1. Biosynthetic sodium (N-acetyl-N-phenylhydroxylamine NO-β-d-glucosid)-uronate is hydrolysed completely by purified mouse urinary β-glucuronidase into the products N-acetyl-N-phenylhydroxylamine and glucuronic acid. The hydrolysis is inhibited by saccharo-(1→4)-lactone. These results not only confirm the identity and purity of the substrate but also establish it as a substrate for β-glucuronidase. 2. Mammalian and bacterial β-glucuronidase preparations hydrolysed the substrate at a rate one-fifth of that for (phenolphthalein β-d-glucosid)uronic acid under the optimum conditions of hydrolysis for each source. 3. The pH optimum is 4·1 and the Michaelis constant, Km, is 3·3×10−4m with purified mouse urinary β-glucuronidase as the enzyme source acting on the NO-β-d-glucosiduronic acid. The aglycone after extraction into chloroform was quantitatively determined spectrophotometrically at its absorption maximum (256mμ). 4. The hydrolysis was studied as a function of time and temperature. 5. From a consideration of the chemical and enzymic properties of this NO-β-d-glucosiduronic acid it is possible to suggest its catabolism in vivo.
