Simple assay for quantifying xanthine oxidase activity.

Area of Science:

• Biochemistry
• Enzyme kinetics
• Analytical chemistry

Background:

• Xanthine oxidase (XO) produces reactive oxygen species, contributing to oxidative stress and related diseases.
• Plant extracts can inhibit XO activity, suggesting therapeutic potential.
• Accurate quantification of XO activity is crucial for disease research and drug development.

Purpose of the Study:

• To develop and validate a sensitive and precise method for assaying xanthine oxidase (XO) enzyme activity.
• To quantify XO activity based on hydrogen peroxide (H2O2) generation using a TMB-H2O2 system.
• To assess the reliability and comparability of the new assay with existing methods.

Main Methods:

• Quantified XO activity by incubating enzyme samples with xanthine.
• Measured H2O2 generation using a 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 system catalyzed by cupric ions.
• Utilized UV-visible spectrometry for optical signal detection and sodium azide to inhibit catalase interference.

Main Results:

• A direct correlation was observed between XO activity and absorbance at 450 nm.
• The assay demonstrated high precision and reliability, with a correlation coefficient of 0.9976 when compared to established methods.
• The developed assay effectively measured XO activity and was validated using Bland-Altman analysis.

Conclusions:

• The TMB-based assay provides a sensitive, precise, and efficient method for quantifying xanthine oxidase activity.
• This novel assay is comparable to existing protocols and suitable for research on oxidative stress and related diseases.
• The method's ability to detect H2O2 generation makes it valuable for biochemical and pharmacological studies involving XO.