Xanthine dehydrogenase has been measured calorimetrically by tetrazolium salt reduction, TTC3 generally being used as electron acceptor (l-5).
Recently, other tetrazolium salts have been introduced that present many advantages over TTC. The present paper deals with the assay of xanthine dehydrogenase by means of INT or Nitro-BT; based on a method developed by Nachlas et al. ( 6) for lactic dehydrogenase.
The present method proved t,o be a simple and sensitive assay for the calorimetric determination of xanthine drhydrogenase, and is also suitable for calorimetric rate studies. METHODS Cream xanthine oxidase (Worthington) was diluted from stock with 0.1 M phosphate, pH 7.8. The incubation mixture has the following composition: INT (Mann) or Nitro-BT
(Sigma, grade III), 4 mg; hypoxanthine or xanthine (Mann), 0.5 ymole; EDTA, 0.1 ymole; gelatin O.l%, 1.0 ml; graded levels of enzyme; phosphate 0.1 M, pH 7.8, to complete 3.50 ml. The reactants were mixed at room temperature, and placed in a Dubnoff shaker at 37Β°C; 0.1 ml PMS (0.2 mg/ml) was added after 5 min, and the reaction was started by the addition of enzyme solution. The mixture was shaken for 15 min at 37", and the reaction was stopped by t'he addition of 0.50 ml of HCl, 0.35M. The