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Purification of Xanthine Oxidase Enzyme and Investigation of Its Immobilization with Glutaraldehyde

Year 2022, Volume: 9 Issue: 3, 314 - 322, 31.10.2022

Yeşim Kaya , Semra Isık , Serap Uzunoglu , Mustafa Oğuzhan Kaya

https://doi.org/10.19159/tutad.1084383

Abstract

In this study, the xanthine oxidase (XO) enzyme was purified by affinity chromatography technique using Sepharose-4B-L-tyrosine-4-aminobenzamidine gel and its immobilization with glutaraldehyde was investigated. Using ammonium sulfate precipitation and affinity gel, xanthine oxidase was purified 643.04-fold in an 11.5% yield. The purity of the enzyme was checked by SDS polyacrylamide gel electrophoresis and a single band around 150 kDa was observed. KM (the Michaelis constant) and VMax (the asymptotic reaction velocity at infinite substrate concentration) of the enzyme were determined at 1.67x10-4 M and 0.56 U/mL.min respectively by using a xanthine compound as a substrate. The in vitro effects of NH4F, NH4Cl, CaCl2, ZnCl2, HgCl2, Hg(NO3)2.H2O compounds and commercially named colchicum dispert, commonly used in the treatment of gout disease in the clinic, were investigated. The IC50 values of compounds showing inhibition effects were determined. Afterward, XO was immobilized with glutaraldehyde. The highest XO activity was observed in the sample of the immobilized enzyme at a rate of 6% glutaraldehyde. The kinetic constants (KM and VMax) of the immobilized enzyme were determined as 5.18x10-4 M and 0.73 U mL-1 min-1 respectively. These values revealed that the catalytic activity of the free enzyme was higher than the immobilized enzyme.

Keywords

Xanthine oxidase affinity chromatography inhibition enzyme immobilization glutaraldehyde

References

  • Arslan, O., 2001. Inhibition of bovine carbonic anhydrase by new sulfonamide compounds. Biochemistry, 66(9): 1206-1208.
  • Beyaztaş S., 2010. Synthesis of a novel affinity gel for purification of xanthine oxidase (XO) enzyme, biotransformation of thiosemicarbazone derivatives via purified enzyme and investigations of the effects of some antibiotics on the enzyme. Ph.D. thesis, Balıkesir University Institute of Science and Technology, Balıkesir, Türkiye. (In Turkish).
  • Beyaztaş, S., Arslan, O., 2011. Affinity effects of some antibiotics on xanthine oxidase enzyme activities in vitro. Hacettepe Journal of Biology and Chemistry, 39(2): 195-205.
  • Beyaztaş, S., Arslan, O., 2015. Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel. Journal of Enzyme Inhibition and Medicinal Chemistry, 30(3): 442-447.
  • Bradford, M.M., 1976. A rapid and sensitive method for the quantitaion of microgram quantites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2): 248-254.
  • Bray, R.C., 1975. The enzymes. In: P.D. Boyer (Ed.), Molybdenum Iron-Sulfur Flauin Hydroxylases and Related Enzymes, 3 rd Edn., Academic Press, New York, pp. 299-419.
  • Cabral, J.M.S., Kennedy, J.F., 2000. Immobilisation techniques for altering thermal stability of enzymes. In: M.N. Gupta (Ed.), Thermostability of Enzymes, Springer, Berlin, pp. 163-179.
  • Coetzee, W.A., Owen, P., Dennis, S.C., Saman, S., Opie, L.H., 1990. Reperfusion damage: free radicals mediate delayed membrane changes rather than early ventricular arrhythmias. Cardiovascular Research, 24(2): 156-164.
  • Dekker, M., 2000. Protein Immobilization: Fundamentals and Applications. Taylor, New York.
  • Dement’ev, A.V., 2013. Xanthine oxidase activity in pig’s blood and oxidative stress assay. Russian Agricultural Sciences, 39(1): 82-85.
  • Fox, P.F., Kelly, A.L., 2006. Indigenous enzymes in milk: Overview and historical aspects-Part 1. International Dairy Journal, 16(6): 500-516.
  • Fridovich, I., 1964. Competitive inhibition of xanthine oxidase by urea and guanidinium ion. Journal of Biological Chemistry, 239(10): 3519-3521.
  • Granger, D.R., Hollwarth, M.E., Parks, D.A., 1986. Ischemia-reperfusion injury: role of oxygen-derived free radicals. Acta Physiologica Scandinavica, 548: 47-63.
  • Harrison, R., 2006. Milk xanthine oxidase: Properties and physiological roles. International Dairy Journal, 16(6): 546-554.
  • Hart, L.J., McGartoll, M.A., Chapman, H.R., Bray, R.C., 1970. The composition of milk xanthine oxidase. Biochemical Journal, 116(5): 851-864.
  • Kenan, T.W., Patton, S., 1995. The structure of milk: implications for sampling and storage. The milk globule membran. In: R.G. Jensen (Ed.), Handbook of Milk Composition, Academic Press, New York, pp. 5-50.
  • Laemelli, U.K., 1970. Cleavege of structural proteins during in assembly of the head of bacteriophage T4. Nature, 227: 680-685.
  • Linder, N., Rapola., J., Raivio, K.O., 1999. Cellular expression of xanthine oxidoreductase protein in normal human tissues. Laboratory Investigation, 79(8): 967-974.
  • Maia, L., Mira, L., 2002. Xanthine oxidase and aldehyde oxidase: A simple procedure for the simultaneous purification from rat liver. Archives of Biochemistry and Biophysics, 400(1): 48-53.
  • Massey, V., Brumby, P.E., Komai, H., 1969. Studies on milk xanthine oxidase: Some spectral and kinetic properties. Journal of Biological Chemistry, 244(7): 16821691.
  • McCord, J.M., Fridovich, I., 1968. The reduction of cytochrome by milk xanthine oxidase. Journal of Biological Chemistry, 243(21): 5753-5760.
  • McManaman, J.L., Bain, D.L., 2002. Structural and conformational analysis of the oxidase to dehydrogenase conversion of xanthine oxidoreductase. Journal of Biological Chemistry, 14: 21261-21268.
  • McManaman, J.L., Neville MC, Wright RM., 1999. Mouse mammary gland xanthine oxidoreductase: purification, characterization, and regulation. Arch Biochem Biophys, 371: 308-316.
  • McManaman, J.L., Shellman, V., Wright, R.M., Repine, J.E., 1996. Purification of rat liver xanthine oxidase and xanthine dehydrogenase by affinity chromatography on benzamidine-sepharose. Archives of Biochemistry and Biophysics, 332(1): 135-141.
  • Metinyurt, G., 2003. Measurement of plasma xanthine oxidase activity by high pressure liquid chromatography (HPLC) and determination of xanthine oxidase levels in patients with hypogonadism. Specialization thesis, Gülhane Military Medical Academy, Ankara, Türkiye. (In Turkish).
  • Moriwaki, Y., Yamamoto, T., Suda, M., Nasako, Y., Takahashi, S., Agdebana, O.E., Hda, T., Higashino, K., 1993. Purification and immunohistochemical tissue localization of human xanthine oxidase. Biochimica et Biophysica Acta, 1164(3): 327-330.
  • Murray, R.K., Granner, D.K., Mayes, P.A., Rodwell, V.W., 2004. Harper Biochemistry. Nobel Medical Bookstores. (In Turkish).
  • Nelson, C.A., Handler, P., 1968. Preparation of bovine xanthine oxidase and the subunit structures of some iron flavoproteins. Journal of Biological Chemistry, 243(20): 5368-5373.
  • Onat, T., Emerk, K., Sözmen, E.Y., 2006. Human Biochemistry, Palme Publishing, Ankara, Türkiye. (In Turkish).
  • Ozer, N., Muftuoglu, M., Ataman, D., Ercan, A., Ogus, I.H., 1999. Simple, high-yield purification of xanthine oxidase from bovine milk. Journal of Biochemical and Biophysical Methods, 39(3): 153-159.
  • Page, S., Powell, D., Benboubetra, M., Stevens, C.R., Blake, D.R., Selase, F., Wolstenholme, A.J., Harrison, R., 1998. Xanthine oxidoreductase in human mammary epithelial cells: activation in response to inflammatory cytokines. Biochimica et Biophysica Acta, 1381(2): 191-202.
  • Parks, D.A., Granger, D.N., 1986. Xanthine oxidase: biochemistry, distribution and physiology. Acta Physiologica Scandinavica, 548: 87-99.
  • Pauff, J.M., Hemann, C.F., Junemann, N., Leimkuhler, S., Hille, R., 2007. The role of arginine 310 in catalysis and substrate specificity in xanthine dehydrogenase from Rhodobacter capsulatus. Journal of Biological Chemistry, 282(17): 12785-12790.
  • Polaina, J., MacCabe, A.P., 2007. Industrial Enzymes. Springer, The Netherlands.
  • Schoutsen, B., De Jong, J.W., Harmsen, E., De Tombe, P.P., Achterberg, P.W., 1983. Myocardial xanthine oxidase/dehdrogenase. Biochemica et Biophysica Acta, 762(4): 519-524.
  • Tubaro, E., Lotti, B., Santiangelli, C., Cavallo, G., 1980. Xanthine oxidase increase in polymorphonuclear leucocytes and macrophages in mice in three pathological situtions. Biochemical Pharmacology, 29(13): 1945-1948.
  • Werns, S.W., Lucchesi, B.R., 1990. Free radical and ischemic tissue injury. Trends in Pharmacological Sciences, 11(4): 161-166.

Purification of Xanthine Oxidase Enzyme and Investigation of Its Immobilization with Glutaraldehyde

Year 2022, Volume: 9 Issue: 3, 314 - 322, 31.10.2022

Yeşim Kaya , Semra Isık , Serap Uzunoglu , Mustafa Oğuzhan Kaya

https://doi.org/10.19159/tutad.1084383

Abstract

In this study, the xanthine oxidase (XO) enzyme was purified by affinity chromatography technique using Sepharose-4B-L-tyrosine-4-aminobenzamidine gel and its immobilization with glutaraldehyde was investigated. Using ammonium sulfate precipitation and affinity gel, xanthine oxidase was purified 643.04-fold in an 11.5% yield. The purity of the enzyme was checked by SDS polyacrylamide gel electrophoresis and a single band around 150 kDa was observed. KM (the Michaelis constant) and VMax (the asymptotic reaction velocity at infinite substrate concentration) of the enzyme were determined at 1.67x10-4 M and 0.56 U/mL.min respectively by using a xanthine compound as a substrate. The in vitro effects of NH4F, NH4Cl, CaCl2, ZnCl2, HgCl2, Hg(NO3)2.H2O compounds and commercially named colchicum dispert, commonly used in the treatment of gout disease in the clinic, were investigated. The IC50 values of compounds showing inhibition effects were determined. Afterward, XO was immobilized with glutaraldehyde. The highest XO activity was observed in the sample of the immobilized enzyme at a rate of 6% glutaraldehyde. The kinetic constants (KM and VMax) of the immobilized enzyme were determined as 5.18x10-4 M and 0.73 U mL-1 min-1 respectively. These values revealed that the catalytic activity of the free enzyme was higher than the immobilized enzyme.

Keywords

affinity chromatography inhibition enzyme immobilization Xanthine oxidase glutaraldehyde

References

  • Arslan, O., 2001. Inhibition of bovine carbonic anhydrase by new sulfonamide compounds. Biochemistry, 66(9): 1206-1208.
  • Beyaztaş S., 2010. Synthesis of a novel affinity gel for purification of xanthine oxidase (XO) enzyme, biotransformation of thiosemicarbazone derivatives via purified enzyme and investigations of the effects of some antibiotics on the enzyme. Ph.D. thesis, Balıkesir University Institute of Science and Technology, Balıkesir, Türkiye. (In Turkish).
  • Beyaztaş, S., Arslan, O., 2011. Affinity effects of some antibiotics on xanthine oxidase enzyme activities in vitro. Hacettepe Journal of Biology and Chemistry, 39(2): 195-205.
  • Beyaztaş, S., Arslan, O., 2015. Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel. Journal of Enzyme Inhibition and Medicinal Chemistry, 30(3): 442-447.
  • Bradford, M.M., 1976. A rapid and sensitive method for the quantitaion of microgram quantites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2): 248-254.
  • Bray, R.C., 1975. The enzymes. In: P.D. Boyer (Ed.), Molybdenum Iron-Sulfur Flauin Hydroxylases and Related Enzymes, 3 rd Edn., Academic Press, New York, pp. 299-419.
  • Cabral, J.M.S., Kennedy, J.F., 2000. Immobilisation techniques for altering thermal stability of enzymes. In: M.N. Gupta (Ed.), Thermostability of Enzymes, Springer, Berlin, pp. 163-179.
  • Coetzee, W.A., Owen, P., Dennis, S.C., Saman, S., Opie, L.H., 1990. Reperfusion damage: free radicals mediate delayed membrane changes rather than early ventricular arrhythmias. Cardiovascular Research, 24(2): 156-164.
  • Dekker, M., 2000. Protein Immobilization: Fundamentals and Applications. Taylor, New York.
  • Dement’ev, A.V., 2013. Xanthine oxidase activity in pig’s blood and oxidative stress assay. Russian Agricultural Sciences, 39(1): 82-85.
  • Fox, P.F., Kelly, A.L., 2006. Indigenous enzymes in milk: Overview and historical aspects-Part 1. International Dairy Journal, 16(6): 500-516.
  • Fridovich, I., 1964. Competitive inhibition of xanthine oxidase by urea and guanidinium ion. Journal of Biological Chemistry, 239(10): 3519-3521.
  • Granger, D.R., Hollwarth, M.E., Parks, D.A., 1986. Ischemia-reperfusion injury: role of oxygen-derived free radicals. Acta Physiologica Scandinavica, 548: 47-63.
  • Harrison, R., 2006. Milk xanthine oxidase: Properties and physiological roles. International Dairy Journal, 16(6): 546-554.
  • Hart, L.J., McGartoll, M.A., Chapman, H.R., Bray, R.C., 1970. The composition of milk xanthine oxidase. Biochemical Journal, 116(5): 851-864.
  • Kenan, T.W., Patton, S., 1995. The structure of milk: implications for sampling and storage. The milk globule membran. In: R.G. Jensen (Ed.), Handbook of Milk Composition, Academic Press, New York, pp. 5-50.
  • Laemelli, U.K., 1970. Cleavege of structural proteins during in assembly of the head of bacteriophage T4. Nature, 227: 680-685.
  • Linder, N., Rapola., J., Raivio, K.O., 1999. Cellular expression of xanthine oxidoreductase protein in normal human tissues. Laboratory Investigation, 79(8): 967-974.
  • Maia, L., Mira, L., 2002. Xanthine oxidase and aldehyde oxidase: A simple procedure for the simultaneous purification from rat liver. Archives of Biochemistry and Biophysics, 400(1): 48-53.
  • Massey, V., Brumby, P.E., Komai, H., 1969. Studies on milk xanthine oxidase: Some spectral and kinetic properties. Journal of Biological Chemistry, 244(7): 16821691.
  • McCord, J.M., Fridovich, I., 1968. The reduction of cytochrome by milk xanthine oxidase. Journal of Biological Chemistry, 243(21): 5753-5760.
  • McManaman, J.L., Bain, D.L., 2002. Structural and conformational analysis of the oxidase to dehydrogenase conversion of xanthine oxidoreductase. Journal of Biological Chemistry, 14: 21261-21268.
  • McManaman, J.L., Neville MC, Wright RM., 1999. Mouse mammary gland xanthine oxidoreductase: purification, characterization, and regulation. Arch Biochem Biophys, 371: 308-316.
  • McManaman, J.L., Shellman, V., Wright, R.M., Repine, J.E., 1996. Purification of rat liver xanthine oxidase and xanthine dehydrogenase by affinity chromatography on benzamidine-sepharose. Archives of Biochemistry and Biophysics, 332(1): 135-141.
  • Metinyurt, G., 2003. Measurement of plasma xanthine oxidase activity by high pressure liquid chromatography (HPLC) and determination of xanthine oxidase levels in patients with hypogonadism. Specialization thesis, Gülhane Military Medical Academy, Ankara, Türkiye. (In Turkish).
  • Moriwaki, Y., Yamamoto, T., Suda, M., Nasako, Y., Takahashi, S., Agdebana, O.E., Hda, T., Higashino, K., 1993. Purification and immunohistochemical tissue localization of human xanthine oxidase. Biochimica et Biophysica Acta, 1164(3): 327-330.
  • Murray, R.K., Granner, D.K., Mayes, P.A., Rodwell, V.W., 2004. Harper Biochemistry. Nobel Medical Bookstores. (In Turkish).
  • Nelson, C.A., Handler, P., 1968. Preparation of bovine xanthine oxidase and the subunit structures of some iron flavoproteins. Journal of Biological Chemistry, 243(20): 5368-5373.
  • Onat, T., Emerk, K., Sözmen, E.Y., 2006. Human Biochemistry, Palme Publishing, Ankara, Türkiye. (In Turkish).
  • Ozer, N., Muftuoglu, M., Ataman, D., Ercan, A., Ogus, I.H., 1999. Simple, high-yield purification of xanthine oxidase from bovine milk. Journal of Biochemical and Biophysical Methods, 39(3): 153-159.
  • Page, S., Powell, D., Benboubetra, M., Stevens, C.R., Blake, D.R., Selase, F., Wolstenholme, A.J., Harrison, R., 1998. Xanthine oxidoreductase in human mammary epithelial cells: activation in response to inflammatory cytokines. Biochimica et Biophysica Acta, 1381(2): 191-202.
  • Parks, D.A., Granger, D.N., 1986. Xanthine oxidase: biochemistry, distribution and physiology. Acta Physiologica Scandinavica, 548: 87-99.
  • Pauff, J.M., Hemann, C.F., Junemann, N., Leimkuhler, S., Hille, R., 2007. The role of arginine 310 in catalysis and substrate specificity in xanthine dehydrogenase from Rhodobacter capsulatus. Journal of Biological Chemistry, 282(17): 12785-12790.
  • Polaina, J., MacCabe, A.P., 2007. Industrial Enzymes. Springer, The Netherlands.
  • Schoutsen, B., De Jong, J.W., Harmsen, E., De Tombe, P.P., Achterberg, P.W., 1983. Myocardial xanthine oxidase/dehdrogenase. Biochemica et Biophysica Acta, 762(4): 519-524.
  • Tubaro, E., Lotti, B., Santiangelli, C., Cavallo, G., 1980. Xanthine oxidase increase in polymorphonuclear leucocytes and macrophages in mice in three pathological situtions. Biochemical Pharmacology, 29(13): 1945-1948.
  • Werns, S.W., Lucchesi, B.R., 1990. Free radical and ischemic tissue injury. Trends in Pharmacological Sciences, 11(4): 161-166.
There are 37 citations in total.

Details

Primary Language English
Journal Section Research Article
Authors

Yeşim Kaya 0000-0001-7038-5338

Semra Isık 0000-0002-3022-0878

Serap Uzunoglu 0000-0003-0460-7873

Mustafa Oğuzhan Kaya 0000-0002-8592-1567

Publication Date October 31, 2022
Published in Issue Year 2022 Volume: 9 Issue: 3

Cite

APA Kaya, Y., Isık, S., Uzunoglu, S., Kaya, M. O. (2022). Purification of Xanthine Oxidase Enzyme and Investigation of Its Immobilization with Glutaraldehyde. Türkiye Tarımsal Araştırmalar Dergisi, 9(3), 314-322. https://doi.org/10.19159/tutad.1084383
AMA Kaya Y, Isık S, Uzunoglu S, Kaya MO. Purification of Xanthine Oxidase Enzyme and Investigation of Its Immobilization with Glutaraldehyde. TÜTAD. October 2022;9(3):314-322. doi:10.19159/tutad.1084383
Chicago Kaya, Yeşim, Semra Isık, Serap Uzunoglu, and Mustafa Oğuzhan Kaya. “Purification of Xanthine Oxidase Enzyme and Investigation of Its Immobilization With Glutaraldehyde”. Türkiye Tarımsal Araştırmalar Dergisi 9, no. 3 (October 2022): 314-22. https://doi.org/10.19159/tutad.1084383.
EndNote Kaya Y, Isık S, Uzunoglu S, Kaya MO (October 1, 2022) Purification of Xanthine Oxidase Enzyme and Investigation of Its Immobilization with Glutaraldehyde. Türkiye Tarımsal Araştırmalar Dergisi 9 3 314–322.
IEEE Y. Kaya, S. Isık, S. Uzunoglu, and M. O. Kaya, “Purification of Xanthine Oxidase Enzyme and Investigation of Its Immobilization with Glutaraldehyde”, TÜTAD, vol. 9, no. 3, pp. 314–322, 2022, doi: 10.19159/tutad.1084383.
ISNAD Kaya, Yeşim et al. “Purification of Xanthine Oxidase Enzyme and Investigation of Its Immobilization With Glutaraldehyde”. Türkiye Tarımsal Araştırmalar Dergisi 9/3 (October 2022), 314-322. https://doi.org/10.19159/tutad.1084383.
JAMA Kaya Y, Isık S, Uzunoglu S, Kaya MO. Purification of Xanthine Oxidase Enzyme and Investigation of Its Immobilization with Glutaraldehyde. TÜTAD. 2022;9:314–322.
MLA Kaya, Yeşim et al. “Purification of Xanthine Oxidase Enzyme and Investigation of Its Immobilization With Glutaraldehyde”. Türkiye Tarımsal Araştırmalar Dergisi, vol. 9, no. 3, 2022, pp. 314-22, doi:10.19159/tutad.1084383.
Vancouver Kaya Y, Isık S, Uzunoglu S, Kaya MO. Purification of Xanthine Oxidase Enzyme and Investigation of Its Immobilization with Glutaraldehyde. TÜTAD. 2022;9(3):314-22.

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