AER  Vol.2 No.1 , March 2014
Purification and Characterisation of Xanthine Oxidoreductases from Local Bovids in Malta
Abstract: 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 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.
Cite this paper: Vella, M. , Hunter, T. , Farrugia, C. , Pearson, A. and Hunter, G. (2014) Purification and Characterisation of Xanthine Oxidoreductases from Local Bovids in Malta. Advances in Enzyme Research, 2, 54-63. doi: 10.4236/aer.2014.21006.

[1]   Hille, R. and Nishino, T. (1995) Xanthine Oxidase and Xanthine Dehydrogenase. The FASEB Journal, 9, 995-1003.

[2]   Okamoto, K., Matsumoto, K., Hille, R., Eger, B.T., Pai, E.F. and Nishino, T. (2004) The Crystal Structure of Xanthine Oxidoreductase during Catalysis: Implications for Reaction Mechanism and Enzyme Inhibition. PNAS, 101, 7931-7936.

[3]   Vorbach, C., Harrison, R. and Capecchi, M.R. (2003) Xanthine Oxidoreductase is Central to the Evolution and Function of the Innate Immune System. Immunology, 24, 512-517.

[4]   Steven, C.R., Millar, T.M., Clinach, J.G., Kanczier, K.M., Bodamyall, T. and Blake, D.R. (2000) Anti-bacterial Properties of Xanthine Oxidase in Human Milk. Lancet, 356, 829.

[5]   Hancock, J.T., Salisbury, V., Ovejero-Boglione, M.C., Cherry, R., Hoare, C., Eisenthal, R. and Harrison, R. (2002) Antimicrobial Properties of Milk: Dependence on Presence of Xanthine Oxidase and Nitrite. Antimicrobial Agents and Chemotherapy, 46, 3308-3310.

[6]   Scopes, R.K. (1987) Protein Purification: Principles and Practice. 2nd Edition, Springer-Verlag, New York, 41-64.

[7]   Enroth, C., Eger, B.T., Okamoto, K., Nishino, T., Nishino, T. and Pai, E.F. (2000) Crystal Structures of Bovine Milk Xanthine Dehydrogenase and Xanthine Oxidase: Structure based Mechanism of Conversion. PNAS, 97, 10723-10728.

[8]   Zikakis, J. (1979) Preparation of High Purity Xanthine Oxidase from Bovine Milk. U.S. Patent No. 806,736.

[9]   Gilbert, D.A. and Bergel, F. (1964) The Chemistry of Xanthine Oxidase: An Improved Method of Preparing the Bovine Milk Enzyme. Biochemical Journal, 90, 350-353.

[10]   Waud, W.R., Brady, F.O., Wiley, R.D. and Rajagopalan, K.V. (1975) A New Purification Procedure for Bovine Milk Xanthine Oxidase: Effect of Proteolysis on the Subunit Structure. Archives of Biochemistry and Biophysics, 169, 695- 701.

[11]   Briley, M.S. and Eisenthal, R. (1975) Association of Xanthine Oxidase with the Bovine Milk-Fat-Globule Membrane: Nature of the Enzyme-Membrane Association. Biochemical Journal, 147, 417-423.

[12]   Nishino, T., Nishino, T. and Tsushima, K. (1981) Purification of Highly Active Milk Xanthine Oxidase by Affinity Chromatography on Sepharose 4B/Folate Gel. The FASEB Journal, 131, 369-372.

[13]   Nishino, T. and Tsushima, K. (1986) Interaction of Milk Xanthine Oxidase with Folic Acid. Inhibition of Milk Xanthine Oxidase by Folic Acid and Separation of the Enzyme into Two Fractions on Sepharose 4B/Folate Gel. The Journal of Biological Chemistry, 261, 11242-11246.

[14]   Fukushima, T., Adachi, T. and Hirano, K. (1995) The Heparin Binding Site of Human Xanthine Oxidase. Biological & Pharmaceutical Bulletin, 84, 156-158.

[15]   Nishino, T., Okamoto, K., Eger, B.T., Pai, E.F. and Nishino, T. (2008) Mammalian Xanthine Oxidoreductase— Mechanism of Transition from Xanthine Dehydrogenase to Xanthine Oxidase. The FASEB Journal, 275, 3278-3289.

[16]   Krenitsky, T.A., Spector, T. and Hall, W.W. (1986) Xanthine Oxidase from Human Liver: Purification and Characterisation. Archives of Biochemistry and Biophysicss, 247, 108-119.

[17]   Moriwaki, Y., Yamamoto, T., Suda, Nasako, Y., Takahashi, S., Agbedana, O., Hada, T. and Higashino, K. (1993) Purification and Immunohistochemical Tissue Localisation of Human Xanthine Oxidase. Biochimica et Biophysica Acta, 1164, 327-330.

[18]   Atmani, D., Benboubetra, M. and Harrison, R. (2003) Goa’s Milk Xanthine Oxidoreductase Is Grossly Deficient in Molybdenum. Journal of Dairy Science, 71, 7-13.

[19]   Benboubetra, M., Baghiani, A., Atmani, D. and Harrison, R. (2004) Physiochemical and Kinetic Properties of Purified Sheep’s Milk Xanthine Oxidoreductase. Journal of Dairy Science, 87, 1580-1584.

[20]   Sanders, S.A., Eisenthal, R. and Harrison, R. (1997) NADH Oxidase Activity of Human Xanthine Oxidoreductase: Generation of Superoxide Anion. European Journal of Biochemistry, 245, 541-548.

[21]   Hunt, J. and Massey, V. (1992) Purification and Properties of Milk Xanthine Dehydrogenase. The Journal of Biological Chemistry, 267, 21479-21485.

[22]   Andrews, P., Bray, R.C., Edwards, P. and Shooter, K.V. (1964) The Chemistry of Xanthine Oxidase: Ultracentrifuge and Gel-Filtration Studies on the Milk Enzyme. Biochemical Journal, 93, 627-632.

[23]   Cheng, S.G., Koch, U. and Brunner, R. (1988) Characteristics of Purified Cows’ Milk Xanthine Oxidase and its Submolecular Characterisitics. Journal of Dairy Science, 71, 901-916.

[24]   Hart, L.I., McGartoll, M.A., Chapman, H.R. and Bray, R.C.R. (1970) The Composition of Milk Xanthine Oxidase. Biochemical Journal, 116, 851-864.

[25]   Bray, R.C. (1975) The Enzymes. 3rd Edition, B, Academic Press, New York, 303-388.

[26]   Carpani, G., Racchi, M., Ghezzi, P., Terao, M. and Garattini, E. (1990) Purification and Characterization of Mouse Liver Xanthine Oxidase. Archives of Biochemistry and Biophysics, 279, 237-241.

[27]   Mangino, M.E. and Brunner, J.R. (1997) Isolation and Partial Characterization of Xanthine Oxidase Associated with the Milk Fat Globule Membrane of Cow’s Milk. Journal of Dairy Science, 60, 841-850.

[28]   McManaman, J.L. and Bain, D.L. (2002) Structural and Conformational Analysis of the Oxidase to Dehydrogenase Conversion of Xanthine Oxidoreductase. The Journal of Biological Chemistry, 277, 21261-21268.

[29]   Guskov, E.P., Kletskii, M.E., Kornienko, I.V., Olekhnovich, L.P., Chistyakov, V.A., Shkurat, T.P., Prokofev, V.N. and Zhdanov, Y. (2002) Allantoin as a Free Radical Scavenger. Archives of Biochemistry and Biophysics, 383, 105-107.

[30]   Komai, H., Massey V. and Palmer, G. (1968) The Preparation and Properties of Deflavo Xanthine Oxidase. The Journal of Biochemistry, 244, 1692-1700.