ELECTROPHORETIC SEPARATION OF NUCLEIC ACID COMPONENTS 271 



constant"' ^^ adenylic acid would appear to have an effective axial ratio 

 of about 5:1. The axial ratio of the dinucleotide must thus lie between 

 2.5:1 and 10:1. Taking the shape to be an oblate ellipsoid, and assuming 

 the resistance to motion to be the same as in translational diffusion, the 

 ratio of the frictional resistance of the dimer to that of the monomer lies 

 between the limits 1.32 and 1.85. The observed relative mobilities on 

 paper of the mono-, di-, and trinucleotides of uridylic acid (after correction 

 for movement due to flow of the buffer solution through the paper) are 

 1:1.4:1.5 at pH 3.5. ^"^ At this pH the net charges on the mono-, di-, and 

 triuridylic acids are 1, 2, and 3 so that if the frictional resistance to motion 

 of a mononucleotide is taken as 1, that of a dinucleotide is 1.43 and of a 

 trinucleotide 2.0. 



Using these figures and the dissociation cur\es, the mobilities of any 

 mono-, di-, or trinucleotide relative to uridylic acid may be calculated for 

 any pH. For example at pH 3.5 the relative mobilities of 



AG = (0.46 + 0.95)/1.43 = 0.99 



of AC = (0.46 + 0.16)/1.43 = 0.43 



and of AAU = (0.46 + 0.46 + 1.0)/2 = 0.96 



times that of uridylic acid. (Here A, G, C, and U represent adenylic, gua- 

 nylic, cytidylic, and uridylic acid residues, respectively.) It is again as- 

 sumed that the mononucleotides have approximately the same size and 

 shape, and that the dissociation constants of individual groups are the same, 

 whether in nucleotides or in polynucleotides. 



Evidently the relative mobilities of the mono-, di-, and trimers of a sin- 

 gle nucleotide form a converging series so that separation of the tetra- and 

 higher polynucleotides by this method is unlikely. 



Remarkably good agreement is found between the mobilities relative to 

 uridylic acid calculated on this basis and those observed in practice with 

 the technique of electrophoresis on paper immersed in carbon tetrachloride 

 (Section II). In Figure 2 are plotted the observed movements of 19 mono- 

 and polynucleotides in 0.05 m ammonium formate buffer pH 3.5 against 

 their calculated mobilities relative to that of uridylic acid. These data were 

 taken from preparative runs in which no particular care was taken to con- 

 trol the voltage gradient to better than ±10%. Under these conditions 

 about 25 % of the movement of uridylic acid is accounted for by flow of the 

 buffer solution due to endosmosis and incomplete saturation of the paper. 

 Table II gives the calculated relative mobilities and observed movement 

 of a number of nucleotides and polynucleotides at pH 3.5. 



" A. H. Gordon and P. Reichard, Biochem. J. 48, 569 (1951). 



»2 G. Schramm, W. Albrecht, and K. Munk, Z. Naturforsch. 7b, 10 (1952). 



