Purification of Urate Oxidase

Abstract

The usefulness of the gel filtration technique as a step in the purification of ox kidney urate oxidase has again been demonstrated. The elution profiles obtained subsequent to gel filtration have shown that urate oxidase activity is contained, broadly speaking, in two peaks, named respectively; Peak I and Peak II. As has also been shown by Tate (116) and James (117), the former peak is characterised by high protein, and low activity values, and the latter peak by high specific activity. Some properties of the two peaks (i.e. Michaelis Constant, Temperature-stability curves, and the nature of bonding), have been examined. The Peak II fractions have also been investigated by quantitative E.S.R. measurements. From these studies it has been concluded that: (i) Urate Oxidase is a cuproprotein containing four Cu2+ atoms per enzyme molecule. The E.S.R. studies have also shown that: (ii) This enzyme-bound copper is reduced by substrate to Cu+, which is then reoxidised to Cu2+ by atmospheric oxygen. It may be that this is a possible mechanism by which the enzyme-catalysed oxidation of urate could occur.