Some Physical Properties of Interfering Substances associated with the Alkaline Hydrolysis of RNA from Plant Leaf Tissue

Abstract

PREVIOUS investigations¹ in our laboratory indicated that treatment of de-fatted, dechlorophyllized leaf tissue with alkali would hydrolyse the RNA into spectrophotometrically measurable nucleotides. Other studies² showed that RNA was preferentially eluted with 0.12 N hydrochloric acid from a column of ‘Dowex 1–8X’ on which mixtures of RNA and DNA had been adsorbed. This shortened exposure to alkali and recovery of RNA from mixtures of nucleic acid suggested the possibility of direct estimation of RNA in neutralized leaf hydrolysates by passage through a column of Dowex and subsequent elution with 0.12 N hydrochloric acid. Such eluates, however, invariably possessed greater absorption at 260 mμ than comparable material treated according to Diener and Lasheen³. The lower maxima/minima (Mx/Mn) ratios (A₂₆₀/A₂₃₄) for the neutralized material indicated that this higher absorbance was due to substances other than RNA. It was found that acidification of these alkaline hydrolysates followed by addition of an equal volume of ethanol and 2 drops of 1.0 M magnesium chloride would precipitate these substances absorbing at 260 mμ. Several portions of the hydrolysates, after removal of the extraneous material by centrifugation, were adjusted to pH's of 5.0, 6.0 or 7.0. Equal volumes of water, alcohol, and alcohol plus 2 drops of magnesium chloride were added to duplicates at each _pH and stored for at least 2 h at 2° C. These were then centrifuged to remove the precipitated substances and brought to volume with water and then 10-ml. portions were passed through the column. After washing the column with 5 ml. of double distilled water, the RNA was eluted with 0.12 N hydrochloric acid. The results of a typical experiment are given in Table 1.