302 ZACHARIAS DISCHE 



of PNA. Proteins and certain lipidic cell constituents can also interfere by 

 forming red products. 



Quantitative determination of PNA in presence of aldohexose: — When the amount 

 of glucose or its polymers reaches a certain level, the absorption at 670 mju due to the 

 brown reaction products can increase the total reading. If the amount of glucose is 

 not too high, it is possible to eliminate this discrepancy by dichromatic readings. 

 With Mejbaum's reagent, the procedure recommended by Brown^^ and by Drury" 

 is used. This consists in the simultaneous determination of pentose and glucose from 

 two equations obtained from two measurements of optical densities at two different 

 wavelengths. Using the procedure of Dische and Schwartz, after the determination 

 of optical density at 665 niju, the optical density is determined at the wavelength 

 around 565 mju, at which the optical density of a glucose standard run simultaneously 

 with the unknown shows the same optical density at 665 m^." The difference of De^b — 

 Dses of the experimental sample divided by this difference in the internal standard is 

 then a measure of the concentration of PNA. This difference is only about 25% lower 

 than Dees so that this procedure does not involve a large decrease in sensitivity of the 

 reaction. It is not possible, however, to use such a procedure in the presence of keto- 

 hexoses or ketoheptoses. 



h. Phloroglucinol reaction of Euler and Hahn** 



Procedure: — To 1 cc. of the unknown containing 2 mg. PNA is added 8 cc. of a 

 0.1% solution of FeCls in a mixture of 1 part of concentrated HCl and 6 parts of glacial 

 acetic acid. After stirring, the tubes are immersed for 50 minutes in a boiling water 

 bath and cooled to room temperature. 1 cc. of a 25% phloroglucinol solution in a mix- 

 ture of 1 part concentrated HCl, 1 part H2O, and 2 parts of glacial acetic acid is then 

 added and kept for 20 minutes at room temperature. The tubes with the reaction mix- 

 ture are then immersed in a boiling water bath for exactly 4 minutes, cooled to room 

 temperature, and so kept from 2 to 24 hours. The maximum of the color intensity ap- 

 pears after 10 hours. The absorption maximum is at 680 m^i read with the Beckman 

 spectrophotometer. 



Specificity of the reaction: — ^DNA does not give any color with this pro- 

 cedure; this, however, could be due to the prolonged heating before the 

 addition of phloroglucinol which, of course, destroys the sugar of the purine 

 nucleotides of DNA. No data were reported as to the reactivity of other 

 sugars and aldehydes. 



c. Reaction of Pentose with Cysteine and HiSOi 



PNA, its nucleotides and nucleosides all react in the general reaction of 

 carbohydrates with cysteine and sulfuric acid. The procedure was de- 

 scribed above under I.l b. The readings have to be carried out about 15 



« A. H. Brown, Arch. Biochem. 11, 269 (1946). 

 « H. F. Drury, Arch. Biochem. 19, 455 (1948). 

 *^ Z. Dische, G. Ehrlich, C. Munoz, and L. von Sallmann, A7n. J. Opthalmol. 36, 54 



(1953). 

 ** H. V. Euler and L. Hahn, Svensk Kem. Tidskr. 58, 251 (1946); Chem. Abstr. 41, 



2108 (1947). 



