EFRAIM RACKER 



may arise. Perhaps the most reUable of all the methods of en- 

 zyme assay in crude systems is one used least frequently, since 

 it requires the availability of auxiliary enzymes of "enzymatic 

 purity." The method consists of adding a large excess of the 

 other enzymes of a multistep assay system to allow a more ac- 

 curate determination of the rate-limiting enzyme. 



Perhaps one should not conclude the discussion of these "prac- 

 tical" aspects of enzymes as reagents without mentioning their 

 usefulness as analytical tools in the determination of chemical 

 structures. This approach, clearly envisaged by Emil Fischer, 

 has served as the key method in the determination of the struc- 

 ture of many coenzymes and is increasingly being used in the 

 elucidation of the structure of proteins and other biological 

 polymers. 



Enzymes as Reactants 



In the early years of enzymology, the oxidation of an alde- 

 hyde by a DPN-linked dehydrogenase was presented as: 



A 1 1 1 1 Enzyme ... ,.-. 



Aldehyde > Acid (1) 



^ DPN 



When DPN became available in gram quantities and was 

 widely used as an hydrogen acceptor instead of as a catalyst, the 

 reaction was written : 



Aldehyde + DPN+ ^"''^"' Acid + DPNH + H+ (2) 



Now that an aldehyde-oxidizing enzyme, glyceraldehyde-3- 

 phosphate dehydrogenase, has become available in gram quanti- 

 ties and the participation of its SH group in the catalysis of alde- 

 hyde oxidation has been demonstrated, the first stages of the 

 reaction sequence can be written as: 



Aldehyde + DPN+ + SH-enzyme > 



Acyl-S-enzyme + DPNH + H+ (3) 



From the physiological point of view, expressions (2) and 

 (3) must be considered as representing Beckman cell artifacts 



228 



