COLOR REACTIONS OF NUCLEIC ACID COMPONENTS 287 



a. Reaction with Diphenylamine 



This reaction was first described in 1930^ and recommended for quanti- 

 tative determinations of DNA in animal tissues by colorimetry in white 

 light. In 1940 Seiberf^ adapted this method for quantitative determination 

 with a photoelectric colorimeter. The sensitivity of the reaction can be still 

 considerably increased by using a photoelectric spectrophotometer. 



Procedure: — The procedure generally used for determinations of DNA is as follows. 

 One volume of the solution containing 50 to 500 ng. DNA per cc. is mixed with a 

 double volume of a reagent, which is prepared by dissolving 1 g. of diphenylamine 

 (twice recrystallized from 70% ethanol or petroleum ether) in 100 cc. of glacial acetic 

 acid of highest purity. 2.75 cc. of concentrated sulfuric acid (reagent grade) is added 

 to 100 cc. of the diphenylamine solution. The reaction mixture is heated at 100° for at 

 least 10 minutes. A blank containing water instead of the unknown is run simulta- 

 neously. The DXA solution gives a blue color which does not change significantly for 

 hours. The absorption curve determined with the Beckman spectrophotometer shows 

 a sharp maximum at 595 m^. 



Influence of time of heating and concentration of the acid on the reaction: — Most 

 authors report that the color intensity reaches its maximum after 10 minutes' heating. 

 The color, however, develops very fast in the beginning, so that for qualitative pur- 

 poses a shorter heating of 3 minutes appears reasonable. Higher concentration of the 

 acid may increase the intensity of the color, but at the same time it decreases the 

 specificity. 



Specificity of the reaction: — ^The blue color developed in the diphenylamine 

 reaction was shown to be produced not only by DNA and 2-deoxyribose, 

 but also by 2-deoxyxylose-7 it is therefore a reaction of 2-deoxypentoses 

 and not only of 2-deoxyribose. Of the constituent mononucleotides of DNA, 

 the two purine nucleotides give the reaction with an intensity twice that of 

 the equivalent amount of DNA.^ Preparations of apurinic acid, prepared 

 and analyzed in Dr. Chargaff's laboratory,^ showed a 4 to 10% lower ab- 

 sorption at 595 mju than equivalent amounts of DNA. [Cf. Chargaff, Chapter 

 10.] This difference appeared already after 3 minutes' heating and did not 

 increase on further heating. It can be assumed that it is due to a slight 

 destruction of the sugar of the purine nucleotide during the preparation 

 of the apurinic acid. Deriaz et al}^ report that a DNA preparation from 

 fish sperm showed a slightly lower color intensity measured with the Spek- 

 ker absorptiometer than one-half of the equivalent amount of 2-deoxyri- 

 bose. Of the pyrimidine nucleotides, thymidylic acid in higher concen- 



6 Z. Dische, Mikrochemie 8, 4 (1930). 



6 F. B. Seibert, J. Biol. Ctiem. 133, 593 (1940). 



7 W. G. Overend, F. Shafizadeh, and M. Stacey, J. Chem. Soc. 1950, 1027. 

 * A. E. Mirsky, Advances in Enzymol. 3, 1 (1943). 



9 C. Tamm, M. E. Hodes, and E. Chargaff, J. Biol. Chem. 195, 49 (1952) . 

 10 R. E. Deriaz, M. Stacey, E. G. Teece, and L. F. Wiggins, J. Chem. Soc. 1949, 1222 



