566 G. SCHMIDT 



Heppel and Whitfeld,^^ ■^*' who found that ribonuclease catalyzes the en- 

 zymic synthesis of benzyl- and of methylcytidylic acid from cyclic 2',3'- 

 cytidylic acid and the corresponding alcohols (which must be present in 

 excess). Similarly, the incubation of a mixture of cyclic 2',3'-cytidylic 

 acid with cytidine results in the formation of the nucleotide-nucleoside-P- 

 diester cytidylyl-cytidine,^" that of cyclic uridylic acid and cytidine in the 

 formation of uridylyl-cytidine. The observed transesterification reactions 

 catalyzed by ribonuclease are not limited to the cyclic nucleotides as 

 substrates, but occur between acyclic P diesters of pyrimidine-3 '-nucleo- 

 tides and other alcohols: for example, the incubation of a mixture of P- 

 methyl-3'-cytidylic acid with cytidine results in the formation of cytidylyl- 

 cytidine and methyl alcohol. Finally, the formation of oligonucleotides by 

 enzymic exchange reactions of cyclic nucleotides with pyrimidine-3'- 

 nucleotides were reported by Heppel et al.^^^ 



The possible occurrence of such exchange reactions must of course be 

 carefully considered in the interpretation of experiments carried out with 

 ribonuclease as a hydrolyzing agent in investigations concerning the 

 structure of PNA. 



Principles of Assay of Ribonuclease. (a) Formation of acid-soluble degrada- 

 tion products of PNA.^^-^"- The mononucleotides and many oligonucleotides 

 are soluble at pH values of 2, whereas PNA is precipitated under these con- 

 ditions. Hydrochloric (0.5 A^), sulfuric, perchloric acids or glacial acetic acid 

 have been used for such partitions. The precipitation of undigested nucleic 

 acid in easily filtrable form is facilitated by the use of alcohol-containing 

 acid solutions.^^ The fine dispersion of nucleic acid precipitates obtained in 

 very dilute substrate solutions with acjueous acids has been used as a 

 principle for a turbidimetric assay method for ribonuclease.^* The acid- 

 soluble hydrolysis products can be determined in the filtrates by phos- 

 phorus determination or by ultraviolet spectrophotometry. Roth and 

 Milstein^' used P'--labeled PNA as substrate and determined the radio- 

 activity of the filtrates obtained after incubation with ribonuclease-con- 

 taining tissue extracts. 



A very suitable reagent for the assay of ribonuclease by determination 

 of its acid-soluble degradation products is 1.5% uranyl chloride in 10% of 

 trichloroacetic acid.^^ According to MacFadyen,'^ this reagent precipitates 



29 L. A. Heppel and P. R. Whitfeld, Biochem. J., 56, Proc. ii (1954). 



30 L. A. Heppel, R. Markham, and R. J. Hilmoe, Nature 171, 1151 (1953). 



'"* L. A. Heppel, P. R. Whitfeld, and R. Markham, Abstr. 126th Meeting Am. Chem. 



Soc, New York p. 52c (1954). 

 3' C. E. Carter and J. P. Greenstein, J. Natl. Cancer Inst. 1, 29 (1946). 

 ^'^ A. Cantero, R. Daoust, and G. de Lamirande, Science, 112, 221 (1950). 

 " J. S. Roth and S. W. Milstein, J. Biol. Chem. 196, 489 (1952). 

 " M. McCarty, J. Expll. Med. 88, 181 (1948). 

 36 D. A. MacFadyen, /. Biol. Chem. 107, 297 (1934). 



