Plasmodium falciparum: development of a transgenic line for screening antimalarials using firefly luciferase as the reporter

High-throughput screening (HTS) of small-molecule libraries against pharmacological targets is a key strategy of contemporary drug discovery. This study reports a simple, robust, and cell-based luminescent method for assaying antimalarial drugs. Using transfection technology, we generated a stable Plasmodium falciparum line with high levels of firefly luciferase expression. A luciferase assay based on this parasite line was optimized in a 96-well plate format and used to compare with the standard [³H] hypoxanthine radioisotope method. The 50% inhibitory concentrations (IC₅₀s) of chloroquine, artesunate, artemether, dihydroartemisinin and curcumin obtained by these two methods were not significantly different (P > 0.05, ANOVA). In addition, this assay could be performed conveniently with a luminescence plate reader using unsynchronized stages within as early as 12 h. Furthermore, the luciferase assay is robust with a Z′ score of 0.77-0.92, which suggests the feasibility for further miniaturization and automation.     

The role of phase separation and feed cycle length in leach beds coupled to methanogenic reactors for digestion of a solid substrate (Part 1): Optimisation of reactors' performance

A two-phase system composed by a leach bed and a methanogenic reactor was modified for the first time to improve volumetric substrate degradation and methane yields from a complex substrate (maize; Zeamays). The system, which was operated for consecutive feed cycles of different durations for 120 days, was highly flexible and its performance improved by altering operational conditions. Daily substrate degradation was higher the shorter the feed cycle, reaching 8.5 g TS_destroyed d⁻¹ (7-day feed cycle) but the overall substrate degradation was higher by up to 55% when longer feed cycles (14 and 28 days) were applied. The same occurred with volumetric methane yields, reaching 0.839 m³ (m³)⁻¹ d⁻¹. The system performed better than others on specific methane yields, reaching 0.434 m³ kg⁻¹ TS_added, in the 14-day and 28-day systems. The UASB and AF designs performed similarly as second stage reactors on methane yields, SCOD and VFA removal efficiencies.     

Synthesis, characterization, X-ray crystallography, and cytotoxicity of a cymantrene keto carboxylic acid for IR labelling of bioactive peptides on a solid support

Cym-CO-CH₂-CH₂-COOH was prepared in good yield by Friedel-Crafts reaction of cymantrene (Cym, CpMn(CO)₃) with succinic anhydride for the IR labelling of peptides and fully characterized, including an X-ray structure analysis (monoclinic space group P2(1)/n, a = 5.727(3) Å, b = 19.865(9) Å, c = 10.518(5) Å, β = 91.211(9)°). The compound was isolated in pure form without the need for chromatographic work-up and subsequently used for solution-phase synthesis of a bioconjugate with phenylalanine methyl ester to allow a complete spectroscopic characterization of this model system. The cymantrene keto carboxylic acid also turned out to be a very robust marker in automated microwave-assisted solid phase peptide synthesis (SPPS). [Leu⁵]-enkephalin (Tyr-Gly-Gly-Phe-Leu) was prepared on a Wang resin and labelled with the cymantrene derivative on the solid support under microwave irradiation in all steps. The metal-carbonyl marker stayed intact during cleavage from the resin with concentrated trifluoroacetic acid. After simple precipitation and lyophilization, the cymantrene-enkephalin bioconjugate could be obtained in analytically pure form without the need of HPLC purification. As required, the compound is non-cytotoxic against MCF-7 cells at up to 100 μM. This protocol thus allows one to introduce organometallic IR spectroscopic labels to peptides in a very straightforward way.     

Modulation of trans-to-cis photoisomerization and photoluminescence of 1,2-bis(4-pyridyl)ethylene or 4-styrylpyridine coordinated to fac-tricarbonyl(5-chloro-1,10-phenathroline)rhenium(I)

Photochemical and photophysical properties of fac-[Re(CO)₃(Clphen)(trans-L)]⁺ complexes, Clphen = 5-chloro-1,10-phenathroline and L = 1,2-bis(4-pyridyl)ethylene, bpe, or 4-styrylpyridine, stpy, were investigated to complement the understanding of intramolecular energy transfer process in tricarbonyl rhenium(I) complexes having an electron withdrawing group attached to polypyridyl ligands. These new compounds were synthesized, characterized and the photoisomerization quantum yields were accurately determined by ¹H NMR spectroscopy. The true quantum yields for fac-[Re(CO)₃(Clphen)(trans-bpe)]⁺ were constant (Φ = 0.55) at all investigated irradiation wavelengths. However, for fac-[Re(CO)₃(Clphen)(trans-stpy)]⁺, similar true quantum yields were observed only at higher energy irradiation (Φ_{313 nm} = 0.53 and Φ_{365 nm} = 0.57), but it decreased significantly at 404 nm (Φ = 0.41). These results indicated different deactivation pathways for the trans-stpy complex photoisomerization. Quantum yields decreased as the ³IL_trans-L and ³MLCT_Re→NN excited states become closer and the behavior was discussed in terms of the excited state energy gaps. Additionally, luminescence properties of photoproducts, fac-[Re(CO)₃(Clphen)(cis-L)]⁺, were also investigated in different environments to analyze the relative energy of the ³MLCT_Re→Clphen excited state for each compound.     

Development of high performance liquid chromatography/electrospray ionization mass spectrometry for assay of ginkgolic acid (15:1) in rat plasma and its application to pharmacokinetics study

A highly sensitive HPLC–ESI-MS method has been developed and validated for the quantification of ginkgolic acid (15:1) in a small quantity of rat plasma (50 μL) using its homologous compound ginkgolic acid (17:1) as an internal standard. GA (15:1) and GA (17:1) were extracted from biological matrix by direct protein precipitation with 5-fold volume of methanol and separated on an Elite hypersil BDS C₁₈ column (2.1 × 100 mm, 3 μm), eluted with acetonitrile:water (92:8, v/v, containing 0.3% glacial acetic acid). Linear range was 8–1000 ng/mL with the square regression coefficient (r²) of 0.996. The lowest concentration (8 ng/mL) in the calibration curve was estimated as LLOQ with both deviation of accuracy and RSD of precision <20% (n = 6). The intra- and inter-day precision ranged from 3.6% to 9.9%, and the intra- and inter-day accuracy was between 89.9% and 101.3%. This method was successfully applied to study pharmacokinetics of GA (15:1) in rats after oral administration at a dose of 10 mg/kg. GA (15:1) pharmacokinetic parameters C_max, T_max, t_1/2, AUC_0–12h are 1552.9 ± 241.0 ng/mL, 0.9 ± 0.7 h, 5.5 ± 2.6 h, 3356.0 ± 795.3 ng h/mL, respectively.     

Synthesis and catalytic performance for olefin epoxidation of a novel dioxomolybdenum (VI) (ferrocenylmethyleneamino)alcoholate complex

A novel dioxomolybdenum (VI) complex of (ferrocenylmethyleneamino)alcoholate was easily synthesized via the reaction of MoO₂Cl₂(THF)₂ with N-hydroxyethyl ferrocenylimine ligand [(η⁵-C₅H₅)Fe{(η⁵-C₅H₄)-CHN-CH₂-CH₂-OH}], and characterized by FR-IR, ¹H NMR, UV-Vis spectroscopy and elemental analysis. In the presence of the catalytic amount of the dioxomolybdenum (VI) complex, good to excellent selectivities of epoxides (77-95%) were obtained for epoxidation of styrene and cyclohexene with tert-butyl hydroperoxide (TBHP) as oxidant in 1,2-dichloroethane at 12 h. Higher initial activity of the complex was observed for the catalyzed styrene epoxidation (451 mol mol_Mo⁻¹ h⁻¹) than that of cyclohexene epoxidation (164 mol mol_Mo⁻¹ h⁻¹) at 1 h. Meanwhile, influences of reaction temperature, solvents, reaction time as well as catalyst amount for olefin epoxidation were also investigated.     

Purification and characterization of two extracellular endochitinases from Massilia timonae

Two extracellular chitinases (designated as Chi-56 and Chi-64) produced by Massilia timonae were purified by ion-exchange chromatography, ammonium sulfate precipitation, and gel-filtration chromatography. The molecular mass of Chi-56 was 56 kDa as determined by both SDS-PAGE and gel-filtration chromatography. On the other hand, Chi-64 showed a molecular mass of 64 kDa by SDS-PAGE and 28 kDa by gel-filtration chromatography suggesting that its properties may be different from those of Chi-56. The optimum temperature, optimum pH, pI, K_m, and V_max of Chi-56 were 55 °C, pH 5.0, pH 8.5, 1.1 mg mL⁻¹, and 0.59 μmol μg⁻¹ h⁻¹, respectively. For Chi-64, these values were 60 °C, pH 5.0, pH 8.5, 1.3 mg mL⁻¹, and 1.36 μmol μg⁻¹ h⁻¹, respectively. Both enzymes were stimulated by Mn²⁺ and inhibited by Hg²⁺, and neither showed exochitinase activity. The N-terminal sequences of Chi-56 and Chi-64 were determined to be Q-T-P-T-Y-T-A-T-L and Q-A-D-F-P-A-P-A-E, respectively.     

Nitrite reduction by Co(II) and Mn(II) substituted myoglobins: towards understanding necessary components of Mb nitrite reductase activity

Nitrite reduction to nitric oxide by heme proteins is drawing increasing attention as a protective mechanism to hypoxic injury in mammalian physiology. Here we probe the nitrite reductase (NiR) activities of manganese(II)- and cobalt(II)-substituted myoglobins, and compare with data obtained previously for the iron(II) analog wt Mb^II. Both Mn^IIMb and Co^IIMb displayed NiR activity, and it was shown that the kinetics are first order each in [protein], [nitrite], and [H⁺], as previously determined for the Fe^II analog wt Mb^II. The second order rate constants (k₂) at pH 7.4 and T = 25 °C, were 0.0066 and 0.015 M^− 1 s^− 1 for Co^IIMb and Mn^IIMb, respectively, both orders of magnitude slower than the k₂ (6 M^− 1 s^− 1) for wt Mb^II. The final reaction products for Mn^IIMb consisted of a mixture of the nitrosyl Mn^IIMb(NO) and Mn^IIIMb, similar to the products from the analogous NiR reaction by wt Mb. In contrast, the products of NiR by Co^IIMb were found to be the nitrito complex Co^IIIMb(ONO⁻) plus roughly an equivalent of free NO. The differences can be attributed in part to the stronger coordination of inorganic nitrite to Co^IIIMb as reflected in the respective M^IIIMb(ONO⁻) formation constants K_nitrite: 2100 M^− 1 (Co^III) and <~0.4 M^− 1 (Mn^III). We also report the formation constants (3.7 and 30 M^− 1, respectively) for the nitrite complexes of the mutant metmyoglobins H64V Mb^III(NO₂⁻) and H64V/V67R Mb^III(ONO⁻) and a K_nitrite revised value (120 M^− 1) for the nitrite complex of wt metMb. The respective K_nitrite values for the three ferric proteins emphasize the importance of a H-bonding residue, such as His64 in the Mb^III distal pocket or the Arg67 in H64V/V67R Mb^III, in stabilizing nitrite coordination. Notably, the NiR activities of the corresponding ferrous Mbs follow a similar sequence suggesting that nitrite binding to these centers are analogously affected by the H-bonding residues.     

Determination and pharmacokinetics of gastrodin in human plasma by HPLC coupled with photodiode array detector

In present study, an HPLC method coupled with photodiode array detector (HPLC-PDA) was established for determination and pharmacokinetics of gastrodin (GAS) in human plasma after an oral administration of GAS capsule. In the method, ethanol and dichloromethane were respectively used for deproteinization and purification during the sample preparation procedure. Separation of GAS was achieved on an AichromBond-AQ C18 column (5 μm, 150 mm × 4.6 mm) with the mobile phase of methanol–0.1% phosphoric acid solution (2:98, v/v) at a flow rate of 0.8 ml/min. The wavelength was set at 220 nm and the injection volume was 20 μl. Under the conditions, the calibration curve was linear within the concentration range of 50–4000 ng/ml with the correlation coefficient (r) of 0.99554 (weight = 1/X²) and the lower limit of quantification (LLOQ) was 50 ng/ml. The inter- and intra-day precisions were less than 11% and the accuracies (%) were within the range of 95.55–103.78%. The extraction recoveries were over 65% with RSDs less than 5.50%. The GAS was proved to be stable under tested conditions. Thus, the method was valid enough to be applied for pharmacokinetic study of GAS in human plasma. The pharmacokinetic parameters of GAS in human plasma after an oral administration of 200 mg GAS capsule were described as: C_max, 1484.55 ± 285.05 ng/ml; T_max, 0.81 ± 0.16 h; t_1/2α, 3.78 ± 2.33 h; t_1/2β, 6.06 ± 3.20 h; t_1/2Ka, 0.18 ± 0.53 h; K₁₂, 0.18 ± 0.41/h; K₂₁, 0.20 ± 0.16/h; K₁₀, 4.11 ± 15.81/h; V1/F, 180.35 ± 89.44 L; CL/F, 62.50 ± 140.03 l/h; AUC_0→t, 5619.41 ± 1972.88 (ng/ml) h; and AUC_0→∞, 7210.26 ± 3472.74 (ng/ml) h, respectively. These will be useful for the clinical application of GAS.     

Electrochemical analysis of the alanine phenylthiohydantoin derivative by cathodic stripping voltammetry

A square-wave cathodic stripping voltammetry method for alanine determination as its phenylthiohydantoin (PTH-alanine) derivative is developed. To this end, all the chemical and instrumental variables affecting the determination of PTH-alanine are optimized. From studies of the mechanisms governing the electrochemical response of PTH-alanine, it was concluded that it is an electrochemically irreversible system with a diffusive-adsorptive reduction phenomenon. Under optimal conditions, the variation of analytical signal (I_p) with PTH-alanine concentration is linear in the 2.4 × 10⁻⁸ − 4.8 × 10⁻⁷ M range, with a LOD of 1.2 × 10⁻⁸ M and a LOQ of 4.2 × 10⁻⁸ M, a RSD (%) less than 11%, and a E_r (%) less than 10%. The optimized method was applied to the determination of PTH-alanine obtained from a synthetic protein after Edman reaction and the results were corroborated by high-performance liquid chromatography with UV detection.     

An efficient catalytic dehydration of fructose and sucrose to 5-hydroxymethylfurfural with protic ionic liquids

The renewable furan-based platform chemical, 5-hydroxymethylfurfural (HMF), has been efficiently synthesized from d-fructose and sucrose in the presence of a catalytic amount of protic ionic liquids. The 1-methylimidazolium-based and N-methylmorpholinium-based ionic liquids are employed. As a result, 74.8% and 47.5% yields of HMF are obtained from d-fructose and sucrose, respectively, at 90 °C for 2 h under nitrogen atmosphere when N-methylmorpholinium methyl sulfonate ([NMM]⁺[CH₃SO₃]⁻) is used as the catalyst in an N,N-dimethylformamide-lithium bromide (DMF-LiBr) system. The acidities of ionic liquids are determined by the Hammett method, and the correlation between acidity and catalytic activity is discussed. Moreover, the effects of reaction temperature and time are investigated, and a plausible reaction mechanism for the dehydration of d-fructose is proposed.     

The potential of the satellite derived green chlorophyll index for estimating midday light use efficiency in maize, coniferous forest and grassland

Light use efficiency (LUE) is an important variable in carbon cycle and climate change research. We present an investigation of remotely estimating midday LUE using the green chlorophyll index (CI_green) derived from the cloud-free Moderate Resolution Imaging Spectroradiometer (MODIS) images in maize, coniferous forest and grassland. Similar temporal patterns are observed in both canopy chlorophyll content and midday LUE which indicates that the chlorophyll content in the maize canopy servers as a proxy of midday LUE (R² = 0.736, p < 0.001). Therefore, the CI_green, tested as a good indicator of canopy chlorophyll content (R² = 0.840, p < 0.001), has been demonstrated to be a reliable candidate in providing reasonable estimates of midday LUE with determination coefficient R² equals to 0.820 and a root mean square error (RMSE) of 0.002 mol CO₂ per mol incident photosynthetic photon flux density (PPFD). Further validation of the prediction model derived from the maize site demonstrates that the CI_green has potential to be applied in the coniferous forest and grassland ecosystems with RMSE of 0.005 and 0.004 mol CO₂ mol⁻¹ PPFD, respectively. A comparison analysis between different vegetation types is included and these results could be helpful in the development of future LUE and terrestrial models.     

High catalytic activities in the norbornene polymerization with neutral palladium complexes containing N4-type tetradentate chelating ligands

Norbornene polymerization catalyzed by new Pd(II) complexes bearing N4-type tetradentate ligands obtained from the reaction between a 6-methyl-2-picolinic acid or picolinic acid and appropriate diamines has been studied. A class of new palladium complexes, [Pd(X¹X²bpb)] and [Pd(X¹X²-6-Me₂bpb)] (X¹ = Me, X² = Me (1 and 4); X¹ = H, X² = H (2 and 5); X¹ = H, X² = NO₂ (3 and 6); bpb = N,N′-(o-phenylene)bis(pyridine-2-carboxamidate); 6-Me₂bpb = N,N′-(o-phenylene)bis(6-methylpyridine-2-carbox-amidate)) were synthesized and characterized. The molecular structure of Pd complex 5 was determined by X-ray crystallography, showing distorted square planar configurations. Using modified methylaluminoxanes (MMAO) as an activator, the palladium complexes exhibited high catalytic activities for the polymerization of norbornene. The catalytic activities up to 4.0 × 10⁶ g of PNBEs/mol_Pd·h and M_w up to 8.34 × 10⁵ g/mol with PDI < 2.53 were observed. Amorphous polynorbornenes (PNBEs) were obtained with good solubility in halogenated aromatic solvents. Interestingly, the structural modification with the methyl groups of pyridyl rings and the strong electron-withdrawing substituents induced improvement in solubility, thermal stability and catalytic activity. FT-IR, ¹H, and ¹³C NMR analyses of the polymers suggest that the catalytic polymerization occurs via vinyl addition mechanism.     

Development of cesium phosphotungstate salt and chitosan composite membrane for direct methanol fuel cells

A novel composite membrane has been developed by doping cesium phosphotungstate salt (Cs_xH_3−xPW₁₂O₄₀ (0 ≤ x ≤3), Cs_x-PTA) into chitosan (CTS/Cs_x-PTA) for application in direct methanol fuel cells (DMFCs). Uniform distribution of Cs_x-PTA nanoparticles has been achieved in the chitosan matrix. The proton conductivity of the composite membrane is significantly affected by the Cs_x-PTA content in the composite membrane as well as the Cs substitution in PTA. The highest proton conductivity for the CTS/Cs_x-PTA membranes was obtained with x = 2 and Cs₂-PTA content of 5 wt%. The value is 6 × 10⁻³ S cm⁻¹ and 1.75 × 10⁻² S cm⁻¹ at 298 K and 353 K, respectively. The methanol permeability of CTS/Cs₂-PTA membrane is about 5.6 × 10⁻⁷, 90% lower than that of Nafion-212 membrane. The highest selectivity factor (φ) was obtained on CTS/Cs₂-PTA-5 wt% composite membrane, 1.1 × 10⁴/S cm⁻³ s. The present study indicates the promising potential of CTS/Cs_x-PTA composite membrane as alternative proton exchange membranes in direct methanol fuel cells.     

Automated immunoassay system for AFP-L3% using on-chip electrokinetic reaction and separation by affinity electrophoresis

Implementation of the on-chip immunoassay for α-fetoprotein (AFP)-L3% was achieved using a fully automated microfluidic instrument platform that will prepare the chip and run the assay with a total assay time of less than 10 min. Reagent/sample mixing, concentration, and reaction in microfluidic channels occur by the electrokinetic analyte transport assay (EATA) technique, enabling the integration of all assay steps on-chip. The determination of AFP-L3%, a biomarker for hepatocellular carcinoma, was achieved by the presence of Lens culinaris agglutinin in the separation channel, causing separation of the fucosylated isoform, AFP-L3, from the nonfucosylated AFP-L1 by lectin affinity electrophoresis. Laser-induced-fluorescence (LIF) detection was used to quantitate the labeled immunocomplexes. The limit of detection (LOD) was 0.1 ng/ml AFP, and assay precision of less than 2% coefficient of variation (CV) was obtained for quantitation from 24 to 922 ng/ml total AFP in spiked serum samples. Assay precision of less than 3% CV was obtained for AFP-L3% measurements from 8.5 to 81%. Furthermore, good correlation of test results for 68 patient serum samples with a commercially available reference method (LiBASys assay for AFP-L3%) was obtained, with r² = 0.981 and slope = 1.03.     

Quantitation of hepcidin in human urine by liquid chromatography-mass spectrometry

Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Serum and urine levels are increased in inflammation and suppressed in hemochromatosis, and they may have diagnostic importance. This study describes the development and validation of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. A stable, isotopically labeled internal standard, [¹⁵N,¹³C₂]Gly12,20-hepcidin, was synthesized and a standard quantity was added to urine samples. Extraction was performed using weak cation exchange magnetic nanoparticles. An ion trap mass spectrometer was used to quantify hepcidin in the samples. The hepcidin assay was validated, and good recovery of hepcidin was obtained. The assay is accurate and precise. Urinary hepcidin levels of 3 to 9 nmol/mmol creatinine⁻¹ were found in healthy controls, with reduced levels in hemochromatosis (P < 0.00006) and elevated levels in inflammation (P < 0.00035). In sickle cell disease, a wide range was found, with the mean value not differing significantly from controls (P < 0.26). In summary, a validated method has been developed for the quantitation of hepcidin using a stable, isotopically labeled internal standard and applied to determine the concentrations of hepcidin in the low nanomolar range in urine samples from patients and controls.     

H NMR spectroscopy as a tool to determine accurate photoisomerization quantum yields of stilbene-like ligands coordinated to rhenium(I) polypyridyl complexes

In this work, the use of proton nuclear magnetic resonance, ¹H NMR, was fully described as a powerful tool to follow a photoreaction and to determine accurate quantum yields, so called true quantum yields (Φ_true), when a reactant and photoproduct absorption overlap. For this, Φ_true for the trans-cis photoisomerization process were determined for rhenium(I) polypyridyl complexes, fac-[Re(CO)₃(NN)(trans-L)]⁺ (NN = 1,10-phenanthroline, phen, or 4,7-diphenyl-1,10-phenanthroline, ph₂phen, and L = 1,2-bis(4-pyridyl)ethylene, bpe, or 4-styrylpyridine, stpy). The true values determined at 365 nm irradiation (e.g. Φ_NMR = 0.80 for fac-[Re(CO)₃(phen)(trans-bpe)]⁺) were much higher than those determined by absorption spectral changes (Φ_UV-Vis = 0.39 for fac-[Re(CO)₃(phen)(trans-bpe)]⁺). Φ_NMR are more accurate in these cases due to the distinct proton signals of trans and cis-isomers, which allow the actual determination of each component concentration under given irradiation time. Nevertheless when the photoproduct or reactant contribution at the probe wavelength is negligible, one can determine Φ_true by regular absorption spectral changes. For instance, Φ_313 nm for free ligand photoisomerization determined both by absorption and ¹H NMR variation are equal within the experimental error (bpe: Φ_UV-Vis = 0.27, Φ_NMR = 0.26; stpy: Φ_UV-Vis = 0.49, Φ_NMR = 0.49). Moreover, ¹H NMR data combined with electronic spectra allowed molar absorptivity determination of difficult to isolate cis-complexes.     

Extraction properties for alkali metal picrates of macrocyclic trinuclear (p-cymene)ruthenium(II) and (pentamethylcyclopentadienyl)rhodium(III) complexes

The solvent extraction properties of macrocyclic trinuclear organometallic complexes, [(p-cymene)Ru(pyO₂)]₃ and [Cp^∗Rh(pyO₂)]₃, for Li⁺, Na⁺, and K⁺ picrates have been investigated in a dichloromethane-water system at 25 °C. The extraction rates of the alkali metal picrates with these macrocyclic complex ligands are unusually slow; the shaking times required to attain equilibrium are at least 1 h for [(p-cymene)Ru(pyO₂)]₃ and 20-40 h for [Cp^∗Rh(pyO₂)]₃. From analysis of the equilibrium data, the extraction constants (K_ex = [ML⁺A⁻]_o/[M⁺][L]_o[A⁻]; M⁺ = alkali metal ion, L = macrocyclic ligand, A⁻ = picrate ion, o = organic phase) have been determined. The log K_ex value varies in the sequences, Li⁺ (5.72) > Na⁺ (4.50) > K⁺ (2.88) for [(p-cymene)Ru(pyO₂)]₃ and Li⁺ (4.79) > Na⁺ (2.70) ≈ K⁺ (2.69) for [Cp^∗Rh(pyO₂)]₃. The K_ex values of 6,6-dibenzyl-14-crown-4 (DBz14C4), which is one of the best Li⁺-selective crown ethers, have also been determined for comparison. It is revealed that [Cp^∗Rh(pyO₂)]₃ is much superior to DBz14C4 both in the extractability for Li⁺ and the selectivity for Li⁺ over Na⁺.     

Biosensor based on the biocatalysis of microperoxidase-11 in nanocomposite material of multiwalled carbon nanotubes/room temperature ionic liquid for amperometric determination of hydrogen peroxide

A novel nanocomposite material of multiwalled carbon nanotubes (MWCNTs) and room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF₆) was explored and used to construct a novel microperoxidase-11 (MP-11) biosensor for the determination of hydrogen peroxide (H₂O₂). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to characterize the performance of the biosensor. Under the optimized experimental conditions, H₂O₂ could be detected in a linear calibration range of 0.5 to 7.0 × 10⁻⁷ mol L⁻¹ with a correlation coefficient of 0.9949 (n = 9) and a detection limit of 3.8 × 10⁻⁹ mol L⁻¹ at 3σ. The modified electrodes displayed excellent electrochemical response, high sensitivity, long-term stability, and good bioactivity and selectivity.     

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

Background

Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C_P) it lacks the resolving Cys (C_R), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.

Methods

Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.

Results

Oxidation of the C_P is fast (k_+ 1 > 10³ M^− 1 s^− 1), however the rate of reduction by GSH is slow (k′_+ 2 = 12.6 M^− 1 s^− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized C_P can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k_+ 1 > 10³ M^− 1 s^− 1), but not by Trx. By surface plasmon resonance analysis, a K_D = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical C_R in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.

Conclusions

GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.

General significance

In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.