130 NITROGEN METABOLISM 



It is therefore natural that spectrophotometric techniques 

 should have been developed for the quantitative estimation 

 of the free bases and the various substances in which they 

 are constituents. The total nucleic acid content of organisms 

 spread in a film on a slide can be determined by ultraviolet 

 spectrophotometric microscopy [10], whilst for cell suspen- 

 sions, there is a technically simpler procedure based on the 

 use of a standard ultraviolet spectrophotometer [40]. By 

 making various assumptions, an average value for the ab- 

 sorption coefficient of a typical nucleic acid can be deduced 

 and thus the absorption measurements interpreted in terms 

 of nucleic acid. The originators of these techniques are well 

 aware that reliable results are only obtained if due account is 

 taken of a large number of variables and that many of the 

 basic premises may not be strictly valid [cf. 10, 40]. Never- 

 theless, even if the results are not entirely accurate in terms 

 of precise quantitative values, they are extremely useful, 

 particularly when the data are used in a comparative manner. 

 The identification and quantitative determination of the 

 various bases and nucleotides present in a nucleic acid 

 necessarily involves hydrolysis of the nucleic acid, and 

 chemical hydrolysis without degradation of one or more of 

 the nitrogenous bases presents some difficulty. Depending 

 on the conditions employed, the products are nucleotides, 

 nucleosides, the free bases or mixtures of these substances. 

 Thus subjecting a PNA to mildly acidic conditions, e.g. 

 N.-HCl at 100° C. for i hour, liberates the purines in the 

 free state, but the pyrimidines still remain in nucleotide 

 combination from which they are freed by more vigorous 

 hydrolysis, e.g. formic acid at 175° C. [see 25]. On the other 

 hand, alkaline hydrolysis of a PNA yields a mixture of the 

 four nucleotides. The deoxypentose nucleic acids behave 

 differently on chemical hydrolysis, and degradation to 

 nucleosides and nucleotides is best achieved by the use of 

 enzymes. In the past, separation of the end-products ob- 

 tained by hydrolysis or enzymic digestion of nucleic acid 

 was accomplished by precipitation as the phosphotungstate 

 or as the uranium or silver salt, or by simply adjusting the 

 pH of the system. More recently, ionophoresis on paper [38] 



