Abstract
This paper reports a sensitive method for assaying xanthine oxidase (XO) enzyme activity. XO produces hydrogen peroxide (H2O2) and superoxide anion radicals (O2•−), promoting the development of oxidative stress-related diseases, and is inhibited by various plant extracts. XO activity is quantified by incubating enzyme samples with an appropriate xanthine concentration as the substrate. The proposed method requires XO activity to be quantified based on H2O2 generation using a 3,3′,5,5′-tetramethylbenzidine (TMB)-H2O2 system catalyzed by cupric ions. After a 30-min incubation at 37 °C, sufficient cupric ion and TMB amounts are added. The assay produces optical signals that can be visually recognized or detected with a UV–visible spectrometer. A direct correlation was found between XO activity and the absorbance at 450 nm of the resulting di-imine (dication) yellow product. The proposed method uses sodium azide to prevent catalase enzyme interference. The new assay's function was confirmed using the TMB-XO assay and a Bland–Altman plot. The resulting correlation coefficient was 0.9976. The innovative assay was relatively precise and comparable to the comparison protocols. In conclusion, the presented method is very efficient at measuring XO activity.
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