[64] Preparation of nucleoside phosphorylase from calf spleen☆

Publisher Summary This chapter discusses the preparation of nucleoside phosphorylase from calf spleen. The assay method is based on the determination of hypoxanthine formed during the phosphorolysis of inosine b y nucleoside phosphorylase. The hypoxanthine is oxidized to uric acid by xanthine oxidase and may be followed by differential spectrophotometry. One unit of nucleoside phosphorylase is defined as the amount of enzyme which under the above conditions causes an increase in optical density of 0.001 per minute at 293 mμ in the initial rate when read in a cuvette with a light path of 1 cm. In the course of assay, 2600 g. of calf spleen is homogenized in a Waring blendor with 2.5 vol. of cold distilled water and filtered through gauze; the residue is washed with 0.5 vol. of water. The chapter describes an alternative preparation of purine nucleoside phosphorylase from beef liver where an acetone powder extract of the liver is subjected to three successive ethanol precipitations followed by fractionation with ammonium sulfate and silica gel. The purity of the preparation is about 200-fold based on the absorption of the various fractions at 280 mμ. Recently this preparation has been used in the synthesis of 4-amino-5-imidazolecarboxamide riboside.