Direct assay of glutathione peroxidase activity using high-performance capillary electrophoresis

Pascual, P.; Martı́nez-Lara, Esther; Bárcena, José Antonio; López-Barea, Juán; Toribio, F.

doi:10.1016/0378-4347(92)80446-w

Cited by 56 publications

(34 citation statements)

References 25 publications

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“…In this assay system, oxidized glutathione (GSSG), which is produced by reaction of GSH with H 2 O 2 , is reduced by NADPH to GSH with the enzymatic function of GR; hence, the reaction progress can be monitored by the decrease in the UV absorbance at 340 nm due to NADPH. [10] NADPH was completely consumed in ca. 600 s in the absence of a catalyst, as shown in Figure 1.…”

Section: Resultsmentioning

confidence: 99%

“…In the NADPH and glutathione reductase (GR)-coupled assay, [10] a test solution was prepared by mixing a 100 m phosphate/6 m EDTA buffer solution (1941 µL) at pH 7.4 containing NADPH (2.0 µmol) and GSH (6.8 µmol) with a GR solution (453 U/mL, 59 µL). An aliquot (300 µL) of the test solution was added to a 1.0 m selenide solution (200 µL) in 100 m phosphate buffer at pH 7.4 and the phosphate buffer solution (430 µL).…”

Section: Discussionmentioning

confidence: 99%

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A Water‐Soluble Cyclic Selenide with Enhanced Glutathione Peroxidase‐Like Catalytic Activities

Kumakura¹,

Mishra²,

et al. 2010

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Antioxidative catalytic activities of trans-3,4-dihydroxyselenolane (DHS red ), a water-soluble cyclic selenide, were investigated in the reaction of hydrogen peroxide with three different thiol substrates, monothiol glutathione (GSH), dithiol dithiothreitol (DTT red ), and polythiol reduced ribonuclease A (RNase A) having eight thiol groups along the polypeptide chain. For all the thiol substrates, DHS red exhibited higher glutathione peroxidase (GPx)-like antioxidative catalytic ac-

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A Water‐Soluble Cyclic Selenide with Enhanced Glutathione Peroxidase‐Like Catalytic Activities

Kumakura¹,

Mishra²,

et al. 2010

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“…Hydroperoxide reduction was followed by the decrease in absorbance of NADPH at 340 nm (39). Activity was evaluated using GSH as the cosubstrate for GSH-Px.…”

Section: Determination Of Glutathione Peroxidase Activitymentioning

confidence: 99%

Decreased platelet inhibition by nitric oxide in two brothers with a history of arterial thrombosis.

Freedman

Loscalzo²,

Benoit³

et al. 1996

J. Clin. Invest.

155

112

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Highly reactive oxygen species rapidly inactivate nitric oxide (NO), an endothelial product which inhibits platelet activation. We studied platelet inhibition by NO in two brothers with a cerebral thrombotic disorder. Both children had hyperreactive platelets, as determined by whole blood platelet aggregometry and flow cytometric analysis of the platelet surface expression of P-selectin. Mixing experiments showed that the patients' platelets behaved normally in control plasma; however, control platelets suspended in patient plasma were not inhibited by NO. As determined by flow cytometry, in the presence of plasma from either patient there was normal inhibition of the thrombin-induced expression of platelet surface P-selectin by prostacyclin, but not NO. Using a scopoletin assay, we measured a 2.7-fold increase in plasma H 2 O 2 generation in one patient and a 3.4-fold increase in the second patient, both compared with control plasma. Glutathione peroxidase (GSH-Px) activity was decreased in the patients' plasmas compared with control plasma. The addition of exogenous GSH-Px led to restoration of platelet inhibition by NO. These data show that, in these patients' plasmas, impaired metabolism of reactive oxygen species reduces the bioavailability of NO and impairs normal platelet inhibitory mechanisms. These findings suggest that attenuated NO-mediated platelet inhibition produced by increased reactive oxygen species or impaired antioxidant defense may cause a thrombotic disorder in humans. ( J. Clin. Invest. 1996. 97:979-987.) Key words: child • cerebral embolism and thrombosis • blood platelet • nitric oxide • glutathione peroxidase

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“…Several procedures for the determination of GSH and GSSG from different biological sources have been reported in the literature, but all require extraction procedures for the determination of glutathione in biological materials. An in situ determination is not possible with chemical (25) and enzymatic (26)(27)(28) methods, HPLC (29)(30)(31)(32)(33)(34)(35)(36)(37)(38), flow cytometry (39)(40)(41) and, more recently, capillary electrophoresis (42,43). GSH and GSSG can be detected by using spectrophotometric (44,45), spectrofluorimetric (46)(47)(48)(49)(50)(51)(52), and electrochemical methods (30,34,37,(53)(54)(55)(56)(57)(58) with levels of sensitivity ranging from nanomolar (33,35) to picomolar (31,32).…”

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confidence: 99%

Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites in vivo

Rompel

Cinco

Latimer

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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The sulfur K-edge x-ray absorption spectra for the amino acids cysteine and methionine and their corresponding oxidized forms cystine and methionine sulfoxide are presented. Distinct differences in the shape of the edge and the inf lection point energy for cysteine and cystine are observed. For methionine sulfoxide the inf lection point energy is 2.8 eV higher compared with methionine. Glutathione, the most abundant thiol in animal cells, also has been investigated. The x-ray absorption near-edge structure spectrum of reduced glutathione resembles that of cysteine, whereas the spectrum of oxidized glutathione resembles that of cystine. The characteristic differences between the thiol and disulfide spectra enable one to determine the redox status (thiol to disulfide ratio) in intact biological systems, such as unbroken cells, where glutathione and cyst(e)ine are the two major sulfurcontaining components. The sulfur K-edge spectra for whole human blood, plasma, and erythrocytes are shown. The erythrocyte sulfur K-edge spectrum is similar to that of fully reduced glutathione. Simulation of the plasma spectrum indicated 32% thiol and 68% disulfide sulfur. The whole blood spectrum can be simulated by a combination of 46% disulfide and 54% thiol sulfur.This paper describes an application of sulfur K-edge x-ray absorption spectroscopy (XAS) to determine the ratio of thiol to disulfide in biological systems. Sulfur is an important element in chemistry and biology, and often it is desirable to assay thiols and disulfide and their ratio in biological samples. There are no spectroscopic techniques available that can distinguish between thiol and disulfide sulfur or sulfur in higher formal oxidation states. This is not surprising, because there are no significant optical markers for sulfur. Also, only one sulfur isotope of Ͻ1% natural abundance has a nuclear magnetic moment; but this isotope possesses an unfavorable nuclear spin with a small magnetic moment and a large quadrupole moment, making NMR unattractive for sulfur analysis.Sulfur K-edge XAS has been shown to exhibit a strong correlation between oxidation state and inflection point energy (IPE) and to have a rich x-ray absorption near-edge structure (XANES) that is related to chemical structure (1-4) and can be used to distinguish between thiol and disulfide sulfur.The two sulfur-containing amino acids found in all cells are cysteine and methionine. Cysteine can be oxidized to the disulfide form cystine. Additionally there are ''mixed'' disulfides (cysteine bound to proteins by formation of a disulfide linkage). Methionine can be reversibly oxidized by HO ⅐ , 1

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Direct assay of glutathione peroxidase activity using high-performance capillary electrophoresis

Cited by 56 publications

References 25 publications

A Water‐Soluble Cyclic Selenide with Enhanced Glutathione Peroxidase‐Like Catalytic Activities

A Water‐Soluble Cyclic Selenide with Enhanced Glutathione Peroxidase‐Like Catalytic Activities

Decreased platelet inhibition by nitric oxide in two brothers with a history of arterial thrombosis.

Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites in vivo

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