Abstract

This protocol clarifies a simple and precise method for measuring the activity of xanthine oxidase (XO) enzyme inhibitor. XO enzyme, which accelerates oxidative stress-related disorders through its capacity to generate hydrogen peroxide and superoxide anion radicals (O₂^•-), has been found to be inhibited by several plant extracts. Enzyme samples were incubated with a suitable buffer containing adequate amounts of xanthine as a substrate to determine XO activity. The method depends on direct measurements of uric acid and hydrogen peroxide production to test XO with and without interference. The CUPRAC reagent (Cu(Nc)₂²⁺) was used to inhibit enzyme reaction after incubation was complete. The generated urate and peroxide reduced the Cu(II)-neocuproine complex (Cu(Nc)₂²⁺) to a brightly colored Cu(I)-neocuproine complex (Cu(Nc)₂⁺), which was assessed with a spectrophotometer at 450 nm. XO activity was found to be directly related to the increased absorbance of the colored Cu(I)-neocuproine complex (Cu(Nc)₂⁺). To eliminate catalase enzyme interference, the proposed method used sodium azide and was validated against XO activity using the UV method in matched samples with t-test analysis. The proposed assay can determine XO activity with high precision, as indicated by the correlation coefficient (R² = 0.9935) from comparison with the reference protocol.