292 ZACHARIAS DISCHE 



idly and the maximum is reached only after about 6 hours. At this moment 

 the molar extinction coefficient corresponds to about 120% of that of DNA. 

 Deoxycy tidy lie acid, on the other hand, does not show any color in the 

 reaction. The reaction of thymidylic acid, however, is only so pronounced 

 when free thymidylic acid is present. Thymidylic acid bound in DNA does 

 not react at all in the cysteine reaction in its original form, as after the 

 destruction of the purine-bound sugar in the apurinic acid by 0.2 A^ alkali, 

 the remaining pyrimidine nucleotides do not show any appreciable color 

 even after hours. While the purine nucleosides react in the Stumpf mod- 

 ification \vith the same intensity as the corresponding nucleotides, this is 

 not the case with the pyrimidine nucleosides. While the molar extinction 

 coefficient of thymidylic acid is reported to be 26 % lower than that of the 

 purine nucleotides, these extinction coefficients are identical for thymidine 

 and the purine nucleosides. Cytidine, on the other hand, shows a much 

 lower color than thymidine and the maximum is reached only after 20 

 hours at room temperature.^^ It must be furthermore noted that the ab- 

 sorption spectrum of the pink color in both modifications of the cysteine 

 reaction is not completely identical in the case of thymidylic acid in DNA, 

 insofar as the absorption curve for the first one is somewhat steeper towards 

 the lower wavelengths than that for the second one. 



According to our own experiments, the reason for this difference in the behavior of 

 the free and bound thymidylic acid is due to the fact that the latter in polynucleotide 

 linkage always reacts in the cysteine reaction like thymidine-3',5'-diphosphate. This 

 difference is particularly striking when the reaction is carried out according to the 

 original procedure with the more dilute acid. While under these circumstances the 

 purine nucleotides give the maximum color alreadj^ 60 minutes after the termination 

 of heating, it takes 24 hours for thymidylic acid to reach the maximum of color; and 

 thymidine diphosphate, isolated from DNA^'* or prepared synthetically, requires 

 more than 72 hours to reach the maximum. When the purine-bound sugar of DNA is 

 destroyed by alkali after being previously split from the purine by mild acid hydroly- 

 sis, the remaining pyrimidine nucleoside diphosphates react in the original form of 

 the cysteine reaction in such a way that the color developed during the first 48 hours 

 at room temperature corresponds to the amount of thymidine diphosphate present 

 in the DNA preparation. This shows that the cytidine diphosphate does not signifi- 

 cantly react, at least during the first 48 hours. With increasing concentrations of 

 thymidine diphosphate the intensity of the color increases disproportionately. Never- 

 theless, it is possible to determine quantitatively the concentration of thymidine 

 diphosphate in a DNA preparation with the cysteine reaction. To this end, the purine 

 sugar is destroyed by 2 minutes' heating at 100° with A' 112804 then adjusting the 

 neutralized hydrolysate to 0.2 A'' NaOH and again heating for 2 minutes. The cysteine 

 reaction is carried out on this preparation simultaneousl}' with a series of standards 

 prepared from a DNA of known pyrimidine composition, the concentration of the 

 standards to differ by no more than 10% from each other. The concentration of thymi- 



'!» The author is greatly indebted to Professor A. R. Todd, University of Cambridge, 

 and to the Biochemical Research Foundation, California, for the preparations of 

 thymidine diphosphate used in these experiments. 



