ENZYMES AS REAGENTS 



termination of ATP. It can be used in an assay for hexokinase 

 or phosphohexose isomerase. In the course of several years 

 during which this enzyme served as a useful reagent in many 

 laboratories, differences in the properties of various preparations 

 probably due to minor variations in the purification procedure 

 were encountered. Thus, some preparations contained traces 

 of a TPNH oxidase or of phosphogluconic dehydrogenase which 

 prevented the achievement of a sharp end point at the time of 

 the complete reaction. Variable amounts of hexokinase, phos- 

 phohexose isomerase, adenylate kinase, and glutathione reduc- 

 tase were found in these preparations. Accurate values for ATP 

 could not be obtained in the presence of adenylate kinase, and 

 an analysis of glucose-6-phosphate or ATP in a sample which 

 contained some oxidized glutathione gave erroneous values owing 

 to glutathione reductase. Occasionally it is possible to make a 

 preparation suitable as a reagent by the use of an inhibitor. 

 For example, treatment with A^-ethyl maleimide has been found 

 to eliminate adenylate kinase activity without inactivating glu- 

 cose-6-phosphate dehydrogenase (25). 



The suitability of an enzyme for quantitative substrate de- 

 termination is dependent on an appropriate substrate affinity 

 and absence of inhibitory side reactions. Preparations of glu- 

 cose-6-phosphate dehydrogenase from Torula yeast, although of 

 much higher specific activity than the preparations from brew- 

 ers' yeast, were found to react much more sluggishly at low glu- 

 cose-6-phosphate concentrations and were inadequate for assay 

 purposes (44). 



ANALYSIS OF MULTIENZYME SYSTEMS 



The determination of an enzyme in crude tissue extracts in 

 which complex chains of metabolically linked reactions take 

 place often poses great difficulties. Measurements of formation 

 of the product or of disappearance of the substrate are compli- 

 cated by the presence of the other members of the multienzyme 

 systems which act on the same compounds. Although blocking 

 of secondary reactions may be successfully used in some instances, 

 in other instances new complications, e.g., "product inhibition," 



227 



