274 HEINZ SCHUSTER 



constant is independent of the pH in the range pH 4-8. Groups having 

 pK values lower than 4 or higher than 8, as is the case with the amino 

 groups or the purine and pyrimidine rings of the nucleic acid, must be 

 responsible for the inactivation with formaldehyde. 97 



If TMV-RNA is treated with 1-2 % formaldehyde at pH 6.8, the UV 

 absorption maximum shows a gradual increase up to 30 % as well as a shift 

 of 3-5 nux towards higher wavelengths. 98 Similar effects were observed 

 with all the RNA derivatives which contain NH 2 groups. Schiff's base 

 formation ( — N=CH2) appears to be the most probable reaction in view 

 of the increased absorption and the lability of the product. After treatment 

 with formaldehyde, native DNA did not show this spectral change. 



The nature of the formaldehyde reaction with RNA was studied in more detail by 

 Staehelin" using C 14 -labeled formaldehyde. He found that isolated RNA is much 

 more sensitive to formaldehyde than intact virus is. The amount of formaldehyde 

 bound at half inactivation is about 400 molecules per intact virus particle and 20 

 molecules per RNA. When the reaction with RNA has reached completion, 50-60 

 molecules of formaldehyde are bound per 100 nucleotides having 71 amino groups. 

 At higher salt concentrations, the formaldehyde binding at RNA is greatly decreased. 

 Since the rate of reaction of adenylic acid with formaldehyde is independent of the 

 salt concentration, the decrease in the reaction rate of formaldehyde with RNA at 

 higher salt concentrations must be due to the aggregation of RNA molecules which 

 results in a decrease of availability of NH 2 groups. Reaction of formaldehyde with 

 RNA proceeds in two steps. The first step leads to a more labile form of binding, 

 whereas most of the formaldehyde becomes firmly bound after a more extensive reac- 

 tion. 



Glyoxal and Kethoxal. Glyoxal and related compounds were found to be 

 antiviral agents. 100 Staehelin 101 observed that glyoxal and Kethoxal (/3-ethoxy- 

 a-ketobutyraldehyde) inactivate TMV as well as its infectious RNA. For 

 equivalent molarities of formaldehyde and Kethoxal, TMV is inactivated 

 more rapidly by formaldehyde than by Kethoxal, whereas the situation is 

 reversed for RNA. These differences may be understood if it is considered 

 that formaldehyde is a relatively small molecule so that the nucleic acid in 

 the virus might be more readily accessible to it than to a larger molecule 

 like Kethoxal. Under conditions which lead to approximately 50 % inactiva- 

 tion of isolated RNA, only 10-15 molecules of Kethoxal are bound by one 

 molecule of RNA of molecular weight 2 X 10 6 , corresponding to the amount 

 of formaldehyde which is bound per RNA molecule for the same degree of 

 inactivation. 



97 T. E. Cartwright, A. E. Ritchie, and M. Lauffer, Virology 2, 689 (1956). 



98 H. Fraenkel-Conrat, Biochim. et Biophys. Acta 15, 307 (1955). 



99 M. Staehelin, Biochim. et Biophys. Acta 29, 410 (1958). 



100 G. E. Underwood and S. D. Weed, Proc. Soc. Exptl. Biol. Med. 93, 421 (1956). 



101 M. Staehelin, Biochim. et Biophys. Acta 31, 448 (1959). 



