VOL. 12 (1953) ENZYMIC SYNTHESIS AND BREAKDOWN OF OROTIC ACID I 227 



The reaction mixture in a Thunberg tube consisted of 24.4 micromoles of glucose, 15 micro- 

 moles of MgCIg, 100 micromoles of potassium phosphate buffer (pH 6.1), 10 micromoles of sodium 

 orotate, 20 micromoles of cysteine (pH 7.0), and 51 units of dihydro-orotic dehydrogenase (specific 

 activity 86). DPN (0.05 micromole) and 500 units of glucose dehydrogenase were placed in the side- 

 arm. The total volume was 3.0 ml. The tube was evacuated, filled with helium, and re-evacuated; 

 the process was then repeated. After tipping, incubation was carried out at 35° C for 180 minutes. A 

 control tube contained no glucose. After incubation, the tubes were placed in a boiling water bath 

 for 2 minutes, centrifuged, and aliquots of the supernatant solutions were used for the estimation 

 of orotate and glucose. The anaerobic atmosphere was provided to avoid interference by DPNH 

 oxidase which contaminated the enzyme preparation. 



With the removal of 5.88 micromoles of orotate, the consumption of 5.50 micro- 

 moles of glucose was observed. 



Isolation of the product of orotate reduction. Radioautography of paper chromato- 

 grams of the product (s) of the action of purified dihydro-orotic dehydrogenase on orotate- 

 2-i^C showed essentially one spot. This substance could be adsorbed from reaction 

 mixtures on Dowex i, formate form, and appeared on elution as a discrete peak in an 

 area distinct from known compounds. 



To obtain a sufficient amount of this compound for its identification, a large scale 

 reaction was carried out with 0.64 millimole of 2-^*C-orotate (1.38-10^ cpm.) and 1420 

 units of purified enzyme (specific activity 152). The reaction mixture also contained the 

 remaining components of the standard assay system, in 50-fold greater amounts. During 

 incubation at 34° C, aliquots were removed at intervals and spectrophotometrically 

 tested for orotate disappearance. When the reaction was complete, 4 N HCl was added 

 until the solution became acid to thymol blue; the precipitated protein was then dis- 

 carded. 



After adjusting the pH of the supernatant solution to 7.0 with i M KOH, the solu- 

 tion was subjected to ion-exchange chromatography on a column of Dowex i, formate 

 form (height, ii.o cm; diameter, 4.2 cm). The eluting fluid was 0.055 M sodium formate 

 solution adjusted to pH 3.2 with formic acid. The product, detected by radioactivity 

 measurement, appeared between 20.8 and 25.8 resin bed volumes of eluant and repre- 

 sented at least 80% of the counts applied to the column. The radioactive fractions were 

 combined (915 ml) and passed through Dowex 50, hydrogen ion form (resin bed volume 

 of 150 ml), to remove the sodium ions. Water and formic acid were removed under 

 reduced pressure (at a water bath temperature of 40-45° C). The residue was dried over 

 KOH in a vacuum desiccator, then dissolved in hot water and crystallized in the cold. 

 Upon recrystallization from water, 53 mg of hard white crystals were obtained represent- 

 ing a 53% recovery from orotate, assuming the product to have the molecular weight 

 of dihydro-orotic acid. 



Elementary analysis of the compound as compared with that of dihydro-orotic acid 

 was as follows : 



C5HJO4N2 Calculated C, 37.97, H 3.79, N 17.72 

 Found C, 37.48, H 3.82, N 17.69. 



The compound melted at 269-271° with some decomposition. No weight loss 

 occurred at 77° in vacuo. Titration of a solution containing i.oo mg of the compound 

 to a phenol red endpoint required 6.06 micromoles of NaOH (o.oio A^ yielding an 

 equivalent weight 96% of that calculated for dihydro-orotic acid. 



On the basis of the evidence thus far presented, the enzymic product has been 

 provisionally considered to be dihydro-orotic acid. 

 References p. 234. 



