- The hunt for 8-oxoguanine deaminase
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(Chemical Equation Presented) An enzyme from Pseudomonas aeruginosa, Pa0142 (gi|9945972), that is able to catalyze the deamination of 8-oxoguanine (8-oxoG) to uric acid has been identified for the first time. 8-Oxoguanine is formed by the oxidation of guanine residues within DNA by reactive oxygen species, and this lesion results in G:C to T:A transversions. The value of k cat/Km for the deamination of 8-oxoG by Pa0142 at pH 8.0 and 30 ° C is 2.0 × 104 M-1 s-1. This enzyme can also catalyze the deamination of isocystosine and guanine at rates that are approximately an order of magnitude lower. The three-dimensional structure of a homologous enzyme (gi|44264246) from the Sargasso Sea has been determined by X-ray diffraction methods to a resolution of 2.2 A (PDB entry ). The enzyme folds as a (β/α)8 barrel and is a member of the amidohydrolase superfamily with a single zinc in the active site. This enzyme catalyzes the deamination of 8-oxoG with a kcat/K m value of 2.7 × 105 M-1 s-1. Computational docking of potential high-energy intermediates for the deamination reaction to the X-ray crystal structure suggests that active-site binding of 8-oxoG is facilitated by hydrogen-bond interactions from a conserved glutamine that follows β-strand 1 with the carbonyl group at C6, a conserved tyrosine that follows β-strand 2 with N7, and a conserved cysteine residue that follows β-strand 4 with the carbonyl group at C8. A bioinformatic analysis of available protein sequences suggests that ～200 other bacteria possess an enzyme capable of catalyzing the deamination of 8-oxoG. Copyright
- Hall, Richard S.,Fedorov, Alexander A.,Marti-Arbona, Ricardo,Fedorov, Elena V.,Kolb, Peter,Sauder, J. Michael,Burley, Stephen K.,Shoichet, Brian K.,Almo, Steven C.,Raushel, Frank M.
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- Xanthine dehydrogenase electrocatalysis: Autocatalysis and novel activity
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The enzyme xanthine dehydrogenase (XDH) from the purple photosynthetic bacterium Rhodobacter capsulatus catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid as part of purine metabolism. The native electron acceptor is NAD+ but herein we show that uric acid in its 2-electron oxidized form is able to act as an artificial electron acceptor from XDH in an electrochemically driven catalytic system. Hypoxanthine oxidation is also observed with the novel production of uric acid in a series of two consecutive 2-electron oxidation reactions via xanthine. XDH exhibits native activity in terms of its pH optimum and inhibition by allopurinol.
- Kalimuthu, Palraj,Leimkühler, Silke,Bernhardt, Paul V.
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- Mixed Inhibition of the Oxidoreductase Activity of Xanthine Oxidase by Pd2+ Ion
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Pd2+ ion shows 100percent inhibition of the oxidoreductase activity of xanthine oxidase, the nature of the inhibition being the uncommon mixed type (competitive-non-competitive); to our knowleage this is the first example of moxed inhibition of xanthine oxidase, and also of the fact that Pd2+ can act as an inhibitor of enzyme activity.
- Sau, Apurba Kumar,Mondal, Madhu Sudan,Mitra, Samaresh
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- Low-potential amperometric enzyme biosensor for xanthine and hypoxanthine
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The bacterial xanthine dehydrogenase (XDH) from Rhodobacter capsulatus was immobilized on an edge-plane pyrolytic graphite (EPG) electrode to construct a hypoxanthine/xanthine biosensor that functions at physiological pH. Phenazine methosulfate (PMS) was used as a mediator which acts as an artificial electron-transfer partner for XDH. The enzyme catalyzes the oxidation of hypoxanthine to xanthine and also xanthine to uric acid by an oxidative hydroxylation mechanism. The present electrochemical biosensor was optimized in terms of applied potential and pH. The electrocatalytic oxidation response showed a linear dependence on the xanthine concentration ranging from 1.0 × 10-5 to 1.8 × 10-3 M with a correlation coefficient of 0.994. The modified electrode shows a very low detection limit for xanthine of 0.25 nM (signal-to-noise ratio = 3) using controlled potential amperometry.
- Kalimuthu, Palraj,Leimkühler, Silke,Bernhardt, Paul V.
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- Tetrathiatriarylmethyl radical with a single aromatic hydrogen as a highly sensitive and specific superoxide probe
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Superoxide (O2?-) plays crucial roles in normal physiology and disease; however, its measurement remains challenging because of the limited sensitivity and/or specificity of prior detection methods. We demonstrate that a tetrathiatriarylmethyl (TAM) radical with a single aromatic hydrogen (CT02-H) can serve as a highly sensitive and specific O 2?- probe. CT02-H is an analogue of the fully substituted TAM radical CT-03 (Finland trityl) with an electron paramagnetic resonance (EPR) doublet signal due to its aromatic hydrogen. Owing to the neutral nature and negligible steric hindrance of the hydrogen, O 2?- preferentially reacts with CT02-H at this site with production of the diamagnetic quinone methide via oxidative dehydrogenation. Upon reaction with O2?-, CT02-H loses its EPR signal and this EPR signal decay can be used to quantitatively measure O2?-. This is accompanied by a change in color from green to purple, with the quinone methide product exhibiting a unique UV-Vis absorbance (ε =15,900 M-1 cm-1) at 540 nm, providing an additional O2?- detection method. More than five-fold higher reactivity of CT02-H for O2?- relative to CT-03 was demonstrated, with a second-order rate constant of 1.7×104 M-1 s-1 compared to 3.1×103 M-1 s-1 for CT-03. CT02-H exhibited high specificity for O2?- as evidenced by its inertness to other oxidoreductants. The O2?- generation rates detected by CT02-H from xanthine/xanthine oxidase were consistent with those measured by cytochrome c reduction but detection sensitivity was 10- to 100-fold higher. EPR detection of CT02-H enabled measurement of very low O2?- flux with a detection limit of 0.34 nM/min over 120 min. HPLC in tandem with electrochemical detection was used to quantitatively detect the stable quinone methide product and is a highly sensitive and specific method for measurement of O2 ?-, with a sensitivity limit of ～2×10-13 mol (10 nM with 20-μl injection volume). Based on the O2-dependent linewidth broadening of its EPR spectrum, CT02-H also enables simultaneous measurement of O2 concentration and O2?- generation and was shown to provide sensitive detection of extracellular O 2?- generation in endothelial cells stimulated either by menadione or with anoxia/reoxygenation. Thus, CT02-H is a unique probe that provides very high sensitivity and specificity for measurement of O 2?- by either EPR or HPLC methods.
- Liu, Yangping,Song, Yuguang,De Pascali, Francesco,Liu, Xiaoping,Villamena, Frederick A.,Zweier, Jay L.
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- Bioluminescence Detection of Superoxide Anion Using Aequorin
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Although the superoxide anion (O2 -·) is generated during normal cellular respiration and has fundamental roles in a wide range of cellular processes, such as cell proliferation, migration, apoptosis, and homeostasis, its dysregulation is associated with a variety of diseases. Regarding these prominent roles in biological systems, the development of accurate methods for quantification of superoxide anion has attracted tremendous research attention. Here, we evaluated aequorin, a calcium-dependent photoprotein, as a potential bioluminescent reporter protein of superoxide anion. The mechanism is based on the measurement of aequorin bioluminescence, where the lower the concentration of coelenterazine under the oxidation of superoxide anion, the lower the amount aequorin regeneration, leading to a decrease in bioluminescence. The bioluminescence intensity of aequorin was proportional to the concentration of superoxide anion in the range from 4 to 40 000 pM with a detection limit (S/N = 3) of 1.2 pM, which was 5000-fold lower than those of the chemiluminescence methods. The proposed method exhibited high sensitivity and has been successfully applied to the determination of superoxide anion in the plant cell samples. The results could suggest a photoprotein-based bioluminescence system as a highly sensitive, specific, and simple bioluminescent probe for in vitro detection of superoxide anion.
- Rahmani, Hossein,Ghavamipour, Fahimeh,Sajedi, Reza H.
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- Inhibition studies of bovine xanthine oxidase by luteolin, silibinin, quercetin, and curcumin
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Xanthine oxidoreductase (XOR) is a molybdenum-containing enzyme that under physiological conditions catalyzes the final two steps in purine catabolism, ultimately generating uric acid for excretion. Here we have investigated four naturally occurring compounds that have been reported to be inhibitors of XOR in order to examine the nature of their inhibition utilizing in vitro steady-state kinetic studies. We find that luteolin and quercetin are competitive inhibitors and that silibinin is a mixed-type inhibitor of the enzyme in vitro, and, unlike allopurinol, the inhibition is not time-dependent. These three natural products also decrease the production of superoxide by the enzyme. In contrast, and contrary to previous reports in the literature based on in vivo and other nonmechanistic studies, we find that curcumin did not inhibit the activity of purified XO nor its superoxide production in vitro.
- Pauff, James M.,Hille, Russ
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- Effects of zinc acexamate (NAS-501) on superoxide radicals and lipid peroxidation of rat gastric mucosa
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Zinc acexamate (NAS-501), an anti-ulcer agent, has been reported to prevent various acute experimental gastric mucosal lesions and duodenal ulcers in rats. In order to clarify the mechanisms by which NAS-501 exhibits the anti-ulcer effects, we investigated the anti-oxidative effects of NAS-501 in vitro and in vivo. NAS-501 significantly reduced the superoxide radical-dependent chemiluminescence, generated by hypoxanthine-xanthine oxidase, rat neutrophils and guinea-pig macrophages in vitro. These in vitro effects were also confirmed by electron spin resonance using a 5,5-dimethyl-1-pyrroline-N-oxide spin-trapping method. In addition, NAS-501 significantly inhibited lipid peroxidation induced by increasing concentrations of Fe2+/ascorbate in rat gastric mucosal homogenate in vitro. Oral administration of NAS-501 (30 mg/kg) significantly inhibited production of thiobarbituric acid-reactive substance in rat gastric mucosa following per os instillation of 60% ethanol in 150 mmol/l HCI in vivo. These results suggest that NAS-501 exhibits the preventive effect from acute gastric mucosal lesions by the anti-oxidative activity.
- Tsutsui, Yasuhiro,Nakamura, Yukio,Yamaguchi, Shiho,Kawanaka, Nobuyo,Sato, Makoto
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- Kinetic model of oxidation catalyzed by xanthine oxidase - The final enzyme in degradation of purine nucleosides and nucleotides
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A new kinetic model is presented for analysis of experimental data of oxidation process catalyzed by milk xanthine oxidase. The kinetics for two substrates, xanthine and its analog 2-chloroadenine, in a broad pH range (5.8-9.0) are best described by an equation which is a rational function of degree 2:3 and 2:2, respectively. Copyright Taylor & Francis, Inc.
- Banach, Kinga,Bojarska, Elzbieta,Kazimierczuk, Zygmunt,Magnowska, Lucyna,Bzowska, Agnieszka
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- Inhibition of xanthine oxidase by flavonoids.
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Various dietary flavonoids were evaluated in vitro for their inhibitory effect on xanthine oxidase, which has been implicated in oxidative injury to tissue by ischemia-reperfusion. Xanthine oxidase activity was determined by directly measuring uric acid formation by HPLC. The structure-activity relationship revealed that the planar flavones and flavonols with a 7-hydroxyl group such as chrysin, luteolin, kaempferol, quercetin, myricetin, and isorhamnetin inhibited xanthine oxidase activity at low concentrations (IC50 values from 0.40 to 5.02 microM) in a mixed-type mode, while the nonplanar flavonoids, isoflavones and anthocyanidins were less inhibitory. These results suggest that certain flavonoids might suppress in vivo the formation of active oxygen species and urate by xanthine oxidase.
- Nagao,Seki,Kobayashi
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- Hydrogen peroxide is the major oxidant product of xanthine oxidase
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Xanthine oxidase (XO) is a critical source of reactive oxygen species (ROS) in inflammatory disease. Focus, however, has centered almost exclusively on XO-derived superoxide (O2?-), whereas direct H2O2 production from XO has been less well investigated. Therefore, we examined the relative quantities of O2?- and H2O2 produced by XO under a range (1-21%) of O2 tensions. At O2 concentrations between 10 and 21%, H2O2 accounted for ～75% of ROS production. As O2 concentrations were lowered, there was a concentration-dependent increase in H2O2 formation, accounting for 90% of ROS production at 1% O2. Alterations in pH between 5.5 and 7.4 did not affect the relative proportions of H2O2 and O2?- formation. Immobilization of XO, by binding to heparin-Sepharose, further enhanced relative H2O2 production by ～30%, under both normoxic and hypoxic conditions. Furthermore, XO bound to glycosaminoglycans on the apical surface of bovine aortic endothelial cells demonstrated a similar ROS production profile. These data establish H2O2 as the dominant (70-95%) reactive product produced by XO under clinically relevant conditions and emphasize the importance of H2O2 as a critical factor when examining the contributory roles of XO-catalyzed ROS in inflammatory processes as well as cellular signaling.
- Kelley, Eric E.,Khoo, Nicholas K.H.,Hundley, Nicholas J.,Malik, Umair Z.,Freeman, Bruce A.,Tarpey, Margaret M.
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- Pd nanoparticles supported on nitrogen, sulfur-doped three-dimensional hierarchical nanostructures as peroxidase-like catalysts for colorimetric detection of xanthine
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Pd nanoparticles supported on nitrogen, sulfur-doped three-dimensional hierarchical nanostructures (Pd/N-S-CS) were successfully prepared by glycol reduction of Pd on nitrogen, sulfur-doped three-dimensional hierarchical nanostructures (N-S-CS) using humic acid (HA) and thiourea as the precursors. Transmission electron microscopy (TEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS) were employed to confirm the characterization of Pd/N-S-CS. The results showed that the obtained Pd/N-S-CS had interesting three-dimensional hierarchical nanostructures which were composed of nanosheets, and Pd nanoparticles with an average diameter of 6 nm were distributed on the surface of the nanosheets. Furthermore, the Pd/N-S-CS exhibited excellent peroxidase-like activity and it can effectively catalyze the classical peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 to produce a color reaction. Based on this finding, a simple, sensitive, and selective colorimetric method for H2O2 and xanthine detection was developed and applied to detect xanthine in urine samples. The detection limit of H2O2 and xanthine were 3.3 × 10-6 M and 2.9 × 10-7 M, respectively. This work is expected to provide a novel and efficient method for the detection of xanthine in the human body. This journal is
- Shi, Weijie,Fan, Hai,Ai, Shiyun,Zhu, Lusheng
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- Structure-activity relationship of xanthones as inhibitors of xanthine oxidase
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Polygala plants contain a large number of xanthones with good physiological activities. In our previous work, 18 xanthones were isolated from Polygala crotalarioides. Extented study of the chemical composition of the other species Polygala sibirica led to the separation of two new xanthones-3-hydroxy-1,2,6,7,8-pentamethoxy xanthone (A) and 6-O--D-glucopyranosyl-1,7- dimethoxy xanthone (C)-together with 14 known xanthones. Among them, some xanthones have a certain xanthine oxidase (XO) inhibitory activity. Furthemore, 14 xanthones as XO inhibitors were selected to develop three-dimensional quantitative structure-activity relationship (3D-QSAR) using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models. The CoMFA model predicted a q2 value of 0.613 and an r2 value of 0.997. The best CoMSIA model predicted a q2 value of 0.608 and an r2 value of 0.997 based on a combination of steric, electrostatic, and hydrophobic effects. The analysis of the contour maps from each model provided insight into the structural requirements for the development of more active XO inhibitors.
- Zhou, Ling-Yun,Peng, Jia-Le,Wang, Jun-Ming,Geng, Yuan-Yuan,Zuo, Zhi-Li,Hua, Yan
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- Tissue metabolomic profiling to reveal the therapeutic mechanism of reduning injection on LPS-induced acute lung injury rats
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Acute lung injury (ALI) is a severe respiratory disease. To date, no medical interventions have been proven effective in improving the outcome. Reduning injection (RDN) showed a potential effect in the therapy of ALI. However, seldom does research concern the holistic pharmacological mechanisms of RDN on ALI. A metabolomic strategy, based on two consecutive extractions of the lung tissue, has been developed to investigate therapeutic mechanisms of RDN on ALI model rat. The extraction procedure was an aqueous extraction with methanol-water followed by organic extraction with dichloromethane-methanol. According to the lipophilicity of extracts, aqueous extracts were analyzed on the T3 column and organic extracts on the C18 column. Partial least-squares discriminant analysis was utilized to identify differences in metabolic profiles of rats. A total of 14 potential biomarkers in lung tissue were identified, which mainly related to phospholipid metabolism, sphingolipid metabolism, nucleotide metabolism and energy metabolism. The combined analytical method provides complementary metabolomics information for exploring the action mechanism of RDN against ALI. And the obtained results indicate metabolomics is a promising tool for understanding the holism and synergism of traditional Chinese medicine.
- Xiong, Zhili,Weng, Yanmin,Lang, Lang,Ma, Shuping,Zhao, Longshan,Xiao, Wei,Wang, Yanjuan
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p. 10023 - 10031
(2018/03/23)
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- Real-Time Monitoring of Enzyme-Catalysed Reactions using Deep UV Resonance Raman Spectroscopy
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For enzyme-catalysed biotransformations, continuous in situ detection methods minimise the need for sample manipulation, ultimately leading to more accurate real-time kinetic determinations of substrate(s) and product(s). We have established for the first time an on-line, real-time quantitative approach to monitor simultaneously multiple biotransformations based on UV resonance Raman (UVRR) spectroscopy. To exemplify the generality and versatility of this approach, multiple substrates and enzyme systems were used involving nitrile hydratase (NHase) and xanthine oxidase (XO), both of which are of industrial and biological significance, and incorporate multistep enzymatic conversions. Multivariate data analysis of the UVRR spectra, involving multivariate curve resolution-alternating least squares (MCR-ALS), was employed to effect absolute quantification of substrate(s) and product(s); repeated benchmarking of UVRR combined with MCR-ALS by HPLC confirmed excellent reproducibility.
- Westley, Chloe,Fisk, Heidi,Xu, Yun,Hollywood, Katherine A.,Carnell, Andrew J.,Micklefield, Jason,Turner, Nicholas J.,Goodacre, Royston
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p. 6983 - 6987
(2017/05/29)
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- Voltammetric determination of epinephrine and xanthine based on sodium dodecyl sulphate assisted tungsten trioxide nanoparticles
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This work reports the voltammetric determination of epinephrine (EP) and xanthine (XA) using gamma irradiated tungsten trioxide (WO3) nanoparticles. WO3 NPs synthesized by anionic surfactant sodium dodecyl sulphate (SDS) assisted template method was subjected to gamma rays under different doses (0-150 kGy). The experimental results revealed that the 50 kGy irradiation on SDS-WO3 NPs led to significant changes in crystallite size and morphology which enriched its electrocatalytic activity greatly. The electrochemical behaviors of EP and XA at 50 kGy gamma irradiated SDS-WO3 modified glassy carbon electrodes (GCE) were studied by cyclic voltammetry and differential pulse voltammetry. The outcomes confirmed that the proposed electrode demonstrate excellent electrocatalytic activity towardsthe oxidation of EP and XA in phosphate buffer solution (PBS, pH 7.0). The fabricated electrode possess lowest detection limit of 1.8?nM and 2.2?nM for EP and XA respectively with the very wide dynamic linear range of 9?nM to 1?mM. Finally, the modified sensor was successfully implemented to detect EP and XA in the human serum samples with excellent selectivity, good stability and reproducibility.
- Anithaa,Asokan,Sekar
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- Enzymatic synthesis of ribo- and 2′-deoxyribonucleosides from glycofuranosyl phosphates: An approach to facilitate isotopic labeling
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Milligram quantities of α-D-ribofuranosyl 1-phosphate (sodium salt) (αR1P) were prepared by the phosphorolysis of inosine, catalyzed by purine nucleoside phosphorylase (PNPase). The αR1P was isolated by chromatography in >95% purity and characterized by 1H and 13C NMR spectroscopy. Aqueous solutions of αR1P were stable at pH 6.4 and 4 °C for several months. The isolated αR1P was N-glycosylated with different nitrogen bases (adenine, guanine and uracil) using PNPase or uridine phosphorylase (UPase) to give the corresponding ribonucleosides in high yield based on the glycosyl phosphate. This methodology is attractive for the preparation of stable isotopically labeled ribo- and 2′-deoxyribonucleosides because of the ease of product purification and convenient use and recycling of nitrogen bases. The approach eliminates the need for separate reactions to prepare individual furanose-labeled ribonucleosides, since only one ribonucleoside (inosine) needs to be labeled, if desired, in the furanose ring, the latter achieved by a high-yield chemical N-glycosylation. 2′-Deoxyribonucleosides were prepared from 2′-deoxyinosine using the same methodology with minor modifications.
- Zhang, Wenhui,Turney, Toby,Surjancev, Ivana,Serianni, Anthony S.
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p. 125 - 133
(2017/08/08)
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- Label-Free Surface Enhanced Raman Scattering Approach for High-Throughput Screening of Biocatalysts
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Biocatalyst discovery and directed evolution are central to many pharmaceutical research programs, yet the lack of robust high-throughput screening methods for large libraries of enzyme variants generated (typically 106-108) has hampered progress and slowed enzyme optimization. We have developed a label-free generally applicable approach based on Raman spectroscopy which results in significant reductions in acquisition times (>30-fold). Surface enhanced Raman scattering (SERS) is employed to monitor the enzyme-catalyzed conversion by xanthine oxidase of hypoxanthine to xanthine to uric acid. This approach measures the substrates and products directly and does not require chromogenic substrates or lengthy chromatography, was successfully benchmarked against HPLC, and shows high levels of accuracy and reproducibility. Furthermore, we demonstrate that this SERS approach has utility in monitoring enzyme inhibition illustrating additional medical significance to this high-throughput screening method.
- Westley, Chloe,Xu, Yun,Carnell, Andrew J.,Turner, Nicholas J.,Goodacre, Royston
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p. 5898 - 5903
(2016/07/06)
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- Simultaneous determination of dopamine and uric acid in the presence of high ascorbic acid concentration using cetyltrimethylammonium bromide-polyaniline/activated charcoal composite
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We describe a simple, low-cost and mass producible composite made up of cetyltrimethylammonium bromide (CTAB) functionalized polyaniline (PANI) and activated charcoal (CTAB-PANI/AC) for simultaneous determination of dopamine (DA) and uric acid (UA). The composite formation was verified through scanning electron microscopy, electrochemical impedance spectroscopy and electrochemical methods. The CTAB-PANI/AC composite was used to modify a glassy carbon electrode (GCE) and the resulting modified electrode displayed excellent electrocatalytic activity to DA and UA and successfully separates their overlapped voltammetric peaks. The composite completely inhibits the AA signal and does not produce any voltammetric signal for AA up to 2 mM. The DA and UA can be selectively detectable up to detection limits of 0.06 (±0.006) μM and 0.20 (±0.008) μM, respectively. The effects of kinetics, analyte concentration and pH of the supporting electrolyte were investigated and optimized. The modified electrode has appreciable stability, repeatability and reproducibility. Besides, the practical feasibility of the sensor is demonstrated in biological samples, which delivered satisfactory recovery results.
- Govindasamy, Mani,Chen, Shen-Ming,Mani, Veerappan,Sathiyan, Anandaraj,Merlin, Johnson Princy,Al-Hemaid, Fahad M. A.,Ali, M. Ajmal
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p. 100605 - 100613
(2016/11/09)
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- The Influencing of Preanodized Inlaying Ultrathin Carbon Paste Electrode on the Oxidation for the Xanthine and Hypoxanthine by the Hydrogen Bond
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In this paper, a pre-anodized inlaying ultrathin carbon paste electrode (PAIUCPE) with 316L as a matrix was constructed by a simple and fast electrochemical pretreatment. Using xanthine (Xa) and hypoxanthine (HXa) as the target compounds, the pH effects compositions of buffer solution, the accumulation times, hydrogen bond catalysis, degree of auxiliary electrode reaction on the size of peak currents (Ip) of Xa and HXa was discussed in detail. Also, it was proposed that Xa and HXa were respectively absorbed at the surface of PAIUCPE through hydrogen bonding. The influencing mechanisms of the PAIUCEP on electrochemical oxidation of Xa and HXa were explained in detail. Moreover, the linear relationships for the Xa and HXa were obtained in the range of 6×10-8-3×10-5 mol/L and 2×10-7-7×10-5 mol/L, respectively. The detection limits for the Xa and HXa were 1.2×10-8 mol/L and 5.7×10-8 mol/L, respectively. Moreover, this proposed method could be applied to determine the Xa and HXa in human urine simultaneously with satisfactory results.
- Qiao, Yue-Chun,Li, Jing,Li, Yao,Li, Quan-Min
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p. 1011 - 1019
(2015/12/01)
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- Identification of Fleeting Electrochemical Reaction Intermediates Using Desorption Electrospray Ionization Mass Spectrometry
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We report a new method for the mass spectrometric detection of fleeting reaction intermediates in electrochemical reactions utilizing a "waterwheel" working electrode setup. This setup takes inspiration from desorption electrospray ionization (DESI) mass spectrometry, where the sampling time is on the order of milliseconds, to sample directly from the surface of a working electrode for mass spectrometric analysis. We present data that show the formation of a diimine intermediate of the electrochemical oxidation of uric acid that has a lifetime in solution of 23 ms as well as data that provide evidence for the formation of a similar diimine species from the electrooxidation of xanthine, which has not been previously observed.
- Brown, Timothy A.,Chen, Hao,Zare, Richard N.
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p. 7274 - 7277
(2015/06/30)
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- Purines oxidation by immobilized xanthine oxidase on magnetic polysiloxane-polyvinyl alcohol composite
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Bovine milk xanthine oxidase (XOD, E.C. 1.17.3.2) was covalently immobilized, via glutaraldehyde, on magnetic polysiloxane-polyvinyl alcohol (mPOS-PVA) particles yielding a preparation containing 9.5 ± 0.5 μg of protein per mg of support and specific activity of 36.3 ± 7.8 mU/mg of protein (55.0 ± 11.7% of the free enzyme). Optimal pH (8.8) and temperature (60 °C) were slightly higher than those established for the free enzyme (8.2 and 55 °C, respectively). No decrease of activity was observed after five reuses and only 17% was lost at the tenth reuse. The apparent Michaelis constant estimated for the mPOS-PVA-XOD (8.86 ± 0.88 μM) was not statistically different from the free enzyme (7.48 ± 1.01 μM). The 6-mercaptopurine oxidation catalyzed by the mPOS-PVA-XOD followed the same pathway described for the free enzyme, namely, 6-mercaptopurine → 6-mercapto-8-hydroxypurine → 6-thiouric acid, and no 6-thioxanthine was formed.
- Neri, David F.M.,Bernardino, Débora P.B.,Beltr?o, Eduardo I.C.,Carvalho Jr., Luiz B.
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experimental part
p. 210 - 214
(2012/03/27)
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- Regulation of xanthine oxidase activity by substrates at active sites via cooperative interactions between catalytic subunits: Implication to drug pharmacokinetics
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Three xanthine oxidase substrates (i.e., xanthine, adenine, and 2-amino-4-hydroxypterin) show a "substrate inhibition" pattern (i.e., slower turnover rates at higher substrate concentrations), whereas another two substrates (i.e., xanthopterin and lumazine) show a "substrate activation" pattern (i.e., higher turnover rates at higher substrate concentrations). Binding of a 6-formylpterin at one of the two xanthine oxidase active sites slows down the turnover rate of xanthine at the adjacent active site from 17.0 s-1 to 10.5 s-1, and converts the V-[S] plot from "substrate inhibition" pattern to a classical Michaelis-Menten hyperbolic saturation pattern. In contrast, binding of xanthine at an active site accelerates the turnover rate of 6-formylpterin at the neighboring active site. The experimental results demonstrate that a substrate can regulate the activity of xanthine oxidase via binding at the active sites; or a xanthine oxidase catalytic subunit can simultaneously serve as a regulatory unit. Theoretical simulation based on the velocity equation derived from the extended Michaelis-Menten model shows that the substrate inhibition and the substrate activation behavior in the V-[S] plots could be obtained by introducing cooperative interactions between two catalytic subunits in homodimeric enzymes. The current work confirms that there exist very strong cooperative interactions between the two catalytic subunits of xanthine oxidase.
- Tai,Hwang
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experimental part
p. 69 - 78
(2012/01/05)
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- HAIR TREATMENT PRODUCTS COMPRISING POLYMERS
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The invention relates to hair treatment products, comprising at least one copolymer made of 0.1 to 50% (in relation to the total number of monomers in the copolymer) monomers of the formula (I), wherein the unknowns are defined as in claim 1, and A2) are monomers from the group of acrylic acid, methacrylic acid and the like, and—optionally non-ionic monomers from the group of acrylamide, vinyl alcohol, and the like, wherein the monomers A2 and A3 together represent 50 to 99.9% (in relation to the total number of monomers in the copolymer) of the copolymer, at least one silicon and at least one selected care product, wherein the products result in advantageous effects for skin and hair.
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- BIOLOGICALLY-ACTIVE STILBENE DERIVATIVES AND COMPOSITIONS THEREOF
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The invention relates to stilbene derivatives displaying a large spectrum of biological activities, particularly anti-cancer, antioxidant and antiinflammatory activities, pharmaceutical and nutraceutical compositions containing such compounds and their uses as therapeutic agents or nutritional supplements.
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Page/Page column 13-14; 19
(2009/03/07)
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- Proposed mechanisms for HOOOH formation in two typical enzyme reactions responsible for superoxide anion production in biological systems
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We investigated the hypoxanthine (HPX)-xanthine oxidase (XOD) reaction by examining the chemiluminescence (CL) response mediated by a luminol analog, 8-amino-5-chloro-7-phen-y]pyrido[3,4-d]pyridazine-1,4-(2H, 3H)-dione sodium salt (1-012). It was found th
- Kohno, Masahiro,Sato, Emiko,Yaekashiwa, Noriko,Mokudai, Takayuki,Niwano, Yoshimi
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experimental part
p. 302 - 307
(2010/01/16)
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- Xanthine oxidase-activated prodrugs of thymidine phosphorylase inhibitors
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Thymidine phosphorylase (TP) is over-expressed in various tumour types and plays an important role in tumour angiogenesis, growth, invasion and metastasis. The enzymatic activity of TP is required for the angiogenic effect of TP, therefore, inhibitors of TP are of significant interest in cancer chemotherapy. A series of xanthine oxidase (XO) activated prodrugs of known inhibitors of TP have been designed and synthesized with the ultimate intent of improving tumour selectivity and pharmacokinetic characteristics. These prodrugs were not inhibitors of TP, but were selectively oxidized by XO at C-2 and/or C-4 of the uracil ring moiety to generate the desired TP inhibitor. Molecular modelling of both the TP inhibitors and XO-activated prodrugs rationalized their binding in the active site of the human TP crystal structure.
- Reigan, Philip,Gbaj, Abdul,Stratford, Ian J.,Bryce, Richard A.,Freeman, Sally
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p. 1248 - 1260
(2008/09/21)
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- Antioxidative activities of galloyl glucopyranosides from the stem-bark of Juglans mandshurica
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Two phenolics, 1,2,6-trigalloylglucose (1) and 1,2,3,6-tetragalloylglucose (2), isolated from the stem-bark of Juglans mandshurica were evaluated for their antioxidative activities. The results showed that compounds 1 and 2 exhibited strong scavenging activities against 1,1′-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzenthiazoline-6-sulphonic) acid (ABTS?+), and superoxide radicals (O2 ?-), and also had a significant inhibitory effect on lipid peroxidation and low-density lipoprotein (LDL) oxidation. The strong superoxide radical scavenging of 1 and 2 resulted from the potential competitive inhibition with xanthine at the active site of xanthine oxidase (OX). In addition, compounds 1 and 2 displayed significant lipoxygenase inhibitory activity, the mode of inhibition also being identified as competitive. In comparison, the antioxidative activities of compounds 1 and 2, together with gallic acid, indicated that the number of galloyl moieties could play an important role in the antioxidative activity.
- Ngoc, Tran Minh,Hung, Tran Manh,Thuong, Phuong Thien,Kim, Jin-Cheon,Choi, Jae Sue,Bae, Kihwan,Hattori, Masao,Choi, Chung-Sig,Lee, Joon Seok,Min, Byung-Sun
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p. 2158 - 2163
(2008/12/23)
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- Photosensitized oxidation of hypoxanthine and xanthine by aluminum phthalocyanine tetrasulfonate. Role of the alkylating quinone 2,5-dichloro-diaziridinyl-1,4-benzoquinone
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Photoirradiation of nitrogen-saturated aqueous solutions containing aluminum phthalocyanine tetrasulfonate (AlPcS4) at 675 nm in the presence of 2,5-dichloro-diaziridinyl-1,4-benzoquinone (AZDClQ) and hypoxanthine (HX) produces the oxidized HX
- Alegria, Antonio E.,Inostroza, Yaritza,Kumar, Ajay
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experimental part
p. 1583 - 1588
(2009/04/06)
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- Comprehensive study of bioanalytical platforms: Xanthine oxidase
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A comprehensive study of a general bioanalytical platform for biosensor applications is presented using xanthine oxidase (XnOx) as a case study within the framework of developing approaches of broad applicability. In this context, emphasis is placed on amperometric biosensors based on XnOx, which has been immobilized by covalent binding to gold electrodes modified with dithiobis-N-succinimidyl propionate. The immobilized XnOx layers have been characterized using atomic force microscopy under liquid conditions and quartz crystal microbalance techniques. In addition, spatially resolved mapping of enzymatic activity has been carried out using scanning electrochemical microscopy. Redox dyes of phenothiazine derivatives, specifically, thionine and methylene blue, have been found to work well as electron acceptors for reduced XnOx. The kinetic parameters and equilibrium constants of the mediated enzymatic oxidation of xanthine in the presence of the above-mentioned redox dyes have been calculated. The response of the enzymatic electrode to varying xanthine concentrations has been obtained in the presence of thionine or methylene blue as redox mediator in solution. Under these conditions, xanthine could be determined amperometrically at +0.2 V versus SSCE.
- Casero,De Quesada, A. Martinez G.,Jin,Quintana,Pariente,Abruna,Vazquez,Lorenzo
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p. 530 - 537
(2008/02/07)
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- Use of hydroxyguanidines
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The invention features pharmaceutical compositions that include hydroxyguanidines. The compositions can be used, for example, for treating or preventing ischemic diseases, hypoxia,or arrhythmias.
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- Composition of matter having bioactive properties
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Particles of coordinated complex comprising a basic, hydrous polymer and a capacitance adding compound, as well as methods for their production, are described. These complexes exhibit a high degree of bioactivity making them suitable for a broad range of applications through their incorporation into conventional vehicles benefiting from antimicrobial and similar properties.
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- Reaction of purines with hydroxyl radical generated by photolysis of mercaptopyridine-N-oxides
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The UV-photolysis of 4-mercaptopyridine-N-oxyde in aqueous solution is shown to be an efficient method of generating HO. radicals, as an alternative to the radiolysis of water. The reaction of adenine with HO. produced by the photolytic method resulted in the formation of 8-hydroxyadenine and 4,6-diamino-5-formamidopyrimidine in conditions and yields similar to those observed when HO. is produced by water radiolysis. ESR studies of the transients formed upon reaction of HO. with xanthine showed the formation of a radical anion in neutral conditions and a radical dianion at high pH values, due to the oxidation of the substrate by the conjugate base of HO., the O.- radical.
- Vieira,Telo,Dias
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p. 318 - 325
(2007/10/03)
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- Catalytic properties of xanthine oxidase immobilized on carbon materials
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The kinetics of catalysis by xanthine oxidase (XOD) immobilized on soot on the oxidation of xanthine obeys the laws of enzyme kinetics. The kinetics of catalysis with immobilized XOD has been determined by calculation of the rate constant (k), the constant of Michaelis (Km), the maximum reaction rate (V), the energy of activation (Ea) and pre-exponential factor (Zo). The values of the kinetic parameters depend on the nature of the adsorbent. The mechanism of action of the enzyme in the immobilized state has been explained on the basis of the calculated isobaric-isothermal potential of activation (ΔG+), enthalpy of activation (ΔH+) and entropy of activation (ΔS+). The polarographic measurements on the electrooxidation of hydrogen peroxide formed in enzymatic oxidation of xanthine with immobilized XOD have provided the evidence that the rate limiting is due to electrochemical polarisation.
- Horozova,Dimcheva,Jordanova,Kirova
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p. 335 - 338
(2007/10/03)
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- Administering particular compounds against various parasites, mycoplasmas, other indications and other infections
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One or more compounds are administered against one or more kinds of parasites and/or one or more diseases caused by such parasites, against one or more kind of Mycoplasma and/or one or more diseases caused by such Mycoplasmas and/or against one or more of the following indications or infections (a) hairy leukoplakia, (b) oral candidosis, (c) mouth ulcerations-aphthous/herpetic/bacterial, (d) fungal candida, (e) human papilloma virus, (f) molluscum contagiosum, (g) squamous oral carcinoma, (h) Kaposi's sarcoma oral lesions, (i) periodontitis, (j) necrotizing gingivitis, (k) orofacial herpes zoster, and (1) rotaviruses. The present invention furthermore provides specific formulations including such compound(s).
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- Oxidizable color producing reagent
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A triarylimidazole derivative of the formula: STR1 wherein X is O or S; Y is an arylsulfonyl group, an alkyl group, etc.; R2, R3 and R4 are independently an aryl group, etc., is stable in water or a buffer solution, high in measuring sensitivity and is suitable as an oxidizable color producing reagent for determining hydrogen peroxide, etc. generated by enzymatic reaction in a living body sample.
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- Electron-transfer reactions of alkyl peroxy radicals
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One-electron-transfer reactions of alkyl peroxy radicals were studied by pulse radiolysis of aqueous solutions. At pH 13, the methyl peroxy radical was found to rapidly, k = 1 × 105-4.9 × 107 s-1, and quantitatively oxidize various organic substrates with E13 = 0.13-0.76 V vs NHE. On the other hand, this radical was unreactive with compounds with E13 ≥ 0.85 V. Consequently, E13 of the methyl peroxy radical is higher than 0.76 V and lower than 0.85 V, which means that E7 is in the range 1.02-1.11 V. At pH 8, the rate constants of the oxidation of four ferrocene derivatives by the alkyl peroxy radicals ranged from 7.1 × 104 M-1 s-1 for ferrocenedicarboxylate (E8 = 0.66 V) to 2.3 × 106 M-1 s-1 for (hydroxymethyl)ferrocene (E8 = 0.42 V). These rate constants were used to evaluate the reduction potential and self-exchange rate of alkyl peroxy radicals in neutral media from the Marcus equation. The calculated E7 = 1.05 V is in excellent agreement with the estimated E7 = 1.02-1.11 V and with one of the perviously published values E7 = 1.0 V, but the value is in excellent agreement higher than the other E7 ～ 0.6 V. It is suggested that the high reorganization energy, λ = 72 kcal mol-1 redox couple originates from the requirement for solvent reorganization due to the solvation of hydroperoxide anion in the transition state. In support of this are the activation parameters of the reaction of the methyl peroxy radical with uric acid. The activation entropy is 9 eu lower at pH 7.3 than it is at pH 13.2, whereas the activation enthalpies are unchanged. The importance of entropy control was verified in the reactions of cyclohexyl peroxy radicals with α- and δ-tocopherol in aerated cyclohexane (ΔH+ ≈ 0 kcal/mol, and ΔS+ = -25 and -26 eu). The implications of these findings on the inactivation of alkyl peroxy radicals in general are discussed.
- Jovanovic, Slobodan V.,Jankovic, Ivana,Josimovic, Ljubica
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p. 9018 - 9021
(2007/10/02)
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- Use of particular compounds for manufacture of pharmaceutical formulations for treatment against various parasites, mycoplasmas, other indications and other infections
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One or more compounds are used for the manufacture of a pharmaceutical formulation for treatment against one or more kinds of parasites and/or one or more diseases caused by such parasites, against one or more kind of Mycoplasma and/or one or more diseases caused by such Mycoplasmas and/or against one or more of the following indications or infections (a) hairy leukoplakia, (b) oral candidosis, (c) mouth ulcerations-aphthous/herpetic/bacterial, (d) fungal candida, (e) human papilloma virus, (f) molluscum contagiosum, (g) squamous oral carcinoma, (h) Kaposi's sarcoma oral lesions, (i) periodontitis, (j) necrotizing gingivitis, (k) orofacial herpes zoster, and (l) rotaviruses. The present invention furthermore provides specific formulations including such compound(s).
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- Viral treatment system
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This invention relates to pharmaceutical formulations of compounds and drugs for treatment against viruses and prions (proteinaceous infectious particles) eg. CMV, Herpes Simplex, Hepatitis B, Scapie Creutzfeldt-Jakob Disease, in particular for drug treatment of persons and animals suffering from certain retroviral infections, and of persons suffering from infection by retroviruses related to human immuno-deficiency viruses (HIV), and for prophylactic drug treatment of persons who may be suffering from such infections. The anti-viral pharmaceutical formulations according to the invention have the general formula:
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- Electrochemical oxidation of xanthine at solid electrodes
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The electrochemical oxidation of xanthine at glassy carbon electrode (GCE) and pyrolytic graphite electrode (PGE) has been studied in the pH range of 1.8-10.7 and found to proceed in a 4e, 4H(+) reaction via the formation of uric acid at both the electrodes.Cyclic voltammetric behaviour, spectral studies, observed first order rate constant and product identification clearly point out that xanthine initially oxidises in a 2e, 2H(+) reaction to give uric acid which on subsequent oxidation gives the final products.The first order decay of UV absorbing intermediate has been monitored and the products have been identified.A plausible mechanism is suggested on the basis of observed experimental behaviour.
- Goyal, R. N.
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p. 467 - 471
(2007/10/02)
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- One-Electron Redox Potentials of Purines and Pyrimidines
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One-electron redox potentials of some purine and pyrimidine derivatives were determined by pulse radiolysis from electron transfer equilibria involving their and other free radicals.The redox potentials were determined at pH 13 by using p-methoxyphenol (E=0.4 V), Trolox C (E=0.19 V), and tryptophan (E=0.56 V) as references.The lowest oxidation potential measured for DNA bases was guanosine (E=0.72 V vs.NHE), and the highest was for 1-methylpyrimidines (E ca. 1.6 V) Uric acid (E=0.26 V) and isobarbituric acid (E=0.13 V) were found to have the lowest potentials.
- Jovanovic, Slobodan V.,Simic, Michael G.
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p. 974 - 978
(2007/10/02)
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- Competitive Reactivity of Nitrenium and Carbenium Ion Contributors of Purinium Cations with "Soft" Bases
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Delocalized cations formed by ionization of "activated" esters of carcinogenic purine N-oxides react with many nucleophiles to yield C-substitution products but afford oxidation-reduction products with others.The present study provides experimental support for the proposals (1) that these two reactivities result from nucleophilic substitutions at different sites of the delocalized cations and (2) that HSAB "hard" bases react only at carbenium ion sites to form C-substitution products, while "soft" bases react preferentially at nitrenium ion contributors to afford adducts that ultimately yield redox products. "Soft" bases showed the following order of reactivity at a purine nitrenium ion: iodide ca. selenourea >> thio amides ca. thio acids ca. biselenide > bisulfide ca. thiols ca. disulfides > thiosulfate.This order differs significantly from that observed for the double SN2 displacement reaction of nucleophiles with compounds of the type NH2-X.This appears to be the first report of differing orders of nucleophilicities of bases involved in SN1 and SN2 reactions at electron-deficient nitrogen centers.No evidence was found for radical intermediates formed by electron transfer.
- Parham, James C.,Templeton, Mary Agnes
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p. 652 - 657
(2007/10/02)
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- Method of obtaining uric acid from natural products
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This invention relates to improvements and methods of beneficiation of uric acid and primary salts of uric acid. The compounds are obtained by treating fowl excrement or waste material by means of first promptly collecting the waste material; removing the moisture therefrom, and then subsequently grinding and fractionating this material according to particle size. The method results in fractions of the original waste material which have increased concentrations of uric acid. The enrichment in uric acid concentration results in a material which is very suitable for final removal and treatment of the contained uric acid. A process for the extraction of high purity uric acid and primary salts of uric acid from the pretreated waste is also disclosed. The invention includes the utilization of the drying and sieving process of the waste material to obtain portions from the original product which is suitable for animal feed and supplements.
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- On the mechanism of reactions of oncogenic n-acyloxypurines-III. Extent of radical participation1 1 This investigation was supported in part by Grants Number CA-08748 and CA-23622, awarded by the National Cancer Institute, DHEW. Ref. 20 is now designated as II in the series and Ref. 16 is designated as I.
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UV irradiation of a model "activated ester" of the oncogen 3-hydroxyxanthine induced homolytic cleavage of the N-O bond and gave products arising by reduction of as well as by recombination of the solvent caged amidyl radical intermediate. Identification of the latter product constitutes the first evidence that a distinct product associated specifically with a radical from an acyloxypurine can be formed. The absence of this product among those formed spontaneously from 3-acetoxyxanthine provides the first indication that an amidyl radical is not an intermediate in the spontaneous reactions of N-acyloxy purines.
- Parham, James C.,Templeton, Mary Agnes
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p. 709 - 713
(2007/10/02)
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- Synthesis of prox-Benzoisoallopurinol
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Pyrazoloquinazolin-9-one (prox-benzoisoallopurinol, 1), an extented analogue of 7-hydroxypyrazolopyrimidine (isoallopurinol, 2) and a potential dimensional probe for substrates of xanthine oxidase, has been synthesized by two independent routes.The title compound, prepared by elaboration of either a suitably substituted indazole or a quinazolinone, was found to be an active substrate for and an alternative-substrate inhibitor of xanthine oxidase.The product of enzymatic oxidation of prox-benzoisoallopurinol has been identified as the corresponding prox-benzoisoalloxanthine.
- Foster, Robert H.,Leonard, Nelson J.
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p. 3072 - 3077
(2007/10/02)
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