A SENSITIVE METHOD FOR THE DETERMINATION OF RIBONUCLEIC ACID IN TISSUES AND MICROORGANISMS

Abstract

The increasing interest in the composition of nucleoproteins has emphasized the need for more specific methods of assay of constituent nucleic acids. In a previous paper (1) a method was reported for the assay of deoxyribonucleic acid (DNA) in tissues and microorganisms based on the reaction of p-nitrophenylhydrazine with deoxyribose following hydrolysis of the biological material with 5 per cent trichloroacetic acid (TCA). The present paper reports a method for ribonucleic acid (RNA) assay with an aliquot of the same TCA hydrolysate. The most commonly employed method for RNA assay is that of Mejbaum (a), in which orcinol is used to develop a blue-green color with furfural formed from ribose when the latter is heated with prescribed amounts of FeCl,-HCl solution. The results must be corrected for DNA interference (3, 4). In a similar method developed by von Euler and Hahn (4) phloroglucinol is employed. DNA does not interfere, but the method is less sensitive than the orcinol procedure. In a third method, used by Davidson and Waymouth (5), the furfural formed from ribose is extracted with xylene and the xylene extract is allowed to react with aniline acetate to form a pink color. Although DNA does not interfere, the method is complicated by the necessity of removing HCl prior to color formation and the photosensitivity of the color (6). However, the extraction procedure offers certain advantages, namely (1) concentration of the furfural formed and (2) elimination or minimization of interfering substances. In an effort to combine these advantages with a more efficient chromogenic reagent, a method has been developed in which p-bromophenylhydrazine (PBPH) reacts quantitatively in acidified xylene-alcohol solution with furfural formed from the liberated ribose of RNA. As seen below, the specific extinction coefficient of the colored product formed with the proposed reagent is about l$ times that of the colored product resulting from the Mejbaum or&no1 procedure when optical densities are taken at their respective wave-lengths of maximal absorption. The color is stable and reproducible. Experiments performed with several carbohy-