Enzymes 149 



following control tests, run alongside the regular method, 

 can be used to rule out nonenzymatic reactions: 



1. Treat the sections prior to incubation with agents 

 known to destroy enzymatic activity (boiling water, strong 

 mineral acid, oxidants, heavy metals, etc.). It must be re- 

 marked that the resistance of enzymes varies widely; some 

 are very easily destroyed, others are surprisingly resistant 

 (e.g., hemoglobin is resistant to high temperatures and acids; 

 myosin to precipitation by trichloroacetic acid). In some 

 cases distinction between true and "pseudo"-enzymes must 

 be arbitrary. 



2. Use specific inhibitors ( cyanide, azide, eserine, etc. ) in 

 an effective concentration. 



3. Leave out the substrate from the incubating mixture. 

 Whatever reaction persists under the conditions of these 



control tests cannot be du,e to an enzymatic effect. Of course, 

 the picture in the regular slide may reveal a combination of 

 enzymatic and nonenzymatic reactions. In such a case only 

 the difference between the regular and the control slide can 

 be attributed to enzymatic activity. 



The specificity of enzymes is rarely an absolute one. As a 

 rule, one enzyme will attack several chemically related sub- 

 strates ( although the optimal conditions of activity may vary 

 with the substrate). Conversely, one substrate may be at- 

 tacked by a number of enzymes (although, again, optimal 

 conditions may vaiy with the enzyme ) . Whether one or sev- 

 eral enzymes, are involved in any given reaction (or in re- 

 lated reactions ) is a problem familiar to research workers in 

 both bio- and histochemistry. In biochemistry the successful 

 separation of a crude extract into fractions with distinctly 

 different enzymatic properties is considered to be the evi- 

 dence for the presence of several enzymes. In histochemistry, 

 different topographical patterns of the distribution of activ- 

 ity, obtained under different conditions (varying the sub- 

 strate, pH; the presence of activators or inhibitors) is as- 

 sumed to be the indication for the involvement of more than 



