Model Substrates for the Rapid Assay of Specific Ribonuclease Activity

Publisher Website
Google Scholar
Abstract
A new procedure is proposed for the assay of specific ribonuclease activity. The substrates are [14C]‐labeled benzyl esters of 3′‐ribonucleotides. Methods are described for preparing pure 3′‐uridylic acid, 3′‐cytidylie acid, 3′‐adenylie acid and 3′‐guanylic acid, and for the synthesis of [14C]phenldiazomethane. By reaction of the latter with the former, the four 3′‐ribonucleotide [14C]benzyl esters are readily obtained.After enzymatic reaction of a ribonuclease preparation with one of the model substrates, the released [14C]benzyl alcohol is simply extracted by mixing with toluene. The radioactivity of the organic phase is then measured by liquid scintillation counting. The method has been used to detect contaminating nuclease activity in pancreatic ribonuclease preparations (specific for 3′‐uridylic acid and 3′‐cyidylic acid esters), and in Aspergillus T1 ribonuclease preparations (specific for 3′‐guanylic acid esters).