Sample (150.0 μL) was mixed with 20.0 μL of EDTA solution, 3.0 μL of glutathione reductase (0.2 U Sigma G3664), 10.0 μL of GSH solution, 3 μL of glutathione peroxidase (0.04 U Sigma G6137), 55.0 μL of H2O2 and 663.0 μL of 50 mM sodium phosphate, pH 7.0). To start the reaction, 10 μL of NADPH solution (N5130) was added and the decrease in the absorbance (340 nm) was read after 5 min of incubation at 25 °C. All solutions were prepared in 50 mM buffer and concentrations of reagents in the final mixture were as follows: 1 mM EDTA, 0.2 U glutathione reductase, 2 mM GSH, 0.04 U glutathione peroxidase, 1.5 mM H2O2 and 0.8 mM NADPH. Blank sample was prepared with buffer instead of the sample and background was measured (mixture containing studied sample and buffer only). The effect on the enzyme was calculated using equation [31 (link)]:
Sigma g3664
Manufactured by Merck Group
Sourced in United States
The Sigma G3664 is a general-purpose laboratory centrifuge. It is designed to separate and isolate different components in a sample by using centrifugal force. The centrifuge has a maximum speed of 6,000 rpm and can accommodate a variety of rotor sizes and sample volumes.
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2 protocols using sigma g3664
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Glutathione Peroxidase Inhibition Assay
2
Glutathione Peroxidase Activity Assay
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The GPx activity measurement was performed by homogenizing 5 g of a sample with 25 mL of 50 mM phosphate buffer (pH 7.0 at 25°C) using a homogenizer at 13,500 rpm for 30 s. The mixture was centrifuged at 1,800×g for 15 min at 2°C then filtered through a Whatman filter paper No 1. A volume of 100 μL of the supernatant was mixed with the following: 0.5 mL of phosphate buffer-0.001 M ethylenediaminetetraacetic acid-0.1 M NaN3, 100 μL of the assay mixture containing 5 units/mL glutathione reductase (Sigma G3664, Sigma-Aldrich, St. Louis, MO, USA) in phosphate buffer, 100 μL of 10 mM glutathione (Sigma G4251, Sigma-Aldrich, USA), 100 μL of 1.5 mM nicotinamide adenine dinucleotide phosphate (Sigma N1630, Sigma-Aldrich, USA), and 100 μL of 1.5 mM H2O2 (Sigma H1009, Sigma-Aldrich, USA). The GPx activity was measured by recording the decrease in absorbance of the incubated mixture at 340 nm over 3 min. The GPx activity was expressed as units/g sample.
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