Ribosome Depurination Assay for Ribosome Inactivating Protein Purification

The ribosome depurination assay has been frequently used to identify new ribosome inactivating proteins(RIPs).The assay measures a ribosomal rRNA fragment generated by acidic analine catalyzed cleavage at the RIP catalyzed depurination site.Using published first order kinetic rate constants for RIP catalyzed depurination,the sensitivity of the depurination assay to RIP concentration has been determined.For a 30 minute incubation time of the RIP with a ribosome containing solution,the sensitivity of the depurination assay depends on the rate constant for the RIP catalyzed ribosome depurination.In addition common sources of false positive and false negative results have been determined.These results indicate that use of the depurination assay as a quantitative analytical method in the commercial manufacture of RIPs is complex.In addition if the depurination assay is to be combined with a well established method such as SDS-PAGE,it is important that the minimum detection levels of the two methods be similar in order to prevent a positive result by one method and not the other.