242 



NUCLEIC ACIDS AND RELATED COMPOUNDS 



regenerated by removing mercury with hydrogen sulfide. Ion exchange chromatography 

 may be carried out using either anion or cation exchangers since at very high pH values 

 most of the bases act as weak anions. The methylated xanthines which occur in such com- 

 mercially important plants as coffee and cocoa may be extracted with such organic sol- 

 vents as ethanol, chloroform ortrichloroethylene and chromatographed on silicic acid col- 

 umns (21). Tannins, which tend to complex with them, may be removed by adsorption on 

 magnesium oxide. Since some of these compounds may be classified as alkaloids, meth- 

 ods discussed in Chapter 12 may be consulted. 



CHARACTERIZATION 



The discussion of characterization for most plant constituents has been directed 

 toward deciding whether or not they are present in a given tissue. No such question 

 arises for the nucleic acids. It is a foregone conclusion that they are present, and the 

 analytical problem may involve quantitative measurement or the determination of their 

 composition in terms of the constituent nitrogen bases. Several methods have been de- 

 veloped for analysis of the nucleic acids, and probably no one of them is completely sat- 

 isfactory for plant tissues. In addition to the general references, reviews on the chemical 

 determination (15, 22, 23) and microbiological assay (24) of nucleic acid have appeared. 

 Kern (25) has discussed the problems peculiar to determining the nucleic acids of green 

 leaves. All methods depend on at least a preliminary purification as described under 

 "Isolation." The next step requires separation of the two types of nucleic acid or else a 

 way of determining pentose and deoxypentose in the presence of each other. A proper 

 discussion of these quantitative procedures cannot be given here. They may be found in 

 the general references and (18). 



For determination of the bases present in a nucleic acid sample acid hydrolysis fol- 

 lowed by paper chromatography is quite satisfactory. If it is desired to separate larger 

 quantities of the bases for additional characterization ion exchange chromatography may 

 be applied to the hydrolysate. RNA is hydrolyzed with normal hydrochloric acid for one 

 hour at 100° C. This yields free purines (adenine and guanine), but the pyrimidine -sugar 

 bond is more resistant so that cytidylic and uridylic acids are formed rather than the free 

 bases. DNA is hydrolyzed with 72% perchloric acid at 100° for 2 hours and yields free 

 bases for both purines and pyrimidines. 



For characterization of the mixture of hydrolysis products from nucleic acid by 

 paper chromatography a spot of acidic hydrolysis mixture is applied directly to paper and 

 run in a solvent composed af 170 ml. z'so-propyl alcohol, 41 ml. concentrated hydrochloric 

 acid, and water to make 250 ml. (26). After drying the paper, spots may be detected by 

 the fact that they absorb ultra-violet light and therefore appear as dark spots against 

 background fluorescence when a U. V. lamp (250-265 mfx) is held behind the paper. 

 Guanine fluoresces in ultra-violet if the paper is strongly acidic. Other nitrogen bases 

 do not. Photographic methods have been developed to provide a permanent record (27). 

 Spray reagents have also been developed with varying specificities. The method of Wade 

 and Morgan (28) detects phosphate esters (i. e. nucleotides). It is described in Chapter 

 2. Wood's method (29) detects most purines and cytosine but not uracil, thymine or cyti- 

 dylic acid. A method specific for adenine derivatives has been described by Gerlack and 

 DOring (30). Paper chromatography and ion exchange chromatography of oligonucleotides 

 are reviewed by Cramer (31). 



Thin layer chromatography on cellulose powder with a plaster of Paris binder is 

 claimed (32) to offer many advantages over paper chromatography for separation of nu- 

 cleotides, nucleosides, purines, and pyrimidines. The method is much faster than chro- 

 matography on paper, and better resolution is attained. 



