COLOR REACTIONS OF NUCLEIC ACID COMPONENTS 301 



determinations and differentiation of various nucleotides by spectrophoto- 

 metric measurements in 1937.'^ Several modifications^'' of this reaction were 

 later developed which differ in the ratio between the solution and the re- 

 agent, and the composition of the latter, as regards the concentration of 

 HCl, FeCls , and orcinol. The original form of the reaction, as proposed in 

 1937, uses a higher HCl concentration than all later modifications and is, 

 therefore, the most sensitive of all. The most widely used modification 

 seems to be that proposed by Mejbaum^* in 1939 which uses 6 N HCl. 



Quantitative determination of PNA according to Dische and Schwartz:^^ — To 1.5 cc. 

 of a solution of ribonucleic acid is added 3 cc. of the reagent which is prepared by dis- 

 solving 100 mg. FeClreHjG in 100 cc. of HCl, sp. gr. 1.19, and adding 3.5 cc. of a 6% 

 solution of orcinol (twice recrystallized from benzene) in ethanol. The reaction mix- 

 ture is heated in a water bath for 3 minutes and cooled in tap water. A standard of 

 PNA and a blank containing water instead of the unknown is run simultaneously. 

 The optical density is measured at 665 m^ with the Beckman spectrophotometer 

 against a blank containing water and the reagent. A solution of PNA containing 40 

 Atg. per cc. shows an optical density of 0.18 which does not differ significantly from the 

 density of a solution of 20 Mg- per cc. of adenosine-3-phosphate. The optical density is 

 proportional to the concentration of PNA in the range between 10 and 100 /ig. per cc. 

 Quantitative determination of PNA according to Mejbaum:^^ — To 1 part of the un- 

 known is added an equal volume of concentrated HCl, sp. gr. 1.19, containing 0.1% 

 FeCl3-6H20 and 0.1% of orcinol. The mixture is heated for 20 minutes, or, according 

 to Albaum and Umbreit,^' for 45 minutes. A standard solution of PNA and a water 

 blank are run simultaneously and the optical density of the solution is measured at 

 the absorption maximum, which with the Beckman spectrophotometer lies at 670 m/x 



Specificity of the reaction: — In all the modifications of the Bial reaction 

 only the purine nucleotides and nucleosides react significantly. A more 

 involved procedure for the determination of pyrimidine nucleotides in 

 which bromination and prolonged heating with acid at 105° precedes the 

 performance of the Bial reaction, was developed by Massart and Hoste.^^ 

 Rial's orcinol reaction has a rather low specificity as not only pentoses, 

 but also 2-deoxyribose and DNA, methylpentose, and hexuronic acids give 

 a green color with an absorption maximum around 670 mju. Certain aldo- 

 heptoses which can occur in bacterial polysaccharides also produce a green 

 color with an absorption maximum around 655 m^.'^^ All these substances 

 can occur in tissue extracts as they are prepared for determination of PNA. 



Interference from other substances: — Aldo- and ketohexoses in free form 

 and in polysaccharides produce, in all modifications of Dial's orcinol re- 

 action, a reddish-brown color which can se^•erely interfere with the reaction 



^* Z. Dische and K. Schwarz, Mikrochim. Acta 2, 13 (1937). 



" G. L. Miller, R. H. Colder, and E. E. Miller, Anal. Chem. 23, 903 (1951). 



38 W. Mejbaum, Z. physiol. Chem. 258, 117 (1939). 



■39 H. G. Albaum and W. W. Umbreit, J. Biol. Chem. 167, 369 (1947). 



" Z. Dische, J. Biol. Chem. 204, 983 (1953). 



