Appefidix: analytical methods 167 



the use of sodium hydroxide to adjust the pH of the extract. It has 

 been reported that under such conditions inorganic phosphate and 

 adenosine triphosphate are incompletely precipitated by barium 

 acetate (Kosterhtz and Ritchie, 1943; Ennor and Stocken, 1945), 

 the precipitation being more complete either at a pH higher than 

 8-2-8-4 or if potassium hydroxide was used to adjust the pH 

 instead of sodium hydroxide. Although it has been considered that 

 the barium fractionation method leaves appreciable quantities of 

 adenosine triphosphate and inorganic phosphate in solution 

 (Stone, 1948; Gurdjian et al., 1947) it has been found that the 

 adjustment of the pH with potassium hydroxide and the addition 

 of a large excess of barium acetate results in a quantitative precipi- 

 tation of adenosine triphosphate (cf. Ennor and Stocken, 1948). 

 With small quantities of tissue (100-400 mg wet wt.) the precipita- 

 tion of inorganic phosphate is incomplete, some 5-10% being 

 carried over into the phosphocreatine fraction. It is now well 

 recognized that removal of inorganic phosphate by precipitation as 

 the magnesium ammonium complex is incomplete and that the 

 precipitate absorbs up to 40% of the adenine nucleotides present 

 (Friedkin and Lehninger, 1949; Abood and Gerard, 1952). Also, 

 since both the calcium and barium procedures do not precipitate 

 all the inorganic phosphate, use of these techniques in the deter- 

 mination of radioactivity of phosphocreatine is likely to lead to high 

 and variable results. 



Nucleotides may be quantitatively precipitated from extracts 

 made in trichloracetic acid, as the mercur}^ salts (Kerr, 1942). The 

 precipitate also contains nucleosides and free purines. Nucleotides 

 alone are precipitated with uranyl acetate (Kerr and Seraiderian, 

 1945«, b), purines and nucleosides being separated from the 

 supernatant solution by precipitation of the silver salts under acid 

 and alkaline conditions. The procedure has been adapted for use 

 with 200 mg of cerebral tissues (Thomas, 1957). It has not been 

 examined from the point of view of precipitation of nucleotides 

 other than those of adenine and guanine, but seems capable of 

 development for the examination of nucleotides in systems meta- 

 bolizing radioactive phosphate since inorganic phosphate is either 

 not precipitated or the traces present can be readily removed. 

 Whatever method of fractionation is selected it is important to 

 keep to the concentrations of reactants and the tissue/extractant 

 ratios recommended by the original authors. The methods are not 



