Purification of Xanthine Oxidase and Investigation of Its Immobilization with Glutaraldehyde

Abstract

Xanthine oxidase (XO), in purine metabolism is a flavoprotein containing molybdenum with a key role. It has biological functions such as regeneration of NAD+, iron absorption and mobilization, reduction of nitrates. In this study, xanthine oxidase enzyme was purified by Sepharose-4B-L-tyrosine-4-aminobenzamidine dihydrochloride gel according to affinity chromatography technique and immobilization on glutaraldehyde was investigated. XO purified by ammonium sulfate precipitation and affinity chromatography was obtained with an 11.5 % yield and 694.04 degrees of purity. The purity of XO was confirmed by SDS-PAGE and a single band of around 150 kDa was observed. Kinetic constants (KM and VMax) of the enzyme were determined 1.67x10-4 M and 0.56 U/mL.min respectively by using 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 effect were determined. Afterward XO was immobilized on glutaraldehyde, which was used as a solid support material. 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.min respectively. These values revealed that the catalytic activity of the free enzyme was higher than the immobilized enzyme.