Partial pruficiation of procine liver uricase
Journal of Biological Chemistry
Uricase, the enzyme which oxidizes uric acid to allantoin, has been prepared from both porcine liver and bovine kidney in highly purified form (l-6). Davidson (1) and Holmberg (2), using acetone-dried porcine liver, reported preparations with Qo, (protein) of approximately 6000. Altman et al. (4) reported preparations from bovine kidney with a Qo, (protein) of 450, and Leone (5) prepared a material from the same tissue with a QoZ (protein) of approximately 700. The methods varied in their
... ried in their complexity and yields. A simple and reproducible new method for the preparation of porcine liver uricase was developed and is described in this report. This procedure was based on the early removal of water-and saltsoluble proteins, including nucleoproteins. Since preliminary experiments showed that uricase had solubility properties similar to some of the nucleoproteins, the removal of pentose nucleoprotein and deoxypentose nucleoprotein from the tissue, before the extraction of the enzyme, was indicated. In this method a soluble, stable enzyme preparation free of nucleoproteins is obtained. The enzyme has been purified 300-fold over an alkaline extract of whole liver in good yields with a final Qo, (protein) of 1200. Cysteine increases the rate of oxidation of uric acid by uricase (3) and the rate is dependent on the concentration of the activator. EXPERIMENTAL Measurement of Enzyme Activity Uricase activity is determined at each step in the procedure by the Warburg manometric method at 37" (1, 2, 7, 8) . The reaction chamber contained 40 pmoles of uric acid as lithium urate in 1.0 ml.,l 0.5 to 1.0 ml. of 0.1 M glycine buffer at pH 9.3, 0 to 1.5 ml. of enzyme in sodium carbonate (or sodium carbonate-glycine buffer) at pH 9.6 to 10.0, and distilled water to a total volume of 3.0 ml. The enzyme solution was always the last reagent added. The center well contained 0.2 ml. of 20 per cent KOH and a small strip of fluted filter paper. The gas phase was air. The mixture 1 672 mg. of uric acid are dissolved in 35 ml. of boiling 0.12 N LiOH and the solution is diluted to 100 ml. with distilled water.