562 G. SCHMIDT 



the bonds between a 5'-pyrimidine phosphoryl group and a 3'-hydroxj^ 

 group of the adjacent purine or pyrimidine nucleotide group are hydro- 

 lyzed. Consequently, after exhaustive incubation with ribonuclease, all 

 3 '-phosphoryl groups attached to pyrimidine nucleoside groups are present 

 in the form of secondarj^ phosphoryls. This observation^ — ^in addition to 

 kinetic observations^ — ^supports the conclusion that the incomplete hydrolj'- 

 sis of the internucleotide bonds by ribonuclease is not due to inhibitory 

 influences on the enzyme, but to its sharply defined specificity toward the 

 internucleotide bonds as defined in this chapter. The products obtained 

 after exhaustive digestion of ribonucleic acids with ribonuclease are 3'- 

 uridylic acid, 3'-cytidylic acid, and a considerable number of oligonucleo- 

 tides of various degrees of polymerization. These oligonucleotides were 

 designated as limit polynucleotides by Schmidt ei al}^ in analogy to the 

 terminology used for the end-products of amylase action on polysaccha- 

 rides. The simpler components of the complex mixture of the limit poly- 

 nucleotides of low molecular weights were successfully separated by Volkin 

 and Cohn^^ on ion-exchange columns and by Markham and Smith^^ by 

 ionophoresis. They are unbranched di-, tri-, and tetranucleotides of differ- 

 ent compositions, but all have in common one structural property: each 

 of these oligonucleotides contains one pyrimidine nucleotide group per 

 molecule. The pyrimidine nucleotide group is always terminal and carries 

 the secondary phosphoryl group of the chain on its 3 '-carbon atom. Be- 

 tween 30 and 49 % of the purines of yeast PN A and between 45 and 50 % 

 of those of liver PNA are found as oligonucleotides containing four or 

 fewer mononucleotide groups after exhaustive digestion with ribonuclease. 

 The remainder are present in the form of polj^nucleotides of higher order 

 which have not been separated as yet. 



A considerable proportion of these oligonucleotides, corresponding to 15 

 to 25 % of the total phosphorus of the substrate, is not dialyzable against 

 water. Until recently this fraction was considered to consist of polynucleo- 

 tides of relatively high molecular weight which have been termed "cores" 

 or "limit nucleic acids" by Zamenhof and Chargaff.^^ Markham and 

 Smith^* showed recently, however, that the diffusibility of oligonucleotides 

 through dialysis membranes was greatly enhanced by the presence of 

 sufficient concentrations of sodium chloride in the solution. 



The description of the internucleotide bonds hydrolyzed by ribonuclease 

 implies that the degree of polymerization is not important for the action 

 of this enzyme.-" This conclusion is borne out by the observations of Merri- 



" E. Volkin and W. E. Cohn, J. Biol. Chem. 205, 767 (1953). 



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



»' S. Zamenhof and E. Chargaff, /. Bwl. Chem. 178, 531 (1949). 



" B. Magasanik and E. Chargaff, Biochim. et Biophys. Acta 7, 396 (1951). 



