Quantifying XOR and XO Activities in HCC

XOR and XO activity in the HCC cells were determined by a Xanthine Oxidase Activity Assay Kit (#MAK078, Sigma), which results in a fluorometric (λex = 535/λem = 587 nm) product. XOR and XO activities in the HCC cells were determined by following the increase of fluorescence by using xanthine as the substrate, in the presence of NAD⁺ (for XOR) or absence of NAD⁺ (for XO), using only molecular oxygen as an electron acceptor, for the fixed time interval. The XDH activity was calculated by subtracting from XOR the XO activity (34 (link), 35 (link)). One unit (U) of XOR was defined as the amount of enzyme that catalyzes the oxidation of xanthine to yield 1.0 μM of UA and hydrogen peroxide per minute at 25°C. XOR activity was normalized by protein concentration.