Rapid and effective protocol to measure glutathione peroxidase activity DOI Creative Commons

Ahmed Abdul Sattar,

Amir Abbas Matin,

Mahmoud Hussein Hadwan

et al.

Bulletin of the National Research Centre/Bulletin of the National Research Center, Journal Year: 2024, Volume and Issue: 48(1)

Published: Oct. 8, 2024

Abstract Background Intracellular hydroperoxides are harmful reactive oxygen species that can cause damage. Cells depend on antioxidant enzymes such as glutathione peroxidase (GPx) to prevent their accumulation. GPx is present in all cell compartments, including the cytosol, mitochondria, and peroxisomes. This article will a rapid reliable method for measuring activity, which has been rigorously tested proven dependable. Results The enzyme samples incubated phosphate buffer containing specific concentrations of (GSH) peroxide then suitable time react before Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) added. method, designed ease use, effective straightforward. When GSH reacts with DTNB, mixed disulfides (GS-S-DTNB) formed, where thiol group replaces one nitrobenzoic acid moieties DTNB. release nitro-2-thiobenzoate anion (TNB − ) creates yellow be measured spectrophotometrically at wavelength 412 nm. intensity color directly proportional unreacted concentration. activity was linked decreased absorbance TNB anion, providing sensitive relatively detecting groups various biological samples. Conclusion study utilized Box–Behnken design (BBD) apply response surface modeling (RSM) determine best conditions activity. efficiency new protocol validated by comparing matched using Bland–Altman plot analysis reference methods. just accurate one, correlation coefficient 0.9991. Graphical abstract

Language: Английский

Discovery of MAGL Inhibition by Lophine Derivatives: An Unexpected Finding from Chemiluminescent Assay Development DOI Creative Commons
Roberta Ottria, Silvana Casati, Ornella Xynomilakis

et al.

Molecules, Journal Year: 2025, Volume and Issue: 30(7), P. 1605 - 1605

Published: April 3, 2025

The inhibitory effects of two novel lophine derivatives were unexpectedly discovered during the development a chemiluminescent monoacylglycerol lipase (MAGL) assay. proposed found to exhibit concentration-dependent on MAGL with octanoic and palmitic acid esters 2-(4-hydroxyphenyl)-4,5-diphenylimidazole showing strongest activity. Reversibility assays using fluorometric method confirmed that these compounds interact in stable, irreversible manner. To further investigate their mode interaction, docking studies performed, supporting hypothesis 3 4 may act as competitive inhibitors. Lophine initially designed synthesized potential chemiluminescence pro-enhancers. However, assay optimization revealed no signal production upon hydrolysis, precluding use probes. These findings suggest is promising candidate for inhibitors, although needed enhance binding affinity selectivity.

Language: Английский

Citations

0
Rapid and effective protocol to measure glutathione peroxidase activity DOI Creative Commons

Ahmed Abdul Sattar,

Amir Abbas Matin,

Mahmoud Hussein Hadwan

et al.

Bulletin of the National Research Centre/Bulletin of the National Research Center, Journal Year: 2024, Volume and Issue: 48(1)

Published: Oct. 8, 2024

Abstract Background Intracellular hydroperoxides are harmful reactive oxygen species that can cause damage. Cells depend on antioxidant enzymes such as glutathione peroxidase (GPx) to prevent their accumulation. GPx is present in all cell compartments, including the cytosol, mitochondria, and peroxisomes. This article will a rapid reliable method for measuring activity, which has been rigorously tested proven dependable. Results The enzyme samples incubated phosphate buffer containing specific concentrations of (GSH) peroxide then suitable time react before Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) added. method, designed ease use, effective straightforward. When GSH reacts with DTNB, mixed disulfides (GS-S-DTNB) formed, where thiol group replaces one nitrobenzoic acid moieties DTNB. release nitro-2-thiobenzoate anion (TNB − ) creates yellow be measured spectrophotometrically at wavelength 412 nm. intensity color directly proportional unreacted concentration. activity was linked decreased absorbance TNB anion, providing sensitive relatively detecting groups various biological samples. Conclusion study utilized Box–Behnken design (BBD) apply response surface modeling (RSM) determine best conditions activity. efficiency new protocol validated by comparing matched using Bland–Altman plot analysis reference methods. just accurate one, correlation coefficient 0.9991. Graphical abstract

Language: Английский

Citations

1