EFRAIM RACKER 



able equilibrium, which leads to incomplete utilization of the 

 substrate, can be overcome by the removal of products with 

 secondary enzymes. The availability of spectrophotometers has 

 fostered the development of many sensitive and rapid assay 

 methods. If an enzyme-catalyzed reaction results in no changes 

 in light absorption, it is usually possible to link it to a reaction 

 with suitable spectral properties. Occasionally several enzymes 

 have to be added in excess, while the enzyme to be assayed is 

 added in limiting amounts. The number of complications due 

 to side reactions increases, however, with the number of auxiliary 

 enzymes used. 



"enzymatic purity" of enzymes 



In the course of these studies the enzymologist became aware 

 that a new kind of purity is required for his analytical enzyme 

 tools, since the criteria of chemical purity had little meaning in 

 this case. An enzyme preparation though homogeneous accord- 

 ing to the most rigid physicochemical measurements may con- 

 tain an enzyme impurity which represents only a fraction of 1 % 

 of the total protein. The preparation may be worthless for his 

 analysis if the contaminating protein happens to be (and fre- 

 quently is) a very active enzyme which catalyzes a side reaction 

 in his assay system. On the other hand, another preparation 

 less pure according to physicochemical determinations may be 

 quite suitable as analytical reagent. Thus, the presence or ab- 

 sence of contaminating enzymes which give rise to side reactions 

 with substrate, product, or coenzyme determines whether or not 

 an enzyme is usable. Therefore, new criteria of "enzymatic 

 purity" must be established for each enzyme reagent. 



An example encountered in our laboratory may serve to 

 illustrate the complexities of some of these assays. A preparation 

 of glucose-6-phosphate dehydrogenase from brewers' yeast (20) 

 has been widely used for the quantitative determination of 

 glucose-6-phosphate and of TPN. The enzyme can be used 

 to regenerate TPNH in TPN-linked oxidation-reductions. In 

 combination with hexokinase and glucose it is used for the de- 



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