194 MANUAL OF MICROBIOLOGICAL METHODS 



1950) and two-dimensional chromatography (Bandurski and Axelrod, 

 1951). Most of the procedures described to date, however, leave room 

 for improvement, and no single, universally satisfactory technic is known. 

 Studies of phosphorus metabolism are now being pursued in which only a 

 few biochemical steps are involved, employing dried cells or enzyme 

 preparations, so that complexity of analysis is greatly reduced. 



The uptake or appearance of inorganic phosphorus is readily measured 

 by the method of Fiske and Subbarow (1925). The usual investigation 

 leads from this type of observation to the finding that a large number of 

 phosphorylated compounds, largely carbohydrate derivatives, are formed 

 or broken down during cellular metabolism. Thus, the amount of 

 inorganic phosphorus released by hydrolysis in A^ HCl at 100°C in 7 min 

 (A7 P) from such compounds as adenosine di- and tri-phosphate, glucose- 

 1-phosphate, etc., is measured and reflects the presence and quantity of 

 such phosphorylated compounds. Similarly, hydrolysis under the same 

 conditions for 180 min helps to characterize other phosphorylated com- 

 pounds: phosphopyruvate, triose phosphate, etc., but care must be exer- 

 cised in drawing conclusions from these results, since partial phosphorus 

 liberation occurs even during shorter periods of hydrolysis and other com- 

 pounds may be involved. Therefore, analysis is also made for the non- 

 phosphorus moiety of the compound: pentose, glucose, fructose, etc. 

 Many of the AT P compounds are referred to as ''high-energy phosphate'^ 

 compounds, since, on hydrolysis, 10,000-15,000 cal are liberated com- 

 pared with the 2,000-4,000 cal released upon hydrolysis of the more stable 

 phosphate esters. 



Lipmann and Tuttle (1945) have described a specific method for acyl 

 phosphate to form the corresponding hydroxamic acid; addition of ferric 

 ions results in the formation of a purple complex which is a measure of the 

 acyl phosphate present. Another type of phosphorylated intermediate 

 which is readily measurable includes the ''alkali-labile" triose phosphates, 

 glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Inor- 

 ganic phosphorus is released from these compounds when placed in 

 N NaOH or KOH for 20 min at room temperature (Meyerhof and 

 Lohmann, 1934) and is measured as increase in inorganic phosphorus. 

 Fresh alkali must be used, since silicates, originating from the walls of the 

 glass container, analyze as inorganic phosphorus. 



Mention should be made of the commercial availability of many phos- 

 phorylated compounds of biochemical importance. Such preparations, 

 albeit of stated limited purity in many cases, are useful in metabolic 

 studies as substrates as well as standards for analytical procedures. A 

 useful recent compilation of the role of phosphorus in the metabolism of 

 organisms has appeared (McElroy and Glass, 1951, 1952), including 

 references to many analytical procedures. 



