A novel glutathione peroxidase mimic with antioxidant activity

Many diseases are associated with the overproduction of hydroperoxides that inflict cell damage. A novel cyclodextrin derivative, 6A,6B-diseleninic acid-6A',6B'-selenium bridged beta-cyclodextrin (6-diSeCD), was synthesized to be a functional mimic of glutathione peroxidase (GPX) that normally removes these hydroperoxides. The mimic had high catalytic GPX activity of 13.5 U/micromol, which is 13.6-fold higher than ebselen (PZ51), and was chemically and biologically stable in vitro. Antioxidant activity was studied by ferrous sulfate/ascorbate-induced mitochondria damage model system. These data show that the mimic has great antioxidant activity. Such mimics may result in better clinical therapies for diseases mediated by hydroperoxides.     

Changes in erythrocyte glutathione peroxidase and glutathione reductase in alloxan diabetes

Glutathione peroxidase and glutathione reductase activities were measured in erythrocytes from control, diabetic and insulin-treated diabetic rats. A significant decrease in the activity of glutathione peroxidase and an increase in the glutathione reductase activity were found with increase in the time of diabetes which may result in the alteration in the activity of the pentose phosphate pathway by the modulation of the levels of NADPH. Insulin administration reverses the change in the activity of glutathione peroxidase but does not reverse the glutathione reductase activity during diabetes. The overall changes may be due to changes in the levels of insulin, triiodothyronine and thyroxine.     

Environmental induction models for the investigation of activity: changes in glutathione peroxidase, a crucial factor of the antioxidant defence

Glutathione peroxidase enzyme superfamily plays significant role in the elimination of reactive oxygen free radicals in the animals. Many characteristics of these proteins have been revealed already, but their regulation is still not known. Several data suggest that some environmental factors have certain regulatory effect, while others propose strict genetic regulation. In this report we present four different environmental induction models in which New Zealand white rabbits were used as experimental animals. In three models, free radical load of different origin, lipidperoxide load, application of a glutathione depletor or a prooxidant agent, was introduced. Beside these negative models a positive model was also constructed in which additive selenium was supplied. Glutathione peroxidase activity was measured in blood serum, erythrocyte haemolysate and liver. Reduced glutathione, and malondialdehyde concentration in the liver were also determined. According to the results, the established models are capable for analysing the enzyme activity x environmental interactions.     

The effect of prolonged oral contraceptive steroid use on erythrocyte glutathione peroxidase activity

A clinical study was undertaken to determine whether oral contraceptives (OCs) affect the activity of the enzyme glutathione peroxidase. OC users recruited for the study were volunteers attending the Redhill Family Planning Clinic in England. Their demographic characteristics were noted. Pre- and postmenopausal comparative subjects were also used. The laboratory procedures involved in the study are described. Findings are tabulated. The average erythrocyte glutathione peroxidase levels of women using OCs for more than 7 months were significantly higher than those of the pre- and postmenopausal subjects. These levels increased progressively with duration of OC use. These levels did not fluctuate with the menstrual cycle in either OC or non-OC users. Levels of erythrocyte selenium and plasma pyridoxal were not significantly altered by OC use. Riboflavin status, however, as estimated by glutathione reductase activity was substantially lower in OC users and was lowest in women who had used OCs for the longest amount of time. Riboflavin status was found to be directly correlated with erythrocyte glutathione peroxidase levels. These findings may be important because selenium is currently believed to offer protective benefits against carcinogenesis, especially breast cancer. All the OCs studied produced the same effects.     

Further electrophoretic studies of erythrocyte glutathione peroxidase.

A new, inexpensive technique has been devised for the detection of glutathione peroxidase after electrophoresis. Surveys of several racial groups have revealed new variant phenotypes at polymorphic frequencies in African and Lebanese populations.     

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/micromol and 28.8U/micromol, 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.     

Age-associated changes in oxidative damage and the activity of antioxidant enzymes in rats with inherited overgeneration of free radicals

Reactive oxygen species have been hypothesized to play an important role in the process of aging. To investigate the correlation between oxidative stress and accumulation of protein and DNA damage, we have compared the age-dependent levels of protein carbonyl groups and the activities of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase in cytosol and mitochondrial extracts from liver cells of Wistar and OXYS rats. The latter strain is characterized by increased sensitivity to free radicals. Faster age-dependent increase in the level of protein carbonyl groups was found in OXYS as compared with Wistar rats. A complicated enzyme-specific pattern of age-dependent changes in the activities of antioxidant enzymes was observed. Long-term uptake of dietary supplements Mirtilene forte (extract from the fruits of Vaccinium myrtillus L.) or Adrusen zinco (vitamin E complex with zinc, copper, selenium and omega-3 polyunsaturated fatty acids) sharply decreased the level of protein oxidation in cytosol and mitochondrial extracts of hepatocytes of Wistar and of OXYS rats. Both dietary supplements increased the activity of catalase in the liver mitochondria of OXYS rats. Our results are in agreement with the shorter life-span of OXYS and with the mitochondrial theory of aging, which postulates that accumulation of DNA and protein lesions leads to mitochondrial dysfunction and accelerates the process of aging.     

Diurnal changes in the total antioxidant activity of human saliva

Twenty-four-hour monitoring of saliva antioxidant activity was performed in eight women and four men 18–60 years old. Its maximum, detected at 6 a.m., was more than twice as high (p < 0.001) as its minimum (at 3 p.m.).     

Plasma and erythrocyte selenium and glutathione peroxidase activity in preterm infants at risk for bronchopulmonary dysplasia

The purpose of this study was to examine the relationships between selenium status, as measured by plasma and erythrocyte selenium and glutathione peroxidase (GPx) activity, and other postnatal factors, including selenium intake, gestational age, and oxygen dependence in preterm infants at risk for bronchopulmonary dysplsia. Eighteen preterm infants of 30 wk gestational age or less were included. At postnatal wk 1 and 4, selenium concentrations and GPx activity were measured and oxygen dependence and daily selenium intakes were determined from the medical chart. Plasma and erythrocyte selenium concentrations decreased from wk 1 to wk 4, whereas erythrocyte GPx activity increased. Increased selenium intakes during wk 1 were associated with increased erythrocyte GPx activity at both time-points, as well as a decreased need for supplemental oxygen on d 28. Preterm infants display increasing erythrocyte GPx activity despite declines in plasma and erythrocyte selenium. GPx activity might be enhanced by very early selenium supplementation.     

A novel dicyclodextrinyl diselenide compound with glutathione peroxidase activity

A 6A,6A'-dicyclohexylamine-6B,6B'-diselenide-bis-beta-cyclodextrin (6-CySeCD) was designed and synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). In this novel GPX model, beta-cyclodextrin provided a hydrophobic environment for substrate binding within its cavity, and a cyclohexylamine group was incorporated into cyclodextrin in proximity to the catalytic selenium in order to increase the stability of the nucleophilic intermediate selenolate. 6-CySeCD exhibits better GPX activity than 6,6'-diselenide-bis-cyclodextrin (6-SeCD) and 2-phenyl-1,2-benzoisoselenazol-3(2H)-one (Ebselen) in the reduction of H(2)O(2), tert-butyl hydroperoxide and cumenyl hydroperoxide by glutathione, respectively. A ping-pong mechanism was observed in steady-state kinetic studies on 6-CySeCD-catalyzed reactions. The enzymatic properties showed that there are two major factors for improving the catalytic efficiency of GPX mimics. First, the substrate-binding site should match the size and shape of the substrate and second, incorporation of an imido-group increases the stability of selenolate in the catalytic cycle. More efficient antioxidant ability compared with 6-SeCD and Ebselen was also seen in the ferrous sulfate/ascorbate-induced mitochondria damage system, and this implies its prospective therapeutic application.     

Decreased glutathione peroxidase activity in sciatic nerve of alloxan-induced diabetic mice and its correlation with blood glucose levels

The effect of alloxan-induced diabetes on glutathione peroxidase (GSH-Px) activity in sciatic nerve of mice has been studied. We have found, 7 days after alloxan treatment, a significant decrease in this enzymatic activity in the cytosol of sciatic nerve of diabetic mice, and moreover, that these changes remained unaltered up to 21 days after alloxan injection. No modification in the glutathione content of sciatic nerve of diabetic mice was observed throughout the experiment when compared with controls. The decrease in GSH-Px activity in this tissue shows a good correlation with the increase of blood glucose levels throughout the experiment. It is hypothesized whether a combination of mechanisms could be involved in this decrease of GSH-Px activity and if oxygen radicals might be the common mediators of these processes.     

Human erythrocyte catechol-O-methyltransferase: correlation with lung and kidney activity

Catechol-O-methyltransferase (COMT) activity measured in homogenates of human lung or renal cortical tissue was approximately an order of magnitude greater than the enzyme activity in human erythrocytes. Lung and blood enzyme activities varied from individual to individual over a 5–7 fold range. There was a highly significant correlation between the COMT activity in erythrocytes and the enzyme activity in lung (r = 0.59, P < .001, N = 29) and kidney (r = 0.81, P < .005, N = 12) of the same subjects. These results suggest that the relative COMT activity in lung and kidney might be predicted by the measurement of the enzyme activity in blood and raise the possibility that individual variation in the metabolism of endogenous catecholamines and catechol drugs in man may result in part from variations in COMT activity.     

A trifunctional enzyme with glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.     

Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro.

Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni. Using partially purified cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII-EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical. Fasciola hepatica cytochrome c peroxidase effectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low specific activity of the final partially purified sample the effects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated. The activity of the partially purified enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially purified enzyme with 1 mM calcium chloride did not have any effect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (.OH). We suggest that the capacity of CcP to inhibit .OH-formation, by efficiently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica.     

77Se NMR characterization of 77Se-labeled ovine erythrocyte glutathione peroxidase

Lambs, maintained on a selenium-deficient diet supplemented with 94 atom % Na2 27SeO3, have been used as a source of 77Se-enriched erythrocyte glutathione peroxidase. After 5 months on this diet, the percentage of selenium in the enzyme derived from the supplement had reached 88%. From each monthly bleeding of two sheep, approximately 20 mg of 77Se-enriched glutathione peroxidase could be isolated in pure form. Although attempts to observe 77Se NMR signals from the native enzyme labeled with 6,6'-[77Se]diselenobis-(3-nitrobenzoic acid) failed, due to the low solubility of the enzyme, two 77Se resonances were observed after unfolding the enzyme with 8 M urea and reaction with iodoacetamide. These resonances, at 195 and 377 ppm, were from the selenoether alkylamide derivative and from protein cross-linked selenide sulfide species, respectively. Relaxation time measurements on the selenoether at 4.7 and 9.4 teslas enabled an estimate of the chemical shift anisotropy to be made. A value of less than or equal to 262 ppm was determined. Reduction of the denatured selenide sulfide species with dithiothreitol gave an observable 77Se resonance from the Se- moiety at pH 8 and from SeH at pH 4.2. The chemical form of the selenocysteine residue in the resting state enzyme most consistent with formation of the acetamide derivative and the selenide sulfide is Se- or SeH. From the magnitudes of the estimated chemical shift anisotropies, it is predicted that direct observation of selenium in the native enzyme will be feasible if the enzyme concentration can be increased to 0.25 mM tetrameric glutathione peroxidase.     

Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activity

In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH-S-2,4-dinitrophenyl t-butyl ester (GSH-S-DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH + VL single-chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv-encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv-B3) in Escherichia coli Rosetta. The scFv-B3 was purified by Ni(2+)-immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0-3.0 mg of proteins from 1 L culture. After the active site serines of scFv-B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se-scFv-B3) with GPX activity of 1288 U/micromol. Copyright (c) 2008 John Wiley & Sons, Ltd.