POSITIVE ZYMOGRAM METHOD FOR RIBONUCLEASE

We have developed a rapid, sensitive, and specific zymogram for detecting ribonuclease (RNase). The method makes use of an agarose gel containing the small substrate UpA [uridylyl (3′ → 5′)-adenosine]. UpA is hydrolyzed by RNase to adenosine, which is deaminated by adenosine deaminase. The inosine so formed is linked by a series of enzymatic reactions (nucleoside phosphorylase, xanthine oxidase) to formation of a blue tetrazolium salt. This method is superior in that it entails a staining reaction only at sites of RNase activity (positive zymogram) rather than clearing of a background of RNA (negative zymogram), a process which is often mimicked by protein devoid of RNase activity. © 1979.