Effects of Ionic Strength on Ribonucleic Acid Structure Akd Ribonuclease Activity

Beef pancreas ribonuclease activity is markedly accelerated by increased ionic strength (14). Since certain physical properties of nucleic acids are known to be altered by this factor, it becomes of interest to determine whether salt concentration affects primarily the enzyme, the substrate, or both. A second factor of importance in ribonuclease (RNase) assays is that of substrate concentration. A suggestion of the influence that substrate concentration may exert on the phosphodiesterase activity of RNase can be gained from a consideration of the sensitivities of three published assays (4-6). As the data of Table I demonstrate, there is an inverse relationship between ribonucleic acid concentration and sensitivity when beef pancreas ribonuclease is assayed at pH 5.0. A quantitative investigation of the effects of ionic strength and ribonucleic acid (RNA) concentration on RNase activity, as determined by two different types of procedure, has been carried out. One assay is based on the determination of t,he hydrolytic products of the reaction (phosphodiesterase activity) (4) and the other on the decrease in absorbance at 300 rnp (A& of an RNA solution as a function of time (spectrophotometric procedure) (7). The result,s indicate that both factors are capable of exerting profound effects on ribonuclease measurements with either type of assay. Differences in the effects of ionic strength and substrate concentration on the spectrophotometric and the phosphodiesterase assays suggested that the reaction being measured was different in the two assays. This hypothesis was strengthened by the observation that even in the absence of RNase the AaoO of ribonucleic acid solutions was immediately decreased when the ionic strengt.h of t,he solution was increased. This effect suggests t,hat some type of non-hydrolytic alteration has occurred. Further work will be necessary to det.ermine whether the decrease in absorbance at 300 rnp caused by ribonuclease or by increased ionic strength is due to the same structural change. Vandendriessche (8) observed that the addition of RNase to a solution of RNA caused a temporary increase in the volume