ELECTROPHORETIC SEPARATION OF NUCLEIC ACID COMPONENTS 279 



Deimel and Maiirer-* have described an electrophoretic method for the 

 separation of FN A and DNA fractions which is based on the Schmidt and 

 Thannhaiiser procedure.-^ After hydrolysis in KOH the solution containing 

 ribonucleotides and DNA is run on paper electrophoresis at pH 8.6, when 

 the ribonucleotides move together and more slowly than DNA. Inorganic 

 phosphate runs just ahead of the DNA. The extent to which these fractions 

 may be contaminated with other phosphorus compounds is not clear. 



3. Isolation of Nucleotides and Polynucleotides from Enzymic or 

 Partial Chemical Hydrolysates of Nucleic Acids 



The paper electrophoretic technique is perhaps shown to best advantage 

 in the isolation of the products of the partial breakdown of nucleic acids 

 by enzymic or chemical hydrolysis. 



A mixture of polynucleotides such as that found in ribonuclease digests 

 contains 30 or more different substances whose direct separation by electro- 

 phoresis would be impossible. In practice complex mixtures of this type 

 are first fractionated by nnining chromatographically on Whatman No. 3 

 mm. paper in 70% isopropanol-water (vol. /vol.) with NH3 in the vapor 

 phase (solvent 1). This separates the substances produced in the incomplete 

 ribonuclease digestion of PNA into at least 6 bands (which for convenience 

 are numbered 1 to 6 in order of increasing Rp value). ^' ^°' " Each of these 

 bands contains a relatively small number of substances, many of which 

 may be isolated by a single electrophoretic run. 



a. Cijclic Ribonucleotides 



The cyclic ribonucleotides (nucleoside-2',3'-monohydrogen phosphates) 

 are intermediates in the acid and alkali breakdown of PNA [Cf. Brown and 

 Todd, Chapter 12, and Baddiley, Chapter 4] and may be isolated in small 

 yields from very weak alkaline hydrolysates of PNA''^ (e-g-, 1 hr. at 100° 

 in water over solid BaCOs). During the course of ribonuclease digestion 

 the cyclic pyrimidine nucleotides are formed as intermediates in the lib- 

 eration of the pjTimidine mononucleotides,^' " while complete ribonuclease 

 digests contain very small amounts of cyclic purine nucleotides, originating 

 from end groups of the nucleic acid." 



As they lack a secondary phosphate group, the cyclic nucleotides move 

 faster than polynucleotides or other mononucleotides on chromatograms 

 run in solvent 1. G! moves just below the cyclic dinucleotides such as AC!, 

 etc. (band 5a), and A!, C !, and U! move together with a higher Rf value 

 (band 6). (The symbol ! is here used to denote a 2',3'-monohydrogen 

 phosphate group.) These bands to some extent overlap with those of the 



2" M. Deimel and W. Maurer, Nalurwissenschaften 39, 489 (1952). 

 " R. Markham and J. D. Smith, Biochem. J. 52, 565 (1952). 



