Ribonuclease: A spectrophotometric assay using acridine orange-RNA complex

Abstract

Bovine pancreatic RNase can be accurately and sensitively assayed with RNA-acridine orange complex (1:1, wt/wt) as substrate. The enzyme is optimally assayed in 0.05 m sodium acetate buffer, pH 5.0, at 37°C with a RNA concentration of 0.300 mg/ml. The acridine orange released after 30 min incubation can be determined, after filtration, with a simple visible light spectrophotometer at 492 nm. The increase in absorbance at 492 nm was linear for final RNase concentrations of 0.05–0.6 μg/ml. The assay is applicable to crude cell-free extracts, but RNase T 1 was inactive in this assay.