ELECTROPHORETIC SEPARATION OF NUCLEIC ACID COMPONENTS 277 



0.3 N KOH for 18 hr. at 37°, the solution brought to pH 1 with 60 % wt./wt. 

 HCIO4 , insoluble KCIO4 removed by centrifugation, and the combined 

 washings and supernatant fluid brought to pH 4. Aliquots of this solution 

 containing about 800 /xg. of ribonucleotides are mn at pH 3.5 in 0.02 M 

 citrate buffer on No. 3 mm. paper (using an apparatus similar to that 

 described by Durrum'^) for 18 hr. at 11 v./cm. The size of the paper strip 

 is 72 X 7 cm. (Actually by using the technique of electrophoresis on paper 

 immersed in CCU^ • ^'^ and with a shorter length of paper and correspond- 

 ingly higher voltage gradient this separation could be achieved in 30 min- 

 utes.) Whatman No. 1 paper may also be used for smaller quantities of 

 material (200 ng.).^ The nucleotides are located in ultraviolet light at 

 260 m/z, either visually^" or photographically/*'^' eluted, and estimated 

 spectrophotometrically and by phosphorus determinations. The phosphorus 

 recovery from runs of individual nucleotides is 96-99%. The PNA phos- 

 phorus recovered after alkaline hydrolysis and precipitation of KCIO4 is 

 about 97%, while that recovered as nucleotide P after electrophoresis is 

 94 % of that in the original PNA. Some of this 6 % loss is probably accounted 

 for by losses during the manipulations, but part has been shown to be due 

 to the presence of small amounts of non-nucleotide P in the PNA speci- 

 mens. 



A similar method may be used for the determination of the composition 

 of deoxyribonucleic acids (DNA) by the electrophoretic separation of the 

 mononucleotides produced on successive hydrolysis with deoxyribonuclease 

 and purified snake venom diesterase. Deoxycytidylic and deoxy-5-methyl- 

 cytidylic acids, however, are not separated in the electrophoretic nm. 



Matthews^^ has used paper electrophoresis in borate buffer pH 9 to sep- 

 arate 8-azaguanylic acid from alkaline hydrolysates of the PNA of tobacco 

 mosaic virus isolated from plants treated with 8-azaguanine. The pK of 

 the enol (OH) group of 8-azaguanine is about 3 pH units lower than that 

 of guanine, so that at pH 9 8-azaguanylic acid has a higher mobility than 

 any other mononucleotide. 



2. Separation of Nucleotides from Other Phosphorus Compounds 



A considerable part of the work on the incorporation of the radioactive 

 isotope P^^ into PNA has been based on the assumption that the ribonucleo- 

 tide fraction obtained by the Schmidt and Thannhauser procedure^^ con- 

 tains only PNA phosphorus. 



By separating this ribonucleotide fraction through paper electrophoresis 

 Davidson and Smellie'' have shown that only about 75% of the P can be 

 accounted for as nucleotides, there being present at least six additional P 



22 R. E. F. Matthews, Nature 171, 1065 (1953). 



2» G. Schmidt and S. J. Thannhauser, J. Biol. Chem. 161, 83 (1945). 



