294 ZACHARIAS DISCHE 



color which differs from the absorption spectrum of the coloration obtained 

 with DNA or furfuryl alcohol. The fact that furfuryl alcohol and arabinal 

 produce less color than 2-deoxyribose does not necessarily contradict this 

 assumption about the mechanism of the reaction, as both these substances 

 enter side reactions which are responsible for the additional two absorption 

 maxima, while the intermediate in the case of 2-deoxyribose could be pro- 

 duced in small quantities during a certain time interval and immediately 

 removed by the action with cysteine without being able to enter into side 

 reactions. This assumption seems to be borne out by the fact that thy- 

 midylic acid in the original form of the cysteine reaction produces even a 

 higher color intensity than equivalent amounts of purine nucleotides, as 

 in this case the slow splitting of the glycosidic linkage should still more 

 slow down the formation of the intermediate. In agreement with that, the 

 steeper slope of the absorption curve towards lower wavelengths in the 

 case of thymidylic acid also indicates that in this case the side reactions 

 which produce the peaks at 450 and 415 mn are still more suppressed than 

 in the case of purine nucleotides. 



Quantitative determination of DNA and its constituents by the cysteine reaction with 

 75 vol.% H2S0i : — The optical density at 490 m/i of the reaction mixture is proportional 

 to the concentration of DNA, purine nucleotides, and thymidylic acid in the range 

 between 25 and 500 iig. per cc. of DNA or the equivalent amount of the nucleotides in 

 both modifications of the cysteine reaction. It should be noted that, while the molar 

 extinction coefficient for DNA is lower in the original form of the reaction by about 

 one-third as compared with the Stumpf modification, it is possible to make determina- 

 tions at lower concentrations of the material as the amount of the unknown is twice 

 that used in the other form. As the pyrimidine nucleotides bound in the DNA do not 

 react at all in the reaction, any preparation of DNA can be used as standard solution 

 for its determination in tissues. In the determination of free thymidylic acid or the 

 pyrimidine nucleosides, however, standards of the substance which is to be deter- 

 mined are preferable. Small amounts of pure proteins and carbohydrates in concen- 

 trations which occur in animal tissues will not interfere with the reaction. How far 

 this is also true of plants and bacteria will have to be investigated. 



(2) Reaction of DNA with Cysteine and Concentrated HiSd y^ 



Procedure: — To 1 cc. of a solution containing 50 to 500 ng. per cc. of DNA, 4 cc. of 

 concentrated sulfuric acid (reagent grade) is added under cooling in tap water. The 

 reaction mixture is left for 1 hour at room temperature to avoid air bubble formation, 

 and then 0.1 cc. of a 3% solution of cysteine hydrochloride is added and shaken. The 

 solution is left from 20 to 48 hours at room temperature, and the absorption is then 

 measured with the Beckman spectrophotometer. 



Absorption curve and specificity of the reaction: — ^Pentoses, hexoses, hep- 

 toses, methylpentoses, and hexuronic acids form, under the conditions of 

 this reaction, furfural or the corresponding homologues which all show 



" Z. Dische, /. Biol. Chem. 181, 379 (1949). 



