Xanthine oxidase from human liver: purification and characterization

Area of Science:

• Biochemistry
• Enzymology

Background:

• Xanthine oxidase (EC 1.2.3.2) is a key enzyme in purine metabolism.
• Understanding human xanthine oxidase is crucial for metabolic studies.

Purpose of the Study:

• To purify and characterize xanthine oxidase from human liver.
• To investigate its substrate specificity and compare it with bovine xanthine oxidase.

Main Methods:

• Affinity chromatography for enzyme purification.
• Gel electrophoresis (nondenaturing and denaturing) for purity and subunit analysis.
• Size-exclusion chromatography for molecular mass determination.
• Enzyme kinetics assays to determine substrate specificity.

Main Results:

• Human liver xanthine oxidase was purified 2000-fold, appearing as two active bands on nondenaturing gels.
• The native enzyme has a molecular mass of 300 kDa, composed of two ~150 kDa subunits, with evidence of partial proteolysis.
• The enzyme efficiently oxidized hypoxanthine, 2-hydroxypurine, xanthine, 2-aminopurine, and adenine.
• 2'-deoxyribonucleosides were better substrates than ribonucleosides or arabinonucleosides.
• Human xanthine oxidase showed similar purine substrate specificity but different nucleoside substrate specificity compared to bovine milk xanthine oxidase.
• 20% of electron flow to xanthine was univalently transferred to oxygen, producing superoxide radicals.

Conclusions:

• Human liver xanthine oxidase is a homodimer with subunits susceptible to partial proteolysis.
• The enzyme exhibits distinct substrate preferences, particularly for nucleosides, when compared to its bovine counterpart.
• A significant portion of electron transfer results in superoxide radical formation.