Xanthine oxidase from human liver: purification and characterization.
Xanthine oxidase [EC 1.2.3.2] was purified 2000-fold from human liver. The last step of the procedure involved affinity chromatography. The resulting preparation showed two closely migrating bands of enzyme activity after gel electrophoresis under nondenaturing conditions. No other proteins were detected on these gels. The average particle mass of the enzyme was 300 kDa as determined by size-exclusion chromatography. This together with results of gel electrophoresis under denaturing conditions suggested that the native enzyme was composed of two subunits of approximately 150 kDa each. The electrophoretic patterns also indicated that a portion of these subunits had undergone partial proteolysis. The substrate specificity of the purified human enzyme was studied using an assay in which phenazine ethosulfate coupled the transfer of electrons from the reduced enzyme to cytochrome c. Hypoxanthine, 2-hydroxypurine, xanthine, 2-aminopurine, and adenine were among the most efficient purine substrates studied. Most purine nucleosides tested were oxidized at detectable rates, but with relatively high Km values. The 2'-deoxyribonucleosides were more efficient substrates than were the corresponding ribonucleosides or arabinonucleosides. In a direct comparison with xanthine oxidase from bovine milk, the human enzyme showed a similar specificity toward purine substrates. However, considerable differences between the bovine and human enzymes were observed with nucleoside substrates. With xanthine as the substrate for the human enzyme, 20% of the total electron flow was univalently transferred to oxygen to produce superoxide radicals.
D000818:Animals; D002130:Calcium Phosphates; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003574:Cytochrome c Group / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008892:Milk / Q000201:enzymology; D011687:Purines / Q000378:metabolism; D013379:Substrate Specificity; D014969:Xanthine Oxidase / Q000302:isolation & purification* / Q000378:metabolism
D002130:Calcium Phosphates; D003574:Cytochrome c Group; D011687:Purines; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D014969:Xanthine Oxidase; C485829:calcium phosphate, dibasic, anhydrous