166 



Appendix: analytical methods 



that the calcium fractionation procedure completely separates 

 radioactive orthophosphate from the fraction containing phospho- 

 creatine. Successful adaptations of this technique for the analysis 

 of phosphocreatine and inorganic phosphate in small quantities 

 (100-200 mg) of tissue have been described (Mcllwain, 1951; 

 Kratzing and Narayanaswami, 1953). 



An alternative fractionation procedure based upon the differing 

 solubility of the barium salts of the phosphates was developed by 

 Le Page (1948, 1957). The essential features are summarized in 

 Fig. 19. Certain points need careful attention. The amount of 

 barium acetate added needs defining for each system studied 

 particularly where the quantities and concentrations of the phos- 

 phates differ from those used by Le Page. A second point concerns 



Trichloracetic acid extract 



Adjust to pH 8-4, 

 add excess barium acetate 



Precipitate 



(Take up in CI iV HCl 



and reprecipitate) 



Solution 



(Add ethanol 

 to 75% v/v 

 Adjust to pH 8-4) 



Precipitate 



(Contains adenosine di- and 



triphosphates, hexose diphosphate,. 



3-phosphoglyceric acid) 



Precipitate Solution 



(Contains phosphocreatine, (Contains propane diol 



hexose monophosphates, phosphate, phosphorylethanol- 



di- and triphosphopyridine nucleotide, amine) 



adenylic acid, triose phosphates) 



Fig. 19. Outline of a scheme for the fractionation of acid-soluble 

 phosphates of brain (Le Page, 1957). 



