Enzymes 195 



For the histochemical demonstration of phosphamidase 

 the substrate used is phophoric acid p-chloroaniHde (p- 

 chloroaniHdophosphonic acid). This compound is relatively 

 stable and easier to prepare than the other phosphamides. 

 There is some doubt concerning its correct formula; Rorig^^ 

 believes that the compound synthesized by the method of 

 Otto^^ is actually a derivative of diamidophosphoric acid. It 

 appears that different batches prepared by the same method 

 are not necessarily identical in composition. 



The data on the optimal conditions of activity (pH, acti- 

 vators, etc.) of this enzyme are rather vague. The histo- 

 chemical method to be described has been worked out by 

 the method of trial and error only. Typical pictures are ob- 

 tained only in the pH range between 5 and 6; in the alkaline 

 range, chloroaniHdophosphonic acid as a substrate gives dis- 

 tribution patterns indistinguishable from the regular alkaline 

 phosphatase reaction. Takamatsu and Sho^^ have used phos- 

 phocreatine as a substrate at an alkaline pH; the description 

 of the results obtained is not clear enough to permit the 

 drawing of conclusions. 



Method^^ 



Fixation in acetone or alcohol. Sections should not be 

 coated with collodion. 



Dissolve 1 g. p-chloroanilidophosphonic acid^^ in an ice- 

 cold mixture of 1 ml. of concentrated ammonia and 40 ml. 

 of distilled water with vigorous shaking. The substance usu- 

 ally does not dissolve completely. Filter. Add 2 drops of a 

 0.5 per cent phenolphthalein solution. Titrate back with M 

 acetic acid to a barely perceptible pink shade (about 3 ml. 

 of acid will be required). Fill up to 50 ml. with distilled 

 water. Keep in the icebox. For use, add 0.8-1 ml. of a 5 per 



80. Rorig, K.: J. Am. Chem. Soc, 71:3561, 1949. 



81. Otto, P.: Ber. d. deutsch. chem. Gesellsch., 28:616, 1895. 



82. Takamatsu, H., and Sho, E.: Tr. Soc. Path. Jap., 32:90, 1942. 



83. Gomori, G.: Proc. Soc. Exper. Biol. & Med., 69:407, 1948. 



84. Obtainable from Dajac Laboratories, 511 Lancaster Ave., Leominster^ 

 Mass. 



