A theoretical investigation of the preferred conformations of glutathione and its constituent amino acid residues

The preferred conformations of the tripeptide glutathione have been investigated by performing quantum mechanical calculations using the PCILO method. A series of model compounds representing fragments of the tripeptide has been studied as well as the complete molecule. The results are compared with the available experimental data.     

Capillary zone electrophoresis for analysis of phytochelatins and other thiol peptides in complex biological samples derivatized with monobromobimane

A new method to improve the analysis of phytochelatins and their precursors (cysteine, gamma-Glu-Cys, and glutathione) derivatized with monobromobimane (mBrB) in complex biological samples by capillary zone electrophoresis is described. The effects of the background electrolyte pH, concentration, and different organic additives (acetonitrile, methanol, and trifluoroethanol) on the separation were studied to achieve optimum resolution and number of theoretical plates of the analyzed compounds in the electropherograms. Optimum separation of the thiol peptides was obtained with 150 mM phosphate buffer at pH 1.60. Separation efficiency was improved when 2.5% v/v methanol was added to the background electrolyte. The electrophoretic conditions were 13 kV and capillary dimensions with 30 cm length from the inlet to the detector (38 cm total length) and 50 microm inner diameter. The injection was by pressure at 50 mbar for 17 s. Under these conditions, the separation between desglycyl-peptides and phytochelatins was also achieved. We also describe the optimum conditions for the derivatization of biological samples with mBrB to increase electrophoretic sensitivity and number of theoretical plates. The improved method was shown to be simple, reproducible, selective, and accurate in measuring thiol peptides in complex biological samples, the detection limit being 2.5 microM glutathione at a wavelength of 390 nm.     

Novel fiber-optic biosensor based on immobilized glutathione S-transferase and sol–gel entrapped bromcresol green for the determination of atrazine

The aim of this work was to investigate, for the first time, the potential of the enzyme glutathione S-transferase I (isoenzyme GST-I) for uses in analytical chemistry. A novel fiber-optic biosensor for the detection and determination of the triazine herbicide atrazine was developed based on maize GST-I expressed in E. coli. The sensing bioactive material was a three-layer mini-sandwich. The enzyme was immobilized on the outer layer that consisted of a hydrophilic polyvinylidenefluoride membrane. This membrane was supported on an inner glass disk by means of an intermediate binder sol–gel layer that incorporated bromcresol green (BCG). The biosensor operated in a static mode at 25 °C and the rate of the enzymatic reaction, using atrazine as a substrate, served as an analytical signal. A calibration curve was obtained for atrazine, with analytically useful concentration range 2.52–125 μM. The sensor detection limit was 0.84 μM. The reproducibility of atrazine sensing was in the order of ±3–5%. The method was successfully applied to the determination of this herbicide in real water samples, without sample preparation steps. Atrazine recovery ranged between 85 and 110%. No interference from other pesticides, such as alachlor and carbaryl was observed in the absence of atrazine. The immobilized enzyme retained about 75% of its original activity after 1 month use. Simply unscrewing the terminal holding ring of the probe and placing a new bioactive sandwich could easily replace a deteriorated mini-sandwich.     

Bioimprinted protein exhibits glutathione peroxidase activity

A strategy for design of bioimprinted proteins with glutathione peroxidase (GPX) activity has been proposed. The proteins imprinted with a glutathione derivative were converted into selenium-containing proteins by chemical modifying the reactive hydroxyl groups of serines followed by sodium hydrogen selenide displacement. These selenium-containing proteins exhibited remarkable GPX activities and the GPX activities of reduction of H₂O₂ by glutathione (GSH) were found to be 101-817 U μmol⁻¹, which approaches the activity of a selenium-containing catalytic antibody elicited by a hapten similar to our template. The steady state kinetic study for imprinted protein catalysis revealed Michaelis-Menten kinetics for both H₂O₂ and GSH, e.g. the pesudo-first-order rate constant k_cat (H₂O₂) and the apparent Michaelis constant K_m (H₂O₂) at 1 mM GSH were calculated to be 784 min⁻¹ and 1.24×10⁻³ M, respectively, and the apparent second-order rate constant k_cat (H₂O₂)/K_m (H₂O₂) was determined to be 6.33×10⁵ (M min)⁻¹. The kinetics and the template inhibition showed that the strategy might be a remarkably efficient one for generating artificial enzyme with GPX activity.     

Renewable sol-gel carbon ceramic electrodes modified with a Ru-complex for the amperometric detection of l-cysteine and glutathione

A renewable three-dimensional chemically modified carbon ceramic electrode containing Ru [(tpy)(bpy)Cl] PF₆ was constructed by sol-gel technique. It exhibits an excellent electro-catalytic activity for oxidation of l-cysteine and glutathione at pH range 2-8. Cyclic voltammetry was employed to characterize the electrochemical behavior of the chemically modified electrode. The electrocatalytic behavior is further exploited as a sensitive detection scheme for l-cysteine and glutathione by hydrodynamic amperometry. Optimum pH value for detection is 2 for both l-cysteine and glutathione. The catalytic rate constants for l-cysteine and glutathione were determined, which were about 2.1×10³ and 2.5×10³ M⁻¹ s⁻¹, respectively. Under the optimized condition the calibration curves are linear in the concentration range 5-685 and 5-700 μM for l-cysteine and glutathione determination, respectively. The detection limit (S/N=3) and sensitivity is 1 μM, 5 nA/μM for l-cysteine and 1 μM, 7.8 nA/μM for glutathione. The relative standard deviation (RSD) for the amperogram's currents with five injections of l-cysteine or glutathione at concentration range of linear calibration is <1.5%. The advantages of this amperometric detector are: high sensitivity, good catalytic effect, short response time (t<3 s), remarkable long-term stability, simplicity of preparation and reproducibility of surface fouling (RSD for six successive polishing is 3.31%). This sensor can be used as a chromatographic detector for analysis of l-cysteine and glutathione.     

Ormosil Encapsulated Pyrroloquinoline Quinone‐Modified Electrochemical Sensor for Thiols

An organically modified sol‐gel glass (ORMOSIL) encapsulating pyrroloquinoline quinone (PQQ)‐modified electrode for the rapid, sensitive and simple determination of thiol‐containing compounds such as cysteine and glutathione is reported. The effect of applied potential, nature of thiol compound and pH on the response of the sensor was examined and optimum conditions were determined. The electrochemical responses and detection limits were found to be sensitive to the nature of thiols and pH. The electrochemical responses for cysteine and glutathione at an applied potential of ?0.2 V (vs. Ag/AgCl) were found to be linear with detection limits of 18 nM for cysteine and 36 nM for glutathione at pH 3.5, whereas the detection limits at pH 8.5 were 0.5 μM for cysteine and 1 μM for glutathione. The electrode retained 95% of the original response for 7 days when stored at 4 °C. The ORMOSIL‐encapsulated PQQ was also characterized by spectrophotometry. The absorbance measurement using 5,5′‐dithiobis(2‐nitrobenzoic acid) at 412 nm justify the PQQ‐mediated oxidation of glutathione whereas fluorescence measurements (excitation wavelength=380 nm; emission wavelength=480 nm) justify the successful encapsulation of PQQ in ORMOSIL matrix.     

Quantification of phytochelatins in plants by reversed-phase HPLC–ESI–MS–MS

An on-line HPLC–ESI–MS–MS method has been developed for determination of glutathione and phytochelatins (PC) in plant tissues. For sample pretreatment, dithiothreitol (DTT) must be added at the very beginning, as an anti-oxidant. Optimization of instrumental conditions i.e. composition of HPLC mobile phase, ionization efficiency of the electrospray interface, and MS–MS detection in the multiple ion-monitoring mode, are the central aspects of this work. A polystyrene-packed column was found to be superior to a standard silica-packed reversed-phase column. A concave quadratic gradient of ammonium formate buffer and acetonitrile was found to be optimum. The limits of quantitation were 0.2 mol kg^–1 plant tissue for glutathione and PC. The method has been applied to analysis of tissue samples from Vicia faba grown in Cd-containing nutrient solutions.Dedicated to the memory of Wilhelm Fresenius     

Investigation of Cd(II), Pb(II) and Cu(I) complexation by glutathione and its component amino acids by ESI-MS and size exclusion chromatography coupled to ICP-MS and ESI-MS

The elucidation of structures of glutathione (GSH) complexes play an important role in the fundamental understanding of biochemical pathways of metal ion deactivation in plants. This article attempts to feature key studies for stoichiometry of metal complexes with glutathione and its constituent amino acids to obtain a better understanding of the different metal affinities of the complexation sites of glutathione. The SEC-ICP-MS experiments have indicated that oxidation process of glutathione was accelerated by metal ion presence in following order Cu⁺, Pb²⁺ and Cd²⁺. The redox activity of metal ions was confirmed by ESI-MS experiments, which allowed to observe formation of glutathione disulphide (GSSG) in time. The stoichiometry of Cd²⁺, Cu⁺ and Pb²⁺ complexes with GSH was defined by observing the isotope pattern of investigated metals and hydrogen loss or transfer during binding. The complexes with metal bound to sulphur of 1:1 and 1:2 stoichiometry were found in case of cadmium and lead. The number of hydrogen atoms lost during metal binding and the SEC-ESI-MS results allowed to elucidate that copper is bound by GSSG in ratio 1:1 and 1:2. Additionally, size exclusion chromatography coupled to electrospray MS allowed to differentiate more stable complexes from weak ones that could be created in the gas phase.     

Comparison of pulsed amperometric detection and integrated voltammetric detection for inorganic sulfur compounds in liquid chromatography

A Variety of potential–time waveforms are useful in pulsed electrochemical detection (PED) when applied for the amperometric detection of numerous polar organic compounds following their separation by liquid chromatography (LC). Here, we compare the waveforms for pulsed amperometric detection (PAD) and integrated voltammetric detection (IVD) applied for detection of organosulfur compounds at Au electrodes in acidic media. In PAD waveforms, electrodes response is measured at a constant detection potentials. In IVD waveforms, electrodes current is integrated throughout a fast cyclic scan of the detection potential. As a consequence of this difference in detection strategy, the background signal for IVD is significantly smaller for PAD in the detection of organosulfur compounds whose response mechanisms require the concomitant formation of surface oxides on Au electrodes. Furthermore, in comparison to Pad, IVD has a larger sensitivity and a diminished system peak from 0₂ dissolved in the sample. Use of a preadsorption step increases detection sensitivity in both PAD and IVD. The limit of detection (S/N=3)for cysteine in LC-IVD is ca. 6 nM for a 50-μl injection (i.e., 300 fmol) using a detection waveform that includes a 1000-ms preadsorption period.     

The extraction,antioxidant and against β-amyloid induced toxicity of polyphenols from Alsophila spinulosa leaves

Alsophila spinulosa is a tree-like fern, and many evidences suggested that plant polyphenols had the potential therapeutic for Alzheimer s disease (AD). Herein, polyphenols (ASP) was isolated from A. spinulosa leaves and its major constituent were isoorientin and vitexin. ASP displayed excellent antioxidant activity and obvious anti-lipid peroxidation capacity in vitro. ASP improved the survival rate of C. elegans under high temperature by enhancing the antioxidant enzymes activities and decreasing the lipid peroxidation level. Moreover, ASP alleviated β-amyloid (Aβ) induced paralysis and reduced Aβ deposition, decreased reactive oxygen species (ROS) accumulation and improved the level of skn-1 mRNA. In addition, ASP decreased the levels of pdk-1 and akt-1 mRNA in P13K/AKT signaling pathway. In conclusion, ASP may be a potential ingredient for the alleviation of AD.