Enzymatic oxygen scavenging for photostability without pH drop in single-molecule experiments.
ABSTRACT: Over the past years, bottom-up bionanotechnology has been developed as a promising tool for future technological applications. Many of these biomolecule-based assemblies are characterized using various single-molecule techniques that require strict anaerobic conditions. The most common oxygen scavengers for single-molecule experiments are glucose oxidase and catalase (GOC) or protocatechuate dioxygenase (PCD). One of the pitfalls of these systems, however, is the production of carboxylic acids. These acids can result in a significant pH drop over the course of experiments and must thus be compensated by an increased buffer strength. Here, we present pyranose oxidase and catalase (POC) as a novel enzymatic system to perform single-molecule experiments in pH-stable conditions at arbitrary buffer strength. We show that POC keeps the pH stable over hours, while GOC and PCD cause an increasing acidity of the buffer system. We further verify in single-molecule fluorescence experiments that POC performs as good as the common oxygen-scavenging systems, but offers long-term pH stability and more freedom in buffer conditions. This enhanced stability allows the observation of bionanotechnological assemblies in aqueous environments under well-defined conditions for an extended time.
Project description:1. At high concentrations, in 10mm-phosphate buffer, pH7.0, the sedimentation coefficient of bacterial catalase varies with concentration according to: [Formula: see text] with S(0) (20,w)=11.30S and k(s)=6.29x10(-3)ml mg(-1). Sedimentation-equilibrium experiments yield a molecular weight of 240000. 2. Parallel studies of changes in sedimentation-velocity behaviour and in electronic spectra of bacterial catalase at pH>11 were made. Dissociation is indicated by the appearance of a slow-moving (2.9S) component in sedimentation patterns and this is accompanied by marked changes in absorption spectrum in the Soret region. Values of R=E(406)/E(355) show a theoretically predictable near-linear dependence on alpha, the degree of dissociation calculated from ultracentrifuge data. 3. The Soret absorption of bacterial catalase subunits is much lower than that of the native enzyme, and it is suggested that dissociation produces an environmental constraint on the prosthetic group that results in distortion of the porphyrin ring.
Project description:Although stimuli-responsive supramolecular self-assemblies have been constructed, the controlled drug delivery induced by morphology transitions of these supramolecular self-assemblies on the basis of host-guest-conjugated monomers (HGCMs) are few reported. In this paper, the self-assembly behaviors of AB<sub>2</sub>-type HGCMs, e.g., ?-cyclodextrin-benzimidazole<sub>2</sub> (?-CD-BM<sub>2</sub>), were investigated at neutral and acidic pH conditions, respectively. Specifically, ?-CD-BM<sub>2</sub> first self-assembled into fan-shaped supramolecular self-assemblies with a hydrodynamic diameter of 163 nm at neutral pH, whereas they were further dissociated into spherical supramolecular self-assemblies with a size of 52 nm under acidic conditions. This morphology transition process was utilized to conduct a two-stage DOX delivery under neutral and acidic pH. Basic cell experiments demonstrated that the drug-loaded ?-CD-BM<sub>2</sub>-based supramolecular self-assemblies with varied morphology could inhibit cancer cell proliferation, indicating their potential application in the field of drug delivery.
Project description:The Plant Ontology Consortium (POC, http://www.plantontology.org) is a collaborative effort among model plant genome databases and plant researchers that aims to create, maintain and facilitate the use of a controlled vocabulary (ontology) for plants. The ontology allows users to ascribe attributes of plant structure (anatomy and morphology) and developmental stages to data types, such as genes and phenotypes, to provide a semantic framework to make meaningful cross-species and database comparisons. The POC builds upon groundbreaking work by the Gene Ontology Consortium (GOC) by adopting and extending the GOC's principles, existing software and database structure. Over the past year, POC has added hundreds of ontology terms to associate with thousands of genes and gene products from Arabidopsis, rice and maize, which are available through a newly updated web-based browser (http://www.plantontology.org/amigo/go.cgi) for viewing, searching and querying. The Consortium has also implemented new functionalities to facilitate the application of PO in genomic research and updated the website to keep the contents current. In this report, we present a brief description of resources available from the website, changes to the interfaces, data updates, community activities and future enhancement.
Project description:Catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1(T), which was isolated from an environment exposed to H(2)O(2) and exhibited high catalase activity, was purified and characterized, and its localization in the cell was determined. Its molecular mass was 230 kDa, and the molecule consisted of four identical subunits. The enzyme, which was not apparently reduced by dithionite, showed a Soret peak at 406 nm in a resting state. The catalytic activity was 527,500 U. mg of protein(-1) under standard reaction conditions at 40 degrees C, 1.5 and 4.3 times faster, respectively, than those of the Micrococcus luteus and bovine catalases examined under the same reaction conditions, and showed a broad optimum pH range (pH 6 to 10). The catalase from strain S-1(T) is located not only in the cytoplasmic space but also in the periplasmic space. There is little difference in the activation energy for the activity between strain S-1(T) catalase and M. luteus and bovine liver catalases. The thermoinstability of the activity of the former catalase were significantly higher than those of the latter catalases. The thermoinstability suggests that the catalase from strain S-1(T) should be categorized as a psychrophilic enzyme. Although the catalase from strain S-1(T) is classified as a mammal type catalase, it exhibits the unique enzymatic properties of high intensity of enzymatic activity and thermoinstability. The results obtained suggest that these unique properties of the enzyme are in accordance with the environmental conditions under which the microorganism lives.
Project description:Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.
Project description:Previously, we reported that when agar is autoclaved with phosphate buffer, hydrogen peroxide (H2O2) is formed in the resulting medium (PT medium), and the colony count on the medium inoculated with environmental samples becomes much lower than that on a medium in which agar and phosphate are autoclaved separately (PS medium) (T. Tanaka et al., Appl Environ Microbiol 80:7659-7666, 2014, https://doi.org/10.1128/AEM.02741-14). However, the physicochemical mechanisms underlying this observation remain largely unknown. Here, we determined the factors affecting H2O2 formation in agar. The H2O2 formation was pH dependent: H2O2 was formed at high concentrations in an alkaline or neutral phosphate buffer but not in an acidic buffer. Ammonium ions enhanced H2O2 formation, implying the involvement of the Maillard reaction catalyzed by phosphate. We found that other gelling agents (e.g., gellan and ?-carrageenan) also produced H2O2 after being autoclaved with phosphate. We then examined the cultivability of microorganisms from a fresh-water sample to test whether catalase and pyruvate, known as H2O2 scavengers, are effective in yielding high colony counts. The colony count on PT medium was only 5.7% of that on PS medium. Catalase treatment effectively restored the colony count of PT medium (to 106% of that on PS medium). In contrast, pyruvate was not as effective as catalase: the colony count on sodium pyruvate-supplemented PT medium was 58% of that on PS medium. Given that both catalase and pyruvate can remove H2O2 from PT medium, these observations indicate that although H2O2 is the main cause of reduced colony count on PT medium, other unknown growth-inhibiting substances that cannot be removed by pyruvate (but can be by catalase) may also be involved.IMPORTANCE The majority of bacteria in natural environments are recalcitrant to laboratory culture techniques. Previously, we demonstrated that one reason for this is the formation of high H2O2 levels in media prepared by autoclaving agar and phosphate buffer together (PT medium). In this study, we investigated the factors affecting H2O2 formation from agar. H2O2 formation is pH dependent, and ammonium ions promote this phosphate-catalyzed H2O2 formation. Amendment of catalase or pyruvate, a well-known H2O2-scavenging agent, effectively eliminated H2O2 Yet results suggest that growth-inhibiting factor(s) that cannot be eliminated by pyruvate (but can be by catalase) are present in PT medium.
Project description:We report here the development of hydrogels formed at physiological conditions using PEG (polyethylene glycol) based polymers modified with boronic acids (BAs) as backbones and the plant derived polyphenols ellagic acid (EA), epigallocatechin gallate (EGCG), tannic acid (TA), nordihydroguaiaretic acid (NDGA), rutin trihydrate (RT), rosmarinic acid (RA) and carminic acid (CA) as linkers. Rheological frequency sweep and single molecule force spectroscopy (SMFS) experiments show that hydrogels linked with EGCG and TA are mechanically stiff, arising from the dynamic covalent bond formed by the polyphenol linker and boronic acid functionalized polymer. Stability tests of the hydrogels in physiological conditions revealed that gels linked with EA, EGCG, and TA are stable. We furthermore showed that EA- and EGCG-linked hydrogels can be formed via in situ gelation in pH 7.4 buffer, and provide long-term steady state release of bioactive EA. In vitro experiments showed that EA-linked hydrogel significantly reduced the viability of CAL-27 human oral cancer cells via gradual release of EA.
Project description:Stabilization is a critical issue in the long term operation of dye-sensitized photoelectrosynthesis cells (DSPECs) for water splitting or CO2 reduction. The cells require a stable binding of the robust molecular chromophores, catalysts, and chromophore/catalyst assemblies on metal oxide semiconductor electrodes under the corresponding (photoelectro)chemical conditions. Here, an efficient stabilization strategy is presented based on functionalization of FTO|nanoTiO2 (mesoporous, nanostructured TiO2 deposited on fluorine-doped tin oxide (FTO) glass) electrodes with a vinylsilane followed by surface reductive electropolymerization of a vinyl-derivatized Ru(II) polypyridyl chromophore. The surface electropolymerization was dominated by a grafting-through mechanism, and rapidly completed within minutes. Chromophore surface coverages were controlled up to three equivalent monolayers by the number of electropolymerization cycles. The silane immobilization and cross-linked polymer network produced highly (photo)stabilized chromophore-grafted FTO|nanoTiO2 electrodes. The electrodes showed significant improvements over structures based on atomic layer deposition and polymer dip-coating stabilization methods in a wide pH range from pH ? 1 to pH ? 12.5 under both dark and light conditions. Under illumination, with hydroquinone added as a sacrificial electron transfer donor, a photoresponse for sustained electron transfer mediation occurred for at least ?20 h in a pH ? 7.5 phosphate buffer (0.1 M NaH2PO4/Na2HPO4, with 0.5 M NaClO4). The overall procedure provides an efficient way to fabricate highly stabilized molecular assemblies on electrode surfaces with potential applications for DSPECs in solar fuels.
Project description:The enzyme porphobilinogen synthase (PBGS) can exist in different nonadditive homooligomeric assemblies, and under appropriate conditions, the distribution of these assemblies can respond to ligands such as metals or substrate. PBGS from most organisms was believed to be octameric until work on a rare allele of human PBGS revealed an alternate hexameric assembly, which is also available to the wild-type enzyme at elevated pH [Breinig, S., et al. (2003) Nat. Struct. Biol. 10, 757-763]. Herein, we establish that the distribution of pea PBGS quaternary structures also contains octamers and hexamers, using both sedimentation velocity and sedimentation equilibrium experiments. We report results in which the octamer dominates under purification conditions and discuss conditions that influence the octamer:hexamer ratio. As predicted by PBGS crystal structures from related organisms, in the absence of magnesium, the octameric assembly is significantly destabilized, and the oligomeric distribution is dominated largely by the hexameric assembly. Although the PBGS hexamer-to-octamer oligomeric rearrangement is well documented under some conditions, both assemblies are very stable (under AU conditions) in the time frame of our ultracentrifuge experiments.
Project description:H2S is a signaling molecule in plants and animals. Here we investigated the effects of H2S on programmed cell death (PCD) in barley (Hordeum vulgare L.) aleurone layers. The H2S donor NaHS significantly delayed PCD in aleurone layers isolated from imbibed embryoless barley grain. NaHS at 0.25?mM effectively reduced the accumulation of superoxide anion (·O2 (-)), hydrogen peroxide (H2O2), and malondialdehyde (MDA), promoted the activity of superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), and decreased those of lipoxygenase (LOX) in isolated aleurone layers. Quantitative-PCR showed that NaHS treatment of aleurone tissue led to enhanced transcript levels of the antioxidant genes HvSOD1, HvAPX, HvCAT1, and HvCAT2 and repressed transcript levels of HvLOX (lipoxygenase gene) and of two cysteine protease genes HvEPA and HvCP3-31. NaHS treatment in gibberellic acid- (GA-) treated aleurone layers also delayed the PCD process, reduced the content of ·O2 (-), and increased POD activity while decreasing LOX activity. Furthermore, ?-amylase secretion in barley aleurone layers was enhanced by NaHS treatment regardless of the presence or absence of GA. These data imply that H2S acted as an antioxidant in delaying PCD and enhances ?-amylase secretion regardless of the presence of GA in barley aleurone layers.