Asymmetric Citric Acid 247 



compound was oxidized the isotope was found to be present 

 only in the carboxyl group adjacent to the carbonyl carbon 

 as shown in Fig. 1 (Evans and Slotin, 1941; Wood, Werkman, 

 Hemingway and Nier, 1942). It was universally agreed that 

 citric acid, a symmetrical molecule, could not have been an 

 intermediate in this process, and hence it was thought not 

 to be a direct intermediate in the cycle. 



The brilliant proposal of Ogston (1948) that isotopic citric 

 acid would probably behave in an asymmetric fashion in 

 enzymatic reactions has led to a resolution of the difficulties. 

 According to this idea, labelled citric acid should exist in 

 two isotopically antipodal forms as illustrated in Fig. 2. This 

 proposition was peculiarly easy to test experimentally. It 

 was only necessary to repeat the earlier experiments, this 



HOOCv^ ^CHr>COOH HOOCCHo- ^COOH 



H00CCH2'^^ ^OH HO-^ "^CHgCOOH 



Fig. 2. Isotopically antipodal forms of citric acid. 



time sub-divided into two steps and with isolation of the 

 citric acid itself. When ^^COg was fixed in a rat kidney 

 homogenate, the citric acid isolated was radioactive, and when 

 it was used as the labelled substrate in a rat liver homogenate, 

 the a-ketoglutarate produced was shown by chemical oxida- 

 tion to have a completely asymmetric distribution of ^*C 

 (see Fig. 3, Potter and Heidelberger, 1949). This result 

 confirmed Ogston's proposal and together with the work of 

 Stern and Ochoa (1949) restored citric acid to its rightful 

 place as a true intermediate in the Krebs cycle. 



In our experiments the labelled substrate is added to a rat 

 kidney homogenate maintaining active phosphorylation in 

 the presence of pools of fumarate, citrate, and with malonate 

 to prevent recycling. The citric acid is isolated and purified 

 by silica-gel partition chromatography (Isherwood, 1946), 

 and its specific activity determined. This citrate is added to 

 a second rat liver homogenate in the presence of arsenite to 



