230 



I. LIEBERMAN, A. KORNBERG 



VOL. 12 (1953) 



2W 



250 



260 



270 



280 



300 



310 



290 

 Wavelength (m/i ) 



Fig. 3. A comparison of the absorption spectra of orotate and the product of dihydro-orotate oxidation. 



The curve represents the absorption spectrum of sodium orotate in phosphate bui^er (0.033 ^^> 



pH 6.4). The circles represent the values obtained upon complete oxidation of dihydro-orotate under 



the conditions described in Fig. 2. 



Fig. 4. The requirement for DPNH removal in 

 dihydro-orotate oxidation. 



The reaction mixtures prepared in each 

 of two cuvettes fitted to Thunberg tubes con- 

 tained in 3.0 ml, 15 micromoles of MgClj, 100 

 micromoles of potassium phosphate buffer 

 (pH 6.1), 20 micromoles of cysteine (pH 7.0), 

 0.03 micromole of DPN, 0.53 micromole of 

 dihydro-orotic acid, and 8.0 units of dihydro- 

 orotic dehydrogenase (Protamine Fraction, 

 see Table I). In addition, 20 micromoles of 

 sodium pyruvate and 0.05 ml of lactic dehy- 

 drogenase were placed in the sidearm of one 

 of the Thunberg tubes. The tubes were eva- 

 cuated, filled with helium, and re-evacuated; 

 this procedure wasthen repeated. The progress 

 of the reaction was followed spectrophoto- 

 metrically at 280 ra/i. At the indicated time, 

 one tube was opened to air, the other, 

 containing the lactic dehydrogenase and 

 pyruvate, was tipped. 



10 



30 



50 

 Time in minutes 



I. Ultraviolet absorption of the fusion product (free acid) shows a peak at 220 m/t 

 with molar extinction coefficients of 2-10* and i-io^ at 220 and 240 mix, respectively. 

 The enzymic compound (free acid) possesses only an end absorption with molar extinc- 

 tion coefficients at 220 and 240 m/x of 2 • 10^ and 3-102, respectively. 2. Melting point of 

 the fusion compound was found to be 264-265° uncor. (lit. 247-249°); the biological 

 product melts at 269-271°. 3. Elementary analyses of the natural and synthetic com- 

 pounds are in close agreement with that calculated for dihydro-orotic acid. 4. Reaction 

 with cysteine of the synthetic compound, i)resumably due to the presence of an olelinic 



References p. 234. 



