Purification and Characterisation of Xanthine Oxidoreductases from Local Bovids in Malta

Marita Vella, Therese Hunter, Claude Farrugia, Arwen R. Pearson, Gary Hunter
2014 Advances in Enzyme Research  
Xanthine oxidoreductase (XOR) is a molybdoflavoprotein mainly involved in purine catabolism. It exists in two forms, the oxidase (XO) and dehydrogenase (XDH) which are inter-convertible within mammalian cells. Although various researchers have reported the extraction of mammalian XOR, no extractions have yet been carried out in Malta and subsequently no characterizations are available. In this study, XOR was successfully purified from bovine, caprine and ovine milk through a multistep
more » ... multistep purification process involving both chemical and chromatographic techniques. The molecular weights of the native enzyme were found to be 295 kDa, 281 kDa and 275 kDa, representing the bovine, caprine and ovine XOR respectively. Western blot showed XOR to be represented on SDS-PAGE by a minimum of three major bands having molecular weights of 151 kDa, 131 kDa and 85 kDa. While all samples showed activity on native PAGE, spectrophotometric assays revealed the bovine XOR to be the most active. Surprisingly, the addition of NAD + to the assay mixture inhibited enzyme activity of the bovine and caprine XOR whereas the ovine XOR doubled its activity in response to NAD + . The latter also showed a lower binding affinity to heparin. Following incubation with trypsin, XOR was irreversibly converted to its oxidase form in all samples as reflected by the observed increase in XO activity.
doi:10.4236/aer.2014.21006 fatcat:u6h3pvozlfbydfpusaxjggmfuq