Purification of Urate Oxidase

Abstract

Soluble urate oxidase was prepared and purified from ox kidney. A method has been described which gives a 20-25 fold purification of the enzyme over the alkaline extract from the acetone powder. However, considerable purification was obtained in the preparation of the acetone powder from the whole kidney. The preparation of purified urate oxidase is shown in the following scheme: 1) Cortical tissue was dissected from the kidney and represented some purification of enzyme; the medulla gives somewhat less active extracts, which have a relatively high content of coloured impurities. 2) The cortex was washed in water to eliminate soluble cell components and smaller particulate matter. This procedure gave a considerable, but unassessable, purification of enzyme. Washing removed the albumins, some globulins, carbohydrates and soluble organic and inorganic matter. The composition of ox kidney is shown below (57): Water 750 g/kg of wet tissue Protein 150 g/kg of wet tissue Carbohydrate 9 g/kg of wet tissue Lipid 44-81 g/kg of wet tissue 3) The 1,000 to 10,000 x g fraction (mitochondrial and heavy microsomal fraction) was isolated from the washed, homogenised cortex. The nuclear, microsomal, and supernatant fractions were discarded as urate oxidase is believed to be associated with microbodies which together with mitochondria and lysosomes, constitute the mitochondrial fraction. 4) An acetone powder was made from the isolated fraction. Treatment of the fraction with acetone and ether eliminated lipid constituents and gave an active powder which could be stored for several months with only slight inactivation and from which the enzyme could be readily extracted. Considerable purification of the enzyme was achieved in steps 3 and 4. In one acetone powder preparation 7.52 gm of powder was prepared from 498 gm of fresh kidney. This represents a 66-fold purification on a weight basis. 5) Soluble urate oxidase was prepared by extraction of the acetone powder with 0.2M ammonia (pH 11.3). In most cases, an insoluble residue remained after alkaline extraction but Truscoe (unpublished) has shown that this residue does not contain significant quantities of enzyme. 6) To the crude extract ammonium sulphate was added to 50% saturation, and this procedure generally raised the specific activity 1.5-2 fold over that of crude extract. 7) The ammonium sulphate precipitate extract was incubated with 10mM DTT for two hours at 37°C. The resulting extract was passed through a Sephadex G-200 column 8 cm2 x 80 cm) and the effluent showed a clear-cut separation of activity and protein peaks. The active fractions gave a 20- to 25-fold purification of the enzyme over the alkaline extract of the acetone powder. 8) The active fractions were concentrated using Carbowax and freeze dried to an active powder, although there was some loss of activity and a loss in stability on lyophilisation. 9) Crystallisation of enzyme from active fractions using ammonium sulphate may have been achieved but this is yet to be confirmed. 10) Ox kidney urate oxidase is a cuproprotein with the copper present in valence state 2. There are 2 gram atoms of copper per 100,000 molecular weight unit of enzyme; the molecular weight of the separated activity peak produced by gel filtration. Purification factors calculated on the basis of 1) copper content per mg of protein and 2) enzyme activity per mg protein, at each step in the procedure adopted in the purification of urate oxidase, were in close agreement.