NUCLEOTIDES I29 



the hydrolysis of cyclic nucleotides which are either present 

 initially in the nucleic acid or are produced during the 

 hydrolysis procedure [38]. The products obtained by the 

 digestion of RNx\ with ribonuclease are mainly mono- and 

 di-nucleotides together with some polynucleotide material 

 which, because it w^ould not dialyse, was at first thought to 

 be the 'core' of the nucleic acid and to be of high molecular 

 weight. Markham and Smith have now shown that the 'core' 

 consists of relatively small polynucleotides whose dialysis 

 depends on the concentration of salt in the system [38]. 



Estimation of nucleic acids and their components 



The procedures for estimating total nucleic acid are based 

 on either ultraviolet spectrophotometry or on determinations 

 of orthophosphate or sugar (pentose and deoxypentose). In 

 the latter chemical methods the cells are first extracted with 

 cold trichloracetic acid (TCA) and a fat solvent to remove 

 acid-soluble compounds and phospholipoids. After the nu- 

 cleic acids have been released from the nucleoproteins 

 by heating the extracted cells with 5% TCA at 90° C. for 

 15 minutes, pentose is estimated colorimetrically by the 

 orcinol method and deoxypentose by the Dische-diphenyl- 

 amine method [48]. In the Schmidt and Thannhauser pro- 

 cedure [47] the extracted cells are treated with N.-KOH at 

 37° C. to hydrolyse the PNA to free nucleotides, and then 

 the undegraded DPNA and protein are precipitated by 

 making the hydrolysate normal to HCl. The DPNA and 

 PNA content of the original material is calculated from the 

 organic and inorganic phosphate in the hydrolysate and 

 the organic phosphate in the DPNA fraction. This method 

 assumes that the only cellular acid-insoluble phosphorus 

 compounds are nucleic acids and phosphoproteins — an 

 assumption now known to be invalid [42]. In both the sugar 

 and phosphate methods, the results are expressed in terms 

 of nucleic acid by using conversion factors based on thymus 

 DNA and yeast RNA as standards. 



A characteristic property of purines and pyrimidines is 

 that they strongly absorb in the ultraviolet region of the 

 spectrum, with a peak absorption in the region of 260 m^«. 



