A new spectrophotometric assay method of xanthine oxidase applicable to the crude tissue homogenate containing uricase was presented in this paper. By adding potassium 2,4-dihydroxy-6-carboxy-1,3,5-triazine (potassium oxonate) (0.1 mM) to the crude xanthine oxidase reaction system, uric acid was stoichiometrically formed from xanthine and detectable allantoin was not formed and the formation of uric acid was not influenced by uricase.
Distribution of xanthine oxidase in various rat tissues was measured by this method, and it was shown that the activity was high in the liver, the small intestine, and the spleen. Uricase was shown to distribute mainly in the liver of rats.
Xanthine oxidase [EC 1.2.3.21 (X0) is an enzyme which catalyses the oxidation of a wide range of substrates including aldehydes, purines, pteridines, pyrimidines, azapurines, and other heterocyclic compounds.
To measure the X0 activity in crude biological fluid manometric (14) or spectrophotometric method ( 5) is commonly used.
Manometric assay method is based on measuring O2 uptake depending on the xanthine added to the medium. High endogenous respiration in crude tissue preparations will disturb accurate measurement of O2 uptake from the substrates,
The spectrophotometric assay method is convenient, rapid, and sensitive for the purified enzyme preparations. Based on the difference in the uv absorption spectrum between hypoxanthine or xanthine and uric acid, xanthine oxidase activity can easily be measured aerobically by the increase in the absorbance at 292 nm on the addition of hypoxanthine. But this method is hardly applicable to the crude homogenate from experimental animals which contains an appreciable amount of uricase (UO) .
UO catalyses oxidation of uric acid to allantoin, which shows no absorption peak at 292 nm. From this reason, apparent X0 activity in crude tissue homogenate cannot be evaluated without taking account of uricase activity coexisted.