40 STUDIES ON ALKALINE PHOSPHATASE 



phenolphthalein in the sites which show a low activity by the 

 calcium phosphate method. 



The distribution obtained by this method is closely similar to 

 that obtained by the use of glycerophosphate in the substrate. 



It might be thought that phenolphthalein has a specific affin- 

 ity for either certain parts of tissues or for the calcium phos- 

 phate which is precipitated. This was shown not to be so by 

 exposing sections to a strong solution of phenolphthalein. There 

 was no specific staining of the section. Also, when to a solution 

 of phenolphthalein in the incubation mixture a little alkaline 

 sodium phosphate solution was added, phenolphthalein is not 

 carried down to a significant extent by the precipitate of cal- 

 cium phosphate which is formed. These two experiments show 

 quite clearly that the precipitation of phenolphthalein as a re- 

 sult of enzyme activity occurs owing to its insolubility and not 

 by adsorption at sites which have a specific affinity for phenol- 

 phthalein. 



As mentioned previously, phenol phosphates do not react di- 

 rectly with diazonium hydroxides. Consequently, when a phenol 

 phosphate and a diazonium hydroxide are mixed in alkaline so- 

 lution, no reaction takes place. But, when a section is placed 

 in this mixture, if alkaline phosphatase is present it splits phenol 

 and phosphate from the ester, and the phenol rapidly reacts 

 with any diazonium hydroxide which is present. Thus by using 

 a suitable diazonium hydroxide and a suitable phenol, it should 

 be possible to secure the precipitation of the phenol released by 

 enzyme action, at the actual site of enzyme action. The most 

 commonly used substances for this purpose are a- or /?-naphthol 

 phosphate and diazotised a-naphthylamine, which react as 

 follows: 



>OP0 3 H 2 < >OH 



■» HOP0 3 H 2 + > < 



enzyme 



diazonium hydroxide 



>N=N^ >OH 



