Purification and Characterization of Xanthine Oxidase from Liver of the Sheep (Ovis Aries)

Abstract

Xanthine oxidase is a commercially important enzyme with wide area of medical applications to develop diagnostic kits. Xanthine oxidase was extracted, purified and characterized from sheep liver (SLXO). The purification procedure involved acetone precipitation and chromatography on DEAE-cellulose and Sephacryl S-300 columns. The sheep liver xanthine oxidase was homogeneously purified 31.8 folds with 3.5 U/mg specific activity and 24.1% recovery. SLXO native molecular weight was 150 kDa and on SDS-PAGE appeared as single major band of 75 kDa representing a homodimer protein. Isoelectric focusing of the purified SLXO resolved into two closely related isoforms with pI values of 5.6 and 5.8. The apparent Km for xanthine oxidase at optimum pH 7.6 was found to be 0.9 mM xanthine. FeCl2 and NiCl2 increased the activity of SLXO, while CuCl2 and ZnCl2 were found to be potent inhibitors of the purified enzyme. Allopurinol inhibits SLXO competitively with one binding site on the purified molecule and Ki value of 0.06 mM.

Review Artikel: Isolasi Dan Pengukuran Aktivitas Enzim Xantin Oksidase

“…FeCl2 dan NiCl2 mampu meningkatkan aktivitas enzim, sementara CuCl2 dan ZnCl2 menjadi inhibitor enzim. Allupurinol juga menjadi inhibitor kompetitif pada enzim dengan satu sisi pengikat dengan harga Ki 0,06 mM [26].…”

“…Produk tersebut dikomplekskan dengan reagen Nitroblu tetrazolium (NTB) kemudian akan membentuk formazan berwarna biru gelap yang menghasilkan serapan pada panjang gelombang 575 nm. Untuk menentukan unit xantin oksidase digunakan 0,02 unit bovine milk xantin oksidase yang tersedia secara komersial sebagai reaksi kontrol [4,26,27].…”