An assay for ribonuclease activity, based on ultraviolet absorption of RNA hydrolysate, using phosphotungstic acid

In the method for the determination of ribonuclease activity that depends on the ultraviolet absorption of the RNA hydrolysate, the uranium reagent (25% perchloric acid solution containing 0.75% uranyl acetate) is commonly used for the efficient precipitation of the unhydrolyzed RNA. However, this reagent is always contaminated by the presence of radioactive isotopes. Radioactive uranium is one of the substances used for atomic nuclear fuel and therefore, at least in Japan, the use of uranium compounds requires permission from the government. We tried to find another efficient and non-radioactive precipitant of RNA to replace the uranium reagent, and have developed a phosphotungsten reagent (25% perchloric acid solution containing 0.75% phosphotungstic acid plus 0.6% bovine serum albumin solution) which functions as efficiently as the uranium reagent in the precipitation of RNA. A cell-free crude extract of Dictyostelium discoideum was used as the source of ribonuclease.