The end-products and intermediates of uric acid oxidation by uricase.

Since the early work on uricase carried out by Schittenhelm (l), Wiechowski (a), and Battelli and Stern (3), and t,he elucidation of the structure of uric acid by the work of Behrend and Roosen (4), Fischer (5), and Biltz (6), the chemical nature of the intermediates and end-products of uricase action has been uncertain. The first indications of the structure of a possible intermediate were offered by Schuler and Reindel (7), who were able to isolate, from a uricase system, a silver salt with properties similar to those of the silver salt of hydroxyacetylenediureinecarboxylic acid (HDC) (Formulas V, VI, Fig. 1) which they (8) had previously isolated during the chemical oxidation of uric acid. The existence and structure of such a symmetrical intermediate had been postulated by Behrend (9), who had not been able to effect its isolation. Klemperer (10) concluded from his studies on uricase action that the primary product of uric acid oxidation is an unstable compound which decomposes non-enzymatically to form allantoin, uroxanic acid, and HDC. The relative amounts of these three compounds were found to depend on the pH and the buffer used. Praetorius (11) obtained spectrophotometric evidence for the formation during the action of uricase on uric acid of an unstable intermediate (maximal absorption, 300 to 330 mp) which, he concluded, was a precursor of HDC. Bentley and Neuberger (la), using 01*, H20r8, and 6-Cr4-labeled uric acid, concluded that uricase catalyzes the transfer of 2 electrons from the urate ion, and proposed a mechanism for the enzymatic oxidation of uric acid leading to HDC. Although Wiechowski (2) had considered uricase to catalyze the oxidation of uric acid quantitatively to allantoin, Schuler (13) could account for only 18 per cent of the expected CO:! production at pH 8.9 in borate buffer, and Davidson (14), using a purified uricase preparation in borate buffer at pH 9, could not account for more than 30 per cent of the added uric acid as allantoin. Klemperer (10) also found that only a fraction of