COLOR REACTIONS OF NUCLEIC ACID COMPONENTS 297 



Quantitative determination of DN A in the presence of other substances: — The color 

 developed in the reaction is proportional to the concentration of DNA in the range of 

 0.01 to 0.05% when determined with a Klett photoelectric colorimeter. Aldehydes, 

 fructose and its derivatives can interfere, however, with the reaction. The protein 

 moiety in nucleoproteins interferes by creating a turbidity and producing unspecific 

 colors due to the protein constituents. The first impediment can be eliminated by 

 filtration; the second by extraction with isoamyl alcohol of a boiling point of 130-132°, 

 which does not extract the color produced by histone or protamine constituents but 

 extracts completely the color due to deoxyribose. The standard solution, of course, 

 must be treated in an identical way. Other proteins, however, may interfere and the 

 extraction with isoamyl alcohol may be of no avail. 



d. Reaction of DNA and 2-deoxyrihose with Indole and HCl 



This reaction was first described in 1929." As the original procedure 

 shows much lower sensitivity than other reactions, Ceriotti^® modified it by 

 increasing the acid concentration. This more sensitive modification, how- 

 ever, appears to be less specific. 



Original procedure: — To 1 part of a solution containing 0.5 to 2.5 mg. per cc. of 

 DNA, are added an equal volume of 1% HCl and 0.1 cc. of a 1% solution of indole in 

 ethanol. The mixture is heated for 5 minutes in a boiling water bath. An intensive 

 reddish-brown color appears. The reaction mixture is thoroughly shaken with an 

 equal volume of chloroform, whereupon the water phase becomes jellow-brown while 

 reddish solid particles accumulate at the interphase. 



Specificity of the reaction: — The reaction is produced only by the purine 

 nucleotides of DNA, as destruction of the sugar of purine nucleotides by 

 protracted heating in 2% sulfuric acid destroys the reactivity of DNA. 

 Pentoses and hexoses even at 2.5%, or ribonucleic acid at 0.25%, do not 

 produce any visible color in the water phase. On the other hand, glucosone 

 and the reaction products obtained by deamination of glucosamine with 

 nitrite (probably 2 , 5-anhydromannose) give the same yellowish-brown 

 color as DNA. 



Procedure of Ceriotti for the quantitative determination of DNA: — To 2 cc. of a solu- 

 tion containing 2.5 to 15 ng. per cc. of DNA, are added 1 cc. of 0.04% indole C.P. 

 solution in distilled water and 1 cc. of concentrated HCl (sp. gr. 1.19). The test tube 

 is immersed for 10 minutes in a boiling water bath and cooled under running water, 

 the solution is extracted 3 times with 4 cc. of chloroform, and the water separated by 

 centrifugation from the chloroform. As the purity of the chloroform is of the utmost 

 importance, it should be purified bj' repeated extraction with concentrated H2SO4 , 

 followed by water extraction, and then freed of water by keeping it for 48 hours over 

 CaCh . Finally, it is distilled to give a product with the boiling point of 61 °C. A 

 yellow color appears in the water phase which is read with the Beckman spectrophotom- 

 eter against a blank treated in an identical manner. A faint pink color appears in the 

 chloroform phase. The color given in the water phase is stable for several hours. On 

 long standing at room temperature, a pink color forms which can be again removed 



" Z. Dische, Biochem. Z. 204, 431 (1929). 

 " A. Ceriotti, J. Biol. Chem. 198, 297 (1952). 



