COLOR REACTIONS OF NUCLEIC ACID COMPONENTS 291 



centration of the acid and temperature of the reaction. In one of these 

 reactions concentrated acid is added to the solution and all carbohydrates 

 form reaction products with characteristic absorption spectra in the ultra- 

 violet and the blue part of the spectrum. The absorption curves, however, 

 differ from one class of saccharides to another. This reaction will be desig- 

 nated, therefore, as cysteine reaction of 2-deoxyribose with concentrated 

 H2SO4 . The second reaction is much more selective and can be regarded as a 

 characteristic color reaction of 2-deoxypentose. In it an H2SO4-H2O mixture 

 containing 75 vol. of H2SO4 in 100 cc. of the mixture is added to the sugar 

 solution. 



(1) Cysteine Reaction of 2-Deoxyrihose and DNA with 75 Vol.7o H2S0^. 

 This reaction was first described in 1944'^ and recommended for quantita- 

 tive determination in white light. Later Stumpf^" modified slightly the 

 procedure and adapted it for spectrophotometric measurements. Every 

 one of these modifications offer certain advantages for special purposes, and 

 both precedures therefore are here described. 



Original procedure of Dische: — 1 cc. of a solution containing 50 to 500 ng per cc. of 

 DNA is mixed with 0.05 cc. of cysteine hydrochloride and 5 cc. of 75 vol.% sulfuric 

 acid (70 vol. of H2SO4 cone, plus 30 vol. of H2O) under cooling in ice. The reaction 

 mixture is then transferred for 5 minutes into a water bath of 40°C. and then cooled 

 in tap water. A pink color develops which after 15 minutes reaches 90% of its maxi- 

 mum, and the maximum after about 1 hour. The absorption maximum is at 490 mfi. 

 The color is stable for hours. 



Modification of Stumpf: — 0.05 cc. of cysteine hydrochloride and 5 cc. of 75 vol.% 

 sulfuric acid are added to 0.5 cc. of a solution containing 25 or more ^tg. per cc. of DNA 

 without cooling. The mixture is allowed to cool off at room temperature. The same 

 pink color with an absorption maximum at 490 m/i develops and reaches its maximum 

 after about 15 minutes. 



Specificity of the reaction: — ^The molar extinction coefficients of 2-deoxyri- 

 bose, deoxyadenylic and -guanylic acids do not differ significantly in 

 Stumpf 's modification. 20 Preparations of apurinic acid, however, which 

 react 10 % weaker in the diphenylamine reaction than an equivalent amount 

 of DNA, react 18 % weaker in either modification of the cysteine reaction. 

 Thymidylic acid, which shows a negligible reaction with diphenylamine, 

 reacts strongly in the cysteine reaction. The molar extinction coefficient 

 for the free thymidylic acid was found^* to be 74 % of that for the purine 

 nucleotides in the Stumpf modification. In the original form of the reaction, 

 however, the thymidylic acid, according to our own observations, shows a 

 completely different behavior. When the optical density is determined 

 immediately after the heating period, it is less than 20 % of that of an equiv- 

 alent amount of DNA. The intensity of the color, however, increases rap- 



>9 Z. Dische, Proc. Soc. Exptl. Biol. Med. 55, 217 (1944). 



20 P. Stumpf, /. Biol. Chem. 169, 367 (1947). 



21 L.A. Manson and J. O. Lampen, J. Biol. Chem. 191, 87 (1951). 



