kumar,Goyal, Sandeep,Mazumdar, Shyamalava
-
-
Read Online
- Highly active fluorogenic oxidase-mimicking NiO nanozymes
-
Oxidase-mimicking nanozymes are attractive since they do not require H2O2, but such examples are quite rare. In particular, few can catalyze oxidation of fluorogenic substrates. We herein communicate that NiO nanoparticles are an oxi
- Li, Dai,Liu, Biwu,Huang, Po-Jung Jimmy,Zhang, Zijie,Liu, Juewen
-
-
Read Online
- Multifunctional linker for orthogonal decoration of gold nanoparticles with DNA and protein
-
A novel trifunctional linker was prepared to allow multiple orthogonal functionalization of gold nanoparticles (AuNPs) with biomolecules. Thiooctic groups contained within the linker act as AuNP surface anchors, while two additional orthogonal functional groups are used for attachment of heme and DNA using amide and copper catalyzed Huisgen cycloaddition, respectively. We demonstrate that heme can act as a platform for reconstitution of a functional heme protein such as myoglobin. Using multifunctional linker, fully functional Au-DNA-Mb hybrids were obtained and such multifunctional constructs expand the synthetic toolbox for nanomaterial tailoring and design of biosensors and novel catalytic materials.
- Kendziora, Dania M.,Ahmed, Ishtiaq,Fruk, Ljiljana
-
-
Read Online
- Immobilization of iron hydroxide/oxide on reduced graphene oxide: Peroxidase-like activity and selective detection of sulfide ions
-
We prepared nanocomposites of amorphous iron hydroxide/oxide immobilized on reduced graphene oxide (FeOxH-rGO) with peroxidase-like activity for the detection of sulfide (S2-) ions. FeOxH-rGO nanocomposites were prepared by reaction of GO (size ～ 300 nm) partially reduced by ultraviolet irradiation with Fe2+in Tris-borate solution (5.0 mM, pH 7.0). The amorphous FeO(OH) and Fe(OH)2were immobilized on rGO to form FeOxH-rGO nanocomposites. The as-prepared FeOxH-rGO nanocomposites exhibited peroxidase-like catalytic activity in the H2O2-mediated oxidation of Amplex Red (AR) to fluorescent resorufin. Our AR/FeOxH-rGO probe allowed the detection of H2O2down to 50 nM within 10 min under microwave irradiation (170 W). The catalytic activity of FeOxH-rGO was significantly suppressed in the presence of S2-because of the formation of FeS on the FeOxH-rGO nanocomposites' surfaces. The H2O2/AR-FeOxH-rGO probe provided a limit of detection (signal-to-noise ratio = 3) of 50 nM for S2-with high selectivity (>100-fold) with respect to other anions. Taking advantage of their high stability and selectivity, we employed our H2O2/AR-FeOxH-rGO probe for the detection of S2-in hot spring samples (75.1-619.5 μM) and the results showed good correlation (r = 0.98) with results from inductively coupled plasma mass spectrometry. This label-free, rapid, and simple sensing system shows great potential for the detection of S2-ions in real samples. This journal is
- Hsu, Kuang-I,Lien, Chia-Wen,Lin, Chia-Hua,Chang, Huan-Tsung,Huang, Chih-Ching
-
-
Read Online
- Selective fluorometric detection of aromatic thiols by a chemosensor containing two electrophilic sites with different local softness
-
A resorufin-dinitrophenyl ether conjugate (1) shows emission enhancement for aromatic thiols in aqueous media with a neutral-basic pH, while being nonemissive for aliphatic thiols. This is achieved by two electrophilic sites with different local softness on compound 1; the respective sites selectively react with aromatic or aliphatic thiolate anions.
- Shiraishi, Yasuhiro,Yamamoto, Kohei,Sumiya, Shigehiro,Hirai, Takayuki
-
-
Read Online
- Identification of intermediates in peroxidase catalytic cycle of a DNAzyme possessing heme
-
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.
- Shinomiya, Ryosuke,Araki, Haruka,Momotake, Atsuya,Kotani, Hiroaki,Kojima, Takahiko,Yamamoto, Yasuhiko
-
-
Read Online
- Hydrogen peroxide supports human and rat cytochrome P450 1A2-catalyzed 2-amino-3-methylimidazo[4,5-f]quinoline bioactivation to mutagenic metabolites: Significance of cytochrome P450 peroxygenase
-
We show that the naturally occurring hydroperoxide hydrogen peroxide is highly effective in supporting the cytochrome P450 1A2 peroxygenase-catalyzed metabolic activation of the heterocyclic aromatic amine 2-amino-3- methylimidazo[4,5-f]quinoline (IQ) to genetoxic metabolites. Mutagenicity was assessed by the Ames assay with Salmonella typhimurium strain YG1012 and an activation system consisting of hydroperoxides plus either 3- methylcholanthrene-induced rat liver microsomes (rP4501A) or human P450 1A2- containing microsomes (hP4501A2). The mutagenic response was dependent on the concentration of microsomal protein, IQ, and hydroperoxides. The addition of hydrogen peroxide or tert-butyl hydroperoxide to rP4501A greatly enhanced the yield of histidine prototrophic (His+) revertants. This increase was inhibited, in a concentration-dependent manner, by α-naphthoflavone, a P450 1A inhibitor. Hydrogen peroxide was the most effective peroxygenase cofactor, particularly with hP4501A2 (K(m) =0.1 μM). The hydroperoxide-supported activation of IQ produced reactive intermediates which bound to 2'- deoxyguanosine; LC/MS analysis of the adducts revealed the same major (protonated) adduct at m/z = 464.4 as previously reported for the DNA adduct formed (in vivo or in vitro) by the mixed function-catalyzed bioactivation system. None of the peroxidase-catalyzed IQ metabolites (nitro-, azo-, or azoxy-IQ) were detected. In conclusion, hydrogen peroxide in the physiological/pathological concentration range may be able to support the metabolic activation of arylamines to genotoxic products through the cytochrome P450 peroxygenase pathway.
- Anari, M. Reza,Josephy, P. David,Henry, Tracey,O'Brien, Peter J.
-
-
Read Online
- Effects of bioflavonoids on hepatic P450 activities
-
1. The effects of tangeretin, green tea flavonoids, and other flavonoids on 7-ethoxyresorufin-O-deethylase (EROD; 450 1A), 7-pentoxyresorufin-O-dealkylase (PROD; P450 2B), p-nitrophenol hydroxylase (PNPH, P450 2E1), and erythromycin-N-demethylase (ERDM; P450 3A) were examined in induced rat liver microsomes. EROD, PNPH, ERDM, and nifedipine oxidase (NIFO; P450 3A4) were examined in human liver microsomes. 2. All flavonoids tested inhibited EROD activity at higher concentrations in liver microsomes. Flavone and tangeretin were potent inhibitors of EROD, with IC(50's) of 0.7 and 0.8 μM respectively in rat liver microsomes and 0.15 and 16 μM respectively in human liver microsomes. The green tea flavonoid (-)-epicatechin-3-gallate (EGG) was the most potent inhibitor of EROD in human liver microsomes (IC50 = 75 μM). The effect of the green tea flavonoids on EROD was complex; in addition to inhibition at high concentrations of flavonoid, moderate activation was seen at lower concentrations. 3. 450 2B-, 2E1- and 3A-dependent activities in rat and human liver microsomes were only moderately inhibited by any of the flavonoids tested, and, in general, ECG was the most potent inhibitor for these activities with IC(50's) ranging from 75 to 300 μM. 4. Tangeretin inhibited EROD activity (P450 1A2) in human liver microsomes in a competitive manner with a K(i) = 68 nM. Tangeretin inhibited NIFO activity (P450 3A4) in human liver microsomes in an uncompetitive manner with K(i) = 72 μM.
- Obermeier,White,Yang
-
-
Read Online
- Silicalite-1/glass fibre substrates for enhancing the photocatalytic activity of TiO2
-
Silicalite-1 (S1) coatings were prepared on silica wool substrates by hydrothermal synthesis and subsequently immersed into a Ti-containing sol at a steady rate of 30 cm min-1. The material was annealed in a furnace at 90 ?°C for 2 h and 550 ?°C for 2 h to create a silica fibre core surrounded by concentric layers of silicalite-1 and TiO2. The resulting samples were characterised by X-ray diffraction (XRD), Raman spectroscopy, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and Brunauer-Emmett-Teller (BET) surface area measurement. The photocatalytic activity of the samples was evaluated using the intelligent ink test and during degradation of stearic acid under UVA light (?? = 365 nm). The new coated-fibres were shown to be substantially better photocatalysts than comparable TiO2 coatings on plain glass fibres. The TiO2/S1/glass fibres have potential use in air/water cleaning applications.
- Ozkan,Quesada-Cabrera,Parkin
-
-
Read Online
- Enzyme-Loaded Nanoreactors Enable the Continuous Regeneration of Nicotinamide Adenine Dinucleotide in Artificial Metabolisms
-
Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme for numerous biocatalytic pathways. While in nature, NAD+ is continuously regenerated from NADH by enzymes, all synthetic NAD+ regeneration strategies require a continuous supply of expensive reagents and generate byproducts, making these strategies unattractive. In contrast, we present an artificial enzyme combination that produces NAD+ from oxygen and water continuously; no additional organic substrates are required once a minimal amount pyruvate is supplied. Three enzymes, i.e., LDH, LOX, and CAT, are covalently encapsulated into a substrate-permeable silica nanoreactor by a mild fluoride-catalyzed sol–gel process. The enzymes retain their activity inside of the nanoreactors and are protected against proteolysis and heat. We successfully used NAD+ from the nanoreactors for the continuous production of NAD+ i) to sense glucose in artificial glucose metabolism, and ii) to reduce the non-oxygen binding methemoglobin to oxygen-binding hemoglobin. This latter conversion might be used for the treatment of Methemoglobinemia. We believe that this versatile tool will allow the design of artificial NAD+-dependent metabolisms or NAD+-mediated redox-reactions.
- Jo, Seong-Min,Wurm, Frederik R.,Landfester, Katharina
-
-
Read Online
- Synthetic Silica Nano-Organelles for Regulation of Cascade Reactions in Multi-Compartmentalized Systems
-
In eukaryotic cells, enzymes are compartmentalized into specific organelles so that different reactions and processes can be performed efficiently and with a high degree of control. In this work, we show that these features can be artificially emulated in
- Jiang, Shuai,Caire da Silva, Lucas,Ivanov, Tsvetomir,Mottola, Milagro,Landfester, Katharina
-
supporting information
(2021/12/30)
-
- Electrocatalytic water oxidation by a water-soluble copper complex with a pentadentate amine-pyridine ligand
-
A water-soluble copper complex with a diamine-tripyridine ligand was synthesized successfully and well characterized. It was found to be catalytically active for the water oxidation reaction under basic conditions. Based on the electrochemical test result, this copper complex displayed an apparent rate constant (kcat) of 0.81 s?1for the oxygen evolution reaction in 0.1 M phosphate buffer solution at pH 11.0. More importantly, the copper complex remained stable over 3 h of a bulk electrolysis experiment at 1.60 V with a Faradaic efficiency of 90.7% for O2evolution, and the decrement of current density was only 1.9%. These results suggest that the pentadentate copper complex is an efficient and durable homogeneous Earth-abundant electrocatalyst for water oxidation.
- Chen, Qi,Shen, Junyu,Wang, Jiayi,Xia, Xin,Xu, Ziyi,Yu, Kaishan,Zhang, Qijian,Zheng, Zilin
-
p. 10888 - 10895
(2021/08/17)
-
- Tuneable Time Delay in the Burst Release from Oxidation-Sensitive Polymersomes Made by PISA
-
Reactive polymersomes represent a versatile artificial cargo carrier system that can facilitate an immediate release in response to a specific stimulus. The herein presented oxidation-sensitive polymersomes feature a time-delayed release mechanism in an oxidative environment, which can be precisely adjusted by either tuning the membrane thickness or partial pre-oxidation. These polymeric vesicles are conveniently prepared by PISA allowing the straightforward and effective in situ encapsulation of cargo molecules, as shown for dyes and enzymes. Kinetic studies revealed a critical degree of oxidation causing the destabilization of the membrane, while no release of the cargo is observed beforehand. The encapsulation of glucose oxidase directly transforms these polymersomes into glucose-sensitive vesicles, as small molecules including sugars can passively penetrate their membrane. Considering the ease of preparation, these polymersomes represent a versatile platform for the confinement and burst release of cargo molecules after a precisely adjustable time span in the presence of specific triggers, such as H2O2 or glucose.
- Bellstedt, Peter,Brendel, Johannes C.,Festag, Grit,Gruschwitz, Franka V.,Hoeppener, Stephanie,Kuchenbrod, Maren T.,Sobotta, Fabian H.
-
supporting information
p. 24716 - 24723
(2021/10/14)
-
- Structure and inhibition of Cryptococcus neoformans sterylglucosidase to develop antifungal agents
-
Pathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3β-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3β-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1’s substrate specificity and enable the rational design of antifungal agents targeting Sgl1.
- Airola, Michael V.,Burke, John E.,Clement, Timothy,Del Poeta, Maurizio,Hoffmann, Reece M.,Kim, Jinwoo,Ojima, Iwao,Pereira de Sa, Nivea,Rizzo, Robert C.,Taouil, Adam
-
-
- A specifically triggered turn-on fluorescent probe platform and its visual imaging of HClO in cells, arthritis and tumors
-
Understanding disease-related processes at the molecular level is of great importance for the prevention and treatment of diseases. However, due to the lack of effective analytical tools, it is challenging to gain insight into the relationships between a specific bioactive molecule and the associated disease. Herein, a rapid turn-on resorufin-based fluorescent probe platform utilizing the HClO-specific oxidative cleavage of the amide was constructed, allowing the visualization of HClO in vitro and in vivo. These probes could quickly respond to HClO ( 50 s) with high selectivity and sensitivity (12–153 nM). The probe REClO-6 had the fastest response (30 s) and the highest sensitivity (12 nM), and was successfully used for the imaging of endogenous and exogenous HClO in cells and zebrafish. Notably, it was also successfully applied to the imaging of HClO in mouse arthritis and solid tumors. This study provided a rapid imaging analysis tool, which would be used to investigate the relationship between HClO and the disease-related physiological processes.
- Ding, Yufan,Hu, Chenxi,Li, Hui-Jing,Li, Qinghao,Wang, Xiao-Bo,Wu, Yan-Chao
-
-
- A molecular approach to rationally constructing specific fluorogenic substrates for the detection of acetylcholinesterase activity in live cells, mice brains and tissues
-
Acetylcholinesterase (AChE) is an extremely critical hydrolase tightly associated with neurological diseases. Currently, developing specific substrates for imaging AChE activity still remains a great challenge due to the interference from butyrylcholinesterase (BChE) and carboxylesterase (CE). Herein, we propose an approach to designing specific substrates for AChE detection by combining dimethylcarbamate choline with a self-immolative scaffold. The representative P10 can effectively eliminate the interference from CE and BChE. The high specificity of P10 has been proved via imaging AChE activity in cells. Moreover, P10 can also be used to successfully map AChE activity in different regions of a normal mouse brain, which may provide important data for AChE evaluation in clinical studies. Such a rational and effective approach can also provide a solid basis for designing probes with different properties to study AChE in biosystems and another way to design specific substrates for other enzymes. This journal is
- Wu, Xiaofeng,An, Jong Min,Shang, Jizhen,Huh, Eugene,Qi, Sujie,Lee, Eunhye,Li, Haidong,Kim, Gyoungmi,Ma, Huimin,Oh, Myung Sook,Kim, Dokyoung,Yoon, Juyoung
-
p. 11285 - 11292
(2020/11/04)
-
- Transition-Metal-Mediated versus Tetrazine-Triggered Bioorthogonal Release Reactions: Direct Comparison and Combinations Thereof
-
Bioorthogonal cleavage reactions are gaining popularity in chemically inducible prodrug activation and in the control of biomolecular functions. Despite similar applications, these reactions were developed and optimized on different substrates and under different experimental conditions. Reported herein is a side-by-side comparison of palladium-, ruthenium- and tetrazine-triggered release reactions, which aims at comparing the reaction kinetics, efficiency and overall advantages and limitations of the methods. In addition, we disclose the possibility of mutual combination of the cleavage reactions. Finally, we compare the efficiency of the bioorthogonal deprotections in cellular experiments, which revealed that among the three methods investigated, the palladium- and the tetrazine-promoted reaction can be used for efficient prodrug activation, but only the tetrazine-triggered reactions proceed efficiently inside cells.
- Dzijak, Rastislav,Mancuso, Francesca,Mertlíková-Kaiserová, Helena,Rahm, Michal,Vrabel, Milan
-
p. 1669 - 1675
(2020/09/02)
-
- Multiplex Optical Urinalysis for Early Detection of Drug-Induced Kidney Injury
-
Drug-induced kidney injury (DIKI) is a significant contributor of both acute and chronic kidney injury and remains a major concern in drug development and clinical care. However, current clinical diagnostic methods often fail to accurately and timely detect nephrotoxicity. This study reports the development of activatable molecular urinary reporters (MURs) that are able to specifically detect urinary biomarkers including γ-glutamyl transferase (GGT), alanine aminopeptidase (AAP), and N-acetyl-β-d-glucosaminidase (NAG). By virtue of their discrete absorption and emission properties, the mixture of MURs can serve as a cocktail sensor for multiplex optical urinalysis in the mouse models of drug-induced acute kidney injury (AKI) and chronic kidney disease (CKD). The MURs cocktail not only detects nephrotoxicity earlier than the tested clinical diagnostic methods in drug-induced AKI and CKD mice models, but also possesses a higher diagnostic accuracy. Therefore, MURs hold great promise for detection of kidney function in both preclinical drug screening and clinical settings.
- Cheng, Penghui,Huang, Jiaguo,Li, Jingchao,Miao, Qingqing,Pu, Kanyi
-
p. 6166 - 6172
(2020/05/19)
-
- Isonitrile-responsive and bioorthogonally removable tetrazine protecting groups
-
In vivo compatible reactions have a broad range of possible applications in chemical biology and the pharmaceutical sciences. Here we report tetrazines that can be removed by exposure to isonitriles under very mild conditions. Tetrazylmethyl derivatives are easily accessible protecting groups for amines and phenols. The isonitrile-induced removal is rapid and near-quantitative. Intriguingly, the deprotection is especially effective with (trimethylsilyl)methyl isocyanide, and serum albumin can catalyze the elimination under physiological conditions. NMR and computational studies revealed that an imine-tautomerization step is often rate limiting, and the unexpected cleavage of the Si-C bond accelerates this step in the case with (trimethylsilyl)methyl isocyanide. Tetrazylmethyl-removal is compatible with use on biomacromolecules, in cellular environments, and in living organisms as demonstrated by cytotoxicity experiments and fluorophore-release studies on proteins and in zebrafish embryos. By combining tetrazylmethyl derivatives with previously reported tetrazine-responsive 3-isocyanopropyl groups, it was possible to liberate two fluorophores in vertebrates from a single bioorthogonal reaction. This chemistry will open new opportunities towards applications involving multiplexed release schemes and is a valuable asset to the growing toolbox of bioorthogonal dissociative reactions.
- Tu, Julian,Svatunek, Dennis,Parvez, Saba,Eckvahl, Hannah J.,Xu, Minghao,Peterson, Randall T.,Houk,Franzini, Raphael M.
-
p. 169 - 179
(2019/12/30)
-
- Nitidine chloride-induced CYP1 enzyme inhibition and alteration of estradiol metabolism
-
The cytochrome P450 (P450) 1 family is an important phase I enzyme involved in carcinogen activation. Nitidine chloride (NC) is a pharmacologically active alkaloid with polyaromatic hydrocarbon found in the roots of Zanthoxylum nitidum (Roxb.) DC, a traditional medicinal herb widely used in China. We examined the inhibitory effects of NC on CYP1A1, 1B1, and 1A2. NC significantly inhibited CYP1A1- and 1B1-catalyzed ethoxyresorufin O-deethylation activity (IC50 5 0.28±0.06 and 0.32±0.02 mM, respectively) in a concentration- dependent manner, but only showed slight inhibition of CYP1A2 activity (IC50 > 50 mM). Kinetic analysis revealed that NC competitively inhibited CYP1B1 with a Ki value of 0.47±0.05 mM, whereas NC caused a mixed type of inhibition on CYP1A1 with Ki and KI values of 0.14±0.04 and 0.19±0.09 mM, respectively. The observed enzyme inhibition neither required NADPH nor revealed time dependency. Molecular docking manifested the generation of strong hydrogen-bonding interactions of Ser116 in CYP1A1 and Ser127 in CYP1B1 with methoxy moiety of NC. Additionally, NC-induced alteration of estradiol (E2) metabolism was also investigated in the present study. Hydroxyestradiols, including 2-hydroxyestradiol [(2-OHE2) nontoxic] and 4-hydroxyestradiol [(4-OHE2) genotoxic] generated in recombinant enzyme incubation systems and cultured MCF-7 cells were analyzed, and NC was found to preferentially inhibit the nontoxic 2-hydroxylation activity of E2 mediated by CYP1A1. In conclusion, NCwas a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. The remarkable inhibition on E2 2-hydroxylation might increase the risk of 4- OHE2-induced genotoxicity.
- Mao, Xu,Wang, Jian,Wang, Qian,Yang, Lan,Li, Yilin,Lin, Hao,Peng, Ying,Zheng, Jiang
-
p. 919 - 927
(2019/09/03)
-
- Identification and Directed Development of Non-Organic Catalysts with Apparent Pan-Enzymatic Mimicry into Nanozymes for Efficient Prodrug Conversion
-
Nanozymes, nanoparticles that mimic the natural activity of enzymes, are intriguing academically and are important in the context of the Origin of Life. However, current nanozymes offer mimicry of a narrow range of mammalian enzymes, near-exclusively performing redox reactions. We present an unexpected discovery of non-proteinaceous enzymes based on metals, metal oxides, 1D/2D-materials, and non-metallic nanomaterials. The specific novelty of these findings lies in the identification of nanozymes with apparent mimicry of diverse mammalian enzymes, including unique pan-glycosidases. Further novelty lies in the identification of the substrate scope for the lead candidates, specifically in the context of bioconversion of glucuronides, that is, human metabolites and privileged prodrugs in the field of enzyme-prodrug therapies. Lastly, nanozymes are employed for conversion of glucuronide prodrugs into marketed anti-inflammatory and antibacterial agents, as well as “nanozyme prodrug therapy” to mediate antibacterial measures.
- Walther, Raoul,Winther, Anna K.,Fruergaard, Anne Sofie,van den Akker, Wouter,S?rensen, Lise,Nielsen, Signe Maria,Jarlstad Olesen, Morten T.,Dai, Yitao,Jeppesen, Henrik S.,Lamagni, Paolo,Savateev, Aleksandr,Pedersen, S?ren Lykke,Frich, Camilla Kaas,Vigier-Carrière, Cécile,Lock, Nina,Singh, Mandeep,Bansal, Vipul,Meyer, Rikke L.,Zelikin, Alexander N.
-
supporting information
p. 278 - 282
(2018/12/05)
-
- 'AND'-based fluorescence scaffold for the detection of ROS/RNS and a second analyte
-
Traditionally, fluorescence probes have focused on the detection of a single biomarker for a specific process. In this work, we set out to develop a number of fluorescence probes that enable the detection of a chosen analyte in the presence of reactive oxygen/nitrogen species (ROS/RNS). These fluorescence probes when activated result in the formation of the highly fluorescent pink dye, resorufin. Therefore, we have labelled these fluorescent probes as -Pinkments'. Our first -Pinkment' was shown to detect biologically relevant concentrations of ONOO- and have an excellent selectivity against other ROS/RNS. Pinkment-OH was developed to provide a core unit which could be easily functionalised to produce a range of -AND' based fluorescence probes for the detection of ROS/RNS and a second analyte. For proof of concept, we synthesised Pinkment-OTBS and Pinkment-OAc. These -AND'-based probes were successfully shown to detect ROS/RNS and F- or esterase, respectively.
- Odyniec, Maria L.,Sedgwick, Adam C.,Swan, Alexander H.,Weber, Maria,Tang, T. M. Simon,Gardiner, Jordan E.,Zhang, Miao,Jiang, Yun-Bao,Kociok-Kohn, Gabriele,Elmes, Robert B. P.,Bull, Steven D.,He, Xiao-Peng,James, Tony D.
-
p. 8466 - 8469
(2018/08/04)
-
- Heme-thiolate sulfenylation of human cytochrome P450 4A11 functions as a redox switch for catalytic inhibition
-
Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear. Here, we found that reducing agents (e.g. dithiothreitol and tris(2-carboxyethyl) phosphine) strongly enhanced the catalytic activity of P450 4A11, but not of 10 other human P450s tested. Conversely, added H2O2 attenuated P450 4A11 catalytic activity. Catalytic roles of five of the potentially eight implicated Cys residues of P450 4A11 were eliminated by site-directed mutagenesis. Using an isotope-coded dimedone/iododimedone-labeling strategy and mass spectrometry of peptides, we demonstrated that the heme-thiolate cysteine (Cys-457) is selectively sulfenylated in an H2O2 concentration-dependent manner. This sulfenylation could be reversed by reducing agents, including dithiothreitol and dithionite. Of note, we observed heme ligand cysteine sulfenylation of P450 4A11 ex vivo in kidneys and livers derived from CYP4A11 transgenic mice. We also detected sulfenylation of murine P450 4a12 and 4b1 heme peptides in kidneys. To our knowledge, reversible oxidation of the heme thiolate has not previously been observed in P450s and may have relevance for 20-HETE-mediated functions.
- Albertolle, Matthew E.,Kim, Donghak,Nagy, Leslie D.,Yun, Chul-Ho,Pozzi, Ambra,Savas, üzen,Johnson, Eric F.,Guengerich, F. Peter
-
p. 11230 - 11242
(2017/08/08)
-
- Functional expression and comparative characterization of four feline P450 cytochromes using fluorescent substrates
-
1.?Cytochrome P450s (CYP) are a major group of metabolizing enzymes for xenobiotics in humans and other mammals. The properties of CYP isoforms in the domestic cat, an obligate carnivore, are largely unknown at present. In this study, we studied relative expression in tissues and enzymatic properties of nine significant feline CYP isoforms. 2.?CYP2E2 transcript was most abundant in the feline liver, followed by CYP2A13 and 2E1. Transcripts of CYP3A131, 1A2 and 1A1 were also present in the liver, while CYP2D6 and 3A132 were only slightly expressed. CYP3A131 was a major transcript in the small intestine. 3.?Four major CYP isoforms in the feline liver and small intestine (CYP1A2, CYP2A13, CYP2E2 and CYP3A131) were heterologously expressed in Escherichia coli to generate functional monooxygenase systems. We carried out screenings of 17 test compounds known to be inhibitors of CYP isoforms in other mammals as well as two anticancer drugs to assess the activity modulation of feline CYP isoforms using fluorogenic substrates. These CYP isoforms showed similar selectivity to counterparts in other mammals against inhibitors as a whole but with many exceptions. 4.?The present study suggests the usefulness of the feline CYP recombinant system to obtain chemical affinity information and possible drug interactions in CYP metabolism of domestic cats.
- Okamatsu, Gaku,Kawakami, Kei,Komatsu, Tetsuya,Kitazawa, Takio,Uno, Yasuhiro,Teraoka, Hiroki
-
p. 951 - 961
(2017/10/07)
-
- Efficient Construction of Well-Defined Multicompartment Porous Systems in a Modular and Chemically Orthogonal Fashion
-
A microfluidic assembly approach was developed for efficiently producing hydrogel spheres with reactive multidomains that can be employed as an advantageous platform to create spherical porous networks in a facile manner with well-defined multicompartments and spatiotemporally controlled functions. This strategy allows for not only large scale fabrication of various robust hydrogel microspheres with controlled size and porosity, but also the domains embedded in hydrogel network could be introduced in a modular manner. Additionally, the number of different domains and their ratio could be widely variable on demand. More importantly, the reactive groups distributed in individual domains could be used as anchor sites to further incorporate functional units in an orthogonal fashion, leading to well-defined multicompartment systems. The strategy provides a new and efficient route to construct well-defined functional multicompartment systems with great flexibility and extendibility.
- Gao, Ning,Tian, Tian,Cui, Jiecheng,Zhang, Wanlin,Yin, Xianpeng,Wang, Shiqiang,Ji, Jingwei,Li, Guangtao
-
supporting information
p. 3880 - 3885
(2017/03/27)
-
- Combinatorial Strategy to Identify Fluorescent Probes for Biothiol and Thiophenol Based on Diversified Pyrimidine Moieties and Their Biological Applications
-
We present a feasible paradigm of developing original fluorescent probes for target biomolecules via combinatorial chemistry. In this developmental program, pyrimidine moieties were investigated and optimized as unique recognition units for thiols for the first time through a parallel synthesis in combination with a rapid screening process. This time-efficient and cost-saving process effectively facilitated the developmental progress and provided detailed structure-reactivity relationships. As a result, Res-Biot and Flu-Pht were identified as optimal fluorescent probes for biothiol and thiophenol, respectively. Their favorable characteristics and superior applicability have been well demonstrated in both chemical and biological contexts. In particular, Res-Biot enables the direct visualization of biothiol fluctuations during oxidative stress and cell apoptosis, indicating its suitability in elucidation of a specific pathophysiological process in both living cells and living animals. Meanwhile, Flu-Pht is competent to visualize thiophenols without the interference from endogenous biothiols in living cells.
- Xie, Xilei,Li, Mengmeng,Tang, Fuyan,Li, Yong,Zhang, Leilei,Jiao, Xiaoyun,Wang, Xu,Tang, Bo
-
p. 3015 - 3020
(2017/08/09)
-
- Tetrazine-Responsive Self-immolative Linkers
-
Molecules that undergo activation or modulation following the addition of benign external small-molecule chemical stimuli have numerous applications. Here, we report the highly efficient “decaging” of a variety of moieties by activation of a “self-immolative” linker, by application of water-soluble and stable tetrazine, including the controlled delivery of doxorubicin in a cellular context.
- Neumann, Kevin,Jain, Sarthak,Gambardella, Alessia,Walker, Sarah E.,Valero, Elsa,Lilienkampf, Annamaria,Bradley, Mark
-
-
- TETRAZINE AS A TRIGGER TO RELEASE CAGED CARGO
-
There is presented a kit comprising a masked active agent and a tetrazine trigger, the masked active agent comprising an active agent connected to a masking moiety comprising a monovinyl ether or an active agent enclosed within a cage moiety comprising a monovinyl ether or an allyl group, wherein the tetrazine trigger is configured to release the active agent from the masking moiety or the cage moiety. Methods of preparing the masked active agents, and uses of the kit are also presented.
- -
-
Page/Page column 44-45
(2017/04/11)
-
- A Dual-Response Fluorescent Probe Reveals the H2O2-Induced H2S Biogenesis through a Cystathionine β-Synthase Pathway
-
The two signaling molecules H2S and H2O2 play key roles in maintaining intracellular redox homeostasis. The biological relationship between H2O2 and H2S remains largely unknown in redox biology. In this study, we rationally designed and synthesized single- and dual-response fluorescent probes for detecting both H2O2 and H2S in living cells. The dual-response probe was shown to be capable of mono- and dual-detection of H2O2 and H2S selectively and sensitively. Detailed bioimaging studies based on the probes revealed that both exogenous and endogenous H2O2 could induce H2S biogenesis in living cells. By using gene-knockdown techniques with bioimaging, the H2S biogenesis was found to be majorly cystathionine β-synthase (CBS)-dependent. Our finding shows the first direct evidence on the biological communication between H2O2 (ROS) and H2S (RSS) in vivo. CBS is responsible: A fluorescent probe with two heads for H2O2 and H2S reveals that both exogenous and endogenous H2O2 molecules can induce endogenous H2S biogenesis on a cystathionine β-synthase (CBS) pathway (see scheme).
- Yi, Long,Wei, Lv,Wang, Runyu,Zhang, Changyu,Zhang, Jie,Tan, Tianwei,Xi, Zhen
-
supporting information
p. 15167 - 15172
(2015/11/02)
-
- Single molecule electro-catalysis of non-fluorescent molecule
-
Single molecule catalysis is very powerful in revealing catalytic mechanism at the single molecule level. But fluorescent molecule is always necessary to take part into the catalysis directly in previous research. In order to study the single molecule electro-catalysis of non-fluorescent molecule (SMECNFM) on nano-catalyst, we couple the SMECNFM with a single molecule fluorescence reaction. A certain number of fluorescent molecules will be generated and detected when the SMECNFM happens. Through this method, we can detect the electro-oxidation reaction of one HCOONa molecule. The stability of Pt nano-catalyst supported on active carbon is studied at the single molecule level by this method. This paper also provides a general way to make ultra-sensitive sensor, and to study the SMECNFM for the molecules, such as formic acid, hydrogen, oxygen, etc., on single nanoparticle.
- Li, Fei-Fei,Gu, Jun-Nan,Zhou, Xiao-Chun
-
p. 1514 - 1517
(2015/12/23)
-
- A simple fluorescent off-on probe for the discrimination of cysteine from glutathione
-
A simple and stable fluorescent off-on probe for discrimination of cysteine (Cys) from glutathione (GSH) has been developed by combining resorufin with 7-nitrobenzofurazan. The probe, displaying distinct emission patterns for Cys and GSH at just one excitation wavelength, can be used for simultaneous determination of Cys and GSH in human plasma. This journal is
- Gao, Xinghui,Li, Xiaohua,Li, Lihong,Zhou, Jin,Ma, Huimin
-
supporting information
p. 9388 - 9390
(2015/06/02)
-
- High-throughput, quantitative enzyme kinetic analysis in microdroplets using stroboscopic epifluorescence imaging
-
Droplet-based microfluidic systems offer a range of advantageous features for the investigation of enzyme kinetics, including high time resolution and the ability to probe extremely large numbers of discrete reactions while consuming low sample volumes. Kinetic measurements within droplet-based microfluidic systems are conventionally performed using single point detection schemes. Unfortunately, such an approach prohibits the measurement of an individual droplet over an extended period of time. Accordingly, we present a novel approach for the extensive characterization of enzyme-inhibitor reaction kinetics within a single experiment by tracking individual and rapidly moving droplets as they pass through an extended microfluidic channel. A series of heterogeneous and pL-volume droplets, containing varying concentrations of the fluorogenic substrate resorufin β-d-galactopyranoside and a constant amount of the enzyme β-galactosidase, is produced at frequencies in excess of 150 Hz. By stroboscopic manipulation of the excitation laser light and adoption of a dual view detection system, "blur-free" images containing up to 150 clearly distinguishable droplets per frame are extracted, which allow extraction of kinetic data from all formed droplets. The efficiency of this approach is demonstrated via a Michaelis-Menten analysis which yields a Michaelis constant, Km, of 353 μM. Additionally, the dissociation constant for the competitive inhibitor isopropyl β-d-1-thiogalactopyranoside is extracted using the same method.
- Hess, David,Rane, Anandkumar,Demello, Andrew J.,Stavrakis, Stavros
-
p. 4965 - 4972
(2015/05/13)
-
- A single molecule perspective on the functional diversity of in vitro evolved β-glucuronidase
-
The mechanisms that drive the evolution of new enzyme activity have been investigated by comparing the kinetics of wild-type and in vitro evolved β-glucuronidase (GUS) at the single molecule level. Several hundred single GUS molecules were separated in large arrays of 62 500 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis-Menten kinetics. The large number of single molecule substrate turnover rates is representative of the activity distribution within an entire enzyme population. Partially evolved GUS displays a much broader activity distribution among individual enzyme molecules than wild-type GUS. The broader activity distribution indicates a functional division of work between individual molecules in a population of partially evolved enzymes that-as so-called generalists-are characterized by their promiscuous activity with many different substrates.
- Liebherr, Raphaela B.,Renner, Max,Gorris, Hans H.
-
supporting information
p. 5949 - 5955
(2014/05/20)
-
