Physicochemical and kinetic properties of purified sheep's milk xanthine oxidoreductase

Abstract

Xanthine oxidoreductase (XOR) was purified for the first time from sheep's milk. The ultraviolet-visible absorption spectrum was essentially identical to those of the corresponding bovine, human, and goats' milk enzymes and showed an A280/A450 ratio of 5.35 ± 0.24, indicating a high degree of purity. Like milk XOR from other species, sheep's milk enzyme showed a single band on SDS-PAGE corresponding to a subunit with approximate M r 150,000. Xanthine oxidase activity of purified sheep's milk XOR (0.69 ± 0.04 μmole urate min-1 mg-1) was low relative to that of the bovine milk enzyme (1.83 ± 0.02 μmole urate min-1 mg-1), but higher than those of human or goats' milk XOR. As in the latter 2 cases, the low activity of sheep's milk XOR can be attributed to its relatively low molybdenum content (0.18 atoms per subunit), compared with that of the bovine milk enzyme (0.56 atoms Mo per subunit). Consistent with this, NADH oxidase activity of sheep's milk XOR was similar to that of enzymes purified from bovine, human, or goats' milk. The presence of desulphoenzyme in sheep's milk XOR was demonstrated by resulfuration experiments, whereby xanthine oxidase activity was increased by approximately 75%.