Glutamine synthetase (GS) is one of the central enzymes in nitrogen metabolism With ATP consumption, GS catalyzes glutamate to produce glutamine. The latter is an amino donor of various nitrogenous substances such as proteins, nucleic acids and various coenzymes. As a conditionally essential amino acid for the human body, the production of glutamine has received extensive attention in recent years. GS catalyzes ammonium ion and glutamic acid to synthesize amide, and produces chromogenic substance under the action of invertase and chromogenic agent. The maximum absorption peak is at 570 nm. The activity of GS in samples is calculated by measuring OD value at 570 nm and standard curve.
Glutathione peroxidase 4(GPX4) is a kind of glutathione peroxidase containing selenocysteine, which can reduce phospholipid hydroperoxide to phosphatidyl alcohol, thus protecting the cell membrane from oxidative damage. GPX4 catalyzes the substrate, and the product consumes the reducing agent with the addition of enzyme reagents.The reducing agent has a maximum absorbance at 340 nm, and the GP4-specific enzyme activity can be calculated by measuring non-specific enzyme activity and total enzyme activity by adding GPX-4 inhibitor to the system.
Glutathione peroxidase (GSH-Px) can promote the reaction of hydrogen peroxide (H2O2) and reduced glutathione to produce H2O and oxidized glutathione (GSSG). The activity of glutathione peroxidase can be expressed by the rate of enzymatic reaction. The activity of glutathione can be calculated by measuring the consumption of reduced glutathione. Hydrogen peroxide (H2O2) and reduced glutathione can react without catalysis of GSH-Px, so the portion of GSH reduction by non-enzymatic reaction should be subtracted. GSH can react with dinitrobenzoic acid to produce 5-thio-dinitrobenzoic acid anion, which showed a stable yellow color. Measure the absorbance at 412nm, and calculate the amount of GSH.
With the coenzyme as hydrogen donor, GSSG can be reduced to GSH under the catalysis of GR. Then the GSH content increased and NADPH decreased. The decrease of NADPH absorbance can be measured at 340 nm. The activity of GR can be calculated by detecting the change of NADPH.
With the reduced coenzyme (NADPH) as hydrogen donor, oxidized glutathione (GSSG) can be reduced to reduced glutathione (GSH). The decrease of NADPH absorbance can be measured at 340 nm. The activity of GR can be calculated by detecting the change of NADPH.
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