Detection of Protein Oxidative Activity Using Reduced RNase A

This assay allows to determine whether proteins possess oxidative activity-the ability to introduce disulfide bond in vitro. The substrate for potential oxidases is a ribonuclease A which, for its activity, needs 4 properly formed disulfide bonds (Raines, 1998).RNase A activity can be detected by: Monitoring the digestion of RNA (Lambert and Freedman, 1983); Methylene Blue assay (Greiner-Stoeffele et al., 1996); Analyzing the cleavage of the cyclic CMP (Lyles and Gilbert, 1991; Lyles and Gilbert, 1991). We here describe method for measurements of oxidative activity, based on the cleavage of cCMP. Oxidative activity will be tested by measuring spectrophotometrically RNase A cleavage of cyclic-2’, 3’-cytidinemonophosphate (cCMP) to 3’-cytidinemonophosphate (3’ CMP), which results in an increase in absorption at 296 nm.The reaction equation: RNase A +2’ 3’-cCMP→RNase A + 3’ CMP.