Glutathione-capped CdTe quantum dots for the sensitive detection of glucose

A simple and sensitive assay system for glucose based on the glutathione (GSH)-capped CdTe quantum dots (QDs) was developed. GSH-capped CdTe QDs exhibit higher sensitivity to H2O2 produced from the glucose oxidase catalyzed oxidation Of glucose, and are also more biocompatible than other thiols-capped QDs. Based on the quenching of H2O2 on GSH-capped QDs, glucose can be detected. The detection conditions containing reaction time, the concentration of glucose oxidase and the sizes of QDs were optimized and the detection limits for glucose was determined to be 0.1 mu M; two detection ranges of glucose from 1.0 mu M to 0.5 mM and from 1.0 mM to 20 mM, respectively Were obtained. The detection limit was almost a 1000 times lower than other QDs-based optical glucose sensing systems. The developed glucose detection system was simple and facile with no need of complicated enzyme immobilization and modification of QDs.

Synthesis and catalytic kinetics of tellurium-modified hyaluronic acid with glutathione peroxidase activity

A novel mimic TeHA was synthesized by modifying hyaluronic acid (HA) with tellurium, whose function is similar to that of glutathione peroxidase (GPX). The structure of TeHA was characterized by means of infrared spectroscopy and nuclear magnetic resonance spectroscopy, showing that the target Te is located at -CH2OH of the N-acetyl-D-glucosamine of HA. The activity of TeHA is 163.6 U/mu mol according to Wilson's method. In contrast to other mimics, TeHA displays a high activity. Moreover, TeHA can use many hydroperoxides as substrates, such as H2O2, cumenyl hydroperoxide, and tert-butyl hydroperoxide, and cumenyl hydroperoxide is the optimal substrate. A ping-pong mechanism was deduced for the reduction reactions catalyzed by TeHA according to the steady-state kinetic studies.

Synthesis and kinetics of a novel mimic with glutathione peroxidase activity - Tellurium-containing hyaluronic acid (TeHA)

A novel mimic was synthesized by modifying hyaluronic acid (HA) with tellurium, whose function is similar to that of glutathione peroxidase (GPX). The structure of TeHA was characterized by means of IR and NMR, the target-Te was located at -CH2OH of the N-acetyl-D-glucosamine of HA. The H2O2 reducing activity of TeHA, by glutathione (GSH), was 163.6 U/mu mol according to Wilson's method. In contrast to other mimics, TeHA displayed the highest activity. Moreover, TeHA accepted many hydroperoxides as its substrates, such as H2O2, cumenyl hydroperoxide (CuOOH) and tert-butyl hydroperoxide (t-BuOOH), and CuOOH was the optimal substrate of TeHA. A ping-pong mechanism was observed in the steady-state kinetic studies of the reactions catalyzed by TeHA.

Studies of soluble human catalytic antibody with glutathione peroxidase activity

By screening the phage-displayed human single chain antibody library, we have got the specific single chain antibody bound to GSH-S-DNP butyl ester as the hapten. The tertiary structure of the protein was analyzed with the aid of computer, and the results showed the CDR3 region located on the surface of the antibody. The soluble antibody was expressed in E. coli. and the active site serine was converted into selenocysteine with the chemical modifying method, which resulted in the catalytic antibody with GPx activity of 80 U/mu mol. Furthermore, the same Ping-Pong mechanism as the natural GPx was observed when the kinetic behavior of the antibody was studied.

Human catalytic antibodies with glutathione peroxidase activity

In order to generate catalytic antibodies with glutathione peroxidase (GPx) activity, we prepared GSH-S-DNP butyl ester and GSH-S-DNP benzyl ester as the haptens. Two ScFvs that bound specifically to the haptens were selected from the human phage-displayed antibody library. The two ScFv genes were highly homologous, consisting of 786 bps and belonging to the same VH family-DP25. In the premise of maintaining the amino acid sequence, mutated plasmids were constructed by use of the mutated primers in PCR, and they were over-expressed in E. coli. After the active site serine was converted into selenocysteine with the chemical modifying method, we obtained two human catalytic antibodies with GPx activity of 72.2U/mu mol and 28.8U/mu mol, respectively. With the aid of computer mimicking, it can be assumed that the antibodies can form dimers and the mutated selenocysteine residue is located in the binding site. Furthermore, the same Ping-Pong mechanism as the natural GPx was observed when the kinetic behavior of the antibody with the higher activity was studied. (C) 2001 Elsevier Science BY. All rights reserved.

Cyclodextrin-catalyzed oxidation of glutathione in solution and in an ion trap

Incubated solutions containing glutathione (GSH) and alpha- or beta-cyclodextrins (CDs) were analyzed using electrospray mass spectrometry and tandem mass spectrometry, The results suggest that both CDs can catalyze oxidation of GSH to the oxidized glutathione (GSSG). The collision-induced dissociation (CID) of the 1:1 and 1:2 (CD/GSH) and 1:1 (CD/GSSG) complexes reveals the strong interactions of the CDs with the peptides tested. The 1:2 (CD/GSH) complex is considered to be the oxidation reaction intermediate, which indicates that the three-dimensional structure of the complexed two GSHs in CD complexes Is different from that of the proton-bound GSH dimer, The oxidation product, GSSG, Is also observed in the CID spectrum of the singly charged 1:1 (CD/GSH) complex, suggesting that a complex ion-complex ion reaction occurs by forming a doubly charged complex dimer, as a result of the ability of ion trap to accumulate and activate ions. The observations indicate that ion trap mass spectrometry can be used to explore cyclodextrin-catalyzed reactions and to carry out complex gaseous chemistry research. Copyright (C) 1999 John Wiley & Sons, Ltd.

A selenium-containing abzyme, the activity of which surpassed the level of native glutathione peroxidase

Using two different glutathione derivatives as hapten, we have prepared two abzymes, which display glutathione peroxidase (GPX) activity. Their GPX activities are 0.2 and 1.6 times that of natural GPX from rabbit liver, respectively. Selenium content analysis indicates that the activity difference between the two abzymes is possibly attributed to the conformation difference of the abzymes.

Hydrophobic pocket modification and humanized catalytic antibodies with glutathione peroxidase activity (I) - Preparation and characterization of haptenes and conjugates

The thiol group of glutathione (GSH) was protected by 2,4-dinitrochlorobenzene (DNCB), the product S-substituted dinitrophenyl GSH(GSH-S-DNP) was alcoholized to obtain haptenes 4 and 5 respectively. As haptenes, the two GSH derivatives were characterized by means of H-1 NMR, MALDI-TOF-MS and IR, followed by individually coupling with bovine serum albumin (BSA) via glutaraldehyde. BSB-Hp4 and BSA-Hp5 were purified by Sephadex G-25 gel filtration chromatography. For each conjugate, the average haptene-BSA ratio was 12 : 1. The electrophoresis analysis showed that the average molecular weight of each conjugate was 76 500. The CD spectrum indicated that the conjugates had more a-helix content than BSA did.

Biochemical characterization of selenium-containing catalytic antibody as a cytosolic glutathione peroxidase mimic

A selenium-containing catalytic antibody (Se-4A4), prepared by converting reactive serine residues of a monoclonal antibody (4A4) raised against a GSH derivative into selenocysteines, acts as a mimic of cytosolic glutathione peroxidase (cGPX). To clarify the mechanism of action of this catalytic antibody, detailed studies on kinetic behaviour and biological activity were carried out. A rate of acceleration (k(cat)/K-m/k(uncat)) 10(7)-fold that of the uncatalytic reaction is observed. Under similar conditions, the turnover number (k(cat)) of Se-4A4 is 42% of that of the natural rabbit liver cGPX. The Se-4A4 reaction involves a Ping Pong mechanism, which is the same as that of the natural cGPX. The selenocysteine residue is located in the binding site of the antibody and is shown to be crucial for this activity. Of the thiol compounds tested, only GSH is able to serve as substrate for Se-4A4. It was demonstrated, using the free-radical-damage system (hypoxanthine/xanthine oxidase) of cardiac mitochondria, that Se-4A4 can protect mitochondria from free-radical damage at least 10(4)-fold more effectively than the natural cGPX.

DETERMINATION OF CYSTEINE AND REDUCED GLUTATHIONE IN HUMAN PLASMA BY LIQUID-CHROMATOGRAPHY WITH PULSED AMPEROMETRIC ELECTROCHEMICAL DETECTION USING A PLATINUM-PARTICLES MODIFIED GLASSY-CARBON ELECTRODE

An assay procedure utilizing pulsed amperometric detection at a platinum-particles modified electrode has been developed for the determination of cysteine and glutathione in blood samples following preliminary separation by reversed-phase liquid chromatography. A chemically modified electrode (CME) constructed by unique electroreduction from a platinum-salt solution to produce dispersed Pt particles on a glassy carbon surface was demonstrated to catalyze the electo-oxidation of sulfhydryl-containing compounds: DL-cysteine (CYS), reduced glutathione (GSH). When used as the sensing electrode in flow-system pulsed-amperometric detection (PAD), electrode fouling could be avoided using a waveform in which the cathodic reactivation process occurred at a potential of - 1.0 V vs. Ag/AgCl to achieve a cathodic desorption of atomic sulfur. A superior detection limit for these free thiols was obtained at a Pt particle-based GC electrode compared with other methods; this novel dispersed Pt particles CME exhibited high electrocatalytic stability and activity when it was employed as an electrochemical detector in FIA and HPLC for the determination of those organo-sulfur compounds.

LIQUID-CHROMATOGRAPHY WITH ELECTROCATALYTIC DETECTION OF CYSTEINE, N-ACETYLCYSTEINE AND GLUTATHIONE BY A PRUSSIAN BLUE FILM-MODIFIED ELECTRODE

Chemically modified electrodes prepared by adsorbing prussian blue on a glassy carbon electrode are shown to catalyse the electro-oxidation of cysteine, N-acetylcysteine and glutathione in acidic media. The catalytic response is evaluated with respect to the potential scan rate, the solution pH, the concentration dependence, and other variables. Covering the electrode with Nafion(R) film improved the stability and reproducibility in liquid chromatography with electrochemical detection to the extent that repetitive sample injections produced relative standard deviations of less than 5% over several hours of operation. The limit of detection was 4 pmol for cysteine, 33 pmol for glutathione and 61 pmol for N-acetylcysteine.

A GLASSY-CARBON ELECTRODE MODIFIED BY DISPERSED ALPHA-ALUMINA AS AN ENHANCED ELECTROCHEMICAL DETECTOR IN LIQUID-CHROMATOGRAPHY FOR CYSTEINE AND GLUTATHIONE

The dispersion of alumina particles on a glassy-carbon surface serving as a modified electrode significantly enhances the amperometric detection of cysteine and glutathione following liquid chromatography. With an applied potential of 0.8 V vs. SCE, the detection limits were 1.2 ng for cysteine and 8 ng for glutathione and the electrode response was linear up to 600 ng for cysteine and 1.8-mu-g for glutathione. The modified electrode displayed high sensitivity and stability and was easy and inexpensive to prepare.

IR AND RAMAN-SPECTRA STUDIES OF THE RARE EARCH COMPLEXES WITH GLUTATHIONE

The complexes of rare earth ions with glutathione were prepared and charactrized by IR and Raman spectroscopy in the solid state. Based on the spectral results, the structure and coordination sites of the ligand in these complexes were determined.

Arginine kinase from Litopenaeus vannamei: Cloning, expression and catalytic properties

Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. in this paper, the full-length cDNA of AI( was cloned from shrimp, Litopenaeus vannamei by using RT-PCR and RACE PCR. It was 1446 bp encoding 356 amino acids, and belongs to the conserved phosphagen kinase family. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of AK with the highest expression in the muscle and the lowest in the skin. The expression of AK after challenge with LIPS was tested in hemocytes and muscle, which indicated that the two peak values were 6.2 times (at 3 h) and 10.14 times (at 24 h) in the hemocytes compared with the control values, respectively (P < 0.05), while the highest expression of AK was 41 times (at 24 h) in the muscle compared with the control (P < 0.05). In addition, AK was expressed in Eschetichia coli by prokaryotic expression plasmid pGEX-4T-2. The recombinant protein was expressed as glutathione s-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. After cleavage from GST by using a site-specific protease, the recombinant protein was identified by ESI-MS and showed AK activity. After treatment with 10 mM ATP, the enzyme activity significantly increased. However, the enzyme activity was inhibited by 10 mM alpha-ketoglutarate, 50 mM glucose and 200 mM ATP. This research suggested that AK might play an important role in the coupling of energy production and utilization and the immune response in shrimps. (C) 2009 Elsevier Ltd. All rights reserved.

cDNA cloning and mRNA expression of a selenium-dependent glutathione peroxidase from Zhikong scallop Chlamys farreri

The glutathione peroxidases are essential enzymes of the cellular antioxidant defence system. In the present study, the full-length cDNA sequence encoding an extracellular glutathione peroxidase (designated CfGPx3) was isolated from Zhikong scallop Chlamys farreri. The complete cDNA was of 1194 bp, containing a 5' untranslated region (UTR) of 50 bp, a 3' UTR of 490 bp and an open reading frame (ORF) of 654 bp encoding a polypeptide of 217 amino acids. CfGPx3 possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase, such as the selenocysteine encoded by stop codon UGA, the GPx signature motif ((96)LGVPCNQFI(103)) and the active site motif ((WNFEKF184)-W-179). The high similarity of CfGPx3 with GPx from other organisms indicated that CfGPx3 should be a new member of the glutathione peroxidase family. By fluorescent quantitative real-time PCR, the CfGPx3 mRNA was universally detected in the tissues of haemocytes, gill, gonad, muscle and hepatopancreas with the highest expression in hepatopancreas. After scallops were challenged by Listonella anguillarum, the expression level of CfGPx3 transcript in haemocytes was significantly up-regulated (P<0.05) at 8 h post challenge. These results suggested that CfGPx3 was potentially involved in the immune response of scallops and perhaps contributed to the protective effects against oxidative stress. (C) 2010 Elsevier Inc. All rights reserved.