34. THE RIBONUCLEIC ACIDS OF VIRUSES 259 



TMV hydrolyzate which had been treated in an analogous fashion. From 

 the quantity of RNA employed in this experiment, it was possible to 

 calculate a protein content of 0.25 %. 58 



The serological test for native TMV protein is significantly more sensi- 

 tive than the color reactions and chemical methods for protein determina- 

 tion. An impurity amounting to 0.02% protein may be detected using 

 TMV antiserum, and this type of test showed that the content of native 

 TMV protein in the RNA preparations was less than 0.02 %. 3 



(ii) Protein content of detergent-extracted RNA. Fraenkel-Conrat et al. 4 

 found an upper limit of protein contamination of 0.5 % for detergent-ex- 

 tracted RNA using the microbiuret test of Holden and Pirie. 62 After hy- 

 drolysis of RNA preparations, a protein content of approximately 0.06 % 

 was calculated from the amino acid content. The fact that those amino 

 acids which make up a large percentage of the virus protein were not found 

 in this analysis led to the conclusion that these trace amounts of amino 

 acids are not in the form of viral protein. Protein determination using the 

 phenol reagent 63 gave an estimate of 0.04-0.4 %. 64 



These analytical studies show that the infectivity of the RNA prepara- 

 tions must clearly be due to the RNA alone, and the presence of viral pro- 

 tein can be definitively excluded. The presence of small amounts of amino 

 acids or peptides cannot be excluded. 



(5) Other Constituents. The diphenylamine test shows that there is less 

 than 0.1 % DNA in preparations of infectious RNA. It is safe to say that 

 there is no contamination with DNA since a microbiological analysis of 

 thymidine shows that the DNA content is less than 0.001 %. 65 



Loring et al. 66 ' 67 studied the metal content of virus and virus RNA 

 preparations. Whereas the content of Cu, Ca, and Mg ions varied, accord- 

 ing to the methods of purification, the concentration of iron remained 

 relatively constant, amounting to 8 mg. per 100 gm. of phenol-extracted 

 RNA, and to 1-2 mg. per 100 gm. of detergent-extracted RNA. It was 

 suggested that metal chelates or relatively stable metal complexes appear 

 in the RNA. The significance of metal ions for RNA infectivity was not 

 studied. 



62 M. Holden and N. W. Pirie, Biochem. J. 60, 46 (1955). 



63 O. H. Lowry, N. J. Rosebrough, A. L. Fair, and R. J. Randall, J. Biol. Chew. 193, 

 265 (1951). 



64 L. K. Ramachandran and H. Fraenkel-Conrat, Arch. Biochem. Biophys. 74, 224 

 (1958). 



66 H. Fraenkel-Conrat and B. Singer, Jfth Intern. Congr. Biochem., Vienna, 1958 

 Symposium No. VII (1958). 



66 H. S. Loring and R. S. Waritz, Science 125, 646 (1957). 



67 H. S. Loring, S. Al-Rawi, and Y. Fujimoto, J.Biol. Chem. 233, 1415 (1958). 



