The methods used to demonstrate enzyme activity associated with 

 cellular structures differ from those described above in that the chromo- 

 genic agent is not combined with a particular molecular group but in- 

 stead operates to either catalyze a specific enzyme reaction or to accept 

 hydrogens (electrons) at a specific point in an enzymatic sequence. The 

 enzymes most extensively studied in terms of their location in cell struc- 

 tures are the esterases (alkaline and acid phosphatase) and oxidases 

 (cytochrome oxidase, succinic dehydrogenase). The cytochemical dem- 

 onstration of alkaline phosphatase depends on the deposition of calcium 

 phosphate at sites of enzyme action when tissues are incubated with an 

 appropriate organic phosphate ester in the presence of calcium ions at 

 alkaline pH. The localization of oxidases is accomplished by incubating 

 tissues in a solution of a colorless tetrazoUwn salt which, in the presence 

 of a suitable substrate, is reduced to a colored formazan by accepting 

 hydrogens from either dehydrogenases or flavoproteins. If succinate is 

 used as the substrate (hydrogen donor) the reaction leading to demon- 

 stration of succinic dehydrogenase may be diagrammed as follows: 



TETRAZOLIUM FORMAZAN 



(colorless) (colored) 



SUCCINATE \^ ^^ DPN-H 



SUCCINIC 

 FUMARATE DEHYDROGENASE DPN + 



In this reaction, succinate functions as the electron donor. The electrons 

 removed from succinate as the result of its oxidation via succinic dehy- 

 drogenase are picked up by the colorless tetrazolium to reduce it to the 

 colored formazan. The tetrazolium reaction is sometimes carried out in 

 the presence of metallic ions which chelate with the formazan as it is 

 produced. This facilitates precipitation of the formazan at sites of enzyme 

 activity. 



Quantitative measurement of the amounts of various proteins in cell 

 structures is usually carried out by photometric analysis of tissues stained 

 by appropriate reagents. Methods which have been used successfully for 

 this purpose include (1 ) the Millon reaction, (2) the alkaline Fast green 

 stain for basic proteins (histones), and (3) the Naphthol yellow S stain 

 for basic groups of lysine, arginine, and histidine. 



The localization of polysaccharides in the cell is usually accomplished 

 by the periodic-acid-Schiff (PAS) reaction. This method involves the 

 hydrolysis of C — C bonds in carbohydrates by periodic acid (HIO4) to 



220 / CHAPTER 11 



