Gsh kit

GSH consumption, Fe²⁺ generation and Fe³⁺ depletion were investigated to detect GSH-like activity of Zn_0.4Mg_0.6Fe₂O₄.^{19,22 (link)} To investigate the consumption of GSH, 475 μL of Zn_0.4Mg_0.6Fe₂O₄ (10 μg mL⁻¹) was incubated with 25 μL of GSH (4 mM) at 37 °C for 6 and 12 h. Zn_0.4Mg_0.6Fe₂O₄ was separated by centrifugation and the supernatant was collected for GSH measurement using the GSH kit (Beyotime Biotechnology). Finally, 10 μL of the supernatant was added to 100 μL of the reaction mixture from the GSH kit for 25 min and the concentration of GSH was measured by UV-vis spectroscopy at 410 nm. To investigate Fe²⁺ generation, different concentrations of Zn_0.4Mg_0.6Fe₂O₄ (0.01, 0.025, 0.05, 0.1, 0.25, and 0.5 mg mL⁻¹) were incubated with/without 4 mM GSH in different buffers (pH 5.8 or pH 7.4) at 25 °C for 1 h, then the solutions were centrifuged (15 000 rpm, 20 min) and the supernatant was collected. A 100-μL volume of saturated 1, 10-phenanthroline solution was added to the supernatant. The absorbance at 512 nm was subsequently monitored. To investigate Fe³⁺ depletion, 10 μg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄ was cultured with 4 mM GSH in acidic buffer (pH = 5.8) at 25 °C for 12, 24, 36, and 48 h. Then the Fe²⁺ generation was quantitatively analyzed according to the standard Fe²⁺ concentration absorbance curve, and the Fe³⁺ depletion was equal to the Fe³⁺ generation.

The AsA kit (Nanjing Jiancheng Bioengineering Institute, China) was used to determine the AsA content in accordance with the manufacturer’s instructions. The kit contains all the necessary reagents, including the AsA standard, for the AsA assay.
GSH and GSSG were extracted from samples and the contents were measured using GSH kit and GSSG kit (Beyotime, China) according to the manufacturer’s instructions. The extraction process was carried out strictly following the guidelines provided by the manufacturer.