290 ZACHARIAS DISCHE 



speed of the development of the blue color in the diphenylamine reaction, 

 even at concentrations corresponding to half of the concentration of DNA. 

 This effect, however, disappears almost completely when the heating is 

 continued until the maximum of the color is reached. Cohen^^ found pre- 

 viously that the nucleoprotein from calf thymus and the corresponding 

 nucleic acid, show identical color intensity when solutions of the two sub- 

 stances contained identical amounts of organic phosphorus.^®* 



Detection of DNA and its constituents by dichromatic readings: — When substances 

 which produce colored products with the diphenylamine reagent are present in solu- 

 tion and obscure the reaction of 2-deoxyribose, it is often, accordingto our experi- 

 ments, possible to detect the presence of the latter by measuring the optical density 

 of the unknown at two different wavelengths. When substances which produce a green 

 color are present, the difference of the density at 595 and 650 m/x will in general be 

 either zero or negative for these substances, while it is positive for 2-deoxyribose. If, 

 therefore, the amount of the latter substance is not too high, a positive Dm — Duo 

 will be obtained even when the blue color is no more clearly recognizable. In the case 

 of acetaldehyde, which produces a brown color, Dm — Deso is almost zero^". In the 

 case of substances which yield purple or yellow colors, the difference of the densities 

 at 595 m/x and lower wavelengths may also be used to advantage for the detection of 

 2-deo.xypentose.i^ 



Quantitative determination of DNA and its constituents: — Dsgs , or its equivalent 

 with photoelectric instruments using filters, is proportional to the concentration of 

 DNA. In general, however, it can be recommended to use Dsgs — i^eso which, as was 

 found in our laboratory, is also proportional to the concentration of DNA as a meas- 

 ure of this concentration. Although this difference is about 40% smaller than Dm , 

 the agreement between duplicate samples was found to be better than when Dggs is 

 used as a measure of the concentration. In mixtures of DNA and its breakdown prod- 

 ucts formed by the liberation of bases, the DNA can be determined by carrying out 

 first the reaction on the tissue extract and then bringing the latter to an alkalinity of 

 about O.l to 0.2 A'^, heating for 2 minutes at 100°, and repeating the determination. 

 The free sugar of the breakdown product, as was found in our laboratory, is com- 

 pletely destroyed by this procedure, while DNA is not affected. The difference be- 

 tween the two determinations gives then the amount of DNA. By the use of a spectro- 

 photometer as little as 50 ^g. of DNA can be determined fairly accurately. Steele 

 et al.^^ described a micromethod which permits the determination of as little as 1.6 

 tig. of DNA by the diphenylamine reaction. 



6. Reactions of 2-deoxyribose, DNA and its nucleotides with cysteine and 

 sulfuric acid 



2-Deoxyribose gives two completely different reactions when treated 

 with sulfuric acid in the presence of cysteine, depending upon the con- 



1" S. S. Cohen, J. Biol. Chem. 167, 691 (1945). 



'** H. V. Euler and L. Hahn reported {Arch, neerl. physiol. 28, 398, 1946) that certain 

 proteins increase the intensity of the diphenylamine reaction when present in 

 amounts corresponding to twice to three times that of DNA. G. Bergold (Z. Nat- 

 urforsch. 36, 406, 1948) could not confirm these results. 



" E. Racker, Nature 167, 408 (1951). 



" R. Steele, S. Theodore, and L. Ottolenghi, /. Biol. Chem. 177, 231 (1949). 



