Enzymes 181 



9 the intensity of the reaction rapidly declines; only sites of 

 highest activities will be stained after short periods, of incu- 

 bation (up to 2 hours), and on greatly prolonged incubation 

 diffusion artifacts may become very disturbing. Almost any 

 buffer with a suitable pK can be used (NH4CI-NH4OH; bar- 

 bital; 2-amino-2-methyl-l,3-propanediol). Borax is better 

 avoided because it inhibits the hydrolysis of glycerophos- 

 phate and of certain other substrates and because of its in- 

 compatibility with higher concentrations of Ca. The concen- 

 tration of the buffer should be 0.05-0.1 M. The cheapest and 

 easiest available substrate is glycerophosphate, any commer- 

 cial brand of which can be used; the recommended concen- 

 tration, 0.01-0.03 M. Instead of glycerophosphate, any phos- 

 phoric monoester, compatible with Ca at pH 9, can be used. 

 In the case of unstable substrates, such as adenosinetriphos- 

 phate or acyl phosphates, supersaturation artifacts are un- 

 avoidable but can be recognized a such because they will be 

 present even in inactivated sections. 



The importance of the concentration of Ca ions, has be- 

 come appreciated only recently. It has been known for some 

 time that nuclei will stain rather intensely around centers of 

 high activity, and opinions have been expressed that this 

 phenomenon is an artifact due to diffusion of Ca phosphate 

 or of the enzyme itself (pp. 144-45). The experiments of 

 Novikoff^^ and Gomori^^ have shown decisively that the alka- 

 line phosphatase reaction of cell nuclei is due to the secondary 

 adsorption of Ca phosphate only; the nuclei do not contain 

 any enzyme either originally or by secondary adsorption ( ex- 

 cept possibly in cases of poor fixation). The latter point is 

 also proved by the invariable lack of any nuclear reaction 

 with the azo dye method (Lorch,^^ Novikoff, and Gomori). 

 In the Ca-Co method, the staining of nuclei is due to the 

 relatively low concentration of Ca ions in the incubating 



38. Novikoff, A. B.: Science, 113:320, 1951. 



39. Gomori, G.: J. Lab. & Clin. Med., 37:526, 1951. 



40. Lorch, J.: Quart. J. Micr. Sc, 88:159, 1947. 



