Appendix: analytical methods 165 



Fractionation of Phosphates in the Extracts 



The methods of fractionation adopted depend largely upon the 

 problem being investigated. Generally interest has concentrated 

 upon inorganic phosphate, phosphocreatine, and adenosine di- and 

 triphosphate. However, with the recognition of the presence of 

 numerous nucleotides and other phosphorylated derivatives the 

 problems of complete fractionation of the phosphates in acid extracts 



Trichloracetic acid extract ^ 



(Ca(0H)2 + ethauol to 10% (v/v)) 



Precipitate 



Solution (Add excess Ca(0H)2) 



Precipitate 

 (Contains adenosine di- and 

 triphosphates, inorganic phosphate, 

 hexose phosphates) 



Solution 

 (Add ethanol to 80% (v/v)) 



Precipitate Solution 



(Contains phosphocreatine and (Ethanol soluble phosphorus) 



unidentified phosphates) 



Fig. 18. Outline of a scheme for the fractionation of acid-soluble 

 phosphates of brain (Stone, 1943). 



have become more acute. Usually such a separation is not necessary ; 

 simple separation into groups being sufficient for most purposes. 

 The early procedures for the separation of the calcium salts of 

 the phosphates developed by Stone (1940, 1943) specifically for use 

 with extracts of cerebral tissues hav^ been widely applied by many 

 workers. The later method is illustrated in Fig. 18. The groups of 

 phosphates obtained are heterogeneous but the separation of 

 adenosine triphosphate -f inorganic phosphate from phospho- 

 creatine is good. The fraction with calcium salts soluble in 10% 

 (w/v) aqueous ethanol is complex and contains many components, 

 the total phosphorus of which exceeds in amount the phospho- 

 creatine present. It is reported (Bodian and Dzeiwiatkowski, 1951) 



