A new isotopic assay for purine nucleoside phosphorylase

We have developed a new assay for purine nucleoside phosphorylase which is based on the release of tritium when [2- 3H]inosine is used as the substrate and the reaction is coupled with xanthine oxidase. After the reaction is terminated, residual [2- 3H]inosine is adsorbed on charcoal and the supernatant solution is assayed for radioactivity by liquid scintillation spectrometry. The new method gave results indistinguishable from those obtained by spectrophotometric determination of uric acid produced by the phosphorylase-xanthine oxidase-coupled reaction or by radioassay of chromatographically isolated [8- 14C]hypoxanthine when [8- 14C]inosine was used as substrate. The new method is faster than those involving chromatographic isolation of products. In comparison with spectrophotometric methods, it not only requires less manual time, but it also has the advantage that it can be used to study inhibitors whose ultraviolet absorption might interfere with spectrophotometric determination of uric acid.