Enzymes 169 



rate of an ionic reaction, even at pH 6.3-6.5. Amines (naph- 

 thylamines ) couple much more slowly, and the sluggishness 

 of the reaction precludes the use of azo-coupling for their 

 histochemical demonstration (diffusion artifacts). 



Any naphthol will not couple indiscriminately with any 

 diazonium compound. Each new combination must be tried 

 out in the test tube before its application to histochemistry 

 can be considered. If coupling does take place, the color of 

 the azo dye formed depends on both the naphtholic and the 

 diazoic components used. The shade produced by any specific 

 combination is not necessarily uniform but depends to some 

 extent on factors such as the concentration of the ingredients, 

 pH, temperature, etc. 



Azo dyes of phenols are not sufficiently insoluble for histo- 

 chemical use; for this reason, only naphtholic compounds 

 are employed. Azo dyes formed from ^-naphthol have very 

 brilhant shades but have a tendency to remain in a colloidal 

 solution for a short time before precipitating. This will cause 

 some loss of fine structural details and false localizations 

 (staining of acidophilic structures) around centers of high 

 activity.^ Even the addition of high concentrations of salt 

 to the incubating mixture (making the dye more insoluble 

 by the "salting-out" effect) does not help much. There is also 

 a possibility that the coupling process itself is a rela- 

 tively slow one in the case of ^-naphthol, especially at pH 

 values lower than about 8.5. On the other hand, a-naphthol 

 and especially certain derivatives of ^-naphthol, e.g., the 

 naphthols of the AS series (chemically, substituted amides 

 of 2-hydroxy-3-naphthoic acid ) , give exceedingly sharp and 

 detailed pictures. The shades given by a-naphthol are some- 

 what dull; those given by the AS naphthols, very brilliant. 



Until recently, the only naphtholic substrates readily avail- 

 able on the market were the acetate of a-naphthol and the 

 benzoate of /5-naphthol. At present, practically all the im- 

 portant substrates are made commercially. The Na salts of 



1. Gomori, G.: J. Lab. & Clin. Med., 33:802, 1950. 



