226 I. LIEBERMAN, A. KORNBERG VOL. 12 (1953) 



centrifugation and upon further addition of 25.2 g of ammonium sulfate to the super- 

 natant solution another precipitate was formed. This precipitate was collected by centri- 

 fugation and dissolved in 120 ml of water (Ammonium sulfate, Table I). 



Acid ammonium stdfate fy action. 30 ml of sodium formate buffer (0.5 M , pH 4.2), 

 and then 36 g of ammonium sulfate were added to the ammonium sulfate fraction with 

 stirring. After 5 minutes the precipitate was removed by centrifugation. 18 g of ammo- 

 nium sulfate were added to the supernatant solution and the precipitate that formed 

 was collected and dissolved in 62 ml of sodium acetate buffer (o.oi M, pH 6.0) (Acid 

 ammonium sulfate, Table I). 



Further purification of the activity (three- to five-fold) could be obtained by sub- 

 jecting the acid ammonium sulfate fraction to column chromatography with Dowex i, 

 formate form, 2% cross-linked, and eluting with phosphate buffer. The yields were too 

 variable to warrant inclusion of this step in the routine purification procedure. 



Stoichiometric relationship of orotate and DPNH. The requirement for DPN, glucose, 

 and glucose dehydrogenase for orotate reduction could be replaced with DPNH. With 

 the purified enzyme preparation, oxidation of DPNH was accompanied b}^ the removal 

 of an equimolar amount of orotate (Table II). The essentially complete utilization of the 



TABLE II 



THE EQUIVALENCE OF DPNH AND OROTATE DISAPPEARANCE 



Exp. I Exp. 



Micromoles 



DPNH added 0.159 0.318 



A Orotic acid — 0147 — 0.329 



The reaction mixtures (in glass stoppered cuvettes) contained in 3.0 ml, 15 micromoles of 

 MgClj, 100 micromoles of potassium phosphate buffer (pH 6.1), 20 micromoles of cysteine (pH 7.0), 

 0.4 micromole of sodium orotate, 4.1 units of enzyme (specific activity 178) and the indicated amount 

 of DPNH. The DPNH was added after repeated flushing with Hg and the reaction was followed 

 spectrophotometrically at 280 m/^. Interference by DPNH oxidase, which contaminated the enzyme 

 preparation, was almost completely eliminated in this anaerobic atmosphere and by the rapid rate 

 of the orotate reduction (complete in 2-4 minutes). 



DPNH by orotate in relatively low concentrations under conditions which appear to 

 limit the reaction to this single step (see below) suggests that the equilibrium of the reac- 

 tion is greatly in favour of orotate reduction. 



Stoichiometric relationship of orotate and glucose. Under the standard conditions 

 of assay for dihydro-orotic dehydrogenase (see Methods) and in large scale experiments, 

 the DPNH was present in catalytic amounts and was constantly regenerated b\^ the 

 oxidation of glucose according to the following equations: 



Glucose -V DPN gl"cose dehydrogenase _^ gluconate + DPNH + H+ (4) 



Orotate -|- DPNH -|- H+ dihydro-orotic dehydrogenase _^ dihydro-orotate + DPN (5) 



Sum: Glucose -|- orotate > gluconate H dihydro-orotate. (6) 



In order to establish that orotate reduction under these conditions involves the 

 utilization of an equimolar amount of glucose, a quantitative experiment was carried 

 out. 



References p. 234. 



