PRINCIPLES OF TRACER METHODOLOGY 49 



1C*00H 



2CH2 'C*OOTT 



3 



i KMn04 I 



CH2 > -CH2 + «C02 (1-42) 



*C=0 ^CHs 



eCOOH ^COOH 



a-ketoglutaric succinic 



acid acid 



The implications of this work may he Hsted as follows: (a) The isotope 

 method was able to reveal the two spatial configurations of citric acid and 

 showed that the enzyme can distinguish between them, thus supporting 

 Ogston's concept of a three-point contact between enzyme and sub- 

 strate. (6) The results were not due to an isotope effect (see Chap. 2). 

 (c) Earlier studies which were taken to indicate that citric acid could not 

 be a primary condensation product in the Krebs cycle could now be 

 reinterpreted to show that citric acid is indeed an initial product of 

 condensation. 



Radiocarbon Dating. The development of the carbon-14 method of 

 age determination by Libby and associates (111 to 114) made an out- 

 standing contribution to the fields of geology, anthropology, archaeology, 

 etc. The procedure is based upon the fact that C^Ms being continually 

 formed in the atmosphere by cosmic-ray-produced neutrons. All mate- 

 rial in the life cycle, as well as all material exchangeable with atmospheric 

 COo, is uniformly labeled with C^^ At death, plants and animals cease 

 to take up new carbon from the atmosphere, and no C" is produced in the 

 inanimate material. Thus at the time of death, or when material is 

 removed from the carbon cycle, the Ci-* present begins to decrease in 

 amount owing to radioactive decay without replenishment. It is 

 assumed that the cosmic-radiation intensity and therefore the C^^ specific 

 activity in the biological cycle have been reasonably constant over the 

 past thousands of years. Using the known half-life of C^^ and comparing 

 the specific activities of a relic and contemporary material, it is possible 

 to calculate the time that has elapsed since the death of the relic. For 

 example, Libby et al. (Ill) found a value of 12.5 ± 0.2 counts/min/g 

 carbon for contemporary material and 7.04 + 0.20 for wood samples 

 from ancient Egyptian tombs that were estimated to be 4600 yr old from 

 other considerations. Using a half -life value of 5720 yr for C^-*, it was 

 calculated that the theoretical specific activity for 4G00-yr-old material 

 should be 7.15 + 0.15, which is in excellent agreement with that observed. 

 Hundreds of samples have now been assayed, and the agreement with 

 independent estimations has supported the basic assumptions. 



