MULTIPLE ENZYME SYSTEMS 



whatever its nature, must lie quite far towards the left and the 

 second equally far to the right. 



What can be done in this situation? An obvious approach 

 at present is to study the exchange of isotopically labeled com- 

 pounds, first to aid in establishing the mechanism and, second, 

 to employ the procedure as a measurement of the unfavorable 

 reaction. It is also possible to divide the system into two 

 enzymes (as in the example of argininosuccinic acid synthesis) 

 and to purify one versus the other using the final product as an 

 assay. After purification the problem may yet be unresolved, 

 since the intermediate would not accumulate. Another en- 

 zyme system coupled to one of the products or a trapping agent 

 could perhaps be employed. 



ISOTOPE STUDIES 



Among the most powerful tools we have at present for de- 

 tecting intermediate compounds are the radioactive isotopes. 

 Thus if a substrate can be labeled and if even a trace of an 

 intermediate is present, it can be found by chromatographing 

 the reaction filtrate either on paper or on columns, measuring 

 the compound by direct radioactivity determinations of the 

 chromatographic fractions, or by exposing paper chromato- 

 grams to x-ray films. 



Once a compound is shown to be an intermediate in a com- 

 plex system, it can be studied by the classical enzyme methods. 

 However, a compound is not necessarily an intermediate on the 

 basis of its becoming labeled by an isotopic substrate. A com- 

 pound, E, may be labeled under the following circumstances. 



A* ==± B* , C* , D* 



E* 



While E becomes labeled from substrate A, and, in turn, labeled 

 E is converted to D, it is not an intermediate as such. Frac- 



555 



