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

Authors : Yeşim KAYA, Semra ISIK, Serap UZUNOGLU, Mustafa Oğuzhan KAYA

Pages : 314-322

Doi:10.19159/tutad.1084383

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Publication Date : 2022-10-31

Article Type : Research Paper

Abstract :In this study, the xanthine oxidase insert ignore into journalissuearticles values(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 insert ignore into journalissuearticles values(the Michaelis constant); and VMax insert ignore into journalissuearticles values(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, Hginsert ignore into journalissuearticles values(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 insert ignore into journalissuearticles values(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